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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/06—Antianaemics

Definitions

• the present invention relates to improved methods of reconstituting, repairing, and regenerating tissue using populations of stem cells enriched for early progenitor cells.
• umbilical cord blood (UCB) transplantation has been shown to be a viable alternative donor stem cell source for hematopoietic cell transplantation in subjects with catastrophic diseases treatable with transplantation therapy.
• UCB cells can cross partially mismatched HLA barriers without intolerable acute or chronic Graft- versus-Host Disease (GvHD) (Wagner et al. (1996) Blood 88(3):795-802; Rubinstein et al. (1998) N EnglJ Med 339(22): 1565- ⁇ 577; Rocha, et al.
• UCB cell dose expressed per kilogram of recipient body weight, is the best predictor of outcomes after UCB transplantation (Kurtzberg I, et al. (1996) N EnglJ Med 335:157-166; Stevens et al. (2002) Blood 100(7):2662-2664). Cell dose thresholds strongly correlating with outcomes have been identified.
• UCB units In infants and children weighing ⁇ 40kg, it is possible to find a sufficiently matched UCB unit that will deliver a dose of cells critical for successful engraftment (defined as 3 x 10e7 nucleated cells/kg) within a reasonable time frame in >90% of subjects. In teenagers and adults weighing >40kg, this is possible 30-50% of the time. Because UCB units contain a relatively fixed number of total nucleated cells, units delivering optimal cell dosing for subjects weighing >70kg will only be identified ⁇ 15% of the time. Attempts to increase the dose of cells available for UCBT have included ex vivo expansion and combined unit transplantation.
• the first stem cell population comprises stem cells derived from umbilical cord.
• the second population of stem cells comprises aldehyde dehydrogenase positive (ALDH br ) cells isolated from umbilical cord wherein the cells are either used without further manipulation following isolation or are primed in culture using a combination of cytokines for about 2 to about 7 days prior to introducing the cells into the subject.
• the second cell population is introduced into the subject between 2 and 24 hours after introduction of the first population of UCB.
• the methods of the invention are particularly useful in accelerating time to neutrophil and/or platelet engraftment and immune reconstitution following myeloablative therapy.
• Figures 1 -4 show interim results for neutrophil engraftment and platelet engraftment for 14 patients undergoing the UCB transplant procedures described herein (labeled "ALDH r "). Patients were enrolled at various timepoints, and the trial is ongoing. Therefore, at the time of the analysis, some of the patients had not reached the engraftment endpoints demonstrated in these figures.
• Figure 1 shows the cumulative incidence of neutrophil engraftment up to day 60 in the treatment group compared to historical controls of 69 patients treated for metabolic diseases in the COBLT study.
• Neutrophil engraftment was defined as reaching an ANC of at least 500 neutrophils/ ⁇ l.
• Figure 2 shows the preliminary cumulative incidence of neutrophil engraftment up to day 60 for 14 patients in the treatment group compared to historical controls of 191 patients treated for malignant diseases in the COBLT study.
• Neutrophil engraftment was defined as reaching an ANC of at least 500 neutrophils/ ⁇ l.
• Figure 3 shows the preliminary cumulative incidence of platelet engraftment up to day 200 for 14 patients in the treatment group compared to historical controls of 69 patients treated for metabolic diseases in the COBLT study.
• Platelet engraftment was defined as maintaining a platelet count of at least 50,000 platelets/ ⁇ l of blood without transfusion support.
• Figure 4 shows the preliminary cumulative incidence of platelet engraftment up to day 200 for 14 patients in the treatment group compared to historical controls of 191 patients treated for malignant diseases in the COBLT study.
• Platelet engraftment was defined as maintaining a platelet count of at least 50,000 platelets/ ⁇ l of blood without transfusion support.
• SPC Stem and progenitor cells reproduce and maintain developmental potential until specific biological signals induce the cells to differentiate into a specific cell type or tissue type.
• APC adult stem and progenitor cells
• stem cell refers to a cell with the capability of differentiation and self-renewal, as well as the capability to regenerate tissue.
• engraftment and “in vivo regeneration” refer to the biological process in which implanted or transplanted stem cells reproduce themselves and/or produce differentiated cell progeny in a host organism, and/or replace lost or damaged cells in the host.
• Allogeneic cell therapy is used to treat a variety of diseases or pathological conditions. Allogeneic cell therapy is an important curative therapy for several types of malignancies and viral diseases. Allogeneic cell therapy involves the infusion or transplant of cells to a subject, whereby the infused or transplanted cells are derived from a donor other than the subject. As used herein, the term "derive" or “derived from” is intended to obtain physical or informational material from a cell or an organism of interest, including isolation from, collection from, and inference from the organism of interest.
