CA2703428A1 - Pooled cord blood units - Google Patents
Pooled cord blood units Download PDFInfo
- Publication number
- CA2703428A1 CA2703428A1 CA 2703428 CA2703428A CA2703428A1 CA 2703428 A1 CA2703428 A1 CA 2703428A1 CA 2703428 CA2703428 CA 2703428 CA 2703428 A CA2703428 A CA 2703428A CA 2703428 A1 CA2703428 A1 CA 2703428A1
- Authority
- CA
- Canada
- Prior art keywords
- cord blood
- umbilical cord
- blood
- cells
- pooled
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 210000004700 fetal blood Anatomy 0.000 title claims abstract description 266
- 210000004027 cell Anatomy 0.000 claims abstract description 154
- 210000004369 blood Anatomy 0.000 claims abstract description 105
- 239000008280 blood Substances 0.000 claims abstract description 105
- 238000000034 method Methods 0.000 claims abstract description 95
- 210000003958 hematopoietic stem cell Anatomy 0.000 claims abstract description 68
- 238000011176 pooling Methods 0.000 claims abstract description 68
- 230000009089 cytolysis Effects 0.000 claims abstract description 14
- 239000008194 pharmaceutical composition Substances 0.000 claims abstract description 8
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 claims description 53
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 claims description 53
- 238000002054 transplantation Methods 0.000 claims description 44
- 239000000427 antigen Substances 0.000 claims description 29
- 102000036639 antigens Human genes 0.000 claims description 29
- 108091007433 antigens Proteins 0.000 claims description 29
- 239000000203 mixture Substances 0.000 claims description 26
- 238000000338 in vitro Methods 0.000 claims description 14
- 238000000746 purification Methods 0.000 claims description 14
- 238000010257 thawing Methods 0.000 claims description 11
- 239000003937 drug carrier Substances 0.000 claims description 3
- 239000003112 inhibitor Substances 0.000 claims description 3
- 230000000779 depleting effect Effects 0.000 claims description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 38
- 210000002381 plasma Anatomy 0.000 description 29
- 238000001802 infusion Methods 0.000 description 28
- 239000000243 solution Substances 0.000 description 25
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 24
- 210000000130 stem cell Anatomy 0.000 description 24
- 210000003743 erythrocyte Anatomy 0.000 description 22
- 201000010099 disease Diseases 0.000 description 20
- 238000012545 processing Methods 0.000 description 20
- 238000010790 dilution Methods 0.000 description 18
- 239000012895 dilution Substances 0.000 description 18
- 210000000265 leukocyte Anatomy 0.000 description 17
- 102000009027 Albumins Human genes 0.000 description 16
- 108010088751 Albumins Proteins 0.000 description 16
- 210000000440 neutrophil Anatomy 0.000 description 16
- 238000005138 cryopreservation Methods 0.000 description 15
- 238000011282 treatment Methods 0.000 description 14
- 208000009329 Graft vs Host Disease Diseases 0.000 description 10
- 108060003951 Immunoglobulin Proteins 0.000 description 10
- 208000024908 graft versus host disease Diseases 0.000 description 10
- 102000018358 immunoglobulin Human genes 0.000 description 10
- 238000012360 testing method Methods 0.000 description 10
- 210000004291 uterus Anatomy 0.000 description 10
- 238000011038 discontinuous diafiltration by volume reduction Methods 0.000 description 9
- 208000032839 leukemia Diseases 0.000 description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 8
- 102000017420 CD3 protein, epsilon/gamma/delta subunit Human genes 0.000 description 8
- 108050005493 CD3 protein, epsilon/gamma/delta subunit Proteins 0.000 description 8
- 108010047041 Complementarity Determining Regions Proteins 0.000 description 8
- 102000008100 Human Serum Albumin Human genes 0.000 description 8
- 108091006905 Human Serum Albumin Proteins 0.000 description 8
- 210000001185 bone marrow Anatomy 0.000 description 8
- 239000002577 cryoprotective agent Substances 0.000 description 8
- 230000008774 maternal effect Effects 0.000 description 8
- 239000000523 sample Substances 0.000 description 8
- 229920001612 Hydroxyethyl starch Polymers 0.000 description 7
- 239000012634 fragment Substances 0.000 description 7
- 238000007710 freezing Methods 0.000 description 7
- 229940050526 hydroxyethylstarch Drugs 0.000 description 7
- 229940072221 immunoglobulins Drugs 0.000 description 7
- 208000002491 severe combined immunodeficiency Diseases 0.000 description 7
- 208000032791 BCR-ABL1 positive chronic myelogenous leukemia Diseases 0.000 description 6
- 208000010833 Chronic myeloid leukaemia Diseases 0.000 description 6
- 206010010099 Combined immunodeficiency Diseases 0.000 description 6
- 206010010356 Congenital anomaly Diseases 0.000 description 6
- CMSMOCZEIVJLDB-UHFFFAOYSA-N Cyclophosphamide Chemical compound ClCCN(CCCl)P1(=O)NCCCO1 CMSMOCZEIVJLDB-UHFFFAOYSA-N 0.000 description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 208000033761 Myelogenous Chronic BCR-ABL Positive Leukemia Diseases 0.000 description 6
- 210000001744 T-lymphocyte Anatomy 0.000 description 6
- 230000001154 acute effect Effects 0.000 description 6
- 238000010322 bone marrow transplantation Methods 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 5
- 206010028980 Neoplasm Diseases 0.000 description 5
- 230000030833 cell death Effects 0.000 description 5
- 208000032852 chronic lymphocytic leukemia Diseases 0.000 description 5
- 238000001816 cooling Methods 0.000 description 5
- 230000000875 corresponding effect Effects 0.000 description 5
- 229960004397 cyclophosphamide Drugs 0.000 description 5
- 230000007423 decrease Effects 0.000 description 5
- 238000010828 elution Methods 0.000 description 5
- 230000008014 freezing Effects 0.000 description 5
- 230000003394 haemopoietic effect Effects 0.000 description 5
- 230000001400 myeloablative effect Effects 0.000 description 5
- 239000008188 pellet Substances 0.000 description 5
- 210000002826 placenta Anatomy 0.000 description 5
- 210000002966 serum Anatomy 0.000 description 5
- 238000003860 storage Methods 0.000 description 5
- 208000011580 syndromic disease Diseases 0.000 description 5
- 238000005303 weighing Methods 0.000 description 5
- RSGFPIWWSCWCFJ-VAXZQHAWSA-N 2-hydroxypropane-1,2,3-tricarboxylic acid;(2r,3s,4r,5r)-2,3,4,5,6-pentahydroxyhexanal;phosphoric acid Chemical compound OP(O)(O)=O.OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O.OC(=O)CC(O)(C(O)=O)CC(O)=O RSGFPIWWSCWCFJ-VAXZQHAWSA-N 0.000 description 4
- 208000036762 Acute promyelocytic leukaemia Diseases 0.000 description 4
- 208000010839 B-cell chronic lymphocytic leukemia Diseases 0.000 description 4
- UHDGCWIWMRVCDJ-CCXZUQQUSA-N Cytarabine Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@@H](O)[C@H](O)[C@@H](CO)O1 UHDGCWIWMRVCDJ-CCXZUQQUSA-N 0.000 description 4
- 208000002250 Hematologic Neoplasms Diseases 0.000 description 4
- 208000033826 Promyelocytic Acute Leukemia Diseases 0.000 description 4
- 230000001668 ameliorated effect Effects 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 230000006378 damage Effects 0.000 description 4
- 230000007812 deficiency Effects 0.000 description 4
- 208000035475 disorder Diseases 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 229960000390 fludarabine Drugs 0.000 description 4
- GIUYCYHIANZCFB-FJFJXFQQSA-N fludarabine phosphate Chemical compound C1=NC=2C(N)=NC(F)=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@@H]1O GIUYCYHIANZCFB-FJFJXFQQSA-N 0.000 description 4
- 210000000777 hematopoietic system Anatomy 0.000 description 4
- 230000003211 malignant effect Effects 0.000 description 4
- GLVAUDGFNGKCSF-UHFFFAOYSA-N mercaptopurine Chemical compound S=C1NC=NC2=C1NC=N2 GLVAUDGFNGKCSF-UHFFFAOYSA-N 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- 230000003169 placental effect Effects 0.000 description 4
- 230000001172 regenerating effect Effects 0.000 description 4
- 230000004083 survival effect Effects 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 238000012546 transfer Methods 0.000 description 4
- 230000035899 viability Effects 0.000 description 4
- 208000024893 Acute lymphoblastic leukemia Diseases 0.000 description 3
- 208000014697 Acute lymphocytic leukaemia Diseases 0.000 description 3
- 208000031261 Acute myeloid leukaemia Diseases 0.000 description 3
- COVZYZSDYWQREU-UHFFFAOYSA-N Busulfan Chemical compound CS(=O)(=O)OCCCCOS(C)(=O)=O COVZYZSDYWQREU-UHFFFAOYSA-N 0.000 description 3
- 206010068051 Chimerism Diseases 0.000 description 3
- 206010053567 Coagulopathies Diseases 0.000 description 3
- 206010053138 Congenital aplastic anaemia Diseases 0.000 description 3
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 3
- 201000004939 Fanconi anemia Diseases 0.000 description 3
- 238000012413 Fluorescence activated cell sorting analysis Methods 0.000 description 3
- 208000036066 Hemophagocytic Lymphohistiocytosis Diseases 0.000 description 3
- 241000282412 Homo Species 0.000 description 3
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 description 3
- 206010052210 Infantile genetic agranulocytosis Diseases 0.000 description 3
- 208000009625 Lesch-Nyhan syndrome Diseases 0.000 description 3
- 208000031422 Lymphocytic Chronic B-Cell Leukemia Diseases 0.000 description 3
- 206010025323 Lymphomas Diseases 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 208000008955 Mucolipidoses Diseases 0.000 description 3
- 206010056886 Mucopolysaccharidosis I Diseases 0.000 description 3
- 208000006664 Precursor Cell Lymphoblastic Leukemia-Lymphoma Diseases 0.000 description 3
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 description 3
- 230000004520 agglutination Effects 0.000 description 3
- 239000003146 anticoagulant agent Substances 0.000 description 3
- 229940127219 anticoagulant drug Drugs 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 229960002092 busulfan Drugs 0.000 description 3
- 230000006037 cell lysis Effects 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 230000035602 clotting Effects 0.000 description 3
- 230000024203 complement activation Effects 0.000 description 3
- 230000003750 conditioning effect Effects 0.000 description 3
- 229960000684 cytarabine Drugs 0.000 description 3
- 208000037765 diseases and disorders Diseases 0.000 description 3
- 239000007789 gas Substances 0.000 description 3
- 210000002360 granulocyte-macrophage progenitor cell Anatomy 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 239000000644 isotonic solution Substances 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 229920001184 polypeptide Polymers 0.000 description 3
- 230000035935 pregnancy Effects 0.000 description 3
- 238000004321 preservation Methods 0.000 description 3
- 102000004196 processed proteins & peptides Human genes 0.000 description 3
- 108090000765 processed proteins & peptides Proteins 0.000 description 3
- 238000003908 quality control method Methods 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000002560 therapeutic procedure Methods 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- KMZHZAAOEWVPSE-UHFFFAOYSA-N 2,3-dihydroxypropyl acetate Chemical compound CC(=O)OCC(O)CO KMZHZAAOEWVPSE-UHFFFAOYSA-N 0.000 description 2
- DLFVBJFMPXGRIB-UHFFFAOYSA-N Acetamide Chemical compound CC(N)=O DLFVBJFMPXGRIB-UHFFFAOYSA-N 0.000 description 2
- 102000007469 Actins Human genes 0.000 description 2
- 108010085238 Actins Proteins 0.000 description 2
- 208000032467 Aplastic anaemia Diseases 0.000 description 2
- 206010068220 Aspartylglucosaminuria Diseases 0.000 description 2
- 208000032800 BCR-ABL1 positive blast phase chronic myelogenous leukemia Diseases 0.000 description 2
- 208000004860 Blast Crisis Diseases 0.000 description 2
- 102000013925 CD34 antigen Human genes 0.000 description 2
- 108050003733 CD34 antigen Proteins 0.000 description 2
- 101710098119 Chaperonin GroEL 2 Proteins 0.000 description 2
- 108010034753 Complement Membrane Attack Complex Proteins 0.000 description 2
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 2
- 201000004449 Diamond-Blackfan anemia Diseases 0.000 description 2
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 2
- 208000015872 Gaucher disease Diseases 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 108090000288 Glycoproteins Proteins 0.000 description 2
- 102000003886 Glycoproteins Human genes 0.000 description 2
- 206010018910 Haemolysis Diseases 0.000 description 2
- 208000017604 Hodgkin disease Diseases 0.000 description 2
- 208000021519 Hodgkin lymphoma Diseases 0.000 description 2
- 208000010747 Hodgkins lymphoma Diseases 0.000 description 2
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 2
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 2
- 208000026350 Inborn Genetic disease Diseases 0.000 description 2
- 239000005517 L01XE01 - Imatinib Substances 0.000 description 2
- 208000030289 Lymphoproliferative disease Diseases 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 208000033776 Myeloid Acute Leukemia Diseases 0.000 description 2
- 208000037538 Myelomonocytic Juvenile Leukemia Diseases 0.000 description 2
- 208000015914 Non-Hodgkin lymphomas Diseases 0.000 description 2
- 206010033661 Pancytopenia Diseases 0.000 description 2
- 206010035226 Plasma cell myeloma Diseases 0.000 description 2
- 101000588258 Taenia solium Paramyosin Proteins 0.000 description 2
- 208000002903 Thalassemia Diseases 0.000 description 2
- 208000035317 Total hypoxanthine-guanine phosphoribosyl transferase deficiency Diseases 0.000 description 2
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 2
- 201000009628 adenosine deaminase deficiency Diseases 0.000 description 2
- 230000000735 allogeneic effect Effects 0.000 description 2
- 125000003275 alpha amino acid group Chemical group 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 230000010056 antibody-dependent cellular cytotoxicity Effects 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 208000036556 autosomal recessive T cell-negative B cell-negative NK cell-negative due to adenosine deaminase deficiency severe combined immunodeficiency Diseases 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- -1 carrier Substances 0.000 description 2
- 238000002659 cell therapy Methods 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000002512 chemotherapy Methods 0.000 description 2
- 230000001684 chronic effect Effects 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 230000002596 correlated effect Effects 0.000 description 2
- 125000000151 cysteine group Chemical class N[C@@H](CS)C(=O)* 0.000 description 2
- 230000003111 delayed effect Effects 0.000 description 2
- 239000008121 dextrose Substances 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 239000000539 dimer Substances 0.000 description 2
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 2
- VJJPUSNTGOMMGY-MRVIYFEKSA-N etoposide Chemical compound COC1=C(O)C(OC)=CC([C@@H]2C3=CC=4OCOC=4C=C3[C@@H](O[C@H]3[C@@H]([C@@H](O)[C@@H]4O[C@H](C)OC[C@H]4O3)O)[C@@H]3[C@@H]2C(OC3)=O)=C1 VJJPUSNTGOMMGY-MRVIYFEKSA-N 0.000 description 2
- 229960005420 etoposide Drugs 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 208000029944 familial hemophagocytic lymphohistiocytosis type 1 Diseases 0.000 description 2
- 208000001031 fetal erythroblastosis Diseases 0.000 description 2
- 239000012467 final product Substances 0.000 description 2
- 208000016361 genetic disease Diseases 0.000 description 2
- 229940080856 gleevec Drugs 0.000 description 2
- KTUFNOKKBVMGRW-UHFFFAOYSA-N imatinib Chemical compound C1CN(C)CCN1CC1=CC=C(C(=O)NC=2C=C(NC=3N=C(C=CN=3)C=3C=NC=CC=3)C(C)=CC=2)C=C1 KTUFNOKKBVMGRW-UHFFFAOYSA-N 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 201000005992 juvenile myelomonocytic leukemia Diseases 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 230000036210 malignancy Effects 0.000 description 2
- 229960001428 mercaptopurine Drugs 0.000 description 2
- 239000011325 microbead Substances 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 206010028537 myelofibrosis Diseases 0.000 description 2
- 230000003204 osmotic effect Effects 0.000 description 2
- 230000002093 peripheral effect Effects 0.000 description 2
- 239000000546 pharmaceutical excipient Substances 0.000 description 2
- 210000004214 philadelphia chromosome Anatomy 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000000135 prohibitive effect Effects 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 201000007153 reticular dysgenesis Diseases 0.000 description 2
- 208000007056 sickle cell anemia Diseases 0.000 description 2
- 210000003491 skin Anatomy 0.000 description 2
- 239000007858 starting material Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 206010043554 thrombocytopenia Diseases 0.000 description 2
- 210000003954 umbilical cord Anatomy 0.000 description 2
- HJTAZXHBEBIQQX-UHFFFAOYSA-N 1,5-bis(chloromethyl)naphthalene Chemical compound C1=CC=C2C(CCl)=CC=CC2=C1CCl HJTAZXHBEBIQQX-UHFFFAOYSA-N 0.000 description 1
- 239000001763 2-hydroxyethyl(trimethyl)azanium Substances 0.000 description 1
- 208000010543 22q11.2 deletion syndrome Diseases 0.000 description 1
- STQGQHZAVUOBTE-UHFFFAOYSA-N 7-Cyan-hept-2t-en-4,6-diinsaeure Natural products C1=2C(O)=C3C(=O)C=4C(OC)=CC=CC=4C(=O)C3=C(O)C=2CC(O)(C(C)=O)CC1OC1CC(N)C(O)C(C)O1 STQGQHZAVUOBTE-UHFFFAOYSA-N 0.000 description 1
- YXHLJMWYDTXDHS-IRFLANFNSA-N 7-aminoactinomycin D Chemical compound C[C@H]1OC(=O)[C@H](C(C)C)N(C)C(=O)CN(C)C(=O)[C@@H]2CCCN2C(=O)[C@@H](C(C)C)NC(=O)[C@H]1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=C(N)C=C3C(=O)N[C@@H]4C(=O)N[C@@H](C(N5CCC[C@H]5C(=O)N(C)CC(=O)N(C)[C@@H](C(C)C)C(=O)O[C@@H]4C)=O)C(C)C)=C3N=C21 YXHLJMWYDTXDHS-IRFLANFNSA-N 0.000 description 1
- 108700012813 7-aminoactinomycin D Proteins 0.000 description 1
- 206010000206 ABO incompatibility Diseases 0.000 description 1
- 102100024643 ATP-binding cassette sub-family D member 1 Human genes 0.000 description 1
- 201000011452 Adrenoleukodystrophy Diseases 0.000 description 1
- 201000010000 Agranulocytosis Diseases 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 206010002965 Aplasia pure red cell Diseases 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 238000012935 Averaging Methods 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- GUBGYTABKSRVRQ-DCSYEGIMSA-N Beta-Lactose Chemical compound OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-DCSYEGIMSA-N 0.000 description 1
- 201000004569 Blindness Diseases 0.000 description 1
- 208000005692 Bloom Syndrome Diseases 0.000 description 1
- 208000010392 Bone Fractures Diseases 0.000 description 1
- 208000018240 Bone Marrow Failure disease Diseases 0.000 description 1
- 208000020084 Bone disease Diseases 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 208000011691 Burkitt lymphomas Diseases 0.000 description 1
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 1
- 108700007522 CCG-141 protocol Proteins 0.000 description 1
- 201000007155 CD40 ligand deficiency Diseases 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 201000009030 Carcinoma Diseases 0.000 description 1
- 235000019743 Choline chloride Nutrition 0.000 description 1
- 206010008909 Chronic Hepatitis Diseases 0.000 description 1
- 208000031879 Chédiak-Higashi syndrome Diseases 0.000 description 1
- 201000003874 Common Variable Immunodeficiency Diseases 0.000 description 1
- 108010069112 Complement System Proteins Proteins 0.000 description 1
- 102000000989 Complement System Proteins Human genes 0.000 description 1
- 206010062759 Congenital dyskeratosis Diseases 0.000 description 1
- 208000034958 Congenital erythropoietic porphyria Diseases 0.000 description 1
- 208000025212 Constitutional neutropenia Diseases 0.000 description 1
- 102100025621 Cytochrome b-245 heavy chain Human genes 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- 206010011878 Deafness Diseases 0.000 description 1
- 206010051396 Delayed engraftment Diseases 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 208000000398 DiGeorge Syndrome Diseases 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- UNXHWFMMPAWVPI-UHFFFAOYSA-N Erythritol Natural products OCC(O)C(O)CO UNXHWFMMPAWVPI-UHFFFAOYSA-N 0.000 description 1
- 208000007209 Erythropoietic Porphyria Diseases 0.000 description 1
- 102000003951 Erythropoietin Human genes 0.000 description 1
- 108090000394 Erythropoietin Proteins 0.000 description 1
- 208000006168 Ewing Sarcoma Diseases 0.000 description 1
- 208000026019 Fanconi renotubular syndrome Diseases 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 208000025499 G6PD deficiency Diseases 0.000 description 1
- 208000009796 Gangliosidoses Diseases 0.000 description 1
- 208000010055 Globoid Cell Leukodystrophy Diseases 0.000 description 1
- 206010018444 Glucose-6-phosphate dehydrogenase deficiency Diseases 0.000 description 1
- 102000004269 Granulocyte Colony-Stimulating Factor Human genes 0.000 description 1
- 108010017080 Granulocyte Colony-Stimulating Factor Proteins 0.000 description 1
- 208000031886 HIV Infections Diseases 0.000 description 1
- 208000037357 HIV infectious disease Diseases 0.000 description 1
- 102000001554 Hemoglobins Human genes 0.000 description 1
- 108010054147 Hemoglobins Proteins 0.000 description 1
- 208000032843 Hemorrhage Diseases 0.000 description 1
- 208000001825 Hereditary elliptocytosis Diseases 0.000 description 1
- 208000032672 Histiocytosis haematophagic Diseases 0.000 description 1
- 101001078143 Homo sapiens Integrin alpha-IIb Proteins 0.000 description 1
- 101001015004 Homo sapiens Integrin beta-3 Proteins 0.000 description 1
- 101001057504 Homo sapiens Interferon-stimulated gene 20 kDa protein Proteins 0.000 description 1
- 101001055144 Homo sapiens Interleukin-2 receptor subunit alpha Proteins 0.000 description 1
- 101000581981 Homo sapiens Neural cell adhesion molecule 1 Proteins 0.000 description 1
- 101000980827 Homo sapiens T-cell surface glycoprotein CD1a Proteins 0.000 description 1
- 101000716149 Homo sapiens T-cell surface glycoprotein CD1b Proteins 0.000 description 1
- 101000716124 Homo sapiens T-cell surface glycoprotein CD1c Proteins 0.000 description 1
- 101001067100 Homo sapiens Uroporphyrinogen-III synthase Proteins 0.000 description 1
- 208000015178 Hurler syndrome Diseases 0.000 description 1
- 208000015204 Hurler-Scheie syndrome Diseases 0.000 description 1
- 208000018121 Hypoxanthine-guanine phosphoribosyltransferase deficiency Diseases 0.000 description 1
- XDXDZDZNSLXDNA-TZNDIEGXSA-N Idarubicin Chemical compound C1[C@H](N)[C@H](O)[C@H](C)O[C@H]1O[C@@H]1C2=C(O)C(C(=O)C3=CC=CC=C3C3=O)=C3C(O)=C2C[C@@](O)(C(C)=O)C1 XDXDZDZNSLXDNA-TZNDIEGXSA-N 0.000 description 1
- XDXDZDZNSLXDNA-UHFFFAOYSA-N Idarubicin Natural products C1C(N)C(O)C(C)OC1OC1C2=C(O)C(C(=O)C3=CC=CC=C3C3=O)=C3C(O)=C2CC(O)(C(C)=O)C1 XDXDZDZNSLXDNA-UHFFFAOYSA-N 0.000 description 1
- 208000001911 Idiopathic aplastic anemia Diseases 0.000 description 1
- 206010061598 Immunodeficiency Diseases 0.000 description 1
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 1
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 1
- 102100025306 Integrin alpha-IIb Human genes 0.000 description 1
- 102100032999 Integrin beta-3 Human genes 0.000 description 1
- 102100027268 Interferon-stimulated gene 20 kDa protein Human genes 0.000 description 1
- 206010022822 Intravascular haemolysis Diseases 0.000 description 1
- 208000012565 Kostmann syndrome Diseases 0.000 description 1
- 208000028226 Krabbe disease Diseases 0.000 description 1
- 239000005536 L01XE08 - Nilotinib Substances 0.000 description 1
- 201000005099 Langerhans cell histiocytosis Diseases 0.000 description 1
- 206010064912 Malignant transformation Diseases 0.000 description 1
- 108010049137 Member 1 Subfamily D ATP Binding Cassette Transporter Proteins 0.000 description 1
- 201000011442 Metachromatic leukodystrophy Diseases 0.000 description 1
- 108091092878 Microsatellite Proteins 0.000 description 1
- 206010072927 Mucolipidosis type I Diseases 0.000 description 1
- 206010072928 Mucolipidosis type II Diseases 0.000 description 1
- 208000002678 Mucopolysaccharidoses Diseases 0.000 description 1
- 206010056893 Mucopolysaccharidosis VII Diseases 0.000 description 1
- 208000025915 Mucopolysaccharidosis type 6 Diseases 0.000 description 1
- 208000006670 Multiple fractures Diseases 0.000 description 1
- 208000034578 Multiple myelomas Diseases 0.000 description 1
- 208000033833 Myelomonocytic Chronic Leukemia Diseases 0.000 description 1
- 208000028389 Nerve injury Diseases 0.000 description 1
- 102100027347 Neural cell adhesion molecule 1 Human genes 0.000 description 1
- 206010029260 Neuroblastoma Diseases 0.000 description 1
- 201000008962 Nezelof syndrome Diseases 0.000 description 1
- 201000007142 Omenn syndrome Diseases 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 206010033128 Ovarian cancer Diseases 0.000 description 1
- 108090000526 Papain Proteins 0.000 description 1
- 208000018737 Parkinson disease Diseases 0.000 description 1
- 208000000733 Paroxysmal Hemoglobinuria Diseases 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 102100036050 Phosphatidylinositol N-acetylglucosaminyltransferase subunit A Human genes 0.000 description 1
