Selection and use of umbilical cord cell fractions suitable for transplantation

Abstract

Methods of selecting umbilical cord blood units for ex-vivo expansion, separation of CD133+/CD34+ positive and uncultured CD133+/CD34+ negative fractions, methods for expanding the selected CD133+/CD34+ fraction, selection of expanded populations of CD133+/CD34+ cord blood cells for transplantation to subjects in need thereof and the therapeutic use of suitable selected, ex-vivo expanded CD133+/CD34+ and unselected CD133/CD34 negative cord blood fractions for transplantation in the clinical setting, for treatment of hematological malignancies are provided. The present invention also envisions kits comprising the expanded and unselected cord blood fractions.

1 . A method of selecting a cryopreserved umbilical cord blood unit for ex-vivo expansion and transplantation into a subject, comprising:
 (a) determining in said umbilical cord blood unit prior to ex-vivo expansion the following pre-cryopreservation parameters:
 (i) about 8×106 to about 15×106 total CD34+ cells; 
 (ii) HLA-matched at at least 4 out of 6 HLA class I (HLA-A and HLA-B, low resolution) and HLA class II (HLA-DRB1, high resolution) loci with said subject; 
 (iii) about 1.8×109 to about 3.0×109 pre-cryopreserved total nucleated cells; 
 (iv) about 1.5×107 to about 3.0×107 pre-cryopreserved total nucleated cells per kilogram subject weight, 
   (b) selecting or excluding said umbilical cord blood unit according to said parameters, thereby selecting an umbilical cord blood unit suitable for ex-vivo expansion and transplantation into the subject.   
     
     
         2 . The method of  claim 1 , wherein (i) is about 8×106 to about 10×106 total CD34+ cells. 
     
     
         3 . (canceled) 
     
     
         4 . A method of treating a hematological disease in a subject in need thereof, the method comprising:
 (a) separating a single umbilical cord blood unit suitable for transplantation into said subject into (i) a first, selected blood cell fraction comprising CD133+/CD34+ selected cells and (ii) a second, unselected blood cell fraction comprising CD133/CD34 negative cells;   (b) ex vivo culturing said first blood cell fraction comprising CD133+/CD34+ selected cells under conditions allowing for cell proliferation, said conditions comprising providing nutrients, serum and a combination of cytokines including each of stem cell factor, thrombopoietin, FLt3 ligand and IL-6 and nicotinamide in an amount between 1.0 mM to 10 mM;   (c) cryopreserving said ex-vivo cultured first, CD133+/CD34+ selected blood cell fraction and said uncultured second, unselected blood cell fraction from step (b),   (d) thawing said ex-vivo cultured first selected and said uncultured second, unselected blood cell fractions, and   (e) transplanting the thawed ex-vivo cultured first selected and uncultured second unselected blood cell fractions from step (d) into a subject in need thereof,   
       thereby treating said hematological disease in said subject, wherein said single umbilical cord blood unit suitable for transplantation is characterized by the following pre-cryopreservation parameters:
 (i) at least 8×106 total CD34+ cells; 
 (ii) HLA-matched at at least 4 out of 6 HLA class I (HLA-A and HLA-B, low resolution) and HLA class II (HLA-DRB1, high resolution) loci with said subject; 
 (iii) about 1.8×109 to about 3.0×109 pre-cryopreserved total nucleated cells, and (iv) about 1.5×107 to about 3.0×107 pre-cryopreserved total nucleated cells per kilogram subject weight. 
 
     
     
         5 - 6 . (canceled) 
     
     
         7 . The method of  claim 4 , wherein said cryopreserved ex-vivo cultured first selected and said uncultured second, unselected blood cell fractions are thawed on the same day of transplantation. 
     
     
         8 . The method of  claim 4 , wherein said cryopreserved ex-vivo cultured first selected and said uncultured second, unselected blood cell fractions are thawed and reconstituted in infusion solution and wherein said transplantation is affected by infusion in an infusion solution into said patient. 
     
     
         9 - 10 . (canceled) 
     
     
         11 . The method of  claim 8 , wherein said ex-vivo cultured first selected blood cell fraction is infused prior to said uncultured second, unselected blood cell fraction. 
     
     
         12 . The method of  claim 8 , wherein said uncultured second, unselected blood cell fraction is infused prior to said ex-vivo cultured first selected blood cell fraction. 
     
     
         13 . The method of  claim 4 , wherein said first ex-vivo cultured blood cell fraction comprises at least 8×108 total viable cells and said second uncultured blood cell fraction comprises at least 4×108 total viable cells. 
     
     
         14 . (canceled) 
     
     
         15 . The method of  claim 4 , wherein said hematological disease is a hematological malignancy selected from the group consisting of chronic myelogenous leukemia (CML), acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML) and myelodysplastic syndrome (MDS). 
     
     
         16 . (canceled) 
     
     
         17 . The method  claim 15 , wherein the subject has received at least one treatment selected from the group consisting of:
 (a) myeloablative therapy or other conditioning regime prior to transplantation;   (b) Graft-versus-host disease (GvHD) prophylaxis regime prior to transplantation; and   (c) Graft-versus-host disease (GvHD) prophylaxis regime following transplantation.   
     