• Types of allogeneic donors that have been utilized for allogeneic cell therapy protocols include: human leukocyte antigen (HLA)-matched siblings, matched biologically unrelated donors, partially matched biologically related donors, biologically related umbilical cord blood donors, and biologically unrelated umbilical cord blood donors.
• HLA human leukocyte antigen
• the allogeneic donor cells are usually obtained by bone marrow harvest, collection of peripheral blood or collection of placental cord blood at birth.
• the methods of the present invention encompass the administration or introduction of two cell preparations (or “populations"), wherein the administration of each is separated in time so as to accelerate hematopoiesis. "Administration" or
• introduction refers to the intravenous introduction of the cell populations described herein into a subject.
• administration of the two cell preparations follows myeloablative therapy.
• one cell preparation is referred to as the "first cell population” and the other cell preparation is referred to as the "second cell population” or "supplement cell population.”
• the second cell population is administered to a subject no more than about 1 hour, no more than about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, about 4 hours, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about
• the first population comprises umbilical cord blood cells.
• the second cell population comprises SPC that are ALDH r , and thus contain most or all of the stem cells present in a stem cell source.
• the first and the second cell population may be obtained or derived from the same or different donors. Where the first and second cell populations are derived from the same donor, the UCB collected from the donor can be apportioned into about an 80%/20%, about a 75%/25%, about a 60%/40%, about a 65%/35%, about a 60%/40%, about a 55%/45%, or about a 50%/50% split for the first and second cell populations, respectively.
• This split can be an apportionment of one batch of cells collected at a particular time (e.g., a single cord unit collected from the donor, split according to the parameters above), or it can be an apportionment of pooled cord blood units collected from one or more donors.
• the number of nucleated cells required for each infusion is discussed elsewhere herein.
• the ALDH r second population of cells is “primed” prior to introducing the cells into a subject.
• “primed” or “priming” is intended that the cells are exposed to cytokines for about 2 to about 7 days before transplantation.
• the cells are primed in culture for about 5 days prior to transplantation in serum free culture medium containing SCF, IL-7, and FLT-3.
• compositions of the present invention comprising a first and a second population of cells derived from umbilical cord blood are useful in a method of reconstituting blood tissue or other stem and progenitor cell function, wherein the method comprises introducing the second population of cells into a subject in need thereof between 2 and 24 hours after the first population of cells.
• at least the second population is an enriched ALDH br stem cell population.
• treatment is an approach for obtaining beneficial or desired clinical results (i.e., "therapeutic response").
• beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilization (i.e., not worsening) of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
• Treatment can also mean prolonging survival as compared to expected survival if not receiving treatment or receiving different treatment (i.e., only a single dose of cells, or multiple doses of cells spaced greater than 24 hours apart, or some other treatment not encompassed herein).
• Treatment refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented. "Alleviating" a disease means that the extent and/or undesirable clinical manifestations of a disease state are lessened and/or the time course of the progression is slowed or shortened, as compared to a situation without treatment or a different treatment.
• the "treatment” entails administering additively effective SPC to the subject to regenerate tissue (particularly hematopoietic cells).
• the cell populations useful in the methods described herein may be utilized in a variety of contexts.
• the cells may be administered to subjects who have decreased hematologic function resulting from one or more diseases, treatments, or a combination thereof, to accelerate hematologic recovery.
• the methods of the invention are useful for the treatment of patients having: diseases resulting from a failure or dysfunction of normal blood cell production and maturation, hyperproliferative stem cell disorders, aplastic anemia, pancytopenia, thrombocytopenia, red cell aplasia, Blackfan-Diamond syndrome due to drugs, radiation, or infection, idiopathic; hematopoietic malignancies, including acute lymphoblastic (lymphocytic) leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, acute malignant myelosclerosis, multiple myeloma, polycythemia vera, agnogenic myelometaplasia, Waldenstrom's macro globulinemia,
• Hodgkin's lymphoma non-Hodgkins's lymphoma
• immunosuppression in subjects with malignant, solid tumors including malignant melanoma, carcinoma of the stomach, ovarian carcinoma, breast carcinoma, small cell lung, carcinoma, retinoblastoma, testicular carcinoma, glioblastoma, rhabdomyosarcoma, neuroblastoma, Ewing's sarcoma, lymphoma
• autoimmune diseases rheumatoid arthritis, diabetes type I, chronic hepatitis, multiple sclerosis, and systemic lupus erythematosus
• genetic (congenital) disorders anemias, familial aplastic, Fanconi's syndrome, Bloom's syndrome, pure red cell aplasia (PRCA), dyskeratosis congenital, Blackfan-Diamond syndrome, congenital dyserythropoietic syndromes I-IV, MPS I, MPS II, MPS III, MPS IV
• the present method is useful for diseases for which reinfusion of stem cells following myeloablative chemotherapy has been described including acute leukemia, Hodgkin's and non-Hodgkin's lymphoma, neuroblastoma, testicular cancer, breast cancer, multiple myeloma, thalassemia, and sickle cell anemia (Cheson et al. ( ⁇ 9%9) Ann. Intern. Med. 30 110:51; Wheeler et al (199O) J. Clin. Oncol. 8:648; Takvorian et al.