- 208000013544 Platelet disease Diseases 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 241000097929 Porphyria Species 0.000 description 1
- 208000010642 Porphyrias Diseases 0.000 description 1
- 208000033766 Prolymphocytic Leukemia Diseases 0.000 description 1
- 208000003670 Pure Red-Cell Aplasia Diseases 0.000 description 1
- 108700017801 Purine Nucleoside Phosphorylase Deficiency Proteins 0.000 description 1
- 108700014121 Pyruvate Kinase Deficiency of Red Cells Proteins 0.000 description 1
- 208000009527 Refractory anemia Diseases 0.000 description 1
- 208000033501 Refractory anemia with excess blasts Diseases 0.000 description 1
- 206010072684 Refractory cytopenia with unilineage dysplasia Diseases 0.000 description 1
- 201000000582 Retinoblastoma Diseases 0.000 description 1
- 201000002883 Scheie syndrome Diseases 0.000 description 1
- 102000007562 Serum Albumin Human genes 0.000 description 1
- 201000004283 Shwachman-Diamond syndrome Diseases 0.000 description 1
- 208000000859 Sickle cell trait Diseases 0.000 description 1
- 241000543375 Sideroxylon Species 0.000 description 1
- 208000021386 Sjogren Syndrome Diseases 0.000 description 1
- 201000001828 Sly syndrome Diseases 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 101000874347 Streptococcus agalactiae IgA FC receptor Proteins 0.000 description 1
- 208000006011 Stroke Diseases 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 102100024219 T-cell surface glycoprotein CD1a Human genes 0.000 description 1
- 208000022292 Tay-Sachs disease Diseases 0.000 description 1
- 108090000992 Transferases Proteins 0.000 description 1
- 102000004357 Transferases Human genes 0.000 description 1
- 206010060872 Transplant failure Diseases 0.000 description 1
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
- 208000006391 Type 1 Hyper-IgM Immunodeficiency Syndrome Diseases 0.000 description 1
- 108700009899 Type 1 Spherocytosis Proteins 0.000 description 1
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 description 1
- 102100034397 Uroporphyrinogen-III synthase Human genes 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 208000033559 Waldenström macroglobulinemia Diseases 0.000 description 1
- 208000006110 Wiskott-Aldrich syndrome Diseases 0.000 description 1
- 208000026589 Wolman disease Diseases 0.000 description 1
- 201000001696 X-linked hyper IgM syndrome Diseases 0.000 description 1
- 208000033779 X-linked lymphoproliferative disease Diseases 0.000 description 1
- LUTSRLYCMSCGCS-BWOMAWGNSA-N [(3s,8r,9s,10r,13s)-10,13-dimethyl-17-oxo-1,2,3,4,7,8,9,11,12,16-decahydrocyclopenta[a]phenanthren-3-yl] acetate Chemical compound C([C@@H]12)C[C@]3(C)C(=O)CC=C3[C@@H]1CC=C1[C@]2(C)CC[C@H](OC(=O)C)C1 LUTSRLYCMSCGCS-BWOMAWGNSA-N 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 208000013685 acquired idiopathic sideroblastic anemia Diseases 0.000 description 1
- 208000017733 acquired polycythemia vera Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 208000024340 acute graft versus host disease Diseases 0.000 description 1
- 208000034107 acute mast cell leukemia Diseases 0.000 description 1
- 208000036676 acute undifferentiated leukemia Diseases 0.000 description 1
- 229960005305 adenosine Drugs 0.000 description 1
- 210000000577 adipose tissue Anatomy 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 210000004504 adult stem cell Anatomy 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 230000000172 allergic effect Effects 0.000 description 1
- 208000006682 alpha 1-Antitrypsin Deficiency Diseases 0.000 description 1
- 201000008333 alpha-mannosidosis Diseases 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 238000002617 apheresis Methods 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- GOLCXWYRSKYTSP-UHFFFAOYSA-N arsenic trioxide Inorganic materials O1[As]2O[As]1O2 GOLCXWYRSKYTSP-UHFFFAOYSA-N 0.000 description 1
- 229960002594 arsenic trioxide Drugs 0.000 description 1
- 208000010668 atopic eczema Diseases 0.000 description 1
- 230000003416 augmentation Effects 0.000 description 1
- 230000003190 augmentative effect Effects 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 239000001045 blue dye Substances 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 230000003683 cardiac damage Effects 0.000 description 1
- 230000006727 cell loss Effects 0.000 description 1
- 239000002771 cell marker Substances 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000035606 childbirth Effects 0.000 description 1
- SGMZJAMFUVOLNK-UHFFFAOYSA-M choline chloride Chemical compound [Cl-].C[N+](C)(C)CCO SGMZJAMFUVOLNK-UHFFFAOYSA-M 0.000 description 1
- 229960003178 choline chloride Drugs 0.000 description 1
- 239000013611 chromosomal DNA Substances 0.000 description 1
- 208000016532 chronic granulomatous disease Diseases 0.000 description 1
- 201000010902 chronic myelomonocytic leukemia Diseases 0.000 description 1
- 230000004154 complement system Effects 0.000 description 1
- 238000007596 consolidation process Methods 0.000 description 1
- 208000015806 constitutional megaloblastic anemia with severe neurologic disease Diseases 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 230000002338 cryopreservative effect Effects 0.000 description 1
- 208000035250 cutaneous malignant susceptibility to 1 melanoma Diseases 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 208000024389 cytopenia Diseases 0.000 description 1
- 229960000975 daunorubicin Drugs 0.000 description 1
- STQGQHZAVUOBTE-VGBVRHCVSA-N daunorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(C)=O)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 STQGQHZAVUOBTE-VGBVRHCVSA-N 0.000 description 1
- 231100000895 deafness Toxicity 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 229910003460 diamond Inorganic materials 0.000 description 1
- 239000010432 diamond Substances 0.000 description 1
- 231100000676 disease causative agent Toxicity 0.000 description 1
- 208000002173 dizziness Diseases 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 229960004679 doxorubicin Drugs 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 208000009356 dyskeratosis congenita Diseases 0.000 description 1
- 210000003981 ectoderm Anatomy 0.000 description 1
- 229960002224 eculizumab Drugs 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 210000002257 embryonic structure Anatomy 0.000 description 1
- 210000001900 endoderm Anatomy 0.000 description 1
- UNXHWFMMPAWVPI-ZXZARUISSA-N erythritol Chemical compound OC[C@H](O)[C@H](O)CO UNXHWFMMPAWVPI-ZXZARUISSA-N 0.000 description 1
- 230000000925 erythroid effect Effects 0.000 description 1
- 230000010437 erythropoiesis Effects 0.000 description 1
- 229940105423 erythropoietin Drugs 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 201000007249 familial juvenile hyperuricemic nephropathy Diseases 0.000 description 1
- 201000008049 fucosidosis Diseases 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 201000006440 gangliosidosis Diseases 0.000 description 1
- 208000010749 gastric carcinoma Diseases 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 102000054766 genetic haplotypes Human genes 0.000 description 1
- 238000003205 genotyping method Methods 0.000 description 1
- 208000005017 glioblastoma Diseases 0.000 description 1
- 208000008605 glucosephosphate dehydrogenase deficiency Diseases 0.000 description 1
- 201000008977 glycoproteinosis Diseases 0.000 description 1
- 210000003714 granulocyte Anatomy 0.000 description 1
- 201000009277 hairy cell leukemia Diseases 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 208000016354 hearing loss disease Diseases 0.000 description 1
- 201000005787 hematologic cancer Diseases 0.000 description 1
- 230000002489 hematologic effect Effects 0.000 description 1
- 208000024200 hematopoietic and lymphoid system neoplasm Diseases 0.000 description 1
- 238000011134 hematopoietic stem cell transplantation Methods 0.000 description 1
- 208000018706 hematopoietic system disease Diseases 0.000 description 1
- 208000034737 hemoglobinopathy Diseases 0.000 description 1
- 201000001505 hemoglobinuria Diseases 0.000 description 1
- 230000008588 hemolysis Effects 0.000 description 1
- 208000007475 hemolytic anemia Diseases 0.000 description 1
- 208000014752 hemophagocytic syndrome Diseases 0.000 description 1
- 230000011132 hemopoiesis Effects 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 201000008298 histiocytosis Diseases 0.000 description 1
- 208000033519 human immunodeficiency virus infectious disease Diseases 0.000 description 1
- 208000026095 hyper-IgM syndrome type 1 Diseases 0.000 description 1
- 229960000908 idarubicin Drugs 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 229960003444 immunosuppressant agent Drugs 0.000 description 1
- 239000003018 immunosuppressive agent Substances 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 208000018337 inherited hemoglobinopathy Diseases 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 210000005061 intracellular organelle Anatomy 0.000 description 1
- 238000007917 intracranial administration Methods 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 238000007913 intrathecal administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 238000007914 intraventricular administration Methods 0.000 description 1
- 239000002555 ionophore Substances 0.000 description 1
- 230000000236 ionophoric effect Effects 0.000 description 1
- FZWBNHMXJMCXLU-BLAUPYHCSA-N isomaltotriose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O)O1 FZWBNHMXJMCXLU-BLAUPYHCSA-N 0.000 description 1
- 238000011005 laboratory method Methods 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 210000002751 lymph Anatomy 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 230000000527 lymphocytic effect Effects 0.000 description 1
- 230000036212 malign transformation Effects 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- HEBKCHPVOIAQTA-UHFFFAOYSA-N meso ribitol Natural products OCC(O)C(O)C(O)CO HEBKCHPVOIAQTA-UHFFFAOYSA-N 0.000 description 1
- 210000003716 mesoderm Anatomy 0.000 description 1
- 208000005135 methemoglobinemia Diseases 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 230000000394 mitotic effect Effects 0.000 description 1
- 201000007769 mucolipidosis Diseases 0.000 description 1
- 208000020460 mucolipidosis II alpha/beta Diseases 0.000 description 1
- 206010028093 mucopolysaccharidosis Diseases 0.000 description 1
- 208000000690 mucopolysaccharidosis VI Diseases 0.000 description 1
- 208000025919 mucopolysaccharidosis type 7 Diseases 0.000 description 1
- 210000002894 multi-fate stem cell Anatomy 0.000 description 1
- 201000006417 multiple sclerosis Diseases 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 208000016586 myelodysplastic syndrome with excess blasts Diseases 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 208000010125 myocardial infarction Diseases 0.000 description 1
- 230000008764 nerve damage Effects 0.000 description 1
- HHZIURLSWUIHRB-UHFFFAOYSA-N nilotinib Chemical compound C1=NC(C)=CN1C1=CC(NC(=O)C=2C=C(NC=3N=C(C=CN=3)C=3C=NC=CC=3)C(C)=CC=2)=CC(C(F)(F)F)=C1 HHZIURLSWUIHRB-UHFFFAOYSA-N 0.000 description 1
- 229960001346 nilotinib Drugs 0.000 description 1
- 229940109551 nipent Drugs 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 230000001662 opsonic effect Effects 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- 208000002865 osteopetrosis Diseases 0.000 description 1
- 230000001314 paroxysmal effect Effects 0.000 description 1
- 201000003045 paroxysmal nocturnal hemoglobinuria Diseases 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- FPVKHBSQESCIEP-JQCXWYLXSA-N pentostatin Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(N=CNC[C@H]2O)=C2N=C1 FPVKHBSQESCIEP-JQCXWYLXSA-N 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 229940021222 peritoneal dialysis isotonic solution Drugs 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- 208000031223 plasma cell leukemia Diseases 0.000 description 1
- 230000036470 plasma concentration Effects 0.000 description 1
- 210000004180 plasmocyte Anatomy 0.000 description 1
- 210000001778 pluripotent stem cell Anatomy 0.000 description 1
- 208000037244 polycythemia vera Diseases 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920006316 polyvinylpyrrolidine Polymers 0.000 description 1
- OXCMYAYHXIHQOA-UHFFFAOYSA-N potassium;[2-butyl-5-chloro-3-[[4-[2-(1,2,4-triaza-3-azanidacyclopenta-1,4-dien-5-yl)phenyl]phenyl]methyl]imidazol-4-yl]methanol Chemical compound [K+].CCCCC1=NC(Cl)=C(CO)N1CC1=CC=C(C=2C(=CC=CC=2)C2=N[N-]N=N2)C=C1 OXCMYAYHXIHQOA-UHFFFAOYSA-N 0.000 description 1
- 238000007781 pre-processing Methods 0.000 description 1
- 229960005205 prednisolone Drugs 0.000 description 1
- OIGNJSKKLXVSLS-VWUMJDOOSA-N prednisolone Chemical compound O=C1C=C[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 OIGNJSKKLXVSLS-VWUMJDOOSA-N 0.000 description 1
- 229960004618 prednisone Drugs 0.000 description 1
- XOFYZVNMUHMLCC-ZPOLXVRWSA-N prednisone Chemical compound O=C1C=C[C@]2(C)[C@H]3C(=O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 XOFYZVNMUHMLCC-ZPOLXVRWSA-N 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 208000003476 primary myelofibrosis Diseases 0.000 description 1
- 238000003672 processing method Methods 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 201000009410 rhabdomyosarcoma Diseases 0.000 description 1
- 206010039073 rheumatoid arthritis Diseases 0.000 description 1
- HEBKCHPVOIAQTA-ZXFHETKHSA-N ribitol Chemical compound OC[C@H](O)[C@H](O)[C@H](O)CO HEBKCHPVOIAQTA-ZXFHETKHSA-N 0.000 description 1
- 229960004641 rituximab Drugs 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000010187 selection method Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 208000027390 severe congenital neutropenia 3 Diseases 0.000 description 1
- 208000011985 sialidosis Diseases 0.000 description 1
- 208000031162 sideroblastic anemia Diseases 0.000 description 1
- 208000000587 small cell lung carcinoma Diseases 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 229940055944 soliris Drugs 0.000 description 1
- 229960002920 sorbitol Drugs 0.000 description 1
- 208000020431 spinal cord injury Diseases 0.000 description 1
- 238000011476 stem cell transplantation Methods 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 238000013190 sterility testing Methods 0.000 description 1
- 150000003431 steroids Chemical class 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 201000000596 systemic lupus erythematosus Diseases 0.000 description 1
- 230000002381 testicular Effects 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 238000012876 topography Methods 0.000 description 1
- 230000001960 triggered effect Effects 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- OGWKCGZFUXNPDA-XQKSVPLYSA-N vincristine Chemical compound C([N@]1C[C@@H](C[C@]2(C(=O)OC)C=3C(=CC4=C([C@]56[C@H]([C@@]([C@H](OC(C)=O)[C@]7(CC)C=CCN([C@H]67)CC5)(O)C(=O)OC)N4C=O)C=3)OC)C[C@@](C1)(O)CC)CC1=C2NC2=CC=CC=C12 OGWKCGZFUXNPDA-XQKSVPLYSA-N 0.000 description 1
- 229960004528 vincristine Drugs 0.000 description 1
- OGWKCGZFUXNPDA-UHFFFAOYSA-N vincristine Natural products C1C(CC)(O)CC(CC2(C(=O)OC)C=3C(=CC4=C(C56C(C(C(OC(C)=O)C7(CC)C=CCN(C67)CC5)(O)C(=O)OC)N4C=O)C=3)OC)CN1CCC1=C2NC2=CC=CC=C12 OGWKCGZFUXNPDA-UHFFFAOYSA-N 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
Classifications
-
- 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
- A61K35/48—Reproductive organs
- A61K35/51—Umbilical cord; Umbilical cord blood; Umbilical stem 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M2202/00—Special media to be introduced, removed or treated
- A61M2202/04—Liquids
- A61M2202/0413—Blood
- A61M2202/0462—Placental blood, umbilical cord blood
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M5/00—Devices for bringing media into the body in a subcutaneous, intra-vascular or intramuscular way; Accessories therefor, e.g. filling or cleaning devices, arm-rests
- A61M5/14—Infusion devices, e.g. infusing by gravity; Blood infusion; Accessories therefor
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Cell Biology (AREA)
- Developmental Biology & Embryology (AREA)
- Hematology (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Virology (AREA)
- Reproductive Health (AREA)
- Microbiology (AREA)
- Pharmacology & Pharmacy (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
A pharmaceutical composition comprising hematopoietic stem cells isolated from pooled umbilical cord blood from separate donors, which can be infused in a patient in need of an umbilical cord blood transplant to increase the speed and success of engraftment. A method and a pooling criteria for minimizing lysis of total nucleated cells in pooled umbilical cord blood from separate donors is also provided. The method involves considering the ABO blood types of the donors of the umbilical cord blood units to be pooled as well as the ABO
blood type of the respective mothers. The present invention also provides a means for increasing the availability of cord blood transplants to adults or individuals greater than 65 or 70 kilograms.
blood type of the respective mothers. The present invention also provides a means for increasing the availability of cord blood transplants to adults or individuals greater than 65 or 70 kilograms.
Description
TITLE OF THE INVENTION
POOLED CORD BLOOD UNITS
FIELD OF THE INVENTION
[0001] The present invention relates generally to the field of regenerative medicine and the use of umbilical cord blood. More particularly, the present invention relates to the transplantation of umbilical cord blood. The present invention also relates to the pooling of umbilical cord blood from separate donors and a pooling criteria for increasing the yield of total nucleated cells, as well as a method for increasing the speed of engraftment and success of cord blood transplantation.
BACKGROUND OF THE INVENTION
POOLED CORD BLOOD UNITS
FIELD OF THE INVENTION
[0001] The present invention relates generally to the field of regenerative medicine and the use of umbilical cord blood. More particularly, the present invention relates to the transplantation of umbilical cord blood. The present invention also relates to the pooling of umbilical cord blood from separate donors and a pooling criteria for increasing the yield of total nucleated cells, as well as a method for increasing the speed of engraftment and success of cord blood transplantation.
BACKGROUND OF THE INVENTION
[0002] Umbilical cord blood (CB) is a major focus of regenerative medicine because it is a rich source of stem cells, including hematopoietic stem cells (HSC), that is relatively easy to harvest and store in a Cord Blood Bank. Thus, cord blood has been used as an alternative source of HSCs to bone marrow (Grischenko et al., 2006) and cord blood transplantation is increasingly being used as a therapy for patients suffering from various hematopoietic diseases.
These diseases include, for example, patients with genetic mutations or whose marrow has been destroyed in the course of treatment for leukemia and other malignancies.
These diseases include, for example, patients with genetic mutations or whose marrow has been destroyed in the course of treatment for leukemia and other malignancies.
[0003] The first successful cord blood transplantation was performed in 1988 in a Fanconi anemia patient using HLA-compatible sibling cord blood (Gluckman et al., 1989). Twenty years later, the field of cord blood transplantation has grown and a better understanding of cord blood biology and transplantation has resulted from an increase in the number of CB transplants as well as efforts by the National Marrow Donor Program (NMDP). In that regard, the NMDP documented a 39% increase in cord blood unit (CBU) transplants since 2007, with 898 transplants in 2008. As of 2009, more than 400,000 CBUs are available cryopreserved from CB banks and more than 20,000 transplants have been performed (Gluckman and Rocha, 2009).
[0004] Studies based on successful previous CBU transplants have led to the observation that infusing an increased number of total nucleated cells (TNC) increases not only the speed of engraftment but also the chances of survival of the adult patients (Bornstein et al., 2005). This observation has led to the development of a selection criteria that increases the likelihood of more rapid and successful engraftment in a recipient. This selection criteria, in addition to sufficient (e.g., at least 4/6 HLA-matched) human leukocyte antigen (HLA)-matching, advises that the CBU to be transplanted or infused contain at least 3.0x10' nucleated cells and more than 2.5x105 CD34-positive cells (which include HSCs) per kilogram of the recipient (Wagner et al, 2006).
Applying this selection criteria to the cryopreserved CBUs from Public Cord Blood Banks, it is estimated that one hundred percent (100%) of the cryopreserved CBU inventory contains enough nucleated cells (NC) to transplant a recipient of 10 kg, yet only four percent (4%) of the inventory contains enough nucleated cells to transplant a recipient of 70 kg or more (Kurtzberg et al., 2005). For a recipient of 80 kg or more, this value is further reduced to two percent (2%). Furthermore, the foregoing percentages consider only the number of nucleated cells in the CBU
and the actual probability of a patient finding a suitable match is significantly lower when you consider that the CBU
must also be 4/6 HLA-matched or better. The requirement for sufficient nucleated cells thus poses an additional hurdle even if a proper HLA match can be found for a potential recipient.
Applying this selection criteria to the cryopreserved CBUs from Public Cord Blood Banks, it is estimated that one hundred percent (100%) of the cryopreserved CBU inventory contains enough nucleated cells (NC) to transplant a recipient of 10 kg, yet only four percent (4%) of the inventory contains enough nucleated cells to transplant a recipient of 70 kg or more (Kurtzberg et al., 2005). For a recipient of 80 kg or more, this value is further reduced to two percent (2%). Furthermore, the foregoing percentages consider only the number of nucleated cells in the CBU
and the actual probability of a patient finding a suitable match is significantly lower when you consider that the CBU
must also be 4/6 HLA-matched or better. The requirement for sufficient nucleated cells thus poses an additional hurdle even if a proper HLA match can be found for a potential recipient.
[0005] Interestingly, a recent study (Querol, 2009) pointed out that after an inventory of a CB bank reaches a certain size, most of the "common" HLA types will be represented. At this point, further increasing the size of the inventory (i.e., banking a greater number of CBUs from a greater number of donors) will increase the probability of finding and banking an HLA type (e.g., an "uncommon" HLA type) which was not already represented, albeit minimally. However, increasing the size of the inventory will not necessarily address the problem of insufficient nucleated cells to transplant adults weighing more than 70 or 80 kg, for example. That is, the fraction of the inventory that will have sufficient nucleated cells to be used for adult transplantation will remain relatively constant (i.e., 4% for recipients of 70 Kg or more, and 2% for recipients of 80 Kg or more). This further illustrates the need for ways of increasing the number CBUs that are suitable for adults.
[0006] Transplantation of bone marrow and peripheral stem cells requires blood group compatibility to reduce complications such as delayed hematopoiesis and particularly delayed erythropoiesis (Mielcarek et al., 2002). The reaction involves the anti-A and anti-B antibodies in the infused patient's serum that bind to the newly engrafted stem cells from the donor CBU, which can lead to hemolysis in the presence of a major incompatibility. Other post transplant complications are due, for example, to HLA incompatibility between donor and recipient. The most frequent complication linked to HLA incompatibility is graft-versus-host-disease (GVHD). Despite the use of immunosuppressants and steroids, reduction of GVHD generally requires an HLA
compatibility of 5 or 6 out of 6 (i.e., 5/6 or 6/6), unless cord blood is used in which case the required HLA
compatibility is 4/6.
compatibility of 5 or 6 out of 6 (i.e., 5/6 or 6/6), unless cord blood is used in which case the required HLA
compatibility is 4/6.
[0007] The chances of complications related to GVHD increase with the number of CBUs that are infused. While GVHD has not yet been reported after infusion of a single CBU into a patient (Takahashi et al., 2004), infusion of two CBUs led to an increase of acute grade 3 or 4 GVHD in 5 to 20% of transplanted patients (Petz et al., 2006; Majhail et al., 2006). This appears mainly related to the increased amounts of CD3-positive T cells that are infused following the infusion of two or more CBUs from separate donors.