     
         18 - 19 . (canceled) 
     
     
         20 . The method of  claim 4 , wherein
 transplantation of said first ex vivo cultured CD133+/CD34+ blood cell fraction and said second uncultured CD133−/CD34− blood cell fraction decreases time from transplantation to neutrophil engraftment in said subject, when compared to transplantation of a single or double unit of unmanipulated cord blood.   
     
     
         21 . (canceled) 
     
     
         22 . The method of  claim 20 , wherein said neutrophil engraftment consists of achieving an absolute neutrophil count (ANC) ≥0.5×109/L on 3 consecutive measurements on different days with subsequent donor chimerism (≤10% host cells by peripheral blood chimerism), on or before 42 days post transplant. 
     
     
         23 . The method of  claim 4 , wherein transplantation of said first ex vivo cultured CD133+/CD34+ blood cell fraction and said second uncultured CD133−/CD34− blood cell fraction decreases time from transplantation to platelet engraftment in said subject, when compared to transplantation of a single or double unit of unmanipulated cord blood. 
     
     
         24 . The method of  claim 23 , wherein transplantation of said first ex vivo cultured CD133+/CD34+ hematopoietic stem/progenitor blood cell fraction and said second uncultured CD133−/CD34− blood cell fraction increases the probability of platelet engraftment in said subject at 42 days post transplantation or decreases risk of non-engraftment by day 42 after transplantation in said subject when compared to transplantation of a single or double unit of unmanipulated cord blood. 
     
     
         25 . (canceled) 
     
     
         26 . The method of  claim 4 , wherein transplantation of said first ex vivo cultured CD133+/CD34+ blood cell fraction and said second uncultured CD133−/CD34− blood cell fraction decreases the risk of non-relapse mortality after transplantation in said subject, when compared to transplantation of a single or double unit of unmanipulated cord blood. 
     
     
         27 . The method of  claim 26 , wherein transplantation of said first ex vivo cultured CD133+/CD34+ blood cell fraction and said second uncultured CD133−/CD34− blood cell fraction decreases the risk of non-relapse mortality at 210 days after transplantation or transplantation related mortality at 1 year. 
     
     
         28 . (canceled) 
     
     
         29 . The method of  claim 4 , wherein transplantation of the first ex vivo cultured CD133+/CD34+ blood cell fraction and said second uncultured CD133−/CD34− blood cell fraction decreases duration of hospitalization in said subject in the first 100 days post-transplantation by 5-30 days, when compared to transplantation of a single or double unit of unmanipulated cord blood. 
     
     
         30 . (canceled) 
     
     
         31 . The method of  claim 4 , wherein transplantation of the first ex vivo cultured CD133+/CD34+ blood cell fraction and said second uncultured CD133−/CD34− blood cell fraction decreases risk of grade 2/3 bacterial or invasive fungal infections post-transplantation in said subject, when compared to transplantation of a single or double unit of unmanipulated cord blood. 
     
     
         32 . The method of  claim 31 , wherein transplantation of the first ex vivo cultured CD133+/CD34+ blood cell fraction and said second uncultured CD133−/CD34− blood cell fraction decreases risk of grade 2/3 bacterial or invasive fungal infections by 100 days post-transplantation in said subject, when compared to transplantation of a single or double unit of unmanipulated cord blood. 
     
     
         33 . The method of  claim 4 , wherein transplantation of the first ex vivo cultured CD133+/CD34+ blood cell fraction and said second uncultured CD133−/CD34− blood cell fraction decreases risk of acute graft-versus-host disease grade III-IV in said subject at 100 days post transplantation, when compared to transplantation of a single or double unit of unmanipulated cord blood. 
     
     
         34 . The method of  claim 4 , wherein transplantation of the first ex vivo cultured CD133+/CD34+ blood cell fraction and said second uncultured CD133−/CD34− blood cell fraction decreases risk of severe chronic graft-versus-host disease in said subject at 180 days post transplantation, when compared to transplantation of a single or double unit of unmanipulated cord blood. 
     
     
         35 . The method of  claim 4 , wherein said first and second blood cell fractions are co-administered in conjunction with an additional treatment for hematological disease. 
     
     
         36 . The method of  claim 35 , wherein said additional treatment is selected from the group consisting of immunosuppressive treatment, chemotherapy and radio-therapy. 
     
     
         37 . An article of manufacture comprising:
 (a) a packaging material and   (b) umbilical cord blood cell fractions comprising:
 (i) a first blood cell fraction suitable for transplantation into a subject in need thereof comprising CD133+/CD34+ selected cord blood cells ex-vivo cultured under conditions allowing for cell proliferation, said conditions comprising providing nutrients, serum and a combination of cytokines including each of stem cell factor, thrombopoietin, FLt3 ligand and IL-6 and nicotinamide in an amount between 1.0 mM to 10 mM; and 
 (ii) a second, uncultured, unselected blood cell fraction suitable for transplantation into a subject in need thereof comprising CD133/CD34 negative cells, 
   and wherein said packaging material comprises a label or package insert indicating that said first and second umbilical cord blood cell populations are for treating a hematological disease in a subject in need thereof.   
     
     
         38 . The article of manufacture of  claim 37 , wherein said first ex-vivo cultured blood cell fraction comprises at least 8×108 total viable cells and said second uncultured blood cell fraction comprises at least 4×108 total viable cells. 
     
     
         39 . (canceled)