• the present invention is useful for improving myeloablative transplant outcomes by accelerating platelet and neutrophil engraftmcnt fo llowin g c hem ot h erapy ///.
• Allogeneic cell therapy is an important curative therapy for several types of malignancies and viral diseases.
• Allogeneic cell therapy involves the infusion or transplant of cells to a subject, whereby the infused or transplanted cells are derived from a donor other than the subject.
• Types of allogeneic donors that have been utilized for allogeneic cell therapy protocols include: HLA-matched siblings, matched unrelated donors, partially matched family member donors, related umbilical cord blood donors, and unrelated umbilical cord blood donors.
• the allogeneic donor cells are usually obtained by bone marrow harvest, collection of peripheral blood or collection of placental cord blood at birth.
• Allogeneic cells preferably are chosen from human leukocyte antigen (HLA)- compatible donors.
• HLA-compatible lymphocytes may be taken from a fully HLA-matched relative such as a parent, brother or sister.
• donor lymphocytes may be sufficiently HLA-compatible with the recipient to obtain the desired result even if a sibling donor is single-locus mismatched.
• the donor lymphocytes are fully HLA matched with the recipient.
• the cells will be obtained from a donor that is HLA-matched at 6/6 loci. In another embodiment, the cells will be obtained from a donor that is HLA-matched at 5/6 loci.
• the cells will be obtained from a donor that is HLA- matched at 4/6 loci. Mismatches at the A locus are preferred over mismatches at the B locus, which are preferred over mismatches at the DR locus. In various embodiments utilizing UCB, it may not be necessary to HLA-type the cells prior to administration Thus, in one embodiment, the invention provides a method of treating an individual comprising administering to the individual a first and a second population of SPC collected from at least one donor. "Donor" in this context means an adult, child, infant, or a placenta.
• the method comprises administering to the individual a first and/or a second population of SPC that has been collected from a plurality of donors and pooled.
• the first and the second population of SPC may be taken from multiple donors separately, and administered separately, e.g., one or more donors is used for the first cell population, and one or more of the same or different donors is used for the second cell population.
• Umbilical cord blood may be collected in any medically or pharmaceutically- acceptable manner. Various methods for the collection of cord blood have been described. See, e.g., Coe, U.S. Pat. No. 6,102,871; Haswell, U.S. Pat. No. 6,179,819 Bl .
• UCB may be collected into, for example, blood bags, transfer bags, or sterile plastic tubes.
• UCB or stem cells derived therefrom may be stored as collected from a single individual (i.e., as a single unit) for administration, or may be pooled with other units for later administration.
• the cells are transferred to an appropriate cryogenic container and the container decreased in temperature to generally from -120 0 C to -196 0 C and maintained at that temperature.
• the temperature of the cells i.e., the temperature of the cryogenic container
• a temperature compatible with introduction into the subject generally from around room temperature to around body temperature, e.g., from about 20 0 C to about 37.6°C, inclusive
• the cells are introduced into a subject as discussed below.
• At least the second cell population comprises ASPC that are ALDH bl .
• ALDH br cells express high levels of the enzyme aldehyde dehydrogenase and give low side scatter signals in flow cytometric analysis. These cells are highly enriched in hematopoietic progenitor cells and comprise about 0.5% of the nucleated cells in freshly isolated human UCB.
• the various properties of ALDH br cell populations and methods of obtaining them are well known in art. See, for example, U.S. Patent No. 6,537,807; U.S. Patent No. 6,627,759; Storms et al. (1999) Proc. Natl. Acad. Sci USA 96:9118; PCT Publication No. WO2005/083061; Storms et al. (2005) Blood 106(l):95-102; and, Hess et al. (2004) Blood 104(6): 1648-55, each of which is herein incorporated by reference in their entirety.