[0008] A phenomenon of "dominant engraftment" has also been observed following infusion of two or more CBUs into a patient. For example, 100 days after the infusion of two CBUs, only the cells from one of the CBUs were found to have engrafted. The factors controlling the determination of the "dominant unit" are not clear even though 76% of time the dominant CBU was the first unit that was infused into the patient.
The TNC and CD34-positive cell dose were not predictors for determining the dominant unit while the role of CD3-positive cells was not investigated (Majhail, 2006).
The TNC and CD34-positive cell dose were not predictors for determining the dominant unit while the role of CD3-positive cells was not investigated (Majhail, 2006).
[0009] Currently, the prior art teaches several ways of increasing the number of nucleated cells (including HSCs) delivered to a recipient, and thus the chances of successful CB
transplantation. One method involves infusing sequentially (i.e., in series) at least two CBUs directly into the patient (Ballen et al., 2007; Lister et al., 2007). It is known that infused CBUs must be matched for at least 4/6 HLA cord blood donor markers, both between the different CBUs and the recipient, to reduce the chances of graft versus host disease (GVHD). A problem associated with this method of sequential infusion of at least two CBUs is the limited statistical probability of finding two or more donor CBUs with the required at least 4/6 HLA marker compatibility. Furthermore, this technique may be prohibitive in terms of cost, with a single CBU currently costing about $35,000 USD. In any event, the 4/6 matching criteria further limits the percentages of CBUs usable for transplantation.
transplantation. One method involves infusing sequentially (i.e., in series) at least two CBUs directly into the patient (Ballen et al., 2007; Lister et al., 2007). It is known that infused CBUs must be matched for at least 4/6 HLA cord blood donor markers, both between the different CBUs and the recipient, to reduce the chances of graft versus host disease (GVHD). A problem associated with this method of sequential infusion of at least two CBUs is the limited statistical probability of finding two or more donor CBUs with the required at least 4/6 HLA marker compatibility. Furthermore, this technique may be prohibitive in terms of cost, with a single CBU currently costing about $35,000 USD. In any event, the 4/6 matching criteria further limits the percentages of CBUs usable for transplantation.
[0010] Another method involves increasing the number of nucleated cells by collecting fresh peripheral stem cells from the blood of a haplo-identical sibling. The sibling's fresh blood is then subject to a CD34-positive selection to isolate cells (including HSCs) expressing the CD34 antigen, followed by infusing the freshly selected cells sequentially after first infusing a sufficiently HLA-matched CBU (Majhail et al., 2006; Magro et al., 2006; Jaroscak et al., 2003; Fernandez et al., 2003). The fresh CD34 selected cells increases the number of nucleated cells infused, resulting in more rapid engraftment comparable to that observed with standard adult grafts. Since the sibling's cells are subjected to a CD34-positive selection, the sibling's T-cells (which do not express the CD34 antigen) are depleted and hence cannot reject the less numerous CBU cells. Accordingly, there were no reported instances of CBU rejection or primary CBU graft failure in patients who were infused with CD34-positive haplo-identical grafts of non-maternal origin. Interestingly, the sibling's CD34 selected cells infused with the HLA-matched CBU are eventually replaced by the growth of the CBU cells, which is referred to as "delayed engraftment". However, a major problem associated with this method is the very limited statistical probability of identifying a haplo-identical sibling/donor, especially with the reduced sizes of modern families (having an average of 1.5 children).
[0011] An additional method for increasing the number of nucleated cells in a CBU involves the in vitro expansion of fresh CBU-derived stem cells (Araki et al., 2006), however, the cost and practicality of this method can be prohibitive. Furthermore, the possibility of lowering the threshold of HLA
compatibility of 4/6 to 3/6 is not ideal given that Ohnuma et al., (2000) clearly showed that reducing the HLA compatibility between cord blood donor and recipient will significantly decrease the probability of event-free survival.
compatibility of 4/6 to 3/6 is not ideal given that Ohnuma et al., (2000) clearly showed that reducing the HLA compatibility between cord blood donor and recipient will significantly decrease the probability of event-free survival.
[0012] It has been reported that storage of single cord blood specimens at 4 C
for 10-21 days in gas permeable bags produced an apparent increase in the percentage of immature cells (CD34, CD117, GPA) and mitotic activity (S+G2/M cells). With similar storage of pooled specimens there was a further increase in the number of immature colonies cultured (e.g., CD34-positive cells). Based on these observations, Ende et al., (2001) postulated that freshly collected cord blood specimens from different donors could be pooled in vitro and stored at 4 C in gas permeable bags to provide larger numbers of stem cells and could be available for clinical use, for example, to treat patients exposed to lethal irradiation. The authors, however, did not show that this pooling of cord blood is effective for treating a patient in need of a cord blood transplant, and even warned that the pooling of cord blood can only be done "provided [the] pooling does not produce a negative effect". Thus, the efficacy of the pooled cord blood as well as potential complications related thereto were not disclosed. Furthermore, the suggested storage of the pooled cord blood at 4 C in gas permeable bags, while not requiring the presence of a cryoprotectant (e.g., DMSO), would severely limit the shelf-life of the pooled cord blood that could be used.
Additionally, the availability of a plurality of unfrozen cord blood units devoid of cryoprotectants is severely limited.
for 10-21 days in gas permeable bags produced an apparent increase in the percentage of immature cells (CD34, CD117, GPA) and mitotic activity (S+G2/M cells). With similar storage of pooled specimens there was a further increase in the number of immature colonies cultured (e.g., CD34-positive cells). Based on these observations, Ende et al., (2001) postulated that freshly collected cord blood specimens from different donors could be pooled in vitro and stored at 4 C in gas permeable bags to provide larger numbers of stem cells and could be available for clinical use, for example, to treat patients exposed to lethal irradiation. The authors, however, did not show that this pooling of cord blood is effective for treating a patient in need of a cord blood transplant, and even warned that the pooling of cord blood can only be done "provided [the] pooling does not produce a negative effect". Thus, the efficacy of the pooled cord blood as well as potential complications related thereto were not disclosed. Furthermore, the suggested storage of the pooled cord blood at 4 C in gas permeable bags, while not requiring the presence of a cryoprotectant (e.g., DMSO), would severely limit the shelf-life of the pooled cord blood that could be used.
Additionally, the availability of a plurality of unfrozen cord blood units devoid of cryoprotectants is severely limited.
[0013] The fact that the majority of collected CBUs fail to meet the standards for cryopreservation set by CB banks must also be considered. Public CB banks select CBU to be cryopreserved based on two specific criteria: (1) a pre-processing CB volume of more than 65 mL; and (2) a total nucleated cell (TNC) count of more than 1x109 per CBU.
CBUs that do not meet these standards are normally discarded and can represent about 60% to 80% of all collected CBUs. Thus, the vast majority of collected CB is wasted.
CBUs that do not meet these standards are normally discarded and can represent about 60% to 80% of all collected CBUs. Thus, the vast majority of collected CB is wasted.
[0014] There thus remains a need to provide a method of CBU transplantation which overcomes problems of the methods of the prior art. For example, there remains a need to provide a more economic, practical and efficient method of transplantation that can not only improve the effectiveness of treatment, but also increase the number of nucleated cells to allow the treatment of adult patients. In addition, there remains a need to provide such a method of transplantation that would enable the use of CBUs which are not usually used or which are currently discarded by cord blood banks (e.g., do not meet the criteria for public banking). The present invention seeks to meet these and other needs.
[0015] The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.
SUMMARY OF THE INVENTION
SUMMARY OF THE INVENTION
[0016] The present invention relates to the pooling of cord blood from separate donors prior to infusion in a patient.
In one embodiment, frozen or cryopreserved cord blood is pooled. The present invention also relates to a specific pooling criteria for combining cord blood from separate donors prior to infusion for increasing the yield of total nucleated cells (TNC). The present invention also relates to a composition or "carrier unit' comprising, for example, CD34-enriched cells obtained from pooled cord blood from separate donors for improving the success of CBU
transplantation. The present invention also relates to a method of utilizing collected cord blood that has failed to meet the cryopreservation standards set by CB banks, so as to increase the number of CBUs available for engraftment, lower the costs involved with such transplants, and increase the chances of successful transplantation (e.g., by increasing the numbers of HLA matches).
In one embodiment, frozen or cryopreserved cord blood is pooled. The present invention also relates to a specific pooling criteria for combining cord blood from separate donors prior to infusion for increasing the yield of total nucleated cells (TNC). The present invention also relates to a composition or "carrier unit' comprising, for example, CD34-enriched cells obtained from pooled cord blood from separate donors for improving the success of CBU
transplantation. The present invention also relates to a method of utilizing collected cord blood that has failed to meet the cryopreservation standards set by CB banks, so as to increase the number of CBUs available for engraftment, lower the costs involved with such transplants, and increase the chances of successful transplantation (e.g., by increasing the numbers of HLA matches).
[0017] In one aspect, the present invention relates to a method for increasing the success of umbilical cord blood transplantation in a patient. This method comprises:
(a) combining in vitro hematopoietic stem cells from the umbilical cord blood from at least two separate donors; and (b) infusing into the patient a composition comprising the combined hematopoietic stem cells from (a) which have been substantially purified, thereby increasing the chances of successful engraftment.
(a) combining in vitro hematopoietic stem cells from the umbilical cord blood from at least two separate donors; and (b) infusing into the patient a composition comprising the combined hematopoietic stem cells from (a) which have been substantially purified, thereby increasing the chances of successful engraftment.
[0018] In one embodiment, the above-mentioned method comprises pooling umbilical cord blood from at least two separate donors. In another embodiment, the above-mentioned hematopoietic stem cells are substantially purified after the pooling of the umbilical cord blood. In another embodiment, hematopoietic stem cells (HSCs) are substantially purified before the above-mentioned step (a). In another embodiment, the hematopoietic stem cells are substantially purified by a CD34-based purification method. In another embodiment, the hematopoietic stem cells are obtained from unqualified umbilical cord blood. In another embodiment, the umbilical cord blood has been previously frozen. In another embodiment, the above-mentioned method increases the success of umbilical cord blood transplantation in a patient by reducing the time of engraftment. In particular aspects, embodiments of the present invention include any combination of at least two of the above mentioned embodiments (e.g., pooling substantially purified HSCs from previously frozen or thawed cord blood from at least two separate donors).
[0019] In another aspect, the present invention relates to a method for pooling umbilical cord blood collected from separate donors, the method comprising thawing frozen umbilical cord blood from at least two separate donors and combining the thawed umbilical cord blood in vitro.
[0020] In another aspect, the present invention relates to a method for improving the yield of total nucleated cells in pooled umbilical cord blood units from at least two separate donors, the method comprising:
(a) considering the ABO blood type of the donors;
(b) considering the ABO blood type of the mothers of the donors and/or the presence or absence of anti-A and/or anti-B IgG antibodies in prospective umbilical cord blood units to be pooled;
(c) pooling umbilical cord blood units from at least two separate donors based on (a) and (b), thereby improving the yield of total nucleated cells in the pooled umbilical cord blood.
(a) considering the ABO blood type of the donors;
(b) considering the ABO blood type of the mothers of the donors and/or the presence or absence of anti-A and/or anti-B IgG antibodies in prospective umbilical cord blood units to be pooled;
(c) pooling umbilical cord blood units from at least two separate donors based on (a) and (b), thereby improving the yield of total nucleated cells in the pooled umbilical cord blood.
[0021] In one embodiment, the above-mentioned methods, and in particular that of paragraph [0020], involve the pooling of unqualified umbilical cord blood units.
[0022] In one embodiment, the methods or parts thereof of paragraphs [0020]
and [0021] are combined with different embodiments of paragraph [0018].
and [0021] are combined with different embodiments of paragraph [0018].
[0023] In another aspect, the present invention relates to a pharmaceutical composition comprising hematopoietic stem cells collected from frozen umbilical cord blood from separate donors and a pharmaceutically acceptable carrier for reducing the time of engraftment in a patient receiving an umbilical cord blood transplant. In one embodiment, the hematopoietic stem cells are substantially purified from pooled umbilical cord blood from separate donors. In another embodiment, the pooled umbilical cord blood includes or is unqualified umbilical cord blood.
[0024] In another aspect, the present invention relates to a method for reducing the lysis of nucleated cells in collected umbilical cord blood by, for example, preventing IgG antibodies from binding to the anti-A and/or anti-B
antigens expressed on the surface of the nucleated cells. In one embodiment, the umbilical cord blood is pooled umbilical cord blood from separate donors. In another embodiment, the method for reducing the lysis of nucleated cells involves substantially removing or depleting the IgG antibodies from the collected umbilical cord blood or administering an IgG-binding inhibitor to same.
antigens expressed on the surface of the nucleated cells. In one embodiment, the umbilical cord blood is pooled umbilical cord blood from separate donors. In another embodiment, the method for reducing the lysis of nucleated cells involves substantially removing or depleting the IgG antibodies from the collected umbilical cord blood or administering an IgG-binding inhibitor to same.
[0025] In another aspect, the present invention provides a kit for reducing the time of engraftment in a patient receiving an umbilical cord blood transplant. In one non-limiting embodiment, the kit can include a container comprising a number of nucleated cells pooled from umbilical cord blood from separate donors.
[0026] Other aspects, objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0027] The present invention relates to the surprising discovery that the pooling of umbilical cord blood from separate donors, and a composition comprising enriched HSCs from the polled cord blood is advantageous for cord blood transplantation. The present invention also relates to the surprising discovery that CBUs that fail to meet the criteria for public banking can be successfully used in transplantations.
[0028] Definitions [0029] Unless defined otherwise, the scientific and technological terms and nomenclature used herein have the same meaning as commonly understood by a person of ordinary skill to which this invention pertains. Commonly understood definitions of molecular biology terms can be found, for example, in Dictionary of Microbiology and Molecular Biology, 2nd ed. (Singleton et al., 1994, John Wiley & Sons, New York, NY), The Harper Collins Dictionary of Biology (Hale & Marham, 1991, Harper Perennial, New York, NY), Rieger et al., Glossary of genetics: Classical and molecular, 5th edition, Springer-Verlag, New-York, 1991; Alberts et al., Molecular Biology of the Cell, 4th edition, Garland science, New-York, 2002; and, Lewin, Genes VII, Oxford University Press, New-York, 2000.
[0030] In the present description, a number of terms are extensively utilized.
In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided.
In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided.
[0031] The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one" but it is also consistent with the meaning of "one or more", "at least one", and "one or more than one".
[0032] Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value. In general, the terminology "about" is meant to designate a possible variation of up to 10%. Therefore, a variation of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 % of a value is included in the term "about".
[0033] As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have"
and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, un-recited elements or method steps.
and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, un-recited elements or method steps.
[0034] The term "stem cell" refers to any cell that has the ability to divide for indefinite periods of time and to give rise to specialized cells. Stem cells emanate from all germinal layers (i.e., ectoderm, mesoderm, and endoderm).
Typical sources of stem cells include embryos, bone marrow, peripheral blood, umbilical cord blood, placental blood, and adipose tissue. Stem cells can be pluripotent, meaning that they are capable of generating most tissues of an organism. For example, pluripotent stem cells can give rise to cells of the skin, liver, blood, muscle, bone, etc. In contrast, multipotent or adult stem cells typically give rise to limited types of cells. For example, hematopoietic stem cells (HCSs) typically give rise to cells of the lymphoid, myeloid, and erythroid lineages. Viable cells are cells that are alive and frequently are capable of growth and division. Those of skill in the art are aware of methods to determine the viability of cells, e.g., by the ability to exclude trypan blue dye. The term "stem cell" as used herein includes progenitor cells unless otherwise noted. The term "stem cell" as used herein may or may not include totipotent stems cells, which retain the ability to differentiate into all the cells of an organism, including extraembryonic tissues.
Typical sources of stem cells include embryos, bone marrow, peripheral blood, umbilical cord blood, placental blood, and adipose tissue. Stem cells can be pluripotent, meaning that they are capable of generating most tissues of an organism. For example, pluripotent stem cells can give rise to cells of the skin, liver, blood, muscle, bone, etc. In contrast, multipotent or adult stem cells typically give rise to limited types of cells. For example, hematopoietic stem cells (HCSs) typically give rise to cells of the lymphoid, myeloid, and erythroid lineages. Viable cells are cells that are alive and frequently are capable of growth and division. Those of skill in the art are aware of methods to determine the viability of cells, e.g., by the ability to exclude trypan blue dye. The term "stem cell" as used herein includes progenitor cells unless otherwise noted. The term "stem cell" as used herein may or may not include totipotent stems cells, which retain the ability to differentiate into all the cells of an organism, including extraembryonic tissues.
[0035] "Nucleated cells" refers to cells that have a nucleus, i.e., an organelle that comprises chromosomal DNA.
Nucleated cells include, e.g., white blood cells and stem cells. "Unnucleated cells" includes, e.g., adult red blood cells.
Nucleated cells include, e.g., white blood cells and stem cells. "Unnucleated cells" includes, e.g., adult red blood cells.
[0036] The phrase "speed to neutrophil engraftment" refers to an indication of how fast the new immune system created by the newly engrafted stem cells are functioning and is typically indicated by the sum of the number of bands and neutrophils per volume of blood in the recipient. Preferably, the patient is immunocompromised prior to neutrophil engraftment. The shorter the speed or time to neutrophil engraftment, the more advantageous it is for the patient. Similarly, the term "speed of platelet engraftment" refers to an indication of how fast the newly engrafted stem cells is functioning in turning out platelets which are important for clotting. Before platelet engraftment, patients are dependent on transfusion of donor platelets so that they do not have any bleeding problems. The shorter the speed or time to platelet engraftment, the more advantageous it is for the patient.
[0037] CBU cells can be matched with the recipient according to HLA typing within a continuum of haplotype differences. For example, all 6 HLA loci can be matched or the cells can be completely mismatched. The practitioner of the invention will decide the degree of matching appropriate for each situation. In the literature it is reported that matching four out of six (4/6) HLA loci in cord blood transplants is equivalent to matching 5 out of 6 (5/6) in bone marrow transplants when assessing success rates and incidence of graft versus host disease.
[0038] By "sufficiently matched CBU unit" is meant an HLA compatibility of at least four out of six (4/6) between the HLA type of the CBU to be infused and the HLA of the recipient. The skilled person would understand that an HLA
compatibility of greater than or equal to 4/6 would be preferable, including an HLA compatibility of 5/6 and 6/6. The HLA A and B compatibility is done at low resolution but the HLA DRbetal must be done at high resolution.
compatibility of greater than or equal to 4/6 would be preferable, including an HLA compatibility of 5/6 and 6/6. The HLA A and B compatibility is done at low resolution but the HLA DRbetal must be done at high resolution.
[0039] The term "subject", "patient" or "recipient" as used herein refers to an animal, preferably a mammal, and most preferably a human who is the object of treatment, observation or experiment.
[0040] "Mammal" includes humans and both domestic animals such as laboratory animals and household pets, (e.g. cats, dogs, swine, cattle, sheep, goats, horses, rabbits), and non-domestic animals such as wildlife and the like.
[0041] As used herein, the term "purified" or "substantially purified" refers to a cell (e.g., a HSC) having been separated from a component of the composition in which it was originally present. The term purified can sometimes be used interchangeably with the terms "isolated" or "enriched". Thus, for example, a "purified, isolated, or enriched HSC" has been purified to a level not found in nature. A "substantially pure"
HSC preparation is one which is lacking in most other components (e.g., 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 100% free of contaminants). By opposition, the term "crude" means molecules that have not been separated from the components of the original composition in which it was present. Therefore, the terms "separating", "enriching", "purifying" or "isolating" refers to methods by which one or more components of the biological sample are removed from one or more other components of the sample. Sample components include other cells such as leukocytes and may include other components, such as proteins, carbohydrates, nucleic acids or lipids. A
separating, enriching or purifying step preferably removes at least about 70% (e.g., 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 100%), more preferably at least about 90% (e.g., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%) and, even more preferably, at least about 95% (e.g., 95, 96, 97, 98, 99, 100%) of the other components present in the sample from the desired component. For example, a CD34-positive selection performed on crude umbilical cord blood can yield a product which is 92-98% pure in HSCs. For the sake of brevity, the above units (e.g., 66, 67...81, 82,...91, 92%....) have not systematically been recited but are considered, nevertheless, within the scope of the present invention.
HSC preparation is one which is lacking in most other components (e.g., 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 100% free of contaminants). By opposition, the term "crude" means molecules that have not been separated from the components of the original composition in which it was present. Therefore, the terms "separating", "enriching", "purifying" or "isolating" refers to methods by which one or more components of the biological sample are removed from one or more other components of the sample. Sample components include other cells such as leukocytes and may include other components, such as proteins, carbohydrates, nucleic acids or lipids. A
separating, enriching or purifying step preferably removes at least about 70% (e.g., 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 100%), more preferably at least about 90% (e.g., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%) and, even more preferably, at least about 95% (e.g., 95, 96, 97, 98, 99, 100%) of the other components present in the sample from the desired component. For example, a CD34-positive selection performed on crude umbilical cord blood can yield a product which is 92-98% pure in HSCs. For the sake of brevity, the above units (e.g., 66, 67...81, 82,...91, 92%....) have not systematically been recited but are considered, nevertheless, within the scope of the present invention.
[0042] As used herein, the term "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to human. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by regulatory agency of the federal or state government or listed in the U.S.
Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the compounds of the present invention may be administered. Sterile water or aqueous saline solutions and aqueous dextrose and glycerol solutions may be employed as carrier, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin.
Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the compounds of the present invention may be administered. Sterile water or aqueous saline solutions and aqueous dextrose and glycerol solutions may be employed as carrier, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin.
[0043] As used herein, the terms "disease" and "disorder" may be used interchangeably or may be different in that the particular malady or condition may not have a known causative agent (so that etiology has not yet been worked out) and it is therefore not yet recognized as a disease but only as an undesirable condition or syndrome, wherein a more or less specific set of symptoms have been identified by clinicians.
[0044] A "disease that can be ameliorated by a CB transplant" or a "disease treatable with a CB transplant" for which compositions of the present invention are suitable include a large number of diseases and disorders such as those diseases and disorders associated with the hematopoietic system. The compositions described herein are also valuable in the area of regenerative medicine, e.g., using stem cells to trigger the healing process or the regrowth of missing or damaged tissue in patients. Non-limiting classes of diseases and disorders that can be treated by administering the compositions of the present invention include malignant diseases such as hematologic malignancies and benign diseases associated with the hematopoietic system.
Hematologic malignancies are a group of neoplasms that arise through malignant transformation of bone marrow derived cells. Examples of hematologic malignancies and other types of malignant diseases include, but are not limited to, leukemias and lymphomas (e.g., acute lymphoblastic leukemia, acute myelogeneous leukemia, chronic myeloid (myelogenous) leukemia (CML), chronic lymphoid (lymphocytic) leukemia (CLL), juvenile chronic myelogenous leukemia, non-Hodgkin's lymphoma, juvenile myelomonocytic leukemia, biphenotypic leukemia, Burkitt's lymphoma, Hodgkin's lymphoma, multiple myeloma, chronic lymphocytic leukemia, acute undifferentiated leukemia, acute malignant myelosclerosis, polycythemia vera, agnogenic myelometaplasia, Waldenstrom's macroglobulinemia, acute bilineage leukemia, acute mast cell leukemia, chronic myelomonocytic leukemia, hairy cell leukemia, plasma cell leukemia, prolymphocytic leukemia, etc.), myelodysplastic disorders (e.g., refractory anemia with or without ringed sideroblasts, refractory anemia with excess blasts, refractory cytopenia, 5q syndrome, etc.), lymphoproliferative disorders, myelofibrosis, malignant tumors (e.g., breast cancer, neuroblastoma, malignant melanoma, carcinoma of the stomach, ovarian carcinoma, breast small cell lung carcinoma, retinoblastoma, testicular carcinoma, glioblastoma, rhabdomyosarcoma, tumors of the central nervous system, Ewing's sarcoma, etc.), and histiocytosis (e.g., Langerhans cell histiocytosis, familial erythrophagocytic lymphohistiocytosis, hemophagocytic lymphohistiocytosis, X-linked lymphoproliferative disease, etc.).
Hematologic malignancies are a group of neoplasms that arise through malignant transformation of bone marrow derived cells. Examples of hematologic malignancies and other types of malignant diseases include, but are not limited to, leukemias and lymphomas (e.g., acute lymphoblastic leukemia, acute myelogeneous leukemia, chronic myeloid (myelogenous) leukemia (CML), chronic lymphoid (lymphocytic) leukemia (CLL), juvenile chronic myelogenous leukemia, non-Hodgkin's lymphoma, juvenile myelomonocytic leukemia, biphenotypic leukemia, Burkitt's lymphoma, Hodgkin's lymphoma, multiple myeloma, chronic lymphocytic leukemia, acute undifferentiated leukemia, acute malignant myelosclerosis, polycythemia vera, agnogenic myelometaplasia, Waldenstrom's macroglobulinemia, acute bilineage leukemia, acute mast cell leukemia, chronic myelomonocytic leukemia, hairy cell leukemia, plasma cell leukemia, prolymphocytic leukemia, etc.), myelodysplastic disorders (e.g., refractory anemia with or without ringed sideroblasts, refractory anemia with excess blasts, refractory cytopenia, 5q syndrome, etc.), lymphoproliferative disorders, myelofibrosis, malignant tumors (e.g., breast cancer, neuroblastoma, malignant melanoma, carcinoma of the stomach, ovarian carcinoma, breast small cell lung carcinoma, retinoblastoma, testicular carcinoma, glioblastoma, rhabdomyosarcoma, tumors of the central nervous system, Ewing's sarcoma, etc.), and histiocytosis (e.g., Langerhans cell histiocytosis, familial erythrophagocytic lymphohistiocytosis, hemophagocytic lymphohistiocytosis, X-linked lymphoproliferative disease, etc.).
[0045] Examples of benign diseases associated with the hematopoietic system include, but are not limited to, hemoglobinopathies (e.g., thalassemia, sickle cell anemia, etc.), bone marrow failure syndromes (e.g., thrombocytopenia, amegakaryocytic thrombocytopenia, Blackfan-Diamond syndrome, dyskeratosis congenita, Fanconi anemia, osteopetrosis, reticular dysgenesis, sideroblastic anemia, Schwachman-Diamond syndrome, severe aplastic anemia, pancytopenia, agranulocytosis, red cell aplasia, idiopathic aplastic anemia, acquired idiopathic sideroblastic anemia, etc.), immune deficiencies (e.g., DiGeorge syndrome, lymphocyte adhesion disease, Nezelof's syndrome, Omenn syndrome, severe combined immune deficiency, Wiskott-Aldrich syndrome, X-linked hyper-IgM
syndrome, alpha 1-antitrypsin deficiency, etc.), metablolic/storage diseases (e.g., aspartylglucosaminuria, adrenoleukodystrophy, alpha-mannosidosis, fucosidosis, Gaucher's disease, gangliosidosis, Hurler syndrome, Hurler-Scheie syndrome, Scheie syndrome, I-Cell disease, infantile ceroid lipofucoscinosis, Krabbe disease, Lesch-Nyhan syndrome, metachromatic leukodystrophy, Maroteaux-Lamy syndrome, sialidosis, Tay Sach disease, Wolman disease, mucopolysaccharidosis, mucolipidosis, etc.), disorders of neutrophils (e.g., chronic granulomatous disease, Chediak-Higashi syndrome, congenital neutropenia, Kostmann's syndrome, etc.), platelet diseases (e.g., Glanzmann's thrombobasthenia, etc.), porphyria (e.g., congenital erythropoietic porphyria, etc.), viral infections (e.g., HIV infection), and autoimmune disorders (e.g., systemic lupus erythematosus, rheumatoid arthritis, Sjogren's syndrome, type I diabetes, multiple sclerosis, chronic hepatitis, inflammatory osteopathies, etc.).
syndrome, alpha 1-antitrypsin deficiency, etc.), metablolic/storage diseases (e.g., aspartylglucosaminuria, adrenoleukodystrophy, alpha-mannosidosis, fucosidosis, Gaucher's disease, gangliosidosis, Hurler syndrome, Hurler-Scheie syndrome, Scheie syndrome, I-Cell disease, infantile ceroid lipofucoscinosis, Krabbe disease, Lesch-Nyhan syndrome, metachromatic leukodystrophy, Maroteaux-Lamy syndrome, sialidosis, Tay Sach disease, Wolman disease, mucopolysaccharidosis, mucolipidosis, etc.), disorders of neutrophils (e.g., chronic granulomatous disease, Chediak-Higashi syndrome, congenital neutropenia, Kostmann's syndrome, etc.), platelet diseases (e.g., Glanzmann's thrombobasthenia, etc.), porphyria (e.g., congenital erythropoietic porphyria, etc.), viral infections (e.g., HIV infection), and autoimmune disorders (e.g., systemic lupus erythematosus, rheumatoid arthritis, Sjogren's syndrome, type I diabetes, multiple sclerosis, chronic hepatitis, inflammatory osteopathies, etc.).
[0046] The compositions of the present invention are also suitable for the treatment of various genetic diseases and disorders affecting cells of the hematopoietic lineage. Examples of such diseases include, without limitation, thalassemia (e.g., alpha, beta, gamma), familial aplastic anemia, Fanconi's syndrome, Bloom's syndrome, pure red cell aplasia, familial erythrophagocytic lymphohistiocytosis, dyskeratosis congenital, Blackfan-Diamond syndrome, congenital dyserythropoietic syndromes I-IV, Chwachmann-Diamond syndrome, dihydrofolate reductase deficiency, formamino transferase deficiency, aspartyl glucosaminidase deficiency, beta-glucuronidase deficiency, hypoxanthine-guanine phosphoribosyltransferase deficiency, Lesch-Nyhan syndrome (juvenile gout) , congenital spherocytosis, congenital elliptocytosis, congenital stomatocytosis, congenital Rh null disease, paroxysmal nocturnal hemoglobinuria, glucose-6-phosphate dehydrogenase deficiency, pyruvate kinase deficiency, congenital erythropoietin sensitivity, sickle cell disease and trait, met-hemoglobinemia, severe combined immunodeficiency disease, severe combined immunodeficiency disease with absence of T and B
cells, severe combined immunodeficiency disease with absence of T cells, severe combined immunodeficiency disease adenosine deaminase deficiency, bare lymphocyte syndrome, combined immunodeficiency, ionophore-responsive combined immunodeficiency, combined immunodeficiency with a capping abnormality, common variable immunodeficiency, nucleoside phosphorylase deficiency, granulocyte actin deficiency, neutrophil actin deficiency, infantile agranulocytosis, Gaucher's disease, adenosine deaminase deficiency, Kostmann's syndrome, reticular dysgenesis, and congenital leukocyte dysfunction syndromes.
cells, severe combined immunodeficiency disease with absence of T cells, severe combined immunodeficiency disease adenosine deaminase deficiency, bare lymphocyte syndrome, combined immunodeficiency, ionophore-responsive combined immunodeficiency, combined immunodeficiency with a capping abnormality, common variable immunodeficiency, nucleoside phosphorylase deficiency, granulocyte actin deficiency, neutrophil actin deficiency, infantile agranulocytosis, Gaucher's disease, adenosine deaminase deficiency, Kostmann's syndrome, reticular dysgenesis, and congenital leukocyte dysfunction syndromes.
[0047] The compositions of the present invention are also suitable for augmenting or improving regenerative therapies, for example, to heal broken bones or to treat bad burns, blindness, deafness, heart damage (e.g., due to heart attack, etc.), nerve damage (e.g., spinal cord injury), stroke, Parkinson's disease, Alzheimer's disease, diabetes (type I or II), and other conditions.
[0048] "Antibodies" (Abs) and "immunoglobulins" (Igs) are glycoproteins having substantially equivalent structural characteristics. While antibodies exhibit binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibody-like molecules that lack antigen specificity.
Polypeptides of the latter kind are, for example, produced at low levels by the lymph system and at increased levels by myelomas.
Polypeptides of the latter kind are, for example, produced at low levels by the lymph system and at increased levels by myelomas.
[0049] "Native antibodies" and "native immunoglobulins" are normally heterotetrameric glycoproteins of about 150,000 daltons, consisting of two identical light (L) polypeptide chains and two identical heavy (H) polypeptide chains linked to one another by disulfide bonds. Each heavy chain has a variable domain (VH) at one end followed by a number of constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light-chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains.
[0050] The term "variable" refers to the fact that certain portions of the variable domains differ extensively in amino acid sequence among antibodies and are usually used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions both in the light-chain and the heavy-chain variable domains. The more highly conserved or less divergent portions of variable domains are called the frame-work (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a [i-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the R-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (Kabat et al., NIH Publ. No. 91-3242, Vol. I, pages 647-669 (1991)). The constant domains are not involved directly in the binding of an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).
[0051] Digesting native antibodies with the cysteine protease papain, which cleaves antibodies in the hinge region N-terminal to the inter-heavy chain disulfide bonds, yields two identical antigen-binding fragments (Fab) with a single antigen-binding site, and a remaining fragment that is readily crystallizeable (Fc). On the other hand, digesting with the enzyme pepsin, which cleaves antibodies C-terminal to the inter-heavy chain disulfide bonds, produces an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen. An Fab fragment contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. An F(ab')2 fragment can be converted into two Fab' fragments using a reducing reagent such as dithiothreitol (DTT). Fab' fragments differ from Fab fragments by the addition of a few amino acid residues at the carboxyl terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' fragments in which the cysteine residues(s) of the constant domains bear a free thiol group.
[0052] The term "Fv" refers to the minimum antibody fragment which contains a complete antigen-recognition and -binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although with a lower affinity than the entire binding site.
[0053] The light chains of antibodies (immunoglobulins) of any vertebrate species can be assigned to one of two clearly distinct types, called kappa (K) and lambda (A), based on the amino acid sequences of their constant domains.
Their heavy chains can be assigned to one of five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these maybe further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, 6, E, y and p, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
Their heavy chains can be assigned to one of five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these maybe further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, 6, E, y and p, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
[0054] Compounds and compositions of the invention may be administered in a pharmaceutical composition.
Pharmaceutical compositions of the present invention comprise a compound or composition that improves the speed of engraftment and/or success of an umbilical cord transplant and a pharmaceutically-acceptable diluent, carrier, or excipient. In one embodiment, such compounds may comprise substantially purified HSCs from umbilical cord blood units from separate donors. Pharmaceutical compositions may be administered in unit dosage form, for example, relative to the weight of the patient to be infused.
Pharmaceutical compositions of the present invention comprise a compound or composition that improves the speed of engraftment and/or success of an umbilical cord transplant and a pharmaceutically-acceptable diluent, carrier, or excipient. In one embodiment, such compounds may comprise substantially purified HSCs from umbilical cord blood units from separate donors. Pharmaceutical compositions may be administered in unit dosage form, for example, relative to the weight of the patient to be infused.
[0055] The skilled artisan will appreciate that certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present.
[0056] An "effective amount" of a carrier unit or composition of the present invention is an amount sufficient to produce the desired effect, e.g., prevention or treatment of a malignant disease or a benign disease associated with the hematopoietic system.
[0057] As used herein, the term "administering" refers to the delivery of a carrier unit or composition of the present invention by any route including, without limitation, oral, intranasal, intravenous, intraosseous, intraperitoneal, intramuscular, intra-articular, intraventricular, intracranial, intralesional, intratracheal, intrathecal, subcutaneous, intradermal, transdermal, or transmucosal administration. In some instances, patients can be infused with one, two, three, or more umbilical cord blood units prior administering compositions of the present invention. Multiple units such as double cord blood units can be administered simultaneously or consecutively (e.g., over the course of several minutes, hours, or days) to a patient. For example, the interval between administrations can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 60, 90, 120 or 180 minutes. For brevity, not all of the possible time intervals are listed here but the skilled person would understand that the interval between administrations can be any reasonable interval and is adaptable to suit the particular needs of the patient and/or individual performing the administration. In certain instances, compositions of the present invention are administered after either myeloablative, reduced intensity, or non-myeloablative therapy to eliminate the diseased bone marrow or after radiation therapy, chemotherapy, or other radiation exposure having ablated the host bone marrow.
[0058] A "cryoprotectant" or "cryopreservation agent" is a substance that is used to protect biological tissue from freezing damage (damage due to ice formation). Cryoprotective agents which can be used include but are not limited to dimethyl sulfoxide (DMSO), glycerol, polyvinylpyrrolidine, polyethylene glycol, albumin, dextran, sucrose, ethylene glycol, i-erythritol, D-ribitol, D-mannitol, D-sorbitol, iinositol, D-lactose, choline chloride, amino acids, methanol, acetamide, glycerol monoacetate, and inorganic salts. In a preferred embodiment, DMSO is used, a liquid which is nontoxic to cells in low concentration. Being a small molecule, DMSO freely permeates the cell and protects intracellular organelles by combining with water to modify its freezability and prevent damage from ice formation. In certain other instances, the cryoprotectant can be a mixture of DMSO and Gentran 40 or DMSO and hydroxyethyl starch (HES). In a preferred embodiment, the cryoprotectant is added as a solution until the final concentration of DMSO is from about 5% to about 10%. The particular choice of cryoprotectant and its concentration can be adapted by the skilled artisan to meet particular needs of cryopreservation and avoid adverse effects upon thawing, processing and administration to the patient.
[0059] An "anticoagulant" is a substance that prevents blood from clotting.
For example, an anticoagulant can contain citric acid, sodium citrate, sodium phosphate, dextrose, and/or adenosine.
For example, an anticoagulant can contain citric acid, sodium citrate, sodium phosphate, dextrose, and/or adenosine.
[0060] Cord blood collection, processing and cryopreservation [0061] Two methods of collecting umbilical cord blood after childbirth currently exist. The in utero method involves collecting the cord blood while the placenta is still in the uterus while the ex utero method involves collecting the cord blood after the placenta has already been delivered. Although most Cord Blood banks use the ex utero method, the in utero method is associated with a 10% higher volume that is collected as well as reduced clotting, which ultimately impacts the yield of viable nucleated cells in the collected CBU (Wong et al., 2001; Solves et al., 2003; Solves et al., 2003a; Ballen et al., 2001). A further advantage of the in utero method is that the risk of contamination of the collected CBU with, for example, maternal blood or bacteria is reduced.
[0062] Regardless of the collection method used, the cord blood is generally received in a sterile collection bag containing an anti-coagulant solution such as citrate-phosphate-dextrose (CPD) to ensure adequate preservation of the cord blood. The volume of cord blood that has been collected is then measured and a sample of the cord blood is withdrawn for quality control purposes. Among the quality control parameters examined are the numbers of total nucleated cells (TNC) and CD34-positive cells present in the collected cord blood. These two parameters are the most important determinants of the quality of the cord blood.
[0063] After collection, the cord blood undergoes processing. The main goal of cord blood processing is to reduce its volume (e.g., to a standard volume) in order to reduce the storage requirements and costs in large-scale cord blood banks. Reducing the volume of the cord blood also minimizes, for example, the amount of dimethyl sulfoxide (DMSO) required in the final CBU. The volume of the collected cord blood is generally reduced by extracting red blood cells and/or plasma to enrich nucleated cells such as leukocytes and HSCs. This is most commonly done by a so-called "two-step method", which is the method used by the first established cord blood bank founded in New York.
The details of the "two-step method" would be familiar to those skilled in the art of cord blood collection and briefly involves adding a solution of hydroxyethyl starch (HES) in a triple bag closed system (M-Reborero et al., 2000) to maximize the isolation of the leukocytes. Another volume reduction method, the top-and-bottom or top-bottom (TB) method, is based on the equal isolation of nucleated cells with or without adding HES, which has been reported to be a hyperoncotic that can cause intravascular volume expansion by about 150%
(Mehta et al., 2007). Another method employed by some cord blood banks involves drawing out only the plasma or red cells. Other methods of volume reduction are known in the art and would be within the grasp of the skilled person. Furthermore, some processing centers an CB banks cryopreserve their CBUs without any substantial volume reduction step, thus reducing the risk of stem cell loss.
The details of the "two-step method" would be familiar to those skilled in the art of cord blood collection and briefly involves adding a solution of hydroxyethyl starch (HES) in a triple bag closed system (M-Reborero et al., 2000) to maximize the isolation of the leukocytes. Another volume reduction method, the top-and-bottom or top-bottom (TB) method, is based on the equal isolation of nucleated cells with or without adding HES, which has been reported to be a hyperoncotic that can cause intravascular volume expansion by about 150%
(Mehta et al., 2007). Another method employed by some cord blood banks involves drawing out only the plasma or red cells. Other methods of volume reduction are known in the art and would be within the grasp of the skilled person. Furthermore, some processing centers an CB banks cryopreserve their CBUs without any substantial volume reduction step, thus reducing the risk of stem cell loss.
[0064] Once the volume of the collected cord blood has been reduced, a cryoprotectant (e.g.,10% DMSO) is added to minimize cell death during the freezing process. The CBU is then eventually frozen usually in liquid nitrogen.
Different cooling methods that control the rate of cooling can be employed. A
controlled cooling rate may be achieved using a device or apparatus specifically designed to lower temperatures at a controlled rate. Alternatively, a more simplified uncontrolled cooling method can be used whereby prepared cells are placed into a mechanical freezer prior to transferring to liquid nitrogen (e.g., "dump-freezing" method). A
"dump-freezing" method such as incubating the CBU in a freezer at -80 C followed by long term cryopreservation in a vapour phase nitrogen cryofreezer has been reported to ensure adequate preservation of nucleated cells compared with a more controlled cooling rate method (Itoh et al., 2003).
Different cooling methods that control the rate of cooling can be employed. A
controlled cooling rate may be achieved using a device or apparatus specifically designed to lower temperatures at a controlled rate. Alternatively, a more simplified uncontrolled cooling method can be used whereby prepared cells are placed into a mechanical freezer prior to transferring to liquid nitrogen (e.g., "dump-freezing" method). A
"dump-freezing" method such as incubating the CBU in a freezer at -80 C followed by long term cryopreservation in a vapour phase nitrogen cryofreezer has been reported to ensure adequate preservation of nucleated cells compared with a more controlled cooling rate method (Itoh et al., 2003).
[0065] Pooling of cryopreserved cord blood [0066] It is known in the art that increasing the number of nucleated cells (including HSCs) delivered to a recipient increases the speed of engraftment and the chances of successful transplant in a patient. It also known that when multiple CBUs are infused into a patent, often the stem cells from only one "dominant unit" will be successfully engrafted. The other CBUs that were infused but were not eventually engrafted can be said to be "carrier units", which increase the success of engraftment of the "dominant unit'. Consistent with these observations, the present invention relates to the surprising discovery that CB, including frozen cord blood, from separate donors can be pooled and stem cells (e.g., CD34-positive HSCs) can be isolated from the pooled CB to produce a composition or "carrier unit" of the present invention. This carrier unit is useful, for example, for improving the speed of engraftment of a sufficiently HLA-matched CBU to be infused in a patient.
[0067] In accordance with the present invention, CB from a sufficient amount of separate donors are pooled in vitro and HSCs from the pooled CB are substantially purified or enriched to yield a composition or carrier unit of the present invention. In addition to enriching the HSCs, this purification step removes unwanted immune cells, such as T-cells, which can lead to GVDH in the patient to be infused. Methods to purify or enrich HSCs are commonly known in the art. For example, HSCs expressing CD34 can be substantially purified or enriched, by performing a CD34-based positive selection. Negative selections can also be performed based on cell surface markers that are not significantly expressed on the surfaces of the desired HSCs. For example, a negative selection based on the T cell marker CD3 could be performed to minimize the chances of complications from GVHD. It should also be understood that the present invention is not limited to the purification or enrichment of HSCs following the pooling of the CB. That is, the purification of HSCs can also be performed prior to the pooling of the CB (e.g., on individual CB units prior to or following cryopreservation). In this case, it is the products of the purification step (i.e., the enriched HSCs) that are combined to form a composition or carrier unit of the present invention [0068] By "pooling" is meant the combining of umbilical cord blood (or HSCs purified therefrom) from separate donors in vitro, but not for example the sequential (i.e., in series) infusion of CBUs into a patient. The CB combined can be freshly processed CB, cryopreserved (frozen) CB, or a combination of the two, and can be obtained for example from either a donor, CB bank or other suitable source. The CB combined may be qualified (i.e., having passed the quality control standards for banking by a CB bank) or unqualified (i.e., having not met all the standards for banking by a CB bank). However, the pooling of unqualified CB may be desirable for practical and cost-effective reasons. The number of qualified or unqualified CBUs combined can be any one of 2, 3, 4, 5, 6, 7, ,8 , 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 26,... 30, 35, 40, 45, 50, or portions thereof. For brevity, all possibilities are not recited here but the person of skill in the art would understand that any practical number of CBUs or portions thereof can be combined.
[0069] In another embodiment, the present invention relates to a method of thawing and pooling separate cryopreserved or frozen CBUs to ensure viable recovery of nucleated cells.
Ideally, a cryopreserved CBU is thawed in a manner to ensure the viability of nucleated cells (including HSCs). When CBUs containing hypertonic cryopreservative solutions are rapidly mixed with large volumes of isotonic solutions or plasma, the viability of nucleated cells (NC) measurably decreases due to osmotic damage inflicted by the severe solute concentration gradient. In accordance with the present invention, the method of Rubinstein (Rubinstein et al., 1995) was modified to minimize cell death of NCs by performing a first dilution (e.g., a 2-fold dilution) after thawing, incubating for a defined period of time, performing a second dilution, followed by centrifugation and resuspension of the cell pellet. This improved method was found to provide almost a total recovery of viable hematopoietic progenitor cells. Basically, the thawing and washing method of the present invention involves adding an equal volume mixture of albumin/gentran to the CBU to achieve a 2-fold dilution, incubating for a defined period of time, and then transferring the diluted CBU
into a bag containing more of the albumin/gentran solution to achieve a second dilution (e.g., a 10-fold dilution). This has the effect of diluting the DMSO present in the thawed CBU from, for example, 10% at the start, down to less than 1% in the final dilution. The cells are then centrifuged to obtain a cell pellet and the pellets from a suitable number of CBUs are pooled in a final bag and prepared for purification (e.g., HSC
purification via CD34 selection). Of course, other means of thawing and diluting CBUs can be carried out, provided that the conditions are chosen so as to minimize cell death. For example, the present invention describes the use of an isotonic solution of albumin and gentran, as commonly used by most transplant physicians, however other buffers may also be used provided that they can also minimize cell death and, in the case for human recipients, are approved by the appropriate regulatory bodies for human use. Cryopreserved nucleated cells when thawed in a isotonic solution can endure, for example, a 145% increase in volume before succumbing to apoptosis or cell lysis. HSCs, in particular, are more robust and can endure a larger percentage of increase in volume, but this percentage has not yet been precisely documented.
Regardless of the buffer used, the increase in volume should be performed as slowly or gradually as possible. In this regard, the aforementioned incubation for a defined period of time (e.g., 5 minutes), ensures proper osmotic exchange and thus helps minimize cell death.
Ideally, a cryopreserved CBU is thawed in a manner to ensure the viability of nucleated cells (including HSCs). When CBUs containing hypertonic cryopreservative solutions are rapidly mixed with large volumes of isotonic solutions or plasma, the viability of nucleated cells (NC) measurably decreases due to osmotic damage inflicted by the severe solute concentration gradient. In accordance with the present invention, the method of Rubinstein (Rubinstein et al., 1995) was modified to minimize cell death of NCs by performing a first dilution (e.g., a 2-fold dilution) after thawing, incubating for a defined period of time, performing a second dilution, followed by centrifugation and resuspension of the cell pellet. This improved method was found to provide almost a total recovery of viable hematopoietic progenitor cells. Basically, the thawing and washing method of the present invention involves adding an equal volume mixture of albumin/gentran to the CBU to achieve a 2-fold dilution, incubating for a defined period of time, and then transferring the diluted CBU
into a bag containing more of the albumin/gentran solution to achieve a second dilution (e.g., a 10-fold dilution). This has the effect of diluting the DMSO present in the thawed CBU from, for example, 10% at the start, down to less than 1% in the final dilution. The cells are then centrifuged to obtain a cell pellet and the pellets from a suitable number of CBUs are pooled in a final bag and prepared for purification (e.g., HSC
purification via CD34 selection). Of course, other means of thawing and diluting CBUs can be carried out, provided that the conditions are chosen so as to minimize cell death. For example, the present invention describes the use of an isotonic solution of albumin and gentran, as commonly used by most transplant physicians, however other buffers may also be used provided that they can also minimize cell death and, in the case for human recipients, are approved by the appropriate regulatory bodies for human use. Cryopreserved nucleated cells when thawed in a isotonic solution can endure, for example, a 145% increase in volume before succumbing to apoptosis or cell lysis. HSCs, in particular, are more robust and can endure a larger percentage of increase in volume, but this percentage has not yet been precisely documented.
Regardless of the buffer used, the increase in volume should be performed as slowly or gradually as possible. In this regard, the aforementioned incubation for a defined period of time (e.g., 5 minutes), ensures proper osmotic exchange and thus helps minimize cell death.