• the second cell population of cells is primed, but not expanded, prior to administration to the subject.
• the ex vivo priming involves incubation of ALDH r UCB in suitable culture medium containing one or more cytokines.
• the cells are ex vivo primed for not more than 7 days, not more than 6 days, not more than 5 days, 4 days, 3 days, or not more than 2 days prior to introduction into the subject.
• cytokines useful in the methods of the present invention are those which have been used for ex vivo expansion of ASPC and are well known in the art.
• the cells are cultured for 5 days prior to infusion with a cytokine cocktail consisting of stem cell factor (SCF), FLT-3, and interleukin 7 (IL-7) in a serum- free medium.
• SCF stem cell factor
• FLT-3 FLT-3
• IL-7 interleukin 7
• concentration of each cytokine can be determined empirically.
• the concentration of each cytokine is about 5 ng/ml, about 10 ng/ml, about 15 ng/ml, about 20 ng/ml, 25 ng/ml, 30 ng/ml, 35 ng/ml, 40 ng/ml, 45 ng/ml, 50 ng/ml,
• IMDM Iscove's modified Dulbecco's Media
• SCGMTM SCGMTM
• Waymouth's MB 752/1 Media Williams Media E, Medium NCTC- 109, neuroplasma medium, BGJb Medium, Brinster's BMOC-3 Medium, CMRL Medium, CO.sub.2- Independent Medium, Leibovitz's L- 15 Media, and the like.
• Antibiotics, antifungals or other contamination preventive compounds can be added to the incubation medium, if desired.
• Exemplary compounds include but are not limited to penicillin, streptomycin, gentamycin, fungizone or others known in the art.
• the cell populations useful in the methods of the present invention have application in a variety of therapies and diagnostic regimens. They are preferably diluted in a suitable carrier such as buffered saline before administration to a subject.
• the cells may be administered in any physiologically acceptable vehicle. Cells are conventionally administered intravascularly by injection, catheter, or the like through a central line to facilitate clinical management of a patient. This route of administration will deliver cells on the first pass circulation through the pulmonary vasculature. Usually, at least about 1x10 5 cells/kg and preferably about 1 xlO 6 cells/kg or more will be administered in the first cell population of cells, or in the combination of the first and second cell population. See, for example, Sezer et al. (2000) J. Clin. Oncol. 18:3319 and Siena et al. (2000) J.
• additional drugs such as 5-fluorouracil and/or growth factors may also be co-introduced.
• Suitable growth factors include, but are not limited to, cytokines such as IL-2, IL-3, IL-6, IL-11, G-CSF, M-CSF, GM-CSF, gamma-interferon, and erythropoietin.
• the cell populations of the invention can be administered in combination with other cell populations that support or enhance engraftment, by any means including but not limited to secretion of beneficial cytokines and/or presentation of cell surface proteins that are capable of delivering signals that induce stem cell growth, homing, or differentiation.
• first and/or second population of stem cells may be conditioned by the removal of red blood cells and/or granulocytes after it has been frozen and thawed using standard methods.
• the first and/or second population of stem cells may be administered to a subject in any pharmaceutically or medically acceptable manner, including by injection or transfusion.
• the cells or supplemented cell populations may contain, or be contained in any pharmaceutically-acceptable carrier.
• pharmaceutical compositions of the present invention may comprise a target cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
• compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.
• buffers such as neutral buffered saline, phosphate buffered saline and the like
• carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol
• proteins polypeptides or amino acids such as glycine
• antioxidants e.g., chelating agents such as EDTA or glutathione
• adjuvants e.g., aluminum hydroxide
• preservatives e.g., aluminum hydroxide
• a cell composition of the present invention should be introduced into a subject, preferably a human, in an amount sufficient to achieve tissue repair or regeneration, or to treat a desired disease or condition.
• a subject preferably a human
• O x 10 7 cells/kg is used for any treatment, either in the first cell population, the second population, or a combination of the first and second population of stem cells. Where cord blood from several donors is used, the number of cord blood stem cells introduced into a subject may be higher.
• the second population may contain significantly fewer cells.
• the second population contains at least about 10 , or at least about 10 5 nucleated cells per kg.
• the methods of the invention may decrease the number of transplanted cells necessary for hematologic recovery. This method is particularly useful when the number of cells available for transplant is limited.
• therapeutically effective amount is indicated, the precise amount of the compositions of the present invention to be administered can be determined by an art worker with consideration of a subject's age, weight, tumor size, extent of infection or metastasis, and condition of the subject.