[0070] Cord blood pooling criteria for increasing TNC yield [0071] Although the present invention encompasses the in vitro pooling of fresh and/or cryopreserved (frozen) cord blood independent of a pooling criteria, the present invention further provides an in vitro pooling criteria for increasing the yield of total nucleated cells (TNC) in the pooled cord blood. Thus, in another embodiment, the present invention relates to the surprising discovery that the yield of TNC in pooled cord blood samples is significantly affected by the ABO blood types of the CB donors and their respective mothers. More particularly, the maternally-derived anti-A and anti-B IgG antibodies that cross into the placenta and are present in the cord blood are herein identified as significant determinants that affect the survival and thus the yield of TNCs in the pooled CBUs. It is known that cells (including nucleated cells) can be killed or lysed by, for example, the complement system via the binding of IgG antibodies to the surface of the cells. The main details of this pathway are known in the art. Briefly, IgG is the most abundant antibody isotype in humans, representing approximately 70 to 75% of all antibodies. It is also known that nearly all of the IgG antibodies found in newborns are of maternal origin and only trace amounts of IgG originate from the newborn (Miller, 1973). IgG antibodies are divided into four main subgroups (IgG1, IgG2, IgG3 and IgG4) and are composed of an Fab region containing heavy and light chains as well as an Fc region. The classical complement pathway can be triggered through the adjacent binding of at least two molecules of IgGl or IgG3 to an antigen on the surface of the cell, followed by the binding of the C1-complex to the Fc portions of the bound antibodies. Cell lysis can then occur, for example, through the action of the membrane attack complex (MAC) which is inserted into the cell membrane and initiates cell lysis. According to the present invention, anti-A
and anti-B IgG antibodies bind to the A
and B antigens expressed on the surface of cells, resulting in their eventual lysis.
and anti-B IgG antibodies bind to the A
and B antigens expressed on the surface of cells, resulting in their eventual lysis.
[0072] The present invention is further supported by the following observations.
[0073] During pregnancy, the placenta is the site where regulated transfer of IgG antibodies occurs between the mother and the newborn (Jauniaux et al., 1995; Kohler et al., 1966). This transfer is more important after 32 weeks of pregnancy when the IgG level in the newborn is actually higher than that of the mother (Klein and Anstee, 2008).
When the newborn's blood group is incompatible with the blood group of the mother, the child can be diagnosed with Hemolytic Disease of the Newborn (HDN) due to the transfer and binding of anti-A and anti-B IgG antibodies from the mother. A minimum concentration of IgG is required to produce agglutination.
In adult blood, the minimal concentration of IgG anti-A required for agglutination is 0.2 g/mL.
When the newborn's blood group is incompatible with the blood group of the mother, the child can be diagnosed with Hemolytic Disease of the Newborn (HDN) due to the transfer and binding of anti-A and anti-B IgG antibodies from the mother. A minimum concentration of IgG is required to produce agglutination.
In adult blood, the minimal concentration of IgG anti-A required for agglutination is 0.2 g/mL.
[0074] Leukocytes and platelets express A and B antigens albeit at lower levels compared to red blood cells (Klein and Anstee, 2008). The majority of red blood cells are generally removed during the processing of freshly collected cord blood prior to cryopreservation yet the IgG molecules remain.
[0075] Furthermore, anti-A and anti-B IgG antibodies have a higher affinity for adult red blood cells than cord red blood cells. In that regard, Klein et al., (2008) reported that "the binding constant of human IgG anti-A for cord Al red cells was found to be 3.3x10' I/mol compared with 5.7-8.7 x 10' I/mol for adult Al red cells and that for cord Al B red cells to be 2.1 x107 I/mol compared with 3.2-7.0 x 107 I/mol for adult Al B
red cells".
red cells".
[0076] Additionally, it has been observed that newborns having blood group A
or B, where the mother is group 0, have higher levels of anti-A and/or anti-B IgG antibodies in their plasma compared with group A or B mothers.
However, even in such cases the maternal anti-A and anti-B IgG antibodies rarely result in binding of complement to the newborn's red blood cells (Petz et al., 2004).
or B, where the mother is group 0, have higher levels of anti-A and/or anti-B IgG antibodies in their plasma compared with group A or B mothers.
However, even in such cases the maternal anti-A and anti-B IgG antibodies rarely result in binding of complement to the newborn's red blood cells (Petz et al., 2004).
[0077] Thus, the umbilical cord blood that is collected and processed, contains relatively high amounts of maternally-derived anti-A and anti-B IgG antibodies and few red blood cells to which these antibodies can bind.
Furthermore, the maternal anti-A and anti-B IgG antibodies have reduced affinity to the few remaining cord red blood cells present. Leukocytes and other cells such as HSCs, however, remain present in the processed CBU and express A and B antigens. The present invention identifies reasons why TNCs are often lysed or killed in the methods of the prior art: maternally-derived anti-A and anti-B IgG antibodies when found in excess, as compared to red blood cells, for example, bind to nucleated cells (including HSCs) in the CB, resulting in the lysis of these cells and thus in the reduction of the TNC yield in pooled CB.
Furthermore, the maternal anti-A and anti-B IgG antibodies have reduced affinity to the few remaining cord red blood cells present. Leukocytes and other cells such as HSCs, however, remain present in the processed CBU and express A and B antigens. The present invention identifies reasons why TNCs are often lysed or killed in the methods of the prior art: maternally-derived anti-A and anti-B IgG antibodies when found in excess, as compared to red blood cells, for example, bind to nucleated cells (including HSCs) in the CB, resulting in the lysis of these cells and thus in the reduction of the TNC yield in pooled CB.
[0078] Consistent with the above, in one embodiment, the present invention provides the skilled person with a screening or pooling criteria to increase the yield of TNC in pooled CBU, which involves considering the ABO blood types of the mothers and newborns of the respective CBUs to be pooled. The presence or absence of anti-A and/or anti-B IgG antibodies in the CBUs can be inferred from the maternal blood types.
[0079] For example, if a sufficiently HLA-matched CBU is found for a prospective transplant patient whose own marrow has been destroyed in the course of treatment for leukemia or other malignancies, and the blood type of the matched CBU is type 0, then the patient receiving the transplant will also necessarily adopt a blood type 0 following infusion and engraftment of the CBU. According to the present invention, a carrier unit comprising substantially purified HSCs from pooled CB from separate donors can also be infused into the patient to increase the speed and chances of successful engraftment. In this example, CB from donors with blood type 0 would be preferentially chosen to match the blood type of the CBU to be infused. In order to maximize the TNC yield in the pooled cord blood, the ABO blood types of the mothers and newborns of the respective CBUs to be pooled are considered. In the present example, the CBUs that are pooled are of type 0, meaning that the cord blood cells express neither A nor B
antigens. Thus, in such a case, the respective mothers can be of any ABO blood type (i.e., A, B, AB, or 0), since the maternally-derived anti-A and/or anti-B IgG antibodies will not bind or result in lysis of the cells of the pooled CBUs, thereby increasing TNC yield. Of course, adapting the pooling criteria of the present invention to CBU blood types other than 0 (i.e., A, B or AB) would be within the grasp of one skilled in the art.
antigens. Thus, in such a case, the respective mothers can be of any ABO blood type (i.e., A, B, AB, or 0), since the maternally-derived anti-A and/or anti-B IgG antibodies will not bind or result in lysis of the cells of the pooled CBUs, thereby increasing TNC yield. Of course, adapting the pooling criteria of the present invention to CBU blood types other than 0 (i.e., A, B or AB) would be within the grasp of one skilled in the art.
[0080] As another example, if a sufficiently HLA-matched CBU is found for the prospective transplant patient mentioned above, and the blood type of the matched CBU is type A, then the patient receiving the transplant will also necessarily adopt a blood type A following infusion and engraftment of the CBU. In this case, the carrier unit of the present invention should comprise substantially purified HSCs from CBUs of blood type A, to match the blood type of the CBU to be infused. In order to maximize the TNC yield in the pooled cord blood, the ABO blood types of the mothers and newborns of the respective CBUs to be pooled are considered. In the present example, the CBUs that are pooled are of type A, meaning that the cord blood cells necessarily express antigen A. Thus, in such a case, the respective mothers should be preferably of blood group A or AB, since these CBUs will not contain significant amounts of maternally-derived anti-A IgG antibodies, which can bind and result in lysis of the cells of the pooled CBUs, thereby increasing TNC yield. Conversely, in this example, the inclusion of a CBU from a mother having blood type B or 0 would be "undesirable" since this CBU would be expected to contain significant amounts of maternally-derived "unwanted" anti-A IgG antibodies, thus lowering TNC yield.
[0081] As another example, if a sufficiently HLA-matched CBU is found for the prospective transplant patient mentioned above, and the blood type of the matched CBU is type B, then the patient receiving the transplant will also necessarily adopt a blood type B following infusion and engraftment of the CBU. In this case, the carrier unit of the present invention should comprise substantially purified HSCs from CBUs of blood type B, to match the blood type of the CBU to be infused. In order to maximize the TNC yield in the pooled cord blood, the ABO blood types of the mothers and newborns of the respective CBUs to be pooled are considered. In the present example, the CBUs that are pooled are of type B, meaning that the cord blood cells necessarily express B antigen. Thus, in such a case, the respective mothers should be preferably of blood group B or AB, since these CBUs will not contain significant amounts of maternally-derived anti-B IgG antibodies, which can bind and result in lysis of the cells of the pooled CBUs, thereby increasing TNC yield. Conversely, in this example, the inclusion of a CBU from a mother having blood type A or 0 would be "undesirable" since this CBU would be expected to contain significant amounts of maternally-derived "unwanted" anti-B IgG antibodies, thus lowering TNC yield.
[0082] As another example, if a sufficiently HLA-matched CBU is found for the prospective transplant patient mentioned above, and the blood type of the matched CBU is type AB, then the patient receiving the transplant will also necessarily adopt a blood type AB following infusion and engraftment of the CBU. In this case, the carrier unit of the present invention should comprise substantially purified HSCs from CBUs of blood type AB, to match the blood type of the CBU to be infused. In order to maximize the TNC yield in the pooled cord blood, the ABO blood types of the mothers and newborns of the respective CBUs to be pooled are considered.
In the present example, the CBUs that are pooled are of type AB, meaning that the cord blood cells necessarily express both A and B antigens. Thus, in such a case, the respective mothers should be preferably of blood group AB, since these CBUs will not contain significant amounts of maternally-derived anti-A or anti-B IgG antibodies, which can bind and result in lysis of the cells of the pooled CBUs, thereby increasing TNC yield. Conversely, in this example, the inclusion of a CBU from a mother having blood type A, B or 0 would be "undesirable" since this CBU would be expected to contain significant amounts of maternally-derived "unwanted" anti-A and/or anti-B IgG antibodies, thus lowering TNC yield.
In the present example, the CBUs that are pooled are of type AB, meaning that the cord blood cells necessarily express both A and B antigens. Thus, in such a case, the respective mothers should be preferably of blood group AB, since these CBUs will not contain significant amounts of maternally-derived anti-A or anti-B IgG antibodies, which can bind and result in lysis of the cells of the pooled CBUs, thereby increasing TNC yield. Conversely, in this example, the inclusion of a CBU from a mother having blood type A, B or 0 would be "undesirable" since this CBU would be expected to contain significant amounts of maternally-derived "unwanted" anti-A and/or anti-B IgG antibodies, thus lowering TNC yield.
[0083] In summary, in accordance with the pooling criteria of the present invention, the cord blood units to be pooled to form the carrier unit of the present invention should be chosen so as to minimize the presence of IgG
antibodies which can bind to the A and/or B antigens expressed on the surface of the cells of the cord blood units. Of course, it is understood that the availabilities of fresh and/or frozen CBUs of the desired blood types will play a role in determining which cord blood units can and will be chosen for pooling. In this regard, the skilled person practicing the pooling criteria of the present invention may decide that it is necessary to include a CBU of an "undesirable" blood group ("undesirable" CBUs), as mentioned in the examples above, in order to obtain a desired total number of nucleated cells needed for optimal transplantation. This situation may arise, for example, due to a lack of availability of fresh and/or frozen CBUs of the appropriate blood type, urgency of treatment, budgetary and/or time constrains.
However, the skilled person would recognize that inclusion of a CBU from an "undesirable" blood group would negatively impact the TNC yield in the pooled cord blood. Thus, in another embodiment the pooling criteria of the present invention can be used to minimize the impact of "undesirable" CBUs during the pooling process, for example, by minimizing the number of "undesirable" CBUs, which are included in the pooled cord blood in order to maximize TNC yield. For example, if the HLA-matched CBU to be infused into the patient is of blood group A, the corresponding carrier unit of the present invention should be obtained from pooled CBUs of blood group A. The respective mothers of these pooled CBUs should most preferably be of blood group A. If for example, not enough of these CBUs are available, then other CBUs should be used to minimize the presence of unwanted anti-A IgG
antibodies in the pooled cord blood. In this regard, the blood groups of the respective mothers would be (in order of preference) blood group AB, followed by blood group B, and finally, if necessary, blood group 0. It would be within the grasp of the skilled person to extrapolate these teachings in the event that the HLA-matched CBU to be infused into the patient is of blood group B, AB or 0. Other factors influencing the choice of CBUs to be pooled may also be considered such as the amount or volume of red blood cells present in the frozen CBUs to be pooled, as explained below. It should be understood that the skilled person will adapt the pooling criteria of the present invention in order to maximize TNC yield while at the same time taking into account the urgency of the situation and CBUs which are available.
antibodies which can bind to the A and/or B antigens expressed on the surface of the cells of the cord blood units. Of course, it is understood that the availabilities of fresh and/or frozen CBUs of the desired blood types will play a role in determining which cord blood units can and will be chosen for pooling. In this regard, the skilled person practicing the pooling criteria of the present invention may decide that it is necessary to include a CBU of an "undesirable" blood group ("undesirable" CBUs), as mentioned in the examples above, in order to obtain a desired total number of nucleated cells needed for optimal transplantation. This situation may arise, for example, due to a lack of availability of fresh and/or frozen CBUs of the appropriate blood type, urgency of treatment, budgetary and/or time constrains.
However, the skilled person would recognize that inclusion of a CBU from an "undesirable" blood group would negatively impact the TNC yield in the pooled cord blood. Thus, in another embodiment the pooling criteria of the present invention can be used to minimize the impact of "undesirable" CBUs during the pooling process, for example, by minimizing the number of "undesirable" CBUs, which are included in the pooled cord blood in order to maximize TNC yield. For example, if the HLA-matched CBU to be infused into the patient is of blood group A, the corresponding carrier unit of the present invention should be obtained from pooled CBUs of blood group A. The respective mothers of these pooled CBUs should most preferably be of blood group A. If for example, not enough of these CBUs are available, then other CBUs should be used to minimize the presence of unwanted anti-A IgG
antibodies in the pooled cord blood. In this regard, the blood groups of the respective mothers would be (in order of preference) blood group AB, followed by blood group B, and finally, if necessary, blood group 0. It would be within the grasp of the skilled person to extrapolate these teachings in the event that the HLA-matched CBU to be infused into the patient is of blood group B, AB or 0. Other factors influencing the choice of CBUs to be pooled may also be considered such as the amount or volume of red blood cells present in the frozen CBUs to be pooled, as explained below. It should be understood that the skilled person will adapt the pooling criteria of the present invention in order to maximize TNC yield while at the same time taking into account the urgency of the situation and CBUs which are available.
[0084] In another embodiment, the pooling criteria of the present invention involves considering the amount of red blood cells present in the CBUs to be pooled, as increased amounts of red blood cells expressing A and/or B
antigens will decrease the impact of the maternally-derived anti-A and anti-B
IgG antibodies on the survival of nucleated cells (Hsu, 1979).
antigens will decrease the impact of the maternally-derived anti-A and anti-B
IgG antibodies on the survival of nucleated cells (Hsu, 1979).
[0085] In another embodiment, the pooling criteria of the present invention requires the selection of enough CBUs to be pooled in order to ensure the presence, as starting material, of at least 0.1-0.3 x 106 cells CD34-positive cells (or HSCs) per kg of the patient's body prior to thawing, washing and HSC
purification (e.g., CD34-positive selection).
purification (e.g., CD34-positive selection).
[0086] The results in Tables 1 and 2 demonstrate the correlation between the ABO blood types of the pooled CBUs, the ABO blood types of their respective mothers, and the percentage yield of total nucleated cells (TNC).
Table 1 shows results from "incompatible" CBU poolings and Table 2 shows results from "compatible" poolings according to the pooling criteria of the present invention. By "compatible CBU
pooling", it is meant that the CBUs that are pooled contain cells having surface A and/or B antigens which are not recognizable by the mother-derived anti-A
and/or anti-B IgG antibodies present in the pooled CBUs, thus maximizing TNC
yield. Conversely, an "incompatible CBU pooling" refers to CBUs that are pooled which contain cells having surface A and/or B antigens which are indeed recognizable by the mother-derived anti-A and/or anti-B IgG antibodies present in the pooled CBUs, thus negatively affecting TNC yield. Accordingly, the anti-A and/or anti-B IgG
antibodies present in the pooled CBUs can be inferred from the ABO blood types of their respective mothers. For example, mothers having blood type A are expected to pass on anti-B IgG antibodies to their respective CBUs. Mothers having blood type B are expected to pass on anti-A IgG antibodies to their respective CBUs. Mothers having blood type AB are expected to pass on neither anti-A nor anti-B IgG antibodies to their respective CBUs. Mothers having blood type 0 are expected to pass on both anti-A and anti-B IgG antibodies to their respective CBUs.
Table 1 shows results from "incompatible" CBU poolings and Table 2 shows results from "compatible" poolings according to the pooling criteria of the present invention. By "compatible CBU
pooling", it is meant that the CBUs that are pooled contain cells having surface A and/or B antigens which are not recognizable by the mother-derived anti-A
and/or anti-B IgG antibodies present in the pooled CBUs, thus maximizing TNC
yield. Conversely, an "incompatible CBU pooling" refers to CBUs that are pooled which contain cells having surface A and/or B antigens which are indeed recognizable by the mother-derived anti-A and/or anti-B IgG antibodies present in the pooled CBUs, thus negatively affecting TNC yield. Accordingly, the anti-A and/or anti-B IgG
antibodies present in the pooled CBUs can be inferred from the ABO blood types of their respective mothers. For example, mothers having blood type A are expected to pass on anti-B IgG antibodies to their respective CBUs. Mothers having blood type B are expected to pass on anti-A IgG antibodies to their respective CBUs. Mothers having blood type AB are expected to pass on neither anti-A nor anti-B IgG antibodies to their respective CBUs. Mothers having blood type 0 are expected to pass on both anti-A and anti-B IgG antibodies to their respective CBUs.
[0087] In the Tables below, "CBU ABO" refers to the blood group of each of the three CBUs (separated by commas) that were pooled; "Mother ABO" refers to the ABO blood groups of their respective mothers; "TNC Yield %"
refers to the final yield of total nucleated cells (TNCs) after pooling of the three CBUs; and "Mean" refers to the average of the "TNC Yield %" for each pooling.
Table 1: Incompatible CBU oolin s Table 2: Compatible CBU oolin s Mother ABO CBU ABO TNC Yield % Mother ABO CBU ABO TNC Yield %
0,0,A A,0,A 62% 0, A,0 0,0,0 108%
0, A,0 B,0,0 83% A,A,A 0,0,0 68%
A,B,0 A,O,AB 71% 0,0,0 0,0,0 100%
A,A,A A,O,AB 98% 0,0,0 0,0,0 90%
A,B,A A, A,0 93% 0,A,B 0,0,0 98%
A, A, B A, A, B 93% A, A, 0 0, 0, 0 88%
0,0,0 B, A, A 99% 0,0,0 0,0,0 90%
A, 0, 0 A, 0, 0 76% B, A, 0 0, 0, 0 140%
B, A, 0 B, A, 0 93% A, A, A A, A, A 100%
A, A, A A, A, B 86% 0,0,0 0,0,0 85%
A, B, A AB, 0, 0 93% Mean: 97%
0, A, A B, 0, 0 66%
A, 0, A 0, B, B 57%
0, B, 0 A, B, B 69%
B, A, 0 A, A, A 41%
B,A,A B, A, AB 65%
A,B,0 0,B,A 29%
Mean: 75%
refers to the final yield of total nucleated cells (TNCs) after pooling of the three CBUs; and "Mean" refers to the average of the "TNC Yield %" for each pooling.
Table 1: Incompatible CBU oolin s Table 2: Compatible CBU oolin s Mother ABO CBU ABO TNC Yield % Mother ABO CBU ABO TNC Yield %
0,0,A A,0,A 62% 0, A,0 0,0,0 108%
0, A,0 B,0,0 83% A,A,A 0,0,0 68%
A,B,0 A,O,AB 71% 0,0,0 0,0,0 100%
A,A,A A,O,AB 98% 0,0,0 0,0,0 90%
A,B,A A, A,0 93% 0,A,B 0,0,0 98%
A, A, B A, A, B 93% A, A, 0 0, 0, 0 88%
0,0,0 B, A, A 99% 0,0,0 0,0,0 90%
A, 0, 0 A, 0, 0 76% B, A, 0 0, 0, 0 140%
B, A, 0 B, A, 0 93% A, A, A A, A, A 100%
A, A, A A, A, B 86% 0,0,0 0,0,0 85%
A, B, A AB, 0, 0 93% Mean: 97%
0, A, A B, 0, 0 66%
A, 0, A 0, B, B 57%
0, B, 0 A, B, B 69%
B, A, 0 A, A, A 41%
B,A,A B, A, AB 65%
A,B,0 0,B,A 29%
Mean: 75%
[0088] As can be seen in Tables 1 and 2, the results from "incompatible" CBU
poolings resulted in a mean TNC
yield of 75% and the results from "compatible" CBU poolings resulted in a mean TNC yield of 97%. These results demonstrate the importance of considering the ABO blood types of the respective mothers of the CBUs that are to be pooled in order to maximize TNC yield. More particularly as demonstrated in Table 2, CBUs having blood group 0 are generally less affected by the mothers' blood group due to their lack of expression of A and B antigens.
Furthermore, consistent with the pooling criteria of the present invention, the pooling of three CBUs of blood type A
(Table 2, values in bold) with mothers also having blood type A resulted in 100% TNC yield. By comparison, in contrast to the pooling criteria of the present invention, the pooling of three CBUs of blood type A having two of three mothers of blood types B and 0 (and thus expressing anti-A IgG antibodies) (Table 1, values in bold) resulted in only 41 % TNC yield.
poolings resulted in a mean TNC
yield of 75% and the results from "compatible" CBU poolings resulted in a mean TNC yield of 97%. These results demonstrate the importance of considering the ABO blood types of the respective mothers of the CBUs that are to be pooled in order to maximize TNC yield. More particularly as demonstrated in Table 2, CBUs having blood group 0 are generally less affected by the mothers' blood group due to their lack of expression of A and B antigens.
Furthermore, consistent with the pooling criteria of the present invention, the pooling of three CBUs of blood type A
(Table 2, values in bold) with mothers also having blood type A resulted in 100% TNC yield. By comparison, in contrast to the pooling criteria of the present invention, the pooling of three CBUs of blood type A having two of three mothers of blood types B and 0 (and thus expressing anti-A IgG antibodies) (Table 1, values in bold) resulted in only 41 % TNC yield.
[0089] Additionally, increasing the level of incompatibility also impacts the TNC yield. As shown in Table 1, major incompatibility between mothers and CBUs may reduce the TNC yield to 62%
(e.g., Mothers: 0, 0, A, having anti-A
and B, anti-A and B, and anti-B IgG antibodies; with CBUs: A, 0, A) and 29%
(e.g., Mothers: A, B, 0, having anti-B, anti-A, and anti-A and B IgG antibodies; CBUs: 0, B, A). In these examples, the cells from two of the three CBUs pooled (i.e., those expressing antigen A or B and not those of blood group 0) would be recognized by the anti-A or B
IgG antibodies present in the pooled cord blood. In contrast, minor incompatibility may only reduce yield to 83% (e.g., Mothers: 0, A, 0, having anti-A and B, anti-B, and anti-A and B IgG
antibodies; CBUs: B, 0, 0). In the latter example with 83% yield, only the cells from one of the three CBUs pooled (i.e., those expressing antigen B and not those of blood group 0) would be recognized by the anti-B IgG antibodies present in the pooled cord blood.
(e.g., Mothers: 0, 0, A, having anti-A
and B, anti-A and B, and anti-B IgG antibodies; with CBUs: A, 0, A) and 29%
(e.g., Mothers: A, B, 0, having anti-B, anti-A, and anti-A and B IgG antibodies; CBUs: 0, B, A). In these examples, the cells from two of the three CBUs pooled (i.e., those expressing antigen A or B and not those of blood group 0) would be recognized by the anti-A or B
IgG antibodies present in the pooled cord blood. In contrast, minor incompatibility may only reduce yield to 83% (e.g., Mothers: 0, A, 0, having anti-A and B, anti-B, and anti-A and B IgG
antibodies; CBUs: B, 0, 0). In the latter example with 83% yield, only the cells from one of the three CBUs pooled (i.e., those expressing antigen B and not those of blood group 0) would be recognized by the anti-B IgG antibodies present in the pooled cord blood.
[0090] Indirect Coombs tests followed by elutions of bound anti-A and anti-B
IgG antibodies (see Example 2.6) were performed on pooled CBUs and the results are shown in Table 3. In Tables 3, `Plasma IgG" and "IgG eluted from leukocytes" refer to the relative amounts (i.e., visually scored following microscopy) of anti-A and/or anti-B IgG
antibodies present in either the plasma of the pooled cord blood or in the fraction eluted from the surface of leukocytes present in the pooled cord blood. The relative scale for scoring the amount of IgG present includes (from lowest to highest): "Neg"; "weak"; "1 +"; "2+"; "3+" and "4+".
IgG antibodies (see Example 2.6) were performed on pooled CBUs and the results are shown in Table 3. In Tables 3, `Plasma IgG" and "IgG eluted from leukocytes" refer to the relative amounts (i.e., visually scored following microscopy) of anti-A and/or anti-B IgG
antibodies present in either the plasma of the pooled cord blood or in the fraction eluted from the surface of leukocytes present in the pooled cord blood. The relative scale for scoring the amount of IgG present includes (from lowest to highest): "Neg"; "weak"; "1 +"; "2+"; "3+" and "4+".
[0091] As shown in Table 3, there is an overall correlation between the presence of significant amounts of anti-A
and/or anti-B IgG antibodies bound to the surface of the leukocytes and the final TNC yield obtained. For example, the CBU pooling having "Mother ABO" of 0, A, 0 and "CBU ABO" of B, A, A
displayed a level of anti-A IgG of "2+"
and a corresponding TNC yield of 72%. In contrast, other CBU poolings displaying "Neg" levels of eluted anti-A and anti-B IgG antibodies generally correlated with a higher TNC yield. As explained above, the anti-A and/or anti-B IgG
antibodies bound to the surface of the leukocytes contributes to the lysis of these cells.
Table 3: Quantification of anti-A/anti-B IgG antibodies eluted from leukocytes from pooled whole CBUs Mother CBU ABO Plasma IgG IgG eluated from TNC
ABO leukocytes Yield %
A, A, A 0,0,0 IgGAl-3+ IgGAl-1+ 68%
IgG B - weak IgG B - Neg A, B, A A, A, 0 IgG Al - 2+ IgG Al - weak 93%
IgG B - weak IgG B - Neg 0,0,0 0,0,0 IgGAl-4+ IgGAl - Neg 100%
IgG B -1+ IgG B - Neg 0, A, 0 B, A, A IgG Al -1+ IgG Al - 2+ 72%
IgG B - weak IgG B - weak A, A, 0 0, 0, 0 IgG Al - 4+ IgG Al - Neg 88%
IgG B - 3+ IgG B - Neg 0,0,0 0,0,0 IgGAl-3+ IgG Al - Neg 90%
IgG B - 4+ IgG B - Neg A, 0, 0 A, 0, 0 IgG Al - 4+ IgG Al - 2+ 87%
IgG B - 3+ IgG B - weak [0092] Soluble anti-A and anti-B IgG antibodies are mostly present in the plasma of the CBU. Thus, according to the pooling criteria of the present invention, increasing the volume of cord blood plasma containing these IgG
antibodies should negatively impact the final TNC yield in pooled CBUs.
Accordingly, as shown in Table 4, three CBUs were thawed and then diluted in three different Dilution Solutions, each containing 50% Gentran 40 and 50%
of three solutions containing different amounts of cord blood plasma (using a solution of 5% human albumin to equalize the volumes). These solutions were (in order of increasing plasma concentration): "Full Albumin" (a solution of 5% human albumin); "Half Plasma/Half Albumin" (a solution of 50% of cord blood plasma and 50% of a 5%
solution of human albumin); and "Full plasma" (only cord blood plasma).
and/or anti-B IgG antibodies bound to the surface of the leukocytes and the final TNC yield obtained. For example, the CBU pooling having "Mother ABO" of 0, A, 0 and "CBU ABO" of B, A, A
displayed a level of anti-A IgG of "2+"
and a corresponding TNC yield of 72%. In contrast, other CBU poolings displaying "Neg" levels of eluted anti-A and anti-B IgG antibodies generally correlated with a higher TNC yield. As explained above, the anti-A and/or anti-B IgG
antibodies bound to the surface of the leukocytes contributes to the lysis of these cells.
Table 3: Quantification of anti-A/anti-B IgG antibodies eluted from leukocytes from pooled whole CBUs Mother CBU ABO Plasma IgG IgG eluated from TNC
ABO leukocytes Yield %
A, A, A 0,0,0 IgGAl-3+ IgGAl-1+ 68%
IgG B - weak IgG B - Neg A, B, A A, A, 0 IgG Al - 2+ IgG Al - weak 93%
IgG B - weak IgG B - Neg 0,0,0 0,0,0 IgGAl-4+ IgGAl - Neg 100%
IgG B -1+ IgG B - Neg 0, A, 0 B, A, A IgG Al -1+ IgG Al - 2+ 72%
IgG B - weak IgG B - weak A, A, 0 0, 0, 0 IgG Al - 4+ IgG Al - Neg 88%
IgG B - 3+ IgG B - Neg 0,0,0 0,0,0 IgGAl-3+ IgG Al - Neg 90%
IgG B - 4+ IgG B - Neg A, 0, 0 A, 0, 0 IgG Al - 4+ IgG Al - 2+ 87%
IgG B - 3+ IgG B - weak [0092] Soluble anti-A and anti-B IgG antibodies are mostly present in the plasma of the CBU. Thus, according to the pooling criteria of the present invention, increasing the volume of cord blood plasma containing these IgG
antibodies should negatively impact the final TNC yield in pooled CBUs.
Accordingly, as shown in Table 4, three CBUs were thawed and then diluted in three different Dilution Solutions, each containing 50% Gentran 40 and 50%
of three solutions containing different amounts of cord blood plasma (using a solution of 5% human albumin to equalize the volumes). These solutions were (in order of increasing plasma concentration): "Full Albumin" (a solution of 5% human albumin); "Half Plasma/Half Albumin" (a solution of 50% of cord blood plasma and 50% of a 5%
solution of human albumin); and "Full plasma" (only cord blood plasma).
[0093] The results in Table 4 show that TNC yield is generally correlated with the volume of cord blood plasma present in the Dilution Solution. That is, a higher volume of cord blood plasma (and thus higher amounts of anti-A
and/or anti-B IgG antibodies) in the Dilution Solution generally resulted in a decrease in TNC yield.
Table 4: CBUs diluted with Dilution Solutions having Gentran 40 and cord blood plasma and/or human albumin Dilution with Gentran Mother ABO CBU ABO 40 and TNC Yield A, B, A AB, 0, 0 Full plasma 90%
Half plasma/Half albumin 95%
Full albumin 93%
0, A, A B, 0, 0 Full plasma 61%
Half Plasma/Half albumin 62%
Full albumin 66%
B, A, 0 A, A, A Full plasma 41%
Half plasma/Half albumin 42%
Full albumin 41%
0'0'0 0'0'0 Full plasma 70.0%
Half plasma/Half albumin 75.1%
Full albumin 84.7%
B, A, A B, A, AB Full plasma 54%
Half plasma/Half albumin 69%
Full albumin 65%
and/or anti-B IgG antibodies) in the Dilution Solution generally resulted in a decrease in TNC yield.
Table 4: CBUs diluted with Dilution Solutions having Gentran 40 and cord blood plasma and/or human albumin Dilution with Gentran Mother ABO CBU ABO 40 and TNC Yield A, B, A AB, 0, 0 Full plasma 90%
Half plasma/Half albumin 95%
Full albumin 93%
0, A, A B, 0, 0 Full plasma 61%
Half Plasma/Half albumin 62%
Full albumin 66%
B, A, 0 A, A, A Full plasma 41%
Half plasma/Half albumin 42%
Full albumin 41%
0'0'0 0'0'0 Full plasma 70.0%
Half plasma/Half albumin 75.1%
Full albumin 84.7%
B, A, A B, A, AB Full plasma 54%
Half plasma/Half albumin 69%
Full albumin 65%
[0094] Methods for reducing the lysis of TNCs in collected umbilical cord blood [0095] In another embodiment, the present invention relates to a method for reducing the lysis of total nucleated cells in collected umbilical cord blood (e.g., pooled cord blood). More particularly, the method involves preventing IgG
antibodies from binding to the A and/or B antigens expressed on the surface of these nucleated cells. For example, the method can involve substantially removing the IgG antibodies from the collected umbilical cord blood. Methods of removing IgG from samples are known in the art. The method can also be performed by administering an IgG-binding inhibitor to the collected umbilical cord blood.
antibodies from binding to the A and/or B antigens expressed on the surface of these nucleated cells. For example, the method can involve substantially removing the IgG antibodies from the collected umbilical cord blood. Methods of removing IgG from samples are known in the art. The method can also be performed by administering an IgG-binding inhibitor to the collected umbilical cord blood.
[0096] Another example includes blocking the activity of complement activation in the pooled cord blood, which would reduce the lysis TNCs. An antibody specific for terminal complement protein C5 could be used to inhibit complement activation. Soliris (eculizumab) represents such an antibody, which has been approved by the FDA for use in patients with paroxysmal nocturial hemoglobinuria (PNH) who suffer from intravascular hemolysis.
[0097] Method of increasing the speed of engraftment of CBU transplantation [0098] In an embodiment, the present invention relates to the surprising discovery that a composition or "carrier unit" comprising purified or substantially purified HSCs from pooled cord blood units from separate donors can improve the success of CB transplantation. More particularly, infusion of a composition or carrier unit of the present invention, in addition to a sufficiently matched CBU unit, increases the speed of engraftment as compared to a standard CB transplant. Speed of engraftment can be judged, for example, by speed of neutrophil engraftment and/or speed of platelet engraftment.
[0099] In accordance with the present invention, a patient (e.g., an adult patient) having or suspected of having a disease that can be ameliorated by a CB transplantation is sufficiently HLA-matched with a CBU to be infused.
Following infusion of the HLA-matched CBU containing, for example, a minimum of 1.75 x 10' TNC per kg of the recipient, a carrier unit of the present invention is infused into the patient within, for example, two hours. In one embodiment, the carrier unit to be infused comprises prior to processing at least 1 x 106 CD34-positive HSCs per Kg of the recipient. The skilled person would be aware that the exact number of CD34-positive HSCs/kg used as starting material (prior to processing) is less important than the final number of CD34-positive cells that will eventually be infused into the patient. Because some CD34-positive cells will inevitable be lost during the processing of the CBUs to arrive at the carrier unit of the present invention, it is preferable to start with as many CD34-positive cells as feasible and/or available for the transfusion. Thus, in another embodiment, the carrier unit to be infused (i.e., after processing) comprises at least 0.1-0.3 x 106 CD34-positive HSCs per kg of the recipient. In yet another embodiment, the carrier unit to be infused (i.e., after processing) comprises at least 0.2-0.3 x 106 CD34-positive HSCs per kg of the recipient. The upper limit for the optimal number of CD34-positive cells/kg to be infused is not precisely known but, in general, a greater number of CD34-positive cells directly correlates with a higher chance of success. The skilled person would be aware that CD34-positive cells from cord blood have a 5-10-fold greater proliferative capacity than corresponding adult cells. Hence, the infusion of 0.1-0.3 x 106 CD34-positive cells/kg from cord blood is expected to be at least equivalent to the infusion of 1 x 106 CD34-positive adult cells.
Following infusion of the HLA-matched CBU containing, for example, a minimum of 1.75 x 10' TNC per kg of the recipient, a carrier unit of the present invention is infused into the patient within, for example, two hours. In one embodiment, the carrier unit to be infused comprises prior to processing at least 1 x 106 CD34-positive HSCs per Kg of the recipient. The skilled person would be aware that the exact number of CD34-positive HSCs/kg used as starting material (prior to processing) is less important than the final number of CD34-positive cells that will eventually be infused into the patient. Because some CD34-positive cells will inevitable be lost during the processing of the CBUs to arrive at the carrier unit of the present invention, it is preferable to start with as many CD34-positive cells as feasible and/or available for the transfusion. Thus, in another embodiment, the carrier unit to be infused (i.e., after processing) comprises at least 0.1-0.3 x 106 CD34-positive HSCs per kg of the recipient. In yet another embodiment, the carrier unit to be infused (i.e., after processing) comprises at least 0.2-0.3 x 106 CD34-positive HSCs per kg of the recipient. The upper limit for the optimal number of CD34-positive cells/kg to be infused is not precisely known but, in general, a greater number of CD34-positive cells directly correlates with a higher chance of success. The skilled person would be aware that CD34-positive cells from cord blood have a 5-10-fold greater proliferative capacity than corresponding adult cells. Hence, the infusion of 0.1-0.3 x 106 CD34-positive cells/kg from cord blood is expected to be at least equivalent to the infusion of 1 x 106 CD34-positive adult cells.
[00100] In another embodiment, the present invention provides a method for treating a patient having a disease that can be ameliorated by a CB transplant. Non-limiting examples of diseases that can be ameliorated by a CB
transplant include chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), chronic myeloid leukemia (CML), acute myeloid leukemia (AML), Hodgkin's and non-Hodgkin's lymphoma, and virtually any hematological cancer.
[00101 ] Increasing the number of CBU suitable for transplantation of adults [00102] The limited number of total nucleated cells in many cryopreserved CBUs precludes their suitability for transplanting adults or other subjects having a weight of more than, e.g., 65 or 70 kg. Thus, in yet another embodiment, the present invention relates to a method of increasing the number of cryopreserved CBUs that are suitable for transplanting adults or subjects weighing more than, e.g., 65 or 70 kg. In that regard, a CBU lacking sufficient total nucleated cells to be suitable to treat a subject of a certain weight can be infused into the subject along with a composition of the present invention.
[00103] Since the majority of CB that is collected fails to meet the standards for banking (i.e., unqualified CB) and is discarded, much CB is wasted. Therefore, in one embodiment, the present invention provides a method for maximizing the use of collected CB that has failed to qualify for banking by CB banks by pooling cord blood collected from separate donors. While the present invention is not limited to the pooling of unqualified CB compared to qualified CB, the skilled person would understand that pooling unqualified CB
is the most cost effective strategy for CB banks or other similar institutions.
[00104] The present invention is illustrated in further details by the following non-limiting examples.
[00105] Example 1: Overview of cord blood collection and processing [00106] The cord blood was collected in utero, after the newborn's umbilical cord was cut but before the placenta was expelled from the uterus, and transferred to a sterile 250 mL collection bag containing 35 mL of citrate-phosphate-dextrose (CPD), which was then labeled and sent at room temperature to the cord blood bank for processing.
[00107] The first part of cord blood processing involves qualification for the Public Bank, which requires at least 65 mL of blood and a total nucleated cell (TNC) count of at least 1 x 109 cells per collection bag. The majority of collected blood fails to meet these standards. For example, during the first 821 days of activity at the Royal Victoria Hospital, only 32.5% of all collected cord blood units qualified for the Public Cord Blood Bank of Quebec and only 43% qualified for the MUHC-Clinical Research Cord Blood Bank (CRCBB). Thus, the majority of collected cord blood is not banked and is either discarded or used for research purposes.
[00108] If the cord blood unit meets the above standards, the second part of processing involves a volume reduction using the Top-Bottom technique with the Baxter Opti-System. After adding DMSO
to the leuco-platelet layer (bully coat) to a final concentration of 10% (v/v) (averaging about 22 mL 2.9 mL), the processed cord blood unit was incubated first for about one hour at -80 C, followed by a cryopreservation at about -150 C until use. Table 5 summarizes the cord blood units that were processed for the CRCBB from the period of October 2007 to January 2010. The average CBU volume processed was 90.8 mL (with a range of 38 mL to 175 mL) with a white blood cell (WBC) yield of 77% (range: 20% to 127%).
Table 5: Cord blood units processed for the CRCBB from October 2007 to January Leuco-platelet layer (what is WBC
Whole Blood Red Blood Plasma frozen) Yield Volume GBI. Hb Htc Plqts Volume GBI. Hb Volume Volume GBI.
ml 10 /unit g/unit % 10 /unit ml g/unit ml ml 10 /unit %
Mean 90.8 1.1 9.9 33.0 0.2 21.8 0.2 0.0 49.1 22.0 0.8 77%
Std.Dev. 24.3 0.5 3.6 6.2 0.1 12.5 0.3 0.0 15.3 2.9 0.4 20%
Median 89.0 0.9 9.5 33.5 0.2 19.0 0.1 0.0 49.0 22.0 0.7 78%
Max 175.0 2.9 25.3 47.8 0.5 74.0 1.3 0.0 94.0 37.0 3.0 127%
Min 38.0 0.1 1.1 7.2 0.0 5.0 0.0 0.0 10.0 12.0 0.0 20%
[00109] Example 2: Laboratory techniques and analysis [00110] Example 2.1: Cord blood unit volume reduction and cryopreservation [00111] Prior to volume reduction, an aliquot was removed from the collected cord blood unit (CBU) to evaluate the number of total nucleated cells (TNC) and nucleated Red Blood Cells (nRBC).
The CBU was then centrifuged at 2689g for 13.5 minutes at 22 C. Using the Fenwal Semiautomated system Optipress II , the centrifuge triple-bag was pressed following specific settings based on the volume: buffy-coat volume of 33 mL, a buffy coat level of 3.5 to 4.5 and a force of 23. Following volume reduction, a sample was removed from the buffy coat bag for use in a colony-forming unit-granulocyte-macrophage (CFU-GM) assay and for evaluation of TNC, CD34-positive, CD45-positive cell counts, as well as cell viability using 7-AAD. Cryopreservation was performed by slowly adding, within 15 minutes, a mixed solution of 50% dimethyl sulfoxide (DMSO) solution and Gentran 40 to ensure a final concentration of 10% DMSO. A sample of the mixed product was removed and distributed in four cryovials, which were then placed along with the CBU at -80 C for one hour and then transferred to a cryofreezer at <_ -150 C until use.
[00112] Example 2.2: Preparation of Samples for Analysis [00113] The volume reduction described in Example 2.1 permits the removal of plasma and red blood cells from the CBU. The plasma was then used to perform virology analysis and at least four 1-mL samples were cryopreserved for future use. The red blood cells were used for a number of tests, for example, the identification of the CBU blood group and rhesus factor, for sterility testing, and for hemoglobin electrophoresis.
[00114] Example 2.3: Cord Blood pooling technique [00115] CBUs were pooled by groups of three in a Dilution Solution composed of 50% Gentran 40 and 5% Human Albumin. Based on the total volume of the three CBUs, the dilution solution represented 90% of that volume. This resulted in a reduction of the DMSO concentration from 10% to 1 %.
[00116] Example 2.4: Thawing, dilution and pooling of the cord blood units [00117] The CBUs that were to be pooled to form the carrier unit of the present invention were processed in groups of three for greater practicality. One frozen CBU was immersed in a 38 C water bath and rapidly thawed. A volume of Dilution Solution corresponding to twice the volume of the CBU was added to the thawed CBU. The bag containing the thawed and diluted CBU was then incubated on ice for 5 minutes without further manipulation. Following the incubation, the contents of the thawed and diluted CBU was transferred to a 600 mL bag and rinsed twice. Two other CBUs to be pooled were processed the same way, and transferred to the 600 mL bag. The 600 mL bag containing the thawed and diluted pooled CBUs was then centrifuged at 1400 rpm for 9 minutes at 4 C.
Depending on the patient to be transplanted, the required number of CBUs were thawed in groups of three, diluted and centrifuged according to the procedure described above. A final pooling of all the thawed and diluted CBUs was done in a 600 mL bag, after ensuring a final volume of 95 mL.
[00118] Example 2.5: Purification of HSCs via a CD34-positive selection and infusion thereof into patient [00119] HSCs were purified from the pooled CBUs according to the standard protocol set forth by the CliniMACS
CD34 MicroBeads purification kit (Miltenyi Biotec , Bergisch Gladbach, Germany). Briefly, the bag containing the pooled CBUs was incubated at 22 C for 60 minutes with CliniMACS CD34 MicroBeads.
After one wash with CliniMACS
PBS/EDTA with 0.5% human albumin, the final product was applied to a CliniMACS
column for a CD34-positive selection. The negative fraction was used for Short Tandem Repeat identification and for verification of bacterial contamination. An aliquot of the CD34-positive selection was used for CD34, CD45, CD25, CD41, CD61, CD3, CD56 and CD1 6-positive cell counts and for use in CFU-GM assay. The CD34-positive fraction (i.e., the carrier unit of the present invention) was then infused into the patient or recipient within two hours of infusion of the HLA-matched CBU.
[00120] Example 2.6: Indirect Coombs test and elution of anti-A and anti-B IgG
antibodies [00121] The indirect Coombs test (also known as indirect antiglobulin test or IAT) is useful for identifying the presence of antibodies that can bind to the surface of red blood cells. For example, the indirect Coombs test can be used to detect antibodies against RBCs that are present in serum. In general, serum is extracted from the blood, and the serum is incubated with RBCs of known antigenicity. If agglutination occurs, the indirect Coombs test is positive.
The indirect Coombs test is commonly known in the art and the details of this technique would be within the grasp of the skilled person. Furthermore, the skilled person would be aware of different elution methods can be used for the elution and identification of anti-A and anti-B IgG antibodies.
[00122] The identification of bound IgG to nucleated cells was done through Heat Elution at 56 C. After incubation at 56 C, the sample was centrifuged at 3700 rpm for 2 minutes. The eluate was subsequently evaluated for content of anti-A and anti-B IgG using A and B red blood cells.
[00123] Example 3: Processing of cord blood [00124] The cord blood processing method applied by Public Banks (Armitage, 1999; Godinho, 2000) was adapted for the processing of low volume cord blood bags which have been rejected by Public Banks. The Top-Bottom method was executed following a hard centrifugation with the Baxter Opti-System. The semi-automated closed system was configurated to separate the centrifuged CBU into three components:
plasma, red blood cells and a leuco-platelets layer of 20 mL 2 mL. The leuco-platelet layer was then rotated for a period of 30 minutes, followed by the addition of a solution of DMSO 50%/Gentran at a final ratio of 1:4 (v:v) to ensure a final DMSO concentration of 10%. The final product was then frozen for one hour at -80 C, followed by long term cryopreservation in a vapour phase nitrogen cryofreezer. This method of freezing ensures adequate preservation of the nucleated cells as previously compared with a control rate freezing method (Itoh, 2003).
[00125] Example 4: Thawing and pooling of cryopreserved cord blood [00126] Upon selection of the required CBUs, a mixed solution containing equal amounts of 5% human albumin and Gentran 40 was prepared. The CBUs were regrouped in groups of three units to ensure a pre-thawing maximum volume of 60 mL. Using a semi-closed system, each CBU was diluted with twice its original volume and incubated for minutes at 4 C. Following the first dilution, all three CBUs were pooled in a bag containing half of the initial solution of mixed 5% human albumin and Gentran 40. A second dilution, which was also used to rinse the collection bag, was done using an equal volume of the CBU. The remaining solution was then used to rinse the collection CBU bags.
After each rinse, the contents were transferred to a unique pooling bag. HSCs were then purified from the pooled cord blood according to a CD34-positive selection using the CliniMACS CD34 Reagent (Miltenyi Biotec , Bergisch Gladbach, Germany) as detailed in Example 2.7. Following purification, an aliquot was taken to measure the total nucleated cell (TNC) and CD34-positive cell yield and to document viability.
[00127] When pooling 12 units, four groups of three units were thawed and diluted by repeating the above. Each pooled bags were then centrifuged at 1400 rpm at 4 C for 9 minutes. The supernatant was removed to ensure a final volume of approximately 20 mL. A sample was taken from the remaining cell pellet to measure the pooling and washing yield. All cell pellets were pooled in a final collection bag and the volume was measured to ensure a volume of about 95 mL 5 mL.
[00128] Example 5: Treatment of a patient having Chronic Lymphoid Leukemia [00129] A 41 year-old male patient or recipient weighing 81 kg, who was diagnosed in 2007 with an unclassified CD5 Chronic Lymphoid Leukemia (CLL). Upon diagnosis, the patient was treated first with CHOP chemotherapy (cyclophosphamide, doxorubicin, vincristine, and prednisone/prednisolone), and then was treated at his first relapse with Nipent, Cytoxan and Rituxan (PCR regimen), followed by high-dose cytarabine (ara-C; HiDAC) upon his second relapse. He was subjected to an umbilical cord blood transplant according to the present invention. The myeloablative conditioning regimen pre-transplantation was composed of Fludarabine, Busulfan and Cyclophosphamide. The patient having blood group A was matched for a CBU
(i.e., the "HLA-matched CBU" or the "definitive CBU") having an HLA compatibility of 4/6, with one mismatch on locus A and one mismatch on locus B.
This HLA-matched CBU happened to be of blood group 0 and contained 2.9 x 107 TNC/kg and 1.1 x 105 CD34-postive cells/kg of the patient's weight.