• the cells can be administered by using infusion or injection techniques that are commonly known in the art.
• first and second population of stem cells in the methods of the invention, one may also treat the host to reduce immunological rejection of the donor cells, such as those described in U.S. Pat. No. 5,800,539, issued Sep. 1, 1998; and U.S. Pat. No. 5,806,529, issued Sep. 15, 1998, both of which are incorporated herein by reference.
• the cells of the present invention are administered to a subject following treatment with an agent such as myeloablative (high dose) chemotherapy, chemotherapy, radiation, immunosuppressive agents, such as antithymocyte globulin (ATG), busulfan, IVIG, melphalan, methylprednisolone, cyclosporin, azathioprine, methotrexate, mycophenylate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies or other antibody therapies, cytoxin, fiudaribine, cyclosporin, FK506, rapamycin, mycophenylic acid, steroids, FR901228, cytokines, and localized or total body irradiation.
• an agent such as myeloablative (high dose) chemotherapy, chemotherapy, radiation, immunosuppressive agents, such as antithymocyte globulin (ATG), busulfan, IVIG, melphalan, methylprednisolone,
• the cell compositions of the present invention are administered to a subject in conjunction with (e.g.
• T cell ablative therapy using either chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH.
• chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH.
• the cell compositions of the present invention are administered following B-cell ablative therapy such as agents that react with CD20, e.g. Rituxan.
• subjects may undergo standard treatment with high dose chemotherapy followed by stem cell transplantation. Following the transplant, subjects receive an infusion of the two cell populations described herein.
• the dosage of the above treatments to be administered to a subject will vary with the precise nature of the condition being treated and the recipient of the treatment.
• the scaling of dosages for human administration can be performed according to art-accepted practices.
• Methods for monitoring therapeutic response in subjects include assessment of one or more of overall and event-free survival, , platelet engraftment, ANC engraftment, relapse of disease, or the like, in a subject. The response to treatment can be compared to an appropriate control. Methods for monitoring these responses are well known in the art and exemplified herein.
• a "subject" refers to an individual that has been administered the cell preparations of the invention.
• the subject can be a human, a non-human primate, a laboratory animal, or the like, but preferably is a human.
• a "control” can include an individual (or group of individuals) that is (are) untreated, sham treated (e.g., the individual is treated with a first and second cell population in which one or both populations do not contain the cell preparations described herein), treated with a similar or distinct method for improving engraftment and/or improving therapeutic response to stem cell transplantation, or treated with a cell preparation that is different from the cell populations described herein, depending on the nature of the observation.
• an appropriate control may include a subject that has been treated with a second cell population that has not been primed, or may include the therapeutic response of a subject whose second cell population has been cultured without using a priming agent.
• controls can be historical controls.
• the response of the subject to the methods of the invention can be compared to the response seen in previously studied populations of subjects undergoing similar or distinct procedures for modulating engraftment and/or improving therapeutic response to stem cell transplantation.
• the methods of the present invention result in a decrease of incidence and/or severity of grade III and/or grade IV acute graft versus host disease (GvHD), in part by eliminating T cell populations.
• GvHD grade III and/or grade IV acute graft versus host disease
• This elimination from the stem cell population of the invention can be expected to reduce the incidence and severity of GvHD in recipients of allogeneic transplants. See, for example, Ho and Soiffer (2001) Blood 98:3192.
• GvHD occurs when donor T-cells react against antigens on normal host cells causing target organ damage, which often leads to death.
• the principal target organs of GvHD are the immune system, skin, liver and intestine.
• GvHD There are two kinds of GvHD: acute and chronic. Acute GvHD appears within the first three months following transplantation. Signs of acute GvHD include a reddish skin rash on the hands and feet that may spread and become more severe, with peeling or blistering skin. GvHD is ranked based on its severity: stage (or grade) 1 is mild, stage (or grade) 4 is severe. Chronic GvHD develops three months or later following transplantation. The symptoms of chronic GvHD are similar to those of acute GvHD, but in addition, chronic GvHD may also affect the mucous glands in the eyes, salivary glands in the mouth, and glands that lubricate the stomach lining and intestines.
• the subject may be monitored for levels of malignant cells, i.e., for evidence of minimal residual disease. Such monitoring may comprise subject follow-up for clinical signs of relapse.
• the monitoring may also include, where appropriate, various molecular or cellular assays to detect or quantify any residual malignant cells. For example, in cases of sex -mismatched donors and recipients, residual host-derived cells may be detected through use of appropriate sex markers such as Y chromosome-specific nucleic acid primers or probes. In cases of single HLA locus mismatches between donors and recipients, residual host cells may be documented by polymerase chain reaction (PCR) analysis of Class I or Class II loci that differ between the donor and recipient.