[00130] To prepare the carrier unit of the present invention, 45 CBUs were selected for pooling, each selected to have blood group 0 with their corresponding mother's blood group also being blood group 0. These CBUs, although cryopreserved, were CBUs that had failed to qualify for public banking but were nevertheless deemed safe for use in transplantation. The 45 CBUs were pooled in vitro and hematopoietic stem cells were substantially purified via a CD34-positive selection, as detailed above, to give the carrier unit of the present invention. The carrier unit had 0.3 x 106 CD34-positive cells/kg of patient's weight and a purity of 53.8% as determined by FACS analysis. The percent purity was calculated by the formula as presented in Braakman (2008): total number of CD34 events in selected product/total number of CD45 in selected product x 100. The CD34-positive selection resulted in a 17-fold depletion in the amount of CD3-positive cells, with only 0.86 x 104 CD3-positive cells/kg of the patient being infused.
[00131] The infusion of the HLA-matched CBU was done 69 minutes prior to the infusion of the carrier unit and the day of the transplantation/infusion was considered as day 0. Follow-up tests revealed that the patient successfully engrafted neutrophils on day +15 and platelets on day +42. The patient was considered to have successfully engrafted neutrophils based on the standard criteria of having a neutrophil cell count of greater than 0.5 x 109 neutrophils cells/L for a period of at least three consecutive days (with the first day thereof being considered as the day of engraftment). The engraftment of neutrophils at day +15 represented a 29% increase in the speed of engraftment compared to that normally expected from a standard double cord blood transplant (i.e., at day +21).
Additionally, the patient exhibited 100% HLA-matched CBU chimerism at day +14.
Between day +35 and day +100, the patient's chimerism fell below 100%, correlating with the appearance of acute Graft versus Host disease grade II/III of the skin and the gut. However, beginning at day +124 and extending through the date that this application was filed, full chimerism of the HLA-matched CBU was restored in the patient, indicating a successful transplantation.
[00132] Example 6: Treatment of a patient having Philadelphia Chromosome-positive Chronic Myeloid Leukemia [00133] A 32 year-old male patient having blood group B and weighing 56 kg was diagnosed in 2009 with Philadelphia Chromosome-positive Chronic Myeloid Leukemia (Ph+ CML). He was treated with 7 + 3 (cytarabine:daunorubicin) and with Gleevec. Soon after, the patient was not responding to Gleevec and was switched to Nilotinib. Another blast crisis presented in 2010 where he was treated with Flag-Ida (fludarabine, cytarabine, idarubicin and G-CSF). Having a second blast crisis in a very short time, the patient was subjected to an umbilical cord blood transplant according to the present invention. The myeloablative conditioning regimen consisted of fludarabine, busulfan and cyclophosphamide. The patient was matched for a CBU
(i.e., the "HLA-matched CBU" or the "definitive CBU") having an HLA compatibility of 5/6 with a single mismatch on locus A. This HLA-matched CBU
happened to be of blood group A and contained 2.74 x 107 TNC/kg and 0.67 x 105 CD34-positive cell/kg of the patient's weight.
[00134] To prepare the carrier unit of the present invention, 30 CBUs having blood group 0 were selected for pooling. These CBUs, although cryopreserved, were CBUs that had failed to qualify for public banking but were nevertheless deemed safe for use in transplantation. The 30 CBUs were pooled in vitro and hematopoietic stem cells were substantially purified via a CD34-positive selection, as detailed above, to give the carrier unit of the present invention. The carrier unit had 0.2 x 106 CD34-positive cells/kg of patient's weight and a purity of 81.7% as determined by FACS analysis. The carrier unit also had 0.14 x 104 CD3-positive cells/kg after purification, representing an 80-fold decrease between the pre- and post-CD43 selection.
[00135] The infusion of the HLA-matched CBU was done 96 minutes prior to the infusion of the carrier unit and the day of the transplantation/infusion was considered as day 0.
[00136] Example 7: Treatment of a patient having Acute Myeloid Lymphoma [00137] A 30 year-old female, blood group 0, weighing 87.5 kg was diagnosed in 2007 with acute promyelocytic leukemia (APL). The Pethema protocol was used for the induction of the APL and was followed with 6-mercaptopurine (6-MO) and Methotraxate as consolidation treatment. In 2009 the patient relapsed with an Acute Myeloid Lymphoma secondary to APL treatment and was treated with High Dose of Arabinosycytosine (Ara-C). In 2010, she relapsed from her AML and was treated with Etoposide (VP-16, cyclophosphamide and arsenic trioxide and subjected to an umbilical cord blood transplant according to the present invention. The myeloablative conditioning regimen consisted of fludarabine, busulfan and cyclophosphamide.
The patient was matched for a CBU
(i.e., the "HLA-matched CBU" or the "definitive CBU") having an HLA
compatibility of 5/6 with a single mismatch on locus A. This HLA-matched CBU happened to be of blood group 0 and contained 2.63 x 10' TNC/kg and 1.63 x 105 CD34-positive cells/kg of the patient's weight.
[00138] To prepare the carrier unit of the present invention, 42 CBUs having blood group 0 were selected for pooling. Of these 42 CBUs, 37 CBUs originated from mothers having blood group 0, 3 CBUs originated from mothers having blood group A, and two CBUs originated from mothers having blood group B. These CBUs, although cryopreserved, were CBUs that had failed to qualify for public banking but were nevertheless deemed safe for use in transplantation. The 42 CBUs were pooled in vitro and hematopoietic stem cells were substantially purified via a CD34-positive selection, as detailed above, to give the carrier unit of the present invention. The carrier unit had 0.25 x 106 CD34-positive cells/kg of patient's weight and a purity of 75.7% as determined by determined FACS analysis.
The carrier unit also had 0.7 x 104 CD3-positive cells/kg, representing a 107-fold depletion after purification.
[00139] The infusion of the HLA-matched CBU was done 60 minutes prior to the infusion of the carrier unit and the day of the transplantation/infusion was considered as day 0. Follow-up tests revealed that the patient successfully engrafted neutrophils on day +19, representing a 10% increase in the speed of engraftment compared to that normally expected from a standard double cord blood transplant (i.e., at day +21). As for Example 5, the patient was considered to have successfully engrafted neutrophils based on the standard criteria of having a neutrophil cell count of greater than 0.5 x 109 neutrophils cells/L for a period of at least three consecutive days (with the first day thereof being considered as the day of engraftment).
[00140] Although the present invention has been described hereinabove by way of specific embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims.
REFERENCES
Armitage S, Fehily D, Dickinson A, Chapman C, Navarrette C and Contreras M
(1999) Cord blood banking: volume reduction of cord blood units using a semi-automated closed system. Bone Marrow Transplantation 23:505-509.
Ballen KK, Wilson M, Wuu J, Ceredone AM, Hsieh C, Stewart FM, Popovsky MA and Quesenberry PJ (2001) Bigger is better: maternal and neonatal predictors of hematopoietic potential of umbilical cord blood units. Bone Marrow Transplantation 27:7-14.
Barker JN, Weisdorf DJ, DeFor TE, Blazar BR, McGlave PB, Miller JS, Verfaillie CM and Wagner JE (2005) Transplantation of 2 partially HLA-matched umbilical cord blood units to enhance engraftment in adults with hematologic malignancy. Blood 105: 1343-1347.
Braakman E., Schuurhuis, GJ., Freijer FWMB., Voermans C., Theunissen K., van Riet I., Fibbe WE., and Slaper-Cortenbach I (2008) Evaluation of 'out-of-specification' CliniMACS CD34-selection procedures of hematopoietic progenitor cell-apheresis products. Cytotherapy 10(1): 83-89.
Ende et al., "Pooled umbilical cord blood as a possible universal donor for marrow reconstitution and use in nuclear accidents." Life Sci. 2001 Aug 17;69(13):1531-9.
Fiumera AC and Asmussen MA (2001) Difficulties in parentage analysis : the probability that an offspring and parent have the same heterozygous genotype. Genet. Res., Camb, 78: 163-170.
Gluckman E, Broxmeyer HE, Auerbach AD et al. Hematopoietic reconstitution in a patient with Fanconi's anemia by means of umbilical cord blood from an HLA-identified sibling. New Engl J Med 1989;321:214-219.
Gluckman, E and Rocha, V (2009) Cord blood transplantation: state of the art.
Haematologica, 94(4): 451-454.
Godinho MI, de Sousa ME, Carvalhais A and Barbosa IL (2000) Umbilical cord blood processing with the Optipress II
blood extractor. Cytotherapy 2(6): 439-443.
Hsu TC, Steinberg J, LeDoux R, and Sawrrsky A, (1979) The Low Ionic Strength Reaction of Human Blood:
Relationship between the Binding of Serum Immunoglobulin and Complement to Red Blood Cells and Surface Charge of the Cells. British Journal of Haematology 42:403-415.
Itoh T, Minegishi M, Fushimi J, Takahashi H, Kudo Y, Suzuki A, Narita A, Sato Y, Akagi K, Wada Y, Saito A, Kikuchi M, Okamura K, Kaku M and Tsuchiya S (2003) A simple controlled-rate freezing method without a rate-controlled programmed freezer provides optimal conditions for both large-scale and small-scale cryopreservation of umbilical cord blood cells. Transfusion 43:1303-1308.
Jaroscak J, Goltry K, Smith A, Waters-Pick B, Martin PL, Driscoll TA, Howrey R, Chao N, Douville J, Burhop S, Fu P, and Kurtzberg J (2003) Augmentation of umbilical cord blood (UCB) transplantation with ex vivo-expanded UCB
cells: results of a phase 1 trial using the AastromReplicell System. BLOOD, 101(12): 5061-5067.
Jauniaux E, Jurkovic D, Gulbis B, Liesnard C, Lees C, and Campbell S (1995).
Materno-fetal immunoglobulin transfer and passive immunity during the first trimester of human pregnancy. Human Reproduction, 10(12): 3297-3300.
Klein HG, Anstee DJ (2008) Mollison's Blood Transfusion in Clinical Medicine.
Eleventh Edition, Blackwell Publishing Kohler PF, Farr RS (1966) Elevation of Cord over Maternal IgG
Immunologlobulin: Evidence for an Active Placental IgG Transport. Nature, 210:1070-1071.
Kumpel BM, van de Winkel JGJ, Westerdaal NAC, Hadley AG, Dugoujon JM and Blancher A (1996) Antigen topography is critical for interaction of IgG2 anti-red-cell antibodies with Fcy receptors. British Journal of Haematology, 94:175-183.
Kurtzberg J, Cairo MS, Fraser JK, Baxter-Lowe L, Cohen G, Carter SH, and Kernan NA (2005) Magro E, Regidor C, Cabrera R, Sanjuan I, Fortis R, Garcia-Marco JA, Ruiz E, Gil S, Bautista G, Millan I, Madrigal A, and Fernandez MN (2006) Early hematopoietic recovery after single unit unrelated cord blood transplantation in adults supported by co-infusion of mobilized stem cells from a third party donor. Haematologica, 91:640-648.
Majhail NS, Brunstein CG and Wagner JE (2006) Double umbilical cord blood transplantation. Current Opinion in Immunology, 18:571-575 Mehta Y, Dhar A, Meharwal SZS, Trehan N (2007) Comparison of New HES (130/0.4) and HES (200/0.5) in OPCAB
Surgery J Anesth Clin Pharmacology 2007; 23(3): 273-278.
Mielcarek M, Torok-Storb B and Storb R (2002) ABO incompatibility and relapse risk in patients undergoing allogeneic marrow transplantation for acute myeloid leukemia. Bone Marrow Transplantation 30, 547-548.
Miler ME, Stiehm ER (1973) Phagocytlc, opsonic and immunoglobulin studies in newborns. Medical Progress Calif Med 119:43-63.
M-Reboredo N, Di'az A, Castro A, and Villaescusa RG (2000) Collection, processing and cryopreservation of umbilical cord blood for unrelated transplantation. Bone Marrow Transplantation 26:1263-1270.
Ohnuma K, Isoyama K, Ikuta K,Toyoda Y, Nakamura J, Nakajima F, and Nishihirah H (2000) The Influence of HLA
Genotyping Compatibility on Clinical Outcome after Cord Blood Transplantation from Unrelated Donors JOURNAL
OF HEMATOTHERAPY & STEM CELL RESEARCH 9:541-550.
Petz, LD and Garratty G (2004) Immune Hemolytic Anemias, Second Edition, Elsevier Publication.
Querol S, Mufti GJ, Marsh SGE, Pagluica A, Little AM, Shaw, BE, Jeffery R, Garcia, J, Goldman JM, and Madrigal JA
(2009) Cord blood stem cells for hematopoietic stem cell transplantation in the UK: how big should the bank be?
Haematologica, 94(4):536-541.
Results of the Cord Blood Transplantation (COBLT) Study unrelated donor banking program. TRANSFUSION
2005;45:842-855 Rubinstein P, Dobrila L, Rosenfield RE, Adamson JW, Mogllaccio G, Migliaccio AR, Taylor PE, AND Stevens CE
(1995) Processing and cryopreservation of placental/umbilical cord blood for unrelated bone marrow reconstitution.
Proc. Nati. Acad. Sci. 92:10119-10122.
Solves P, Moraga R, Mirabet V, Larrea L, and Soler MA (2003) In utero or ex utero cord blood collection: an unresolved question. TRANSFUSION 43:1174-1176.
Solves P, Moraga R, Saucedo E, Perales A, Soler MA, Larrea L, Mirabet V, Planelles D, Carbonell-Uberos F, Monleo'n J, Planells T, Guille'n M, Andre s A and Franco E (2003a) Cord blood stem cells: Comparison between two strategies for umbilical cord blood collection. Bone Marrow Transplantation 31: 269-273.
Takahashi S, Iseki T, Ooi J, Tomonari A, Takasugi K, Shimohakamada Y, Yamada T, Uchimaru K, Tojo A, Shirafuji N, Kodo H, Tani K, Takahashi T, Yamaguchi T, and Asano S (2004) Single-institute comparative analysis of unrelated bone marrow transplantation and cord blood transplantation for adult patients with hematologic malignancies. Blood, 104(12):3813-3820.
Takahashi, S (2007) Leukemia: Cord blood for allogeneic stem cell transplantation. Current Opinion in Oncology, 19:667-672.
Wagner J,Champlin R, and Petz LD (2006) Symposium Summary Fourth Annual International Umbilical Cord Blood Transplantation Symposium, Los Angeles, California, May 19-20, 2006 Biology of Blood and Marrow Transplantation 12:1206-1217 (2006).
Wong A, Yuen PMP, Li K, Yu ALM and Tsoi WC (2001) Cord blood collection before and after placental delivery:
levels of nucleated cells, haematopoietic progenitor cells, leucocyte subpopulations and macroscopic clots. Bone Marrow Transplantation 27:133-138.
transplant include chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), chronic myeloid leukemia (CML), acute myeloid leukemia (AML), Hodgkin's and non-Hodgkin's lymphoma, and virtually any hematological cancer.
[00101 ] Increasing the number of CBU suitable for transplantation of adults [00102] The limited number of total nucleated cells in many cryopreserved CBUs precludes their suitability for transplanting adults or other subjects having a weight of more than, e.g., 65 or 70 kg. Thus, in yet another embodiment, the present invention relates to a method of increasing the number of cryopreserved CBUs that are suitable for transplanting adults or subjects weighing more than, e.g., 65 or 70 kg. In that regard, a CBU lacking sufficient total nucleated cells to be suitable to treat a subject of a certain weight can be infused into the subject along with a composition of the present invention.
[00103] Since the majority of CB that is collected fails to meet the standards for banking (i.e., unqualified CB) and is discarded, much CB is wasted. Therefore, in one embodiment, the present invention provides a method for maximizing the use of collected CB that has failed to qualify for banking by CB banks by pooling cord blood collected from separate donors. While the present invention is not limited to the pooling of unqualified CB compared to qualified CB, the skilled person would understand that pooling unqualified CB
is the most cost effective strategy for CB banks or other similar institutions.
[00104] The present invention is illustrated in further details by the following non-limiting examples.
[00105] Example 1: Overview of cord blood collection and processing [00106] The cord blood was collected in utero, after the newborn's umbilical cord was cut but before the placenta was expelled from the uterus, and transferred to a sterile 250 mL collection bag containing 35 mL of citrate-phosphate-dextrose (CPD), which was then labeled and sent at room temperature to the cord blood bank for processing.
[00107] The first part of cord blood processing involves qualification for the Public Bank, which requires at least 65 mL of blood and a total nucleated cell (TNC) count of at least 1 x 109 cells per collection bag. The majority of collected blood fails to meet these standards. For example, during the first 821 days of activity at the Royal Victoria Hospital, only 32.5% of all collected cord blood units qualified for the Public Cord Blood Bank of Quebec and only 43% qualified for the MUHC-Clinical Research Cord Blood Bank (CRCBB). Thus, the majority of collected cord blood is not banked and is either discarded or used for research purposes.
[00108] If the cord blood unit meets the above standards, the second part of processing involves a volume reduction using the Top-Bottom technique with the Baxter Opti-System. After adding DMSO
to the leuco-platelet layer (bully coat) to a final concentration of 10% (v/v) (averaging about 22 mL 2.9 mL), the processed cord blood unit was incubated first for about one hour at -80 C, followed by a cryopreservation at about -150 C until use. Table 5 summarizes the cord blood units that were processed for the CRCBB from the period of October 2007 to January 2010. The average CBU volume processed was 90.8 mL (with a range of 38 mL to 175 mL) with a white blood cell (WBC) yield of 77% (range: 20% to 127%).
Table 5: Cord blood units processed for the CRCBB from October 2007 to January Leuco-platelet layer (what is WBC
Whole Blood Red Blood Plasma frozen) Yield Volume GBI. Hb Htc Plqts Volume GBI. Hb Volume Volume GBI.
ml 10 /unit g/unit % 10 /unit ml g/unit ml ml 10 /unit %
Mean 90.8 1.1 9.9 33.0 0.2 21.8 0.2 0.0 49.1 22.0 0.8 77%
Std.Dev. 24.3 0.5 3.6 6.2 0.1 12.5 0.3 0.0 15.3 2.9 0.4 20%
Median 89.0 0.9 9.5 33.5 0.2 19.0 0.1 0.0 49.0 22.0 0.7 78%
Max 175.0 2.9 25.3 47.8 0.5 74.0 1.3 0.0 94.0 37.0 3.0 127%
Min 38.0 0.1 1.1 7.2 0.0 5.0 0.0 0.0 10.0 12.0 0.0 20%
[00109] Example 2: Laboratory techniques and analysis [00110] Example 2.1: Cord blood unit volume reduction and cryopreservation [00111] Prior to volume reduction, an aliquot was removed from the collected cord blood unit (CBU) to evaluate the number of total nucleated cells (TNC) and nucleated Red Blood Cells (nRBC).
The CBU was then centrifuged at 2689g for 13.5 minutes at 22 C. Using the Fenwal Semiautomated system Optipress II , the centrifuge triple-bag was pressed following specific settings based on the volume: buffy-coat volume of 33 mL, a buffy coat level of 3.5 to 4.5 and a force of 23. Following volume reduction, a sample was removed from the buffy coat bag for use in a colony-forming unit-granulocyte-macrophage (CFU-GM) assay and for evaluation of TNC, CD34-positive, CD45-positive cell counts, as well as cell viability using 7-AAD. Cryopreservation was performed by slowly adding, within 15 minutes, a mixed solution of 50% dimethyl sulfoxide (DMSO) solution and Gentran 40 to ensure a final concentration of 10% DMSO. A sample of the mixed product was removed and distributed in four cryovials, which were then placed along with the CBU at -80 C for one hour and then transferred to a cryofreezer at <_ -150 C until use.
[00112] Example 2.2: Preparation of Samples for Analysis [00113] The volume reduction described in Example 2.1 permits the removal of plasma and red blood cells from the CBU. The plasma was then used to perform virology analysis and at least four 1-mL samples were cryopreserved for future use. The red blood cells were used for a number of tests, for example, the identification of the CBU blood group and rhesus factor, for sterility testing, and for hemoglobin electrophoresis.
[00114] Example 2.3: Cord Blood pooling technique [00115] CBUs were pooled by groups of three in a Dilution Solution composed of 50% Gentran 40 and 5% Human Albumin. Based on the total volume of the three CBUs, the dilution solution represented 90% of that volume. This resulted in a reduction of the DMSO concentration from 10% to 1 %.
[00116] Example 2.4: Thawing, dilution and pooling of the cord blood units [00117] The CBUs that were to be pooled to form the carrier unit of the present invention were processed in groups of three for greater practicality. One frozen CBU was immersed in a 38 C water bath and rapidly thawed. A volume of Dilution Solution corresponding to twice the volume of the CBU was added to the thawed CBU. The bag containing the thawed and diluted CBU was then incubated on ice for 5 minutes without further manipulation. Following the incubation, the contents of the thawed and diluted CBU was transferred to a 600 mL bag and rinsed twice. Two other CBUs to be pooled were processed the same way, and transferred to the 600 mL bag. The 600 mL bag containing the thawed and diluted pooled CBUs was then centrifuged at 1400 rpm for 9 minutes at 4 C.
Depending on the patient to be transplanted, the required number of CBUs were thawed in groups of three, diluted and centrifuged according to the procedure described above. A final pooling of all the thawed and diluted CBUs was done in a 600 mL bag, after ensuring a final volume of 95 mL.
[00118] Example 2.5: Purification of HSCs via a CD34-positive selection and infusion thereof into patient [00119] HSCs were purified from the pooled CBUs according to the standard protocol set forth by the CliniMACS
CD34 MicroBeads purification kit (Miltenyi Biotec , Bergisch Gladbach, Germany). Briefly, the bag containing the pooled CBUs was incubated at 22 C for 60 minutes with CliniMACS CD34 MicroBeads.
After one wash with CliniMACS
PBS/EDTA with 0.5% human albumin, the final product was applied to a CliniMACS
column for a CD34-positive selection. The negative fraction was used for Short Tandem Repeat identification and for verification of bacterial contamination. An aliquot of the CD34-positive selection was used for CD34, CD45, CD25, CD41, CD61, CD3, CD56 and CD1 6-positive cell counts and for use in CFU-GM assay. The CD34-positive fraction (i.e., the carrier unit of the present invention) was then infused into the patient or recipient within two hours of infusion of the HLA-matched CBU.
[00120] Example 2.6: Indirect Coombs test and elution of anti-A and anti-B IgG
antibodies [00121] The indirect Coombs test (also known as indirect antiglobulin test or IAT) is useful for identifying the presence of antibodies that can bind to the surface of red blood cells. For example, the indirect Coombs test can be used to detect antibodies against RBCs that are present in serum. In general, serum is extracted from the blood, and the serum is incubated with RBCs of known antigenicity. If agglutination occurs, the indirect Coombs test is positive.
The indirect Coombs test is commonly known in the art and the details of this technique would be within the grasp of the skilled person. Furthermore, the skilled person would be aware of different elution methods can be used for the elution and identification of anti-A and anti-B IgG antibodies.
[00122] The identification of bound IgG to nucleated cells was done through Heat Elution at 56 C. After incubation at 56 C, the sample was centrifuged at 3700 rpm for 2 minutes. The eluate was subsequently evaluated for content of anti-A and anti-B IgG using A and B red blood cells.
[00123] Example 3: Processing of cord blood [00124] The cord blood processing method applied by Public Banks (Armitage, 1999; Godinho, 2000) was adapted for the processing of low volume cord blood bags which have been rejected by Public Banks. The Top-Bottom method was executed following a hard centrifugation with the Baxter Opti-System. The semi-automated closed system was configurated to separate the centrifuged CBU into three components:
plasma, red blood cells and a leuco-platelets layer of 20 mL 2 mL. The leuco-platelet layer was then rotated for a period of 30 minutes, followed by the addition of a solution of DMSO 50%/Gentran at a final ratio of 1:4 (v:v) to ensure a final DMSO concentration of 10%. The final product was then frozen for one hour at -80 C, followed by long term cryopreservation in a vapour phase nitrogen cryofreezer. This method of freezing ensures adequate preservation of the nucleated cells as previously compared with a control rate freezing method (Itoh, 2003).
[00125] Example 4: Thawing and pooling of cryopreserved cord blood [00126] Upon selection of the required CBUs, a mixed solution containing equal amounts of 5% human albumin and Gentran 40 was prepared. The CBUs were regrouped in groups of three units to ensure a pre-thawing maximum volume of 60 mL. Using a semi-closed system, each CBU was diluted with twice its original volume and incubated for minutes at 4 C. Following the first dilution, all three CBUs were pooled in a bag containing half of the initial solution of mixed 5% human albumin and Gentran 40. A second dilution, which was also used to rinse the collection bag, was done using an equal volume of the CBU. The remaining solution was then used to rinse the collection CBU bags.
After each rinse, the contents were transferred to a unique pooling bag. HSCs were then purified from the pooled cord blood according to a CD34-positive selection using the CliniMACS CD34 Reagent (Miltenyi Biotec , Bergisch Gladbach, Germany) as detailed in Example 2.7. Following purification, an aliquot was taken to measure the total nucleated cell (TNC) and CD34-positive cell yield and to document viability.