• PCR polymerase chain reaction
• nucleic acid primers and/or probes specific for the bcr/abl translocation in chronic myelogenous leukemia, for other oncogenes active in various tumors, for inactivated tumor suppressor genes, other tumor-specific genes, or any other assay reagents known to be specific for tumor cells may be employed. Any of these or functionally comparable procedures may be used to monitor the subject for evidence of residual malignant cells.
• the methods of the present invention result in at least about a 10%, at least about a 15%, at least about a 20%, about a 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or at least about a 100% decrease in the presence of malignant cells when compared to a control.
• Treatment of a subject according to the methods of the present invention may be considered efficacious if the disease, disorder or condition is measurably improved in any way. Such improvement may be shown by a number of indicators.
• Measurable indicators include, for example, detectable changes in a physiological condition or set of physiological conditions associated with a particular disease, disorder or condition (including, but not limited to, blood pressure, heart rate, respiratory rate, counts of various blood cell types, levels in the blood of certain proteins, carbohydrates, lipids or cytokines or modulated expression of genetic markers associated with the disease, disorder or condition).
• Treatment of an individual with the stem cells or supplemented cell populations of the invention would be considered effective if any one of such indicators responds to such treatment by changing to a value that is within, or closer to, the normal value.
• the normal value may be established by normal ranges that are known in the art for various indicators, or by comparison to such values in a control.
• the efficacy of a treatment is also often characterized in terms of an individual's impressions and subjective feeling of the individual's state of health. Improvement therefore may also be characterized by subjective indicators, such as the individual's subjective feeling of improvement, increased well-being, increased state of health, improved level of energy, or the like, after administration of the cell populations of the invention.
• the methods of the present invention result in at least about a 30%, at least about a 35%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 125%, about 150%, about 175%, about 200%, about 250%, at least about a 300%, or greater improvement in one or more of the clinical indicators described above when compared to a control.
• neutrophil count The primary measure of hematologic recovery is neutrophil count.
• Neutrophils usually constitute about 45 to 75% of all white blood cells in the bloodstream.
• the risk of infection increases somewhat; when it falls below 500 cells per microliter, the risk of infection increases greatly.
• controlling infections is problematic and subjects are at risk of dying from an infection. Accordingly, in clinical settings, such as transplant settings, the sooner neutrophil counts recover, the sooner a subject can be released from the hospital. Accordingly, any decrease in time that it takes to achieve clinically relevant levels of neutrophils is beneficial to the subject and contemplated herein as acceleration of hematologic recovery.
• neutrophil engraftment is defined as an absolute neutrophil count (ANC) of at least 500 neutrophils/ ⁇ l.
• the neutrophil count may be reported as a date that an individual subject (or an average of multiple subjects) reaches the ANC threshold, or a percentage of the subjects having an ANC of 500 neutrophils/ ⁇ l by a particular day post- transplant, usually around day 42, or the probability that an individual will reach a certain threshold by a certain date.
• the methods of the present invention result in neutrophil engraftment on or before day 10, day 11, day 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,
• the day that patients achieve a benchmark ANC count deemed to be normal will be accelerated by 5 days, 6 to 10 days, 11-20 days, or greater than 20 days relative to a control group of patients.
• Hematologic recovery can also be measured by a clinically relevant recovery of platelets (as would be recognized by the skilled artisan, there are normally between 150,000-450,000 platelets in each microliter of blood).
• platelet engraftment is defined as maintenance of platelet counts of at least 50,000 platelets/ ⁇ l of blood without transfusion support.
• the platelet count may be reported as a date that an individual subject (or an average of multiple subjects) reaches the platelet count threshold, or as a percentage of the subjects having (or probability of a subject reaching) a platelet count of at least 50,000 platelets/ ⁇ l of blood by a particular day post-transplant, usually around day 180.
• the methods of the present invention result in platelet engraftment on or before day 50, day 55, day 60, 65, 70, 75, 80, 85, 90, 95, or on or before day 100.
• the day that patients achieve a benchmark platelet count deemed to be normal will be accelerated by 5 days, 6 to 10 days, 11-20 days, or greater than 20 days relative to a control group of patients.
• rapidity in T cell recovery is also an indicator of accelerated hematologic recovery.
• An indicator of T cell recovery can include response to PHA-induced profileration and/or an increase in the number of CD4+ cells in the subject.
• the CD4+ counts may be reported as a date that an individual subject (or an average of multiple subjects) reaches a CD4+ count threshold, or as a percentage of the subjects having (or the probability of subject reaching) a threshold CD4+ count by a particular benchmark day post-transplant, usually around day 100.
• the methods of the present invention result in T cell counts at day 100 that are at least about 25 to 100% or greater than counts in patients in a control population.
• the post-transplant day that a patient achieves a benchmark CD4 count is about 10 to about 20, about 20 to about 30, about 30 to about 40, about 40 to about 50, or greater than 50 days earlier than the day that patients in a control group achieve the same benchmark CD4 count.
• a therapeutic response can also be measured in terms of overall and/or event free survival.
• Event free survival is defined as the time from transplantation to the day of the first event. Events are defined as graft failure, autologous reconstitution, relapse, or death. Relapse in leukemic subjects is determined by standard criteria. Tertiary end points include description of the incidence of acute GvHD, and other measures of nonrelapse mortality. GvHD is scored according to standard criteria (Przepiorka et al. (1995) Bone Marrow Transplant. 15: 825-828).
• the methods of the present invention result in overall and/or event-free survival that is at least about 30%, at least about 35%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 250%, at least about 300%, or greater % improved over controls (e.g., fewer or no incidences of events reported (particularly grade III and/or grade IV acute GvHD), increased number of days of survival, and/or higher numbers of patients surviving to a certain date post- transplant when compared to a control population).
• Another global measure of therapeutic response is overall survival at 180 days.
• survival in the group of patients transplanted according to the present invention is compared to overall survival in a control group treated by conventional methods.
• patients show an improved overall survival of at least about 5%, of at least about 6-10%, of at least about 11-15%, of at least about 16-20%, or of great than 20% compared to control patients.
• the following examples are offered by way of illustration and not by way of limitation.
• MPS I Major developmental disorder characterized by a common protocol for the preparative regimen (busulfan, cyclophosphamide, ATG) and GvHD prophylaxis (cyclosporine, steroids).
• CBUs were screened for enzyme activity to prevent use of carrier donors. Sixty-four percent of patients were male and 77% were Caucasian. Nearly half the patients (48%) received a UCB units matching at 4/6 HLA loci as measured by low resolution typing at HLA Class I A&B and high resolution typing at HLA Class II DRB 1.
• the cumulative incidence of neutrophil engraftment was 78%, occurring in a median of 26 days.
• Levels of HLA disparity between recipient and donor did not influence engraftment, GvHD or overall survival.
• the surviving patients with MPS, TSD, GLD, and MLD all stabilized and/or gained skills post transplant.
• Three of 8 patients with ALD all of whom had mild to moderate clinical symptoms at the time of referral for transplant, experienced disease progression with neurologic deterioration before stabilization.
• the cord blood unit selected for transplantation was stored in a 2 compartment cryopreservation bag (20%/80% split) in a total of 25ml of cells, hespan and 10% DMSO.
• the 20% (5ml) fraction was removed from liquid nitrogen (procedure 5D.160.01), and thawed in a 37 degree C waterbath to a slushy consistency.
• Dextran/Albumin was added to dilute to 4x the initial volume, the cells were washed, pelleted and resuspended in ALDESORT® assay buffer/ 100U/ml DNase I (Aldagen, Inc., Durham, NC). Red blood cell to white blood cell ration was adjusted to ⁇ lxl ⁇ e8 cells/ml and the cells were lineage depleted with EASYSEP® (StemCell Technologies) anti- glycophorin A and CD 14 cocktails to label cells.
• the labeled cells were mixed with EASYSEP® magnetic nanoparticles and incubated at room temperature for 10 minutes. The sample was then exposed to the EASYSEP® magnetic which will remove lineage positive cells. The residual lineage depleted cells were gently aspirated into a conical tube. RBC: WBC ratio was checked and must have been ⁇ 1 : 10. If it was higher, the EASYSEP® depletion was repeated.
• Example 4 Isolation of ALDH br UCB cells by high speed FACS sorting
• the lineage depleted cells were stained with activated ALDESORT® reagent and incubated at 37 degrees C for 15 minutes. The reaction was stopped, controls were prepared and the ALDH br cells were isolated by high speed flow sorting on the FACSAria sorter (BD Biosciences). Methods for isolating ALDH br cells are more fully described in Storms et al, 1999, supra and PCT Publication No. WO 2005/083061, both of which are herein incorporated by reference in their entirety. The cells may be frozen, infused, or further primed as described in Example 5.