[00127] When pooling 12 units, four groups of three units were thawed and diluted by repeating the above. Each pooled bags were then centrifuged at 1400 rpm at 4 C for 9 minutes. The supernatant was removed to ensure a final volume of approximately 20 mL. A sample was taken from the remaining cell pellet to measure the pooling and washing yield. All cell pellets were pooled in a final collection bag and the volume was measured to ensure a volume of about 95 mL 5 mL.
[00128] Example 5: Treatment of a patient having Chronic Lymphoid Leukemia [00129] A 41 year-old male patient or recipient weighing 81 kg, who was diagnosed in 2007 with an unclassified CD5 Chronic Lymphoid Leukemia (CLL). Upon diagnosis, the patient was treated first with CHOP chemotherapy (cyclophosphamide, doxorubicin, vincristine, and prednisone/prednisolone), and then was treated at his first relapse with Nipent, Cytoxan and Rituxan (PCR regimen), followed by high-dose cytarabine (ara-C; HiDAC) upon his second relapse. He was subjected to an umbilical cord blood transplant according to the present invention. The myeloablative conditioning regimen pre-transplantation was composed of Fludarabine, Busulfan and Cyclophosphamide. The patient having blood group A was matched for a CBU
(i.e., the "HLA-matched CBU" or the "definitive CBU") having an HLA compatibility of 4/6, with one mismatch on locus A and one mismatch on locus B.
This HLA-matched CBU happened to be of blood group 0 and contained 2.9 x 107 TNC/kg and 1.1 x 105 CD34-postive cells/kg of the patient's weight.
[00130] To prepare the carrier unit of the present invention, 45 CBUs were selected for pooling, each selected to have blood group 0 with their corresponding mother's blood group also being blood group 0. These CBUs, although cryopreserved, were CBUs that had failed to qualify for public banking but were nevertheless deemed safe for use in transplantation. The 45 CBUs were pooled in vitro and hematopoietic stem cells were substantially purified via a CD34-positive selection, as detailed above, to give the carrier unit of the present invention. The carrier unit had 0.3 x 106 CD34-positive cells/kg of patient's weight and a purity of 53.8% as determined by FACS analysis. The percent purity was calculated by the formula as presented in Braakman (2008): total number of CD34 events in selected product/total number of CD45 in selected product x 100. The CD34-positive selection resulted in a 17-fold depletion in the amount of CD3-positive cells, with only 0.86 x 104 CD3-positive cells/kg of the patient being infused.
[00131] The infusion of the HLA-matched CBU was done 69 minutes prior to the infusion of the carrier unit and the day of the transplantation/infusion was considered as day 0. Follow-up tests revealed that the patient successfully engrafted neutrophils on day +15 and platelets on day +42. The patient was considered to have successfully engrafted neutrophils based on the standard criteria of having a neutrophil cell count of greater than 0.5 x 109 neutrophils cells/L for a period of at least three consecutive days (with the first day thereof being considered as the day of engraftment). The engraftment of neutrophils at day +15 represented a 29% increase in the speed of engraftment compared to that normally expected from a standard double cord blood transplant (i.e., at day +21).
Additionally, the patient exhibited 100% HLA-matched CBU chimerism at day +14.
Between day +35 and day +100, the patient's chimerism fell below 100%, correlating with the appearance of acute Graft versus Host disease grade II/III of the skin and the gut. However, beginning at day +124 and extending through the date that this application was filed, full chimerism of the HLA-matched CBU was restored in the patient, indicating a successful transplantation.
[00132] Example 6: Treatment of a patient having Philadelphia Chromosome-positive Chronic Myeloid Leukemia [00133] A 32 year-old male patient having blood group B and weighing 56 kg was diagnosed in 2009 with Philadelphia Chromosome-positive Chronic Myeloid Leukemia (Ph+ CML). He was treated with 7 + 3 (cytarabine:daunorubicin) and with Gleevec. Soon after, the patient was not responding to Gleevec and was switched to Nilotinib. Another blast crisis presented in 2010 where he was treated with Flag-Ida (fludarabine, cytarabine, idarubicin and G-CSF). Having a second blast crisis in a very short time, the patient was subjected to an umbilical cord blood transplant according to the present invention. The myeloablative conditioning regimen consisted of fludarabine, busulfan and cyclophosphamide. The patient was matched for a CBU
(i.e., the "HLA-matched CBU" or the "definitive CBU") having an HLA compatibility of 5/6 with a single mismatch on locus A. This HLA-matched CBU
happened to be of blood group A and contained 2.74 x 107 TNC/kg and 0.67 x 105 CD34-positive cell/kg of the patient's weight.
[00134] To prepare the carrier unit of the present invention, 30 CBUs having blood group 0 were selected for pooling. These CBUs, although cryopreserved, were CBUs that had failed to qualify for public banking but were nevertheless deemed safe for use in transplantation. The 30 CBUs were pooled in vitro and hematopoietic stem cells were substantially purified via a CD34-positive selection, as detailed above, to give the carrier unit of the present invention. The carrier unit had 0.2 x 106 CD34-positive cells/kg of patient's weight and a purity of 81.7% as determined by FACS analysis. The carrier unit also had 0.14 x 104 CD3-positive cells/kg after purification, representing an 80-fold decrease between the pre- and post-CD43 selection.
[00135] The infusion of the HLA-matched CBU was done 96 minutes prior to the infusion of the carrier unit and the day of the transplantation/infusion was considered as day 0.
[00136] Example 7: Treatment of a patient having Acute Myeloid Lymphoma [00137] A 30 year-old female, blood group 0, weighing 87.5 kg was diagnosed in 2007 with acute promyelocytic leukemia (APL). The Pethema protocol was used for the induction of the APL and was followed with 6-mercaptopurine (6-MO) and Methotraxate as consolidation treatment. In 2009 the patient relapsed with an Acute Myeloid Lymphoma secondary to APL treatment and was treated with High Dose of Arabinosycytosine (Ara-C). In 2010, she relapsed from her AML and was treated with Etoposide (VP-16, cyclophosphamide and arsenic trioxide and subjected to an umbilical cord blood transplant according to the present invention. The myeloablative conditioning regimen consisted of fludarabine, busulfan and cyclophosphamide.
The patient was matched for a CBU
(i.e., the "HLA-matched CBU" or the "definitive CBU") having an HLA
compatibility of 5/6 with a single mismatch on locus A. This HLA-matched CBU happened to be of blood group 0 and contained 2.63 x 10' TNC/kg and 1.63 x 105 CD34-positive cells/kg of the patient's weight.
[00138] To prepare the carrier unit of the present invention, 42 CBUs having blood group 0 were selected for pooling. Of these 42 CBUs, 37 CBUs originated from mothers having blood group 0, 3 CBUs originated from mothers having blood group A, and two CBUs originated from mothers having blood group B. These CBUs, although cryopreserved, were CBUs that had failed to qualify for public banking but were nevertheless deemed safe for use in transplantation. The 42 CBUs were pooled in vitro and hematopoietic stem cells were substantially purified via a CD34-positive selection, as detailed above, to give the carrier unit of the present invention. The carrier unit had 0.25 x 106 CD34-positive cells/kg of patient's weight and a purity of 75.7% as determined by determined FACS analysis.
The carrier unit also had 0.7 x 104 CD3-positive cells/kg, representing a 107-fold depletion after purification.
[00139] The infusion of the HLA-matched CBU was done 60 minutes prior to the infusion of the carrier unit and the day of the transplantation/infusion was considered as day 0. Follow-up tests revealed that the patient successfully engrafted neutrophils on day +19, representing a 10% increase in the speed of engraftment compared to that normally expected from a standard double cord blood transplant (i.e., at day +21). As for Example 5, the patient was considered to have successfully engrafted neutrophils based on the standard criteria of having a neutrophil cell count of greater than 0.5 x 109 neutrophils cells/L for a period of at least three consecutive days (with the first day thereof being considered as the day of engraftment).
[00140] Although the present invention has been described hereinabove by way of specific embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims.
REFERENCES
Armitage S, Fehily D, Dickinson A, Chapman C, Navarrette C and Contreras M
(1999) Cord blood banking: volume reduction of cord blood units using a semi-automated closed system. Bone Marrow Transplantation 23:505-509.
Ballen KK, Wilson M, Wuu J, Ceredone AM, Hsieh C, Stewart FM, Popovsky MA and Quesenberry PJ (2001) Bigger is better: maternal and neonatal predictors of hematopoietic potential of umbilical cord blood units. Bone Marrow Transplantation 27:7-14.
Barker JN, Weisdorf DJ, DeFor TE, Blazar BR, McGlave PB, Miller JS, Verfaillie CM and Wagner JE (2005) Transplantation of 2 partially HLA-matched umbilical cord blood units to enhance engraftment in adults with hematologic malignancy. Blood 105: 1343-1347.
Braakman E., Schuurhuis, GJ., Freijer FWMB., Voermans C., Theunissen K., van Riet I., Fibbe WE., and Slaper-Cortenbach I (2008) Evaluation of 'out-of-specification' CliniMACS CD34-selection procedures of hematopoietic progenitor cell-apheresis products. Cytotherapy 10(1): 83-89.
Ende et al., "Pooled umbilical cord blood as a possible universal donor for marrow reconstitution and use in nuclear accidents." Life Sci. 2001 Aug 17;69(13):1531-9.
Fiumera AC and Asmussen MA (2001) Difficulties in parentage analysis : the probability that an offspring and parent have the same heterozygous genotype. Genet. Res., Camb, 78: 163-170.
Gluckman E, Broxmeyer HE, Auerbach AD et al. Hematopoietic reconstitution in a patient with Fanconi's anemia by means of umbilical cord blood from an HLA-identified sibling. New Engl J Med 1989;321:214-219.
Gluckman, E and Rocha, V (2009) Cord blood transplantation: state of the art.
Haematologica, 94(4): 451-454.
Godinho MI, de Sousa ME, Carvalhais A and Barbosa IL (2000) Umbilical cord blood processing with the Optipress II
blood extractor. Cytotherapy 2(6): 439-443.
Hsu TC, Steinberg J, LeDoux R, and Sawrrsky A, (1979) The Low Ionic Strength Reaction of Human Blood:
Relationship between the Binding of Serum Immunoglobulin and Complement to Red Blood Cells and Surface Charge of the Cells. British Journal of Haematology 42:403-415.
Itoh T, Minegishi M, Fushimi J, Takahashi H, Kudo Y, Suzuki A, Narita A, Sato Y, Akagi K, Wada Y, Saito A, Kikuchi M, Okamura K, Kaku M and Tsuchiya S (2003) A simple controlled-rate freezing method without a rate-controlled programmed freezer provides optimal conditions for both large-scale and small-scale cryopreservation of umbilical cord blood cells. Transfusion 43:1303-1308.
Jaroscak J, Goltry K, Smith A, Waters-Pick B, Martin PL, Driscoll TA, Howrey R, Chao N, Douville J, Burhop S, Fu P, and Kurtzberg J (2003) Augmentation of umbilical cord blood (UCB) transplantation with ex vivo-expanded UCB
cells: results of a phase 1 trial using the AastromReplicell System. BLOOD, 101(12): 5061-5067.
Jauniaux E, Jurkovic D, Gulbis B, Liesnard C, Lees C, and Campbell S (1995).
Materno-fetal immunoglobulin transfer and passive immunity during the first trimester of human pregnancy. Human Reproduction, 10(12): 3297-3300.
Klein HG, Anstee DJ (2008) Mollison's Blood Transfusion in Clinical Medicine.
Eleventh Edition, Blackwell Publishing Kohler PF, Farr RS (1966) Elevation of Cord over Maternal IgG
Immunologlobulin: Evidence for an Active Placental IgG Transport. Nature, 210:1070-1071.
Kumpel BM, van de Winkel JGJ, Westerdaal NAC, Hadley AG, Dugoujon JM and Blancher A (1996) Antigen topography is critical for interaction of IgG2 anti-red-cell antibodies with Fcy receptors. British Journal of Haematology, 94:175-183.
Kurtzberg J, Cairo MS, Fraser JK, Baxter-Lowe L, Cohen G, Carter SH, and Kernan NA (2005) Magro E, Regidor C, Cabrera R, Sanjuan I, Fortis R, Garcia-Marco JA, Ruiz E, Gil S, Bautista G, Millan I, Madrigal A, and Fernandez MN (2006) Early hematopoietic recovery after single unit unrelated cord blood transplantation in adults supported by co-infusion of mobilized stem cells from a third party donor. Haematologica, 91:640-648.
Majhail NS, Brunstein CG and Wagner JE (2006) Double umbilical cord blood transplantation. Current Opinion in Immunology, 18:571-575 Mehta Y, Dhar A, Meharwal SZS, Trehan N (2007) Comparison of New HES (130/0.4) and HES (200/0.5) in OPCAB
Surgery J Anesth Clin Pharmacology 2007; 23(3): 273-278.
Mielcarek M, Torok-Storb B and Storb R (2002) ABO incompatibility and relapse risk in patients undergoing allogeneic marrow transplantation for acute myeloid leukemia. Bone Marrow Transplantation 30, 547-548.
Miler ME, Stiehm ER (1973) Phagocytlc, opsonic and immunoglobulin studies in newborns. Medical Progress Calif Med 119:43-63.
M-Reboredo N, Di'az A, Castro A, and Villaescusa RG (2000) Collection, processing and cryopreservation of umbilical cord blood for unrelated transplantation. Bone Marrow Transplantation 26:1263-1270.
Ohnuma K, Isoyama K, Ikuta K,Toyoda Y, Nakamura J, Nakajima F, and Nishihirah H (2000) The Influence of HLA
Genotyping Compatibility on Clinical Outcome after Cord Blood Transplantation from Unrelated Donors JOURNAL
OF HEMATOTHERAPY & STEM CELL RESEARCH 9:541-550.
Petz, LD and Garratty G (2004) Immune Hemolytic Anemias, Second Edition, Elsevier Publication.
Querol S, Mufti GJ, Marsh SGE, Pagluica A, Little AM, Shaw, BE, Jeffery R, Garcia, J, Goldman JM, and Madrigal JA
(2009) Cord blood stem cells for hematopoietic stem cell transplantation in the UK: how big should the bank be?
Haematologica, 94(4):536-541.
Results of the Cord Blood Transplantation (COBLT) Study unrelated donor banking program. TRANSFUSION
2005;45:842-855 Rubinstein P, Dobrila L, Rosenfield RE, Adamson JW, Mogllaccio G, Migliaccio AR, Taylor PE, AND Stevens CE
(1995) Processing and cryopreservation of placental/umbilical cord blood for unrelated bone marrow reconstitution.
Proc. Nati. Acad. Sci. 92:10119-10122.
Solves P, Moraga R, Mirabet V, Larrea L, and Soler MA (2003) In utero or ex utero cord blood collection: an unresolved question. TRANSFUSION 43:1174-1176.
Solves P, Moraga R, Saucedo E, Perales A, Soler MA, Larrea L, Mirabet V, Planelles D, Carbonell-Uberos F, Monleo'n J, Planells T, Guille'n M, Andre s A and Franco E (2003a) Cord blood stem cells: Comparison between two strategies for umbilical cord blood collection. Bone Marrow Transplantation 31: 269-273.
Takahashi S, Iseki T, Ooi J, Tomonari A, Takasugi K, Shimohakamada Y, Yamada T, Uchimaru K, Tojo A, Shirafuji N, Kodo H, Tani K, Takahashi T, Yamaguchi T, and Asano S (2004) Single-institute comparative analysis of unrelated bone marrow transplantation and cord blood transplantation for adult patients with hematologic malignancies. Blood, 104(12):3813-3820.
Takahashi, S (2007) Leukemia: Cord blood for allogeneic stem cell transplantation. Current Opinion in Oncology, 19:667-672.
Wagner J,Champlin R, and Petz LD (2006) Symposium Summary Fourth Annual International Umbilical Cord Blood Transplantation Symposium, Los Angeles, California, May 19-20, 2006 Biology of Blood and Marrow Transplantation 12:1206-1217 (2006).
Wong A, Yuen PMP, Li K, Yu ALM and Tsoi WC (2001) Cord blood collection before and after placental delivery:
levels of nucleated cells, haematopoietic progenitor cells, leucocyte subpopulations and macroscopic clots. Bone Marrow Transplantation 27:133-138.
Claims (19)
1. A method for increasing the success of umbilical cord blood transplantation in a patient, said method comprising:
(a) combining in vitro hematopoietic stem cells from the umbilical cord blood from at least two separate donors;
(b) infusing into said patient a composition comprising the combined hematopoietic stem cells from (a) which have been substantially purified, thereby increasing the chances of successful engraftment.
(a) combining in vitro hematopoietic stem cells from the umbilical cord blood from at least two separate donors;
(b) infusing into said patient a composition comprising the combined hematopoietic stem cells from (a) which have been substantially purified, thereby increasing the chances of successful engraftment.
2. The method of claim 1, wherein said combining in vitro comprises pooling umbilical cord blood from at least two separate donors.
3. The method of claim 2, wherein said hematopoietic stem cells are substantially purified after said pooling of umbilical cord blood.
4. The method of claim 1, wherein said hematopoietic stem cells are substantially purified before (a).
5. The method of claim 1, wherein said hematopoietic stem cells are substantially purified by a CD34-based purification method.
6. The method of claim 1, wherein said hematopoietic stem cells are obtained from umbilical cord blood comprising unqualified umbilical cord blood.
7. The method of claim 1, wherein said umbilical cord blood had been previously frozen.
8. The method of claim 1, wherein said increasing the success of umbilical cord blood transplantation comprises reducing the time of engraftment.
9. A method for pooling umbilical cord blood collected from separate donors, said method comprising thawing frozen umbilical cord blood from at least two separate donors and combining said umbilical cord blood in vitro.
10. A method for improving the yield of total nucleated cells in pooled umbilical cord blood units from at least two separate donors, said method comprising:
(a) considering the ABO blood type of said donors;
(b) considering the ABO blood type of the mothers of said donors and/or the presence or absence of anti-A
and/or anti-B IgG antibodies in prospective umbilical cord blood units to be pooled;
(c) pooling umbilical cord blood units from at least two separate donors based on (a) and (b), thereby improving the yield of total nucleated cells in said pooled umbilical cord blood.
(a) considering the ABO blood type of said donors;
(b) considering the ABO blood type of the mothers of said donors and/or the presence or absence of anti-A
and/or anti-B IgG antibodies in prospective umbilical cord blood units to be pooled;
(c) pooling umbilical cord blood units from at least two separate donors based on (a) and (b), thereby improving the yield of total nucleated cells in said pooled umbilical cord blood.
11. The method of claim 10, wherein said umbilical cord blood units to be pooled comprise unqualified umbilical cord blood.
12. A pharmaceutical composition comprising hematopoietic stem cells collected from frozen umbilical cord blood from separate donors and a pharmaceutically acceptable carrier for reducing the time of engraftment in a patient receiving an umbilical cord blood transplant.
13. The pharmaceutical composition of claim 12, wherein said hematopoietic stem cells are substantially purified from pooled umbilical cord blood from separate donors.
14. The pharmaceutical composition of claim 13, wherein said pooled umbilical cord blood comprises unqualified umbilical cord blood.
15. A method for reducing the lysis of nucleated cells in collected umbilical cord blood, said method comprising preventing IgG antibodies from binding to the anti-A and/or anti-B antigens expressed on the surface of said nucleated cells.
16. The method of claim 15, wherein said umbilical cord blood is pooled umbilical cord blood from separate donors.
17. The method of claim 15, wherein said preventing comprises substantially removing or depleting said IgG
antibodies from said collected umbilical cord blood.
antibodies from said collected umbilical cord blood.
18. The method of claim 15, wherein said preventing comprises administering an IgG-binding inhibitor to said collected umbilical cord blood.
19. A kit for reducing the time of engraftment in a patient receiving an umbilical cord blood transplant, said kit comprising a container comprising a number of nucleated cells pooled from umbilical cord blood from separate donors.
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA 2703428 CA2703428A1 (en) | 2010-05-11 | 2010-05-11 | Pooled cord blood units |
PCT/CA2011/050288 WO2011140654A1 (en) | 2010-05-11 | 2011-05-10 | Pooled cord blood units |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA 2703428 CA2703428A1 (en) | 2010-05-11 | 2010-05-11 | Pooled cord blood units |
Publications (1)
Publication Number | Publication Date |
---|---|
CA2703428A1 true CA2703428A1 (en) | 2011-11-11 |
Family
ID=44912971
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA 2703428 Abandoned CA2703428A1 (en) | 2010-05-11 | 2010-05-11 | Pooled cord blood units |
Country Status (2)
Country | Link |
---|---|
CA (1) | CA2703428A1 (en) |
WO (1) | WO2011140654A1 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2015132415A1 (en) * | 2014-03-07 | 2015-09-11 | Emercell Sas | Pooled nk cells from ombilical cord blood and their uses for the treatment of cancer and chronic infectious disease |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP3717635A1 (en) * | 2017-11-28 | 2020-10-07 | The Board of Trustees of the University of Illinois | Multi-chimeric cell and therapy for transplantation and treatment of immune deficiencies and genetic disorders |
WO2021108806A1 (en) * | 2019-11-29 | 2021-06-03 | Rutgers, The State University Of New Jersey | Compositions, kits and methods for storage of blood products and methods of use thereof |
-
2010
- 2010-05-11 CA CA 2703428 patent/CA2703428A1/en not_active Abandoned
-
2011
- 2011-05-10 WO PCT/CA2011/050288 patent/WO2011140654A1/en active Application Filing
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2015132415A1 (en) * | 2014-03-07 | 2015-09-11 | Emercell Sas | Pooled nk cells from ombilical cord blood and their uses for the treatment of cancer and chronic infectious disease |
CN106164256A (en) * | 2014-03-07 | 2016-11-23 | 艾美尔塞勒公司 | From the NK cell collected of Cord blood and the application in treatment cancer and chronic infection disease |
JP2017508479A (en) * | 2014-03-07 | 2017-03-30 | エメルセル エスエーエス | Pooled NK cells derived from cord blood and their use for the treatment of cancer and chronic infections |
JP2020114248A (en) * | 2014-03-07 | 2020-07-30 | エメルセル エスエーエス | Pooled nk cell derived from umbilical cord, and application of the same for therapy of cancer and chronic infectious disease |
CN106164256B (en) * | 2014-03-07 | 2021-08-10 | 艾美尔塞勒公司 | Pooled NK cells from umbilical cord blood and use in the treatment of cancer and chronic infectious diseases |
Also Published As
Publication number | Publication date |
---|---|
WO2011140654A1 (en) | 2011-11-17 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11905333B2 (en) | Selective immunodepletion of endogenous stem cell niche for engraftment | |
CA2642020C (en) | Methods and compositions for enhancing engraftment of hematopoietic stem cells | |
JP2022088551A (en) | Uses of expanded populations of hematopoietic stem/progenitor cells | |
JP7321934B2 (en) | Non-genotoxic transplant conditioning regimens for stem cell transplantation | |
EP2943566A1 (en) | Compositions and methods for expansion of embryonic hematopoietic stem cells | |
EP1906729B1 (en) | Plasma-depleted, non-red blood cell-depleted cord blood compositions and methods of use | |
CN113853204A (en) | Expansion of expanded NK cell fractions suitable for transplantation combination therapy and uses thereof | |
JP2006528627A (en) | Methods and compositions for increasing the effectiveness of therapeutic antibodies using alloreactive natural killer cells | |
US20080008693A1 (en) | Plasma-depleted, non-red blood cell-depleted cord blood compositions and methods of use | |
US12023357B2 (en) | Rejuvenated aged hematopoietic stem cells and methods of use | |
CA2703428A1 (en) | Pooled cord blood units | |
JP7295810B2 (en) | Selection and use of umbilical cord cell fractions suitable for transplantation | |
US7544355B2 (en) | Methods and compositions for allogeneic transplantation | |
US12091684B2 (en) | Platelet-derived mitochondria treatment and method of generating multipotent cells | |
WO2012135838A1 (en) | Methods and compositions for large-scale isolation of very small embryonic-like (vsel) stem cells | |
O’Donoghue | Implications of fetal stem cell trafficking in pregnancy | |
Scaradavou et al. | Combined neonatal blood transplants in a parent‐to‐F1 mouse model: Improved survival rates and stable long‐term engraftment | |
JP2024526837A (en) | Therapeutic NK cell populations | |
CA2668608A1 (en) | Methods for using aldhbr cells to supplement stem cell transplantation | |
Liuba | Hematopoietic stem and progenitor cells and potentials for application in fetal cell replacement therapy | |
Lindton | Experimental studies of human fetal liver cells: in regard to in utero hematopoietic stem cell transplantation |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
FZDE | Dead |
Effective date: 20140513 |