• Example 5. Thawing, sorting, priming, and infusion of the ALDH r cells
• the UCB cells were thawed, ALDH r sorted and cytokine primed 5 days prior to the scheduled conventional UCB transplant (UCBT). Briefly, the 20% fraction of the UCB unit was removed from storage, thawed in a 37°C degree water-bath, mixed with dextran and albumin and washed. The resulting cell pellet was resuspended in
• EASYSEP® medium (Stem Cell Technologies) to remove lineage positive cells.
• the residual lineage negative cells were RBC cell depleted to achieve a WBC:RBC ratio of ⁇ 1 :10.
• This cell population was sorted on a FACSaria (Becton Dickenson) to isolate a purified population of ALDH r cells.
• the ALDH r cells was placed in culture with a cytokine cocktail consisting of SCF 50ng/ml, FLT-3 lOng/ml and IL-7 lOng/ml in serum- free medium (Cellgenix SCGM) and incubated in 5% CO2 at 37 degrees C in diffusion exchange bags (American Fluoseal) for 5 days.
• ALDH r primed cells were transferred to a standard transfer pack with an attached bag of normal saline for infusion. On day 0, transplant day, approximately 4 hours after infusion of the conventional
• the cytokine primed ALDH br UCB cells were harvested, counted, checked for viability and gram stain, connected to the infusion set and transported to the bone marrow transplant unit for infusion.
• Example 6 UCB Thawing and Infusion for the conventional, unmanipulated graft (first cell population)
• Bags of UCB were thawed in the laboratory using sterile technique under a hood.
• the UCB was thawed in a 37°C waterbath, and diluted by 1 : 1 volume using a 5% albumin /dextran solution [albumin 25% (12.5 gms/50 ml) 25 gms in 500 ml dextran] to preserve cell viability.
• the 5% albumin /dextran solution was added slowly to the thawed
• UCB UCB using transfer bags with stopcocks and mixed gently. The thawed and diluted UCB was next weighed and centrifuged (2000 rpm x 20 min at 4° C). Specimens were obtained for cell count and viability, culture, clonogenic assays, and phenotype. Supernatant containing DMSO and the albumin/ dextran solution was removed, and the UCB pellet resuspended again by a 1 : 1 volume using a 5% albumin/dextran solution.
• the UCB was labeled with patient identification information and transferred to the bedside for infusion.
• the UCB was infused via the patient's central venous catheter at a rate of 1-3 ml/min.
• UCB was infused without an in-line filter and was not irradiated. If the patient developed chest tightness or other symptoms, a brief rest (1-2 minutes) was allowed before proceeding with the remainder of the infusion. If a large volume of UCB (>15 ml/kg) was to be infused, half the UCB may have been infused, followed by a 30 minute rest period, and then infusion of the remainder of the UCB. Vital signs were taken every 15 minutes until 2 hours after completion of the infusion. Hydration (2.5-3.0 ml/kg/hr) was maintained for 12 hours after UCB infusion was completed. Furosemide
• Standard cytoreduction for patients with ALL undergoing allogeneic BMT includes cyclophosphamide (100-200 mg/kg) and total body irradiation (TBI, 1,000-1440 cGy).
• TBI total body irradiation
• event-free survival rates can be achieved in 20-45% of children and 20% of adults with ALL in 2nd remission, and up to 60% of patients with ANLL undergoing matched-related allogeneic BMT.
• event- free survival decreases with only 8% of patients cured when transplanted in relapse.
• ATG was used for additional immunosuppressive therapy; methylprednisolone was substituted if patients could not tolerate ATG.
• Standard cytoreduction for patients with non-malignant conditions undergoing allogeneic BMT includes busulfan 16 mg/kg over 4 days (adjusted for pediatric patients to dosing per m2 and followed with targeted levels with first dose PK), cyclophosphamide 200 mg/kg over 4 days and ATG 90 mg/kg over 3 days. Engraftment rates with unrelated donor umbilical cord blood using this regimen ranges between 80- 90%. TBI was avoided to minimize late adverse events such as growth retardation, endocrine failure, cognitive deficits, chronic lung disease or cardiomyopathy.
• Peripheral blood samples were tested on or about days + 30, 60 and 100 for chimerism.
• a bone marrow aspirate and biopsy for cellularity and donor chimerism was performed between days 41-44 if the patient had not demonstrated neutrophil recovery by this time.
• Platelet counts, ANC, and various other clinical indicators of successful engraftment were evaluated as known in the art.
• the results for primed and unprimed samples were combined for statistical evaluation of engraftment.
• the rate of neutrophil engraftment is shown in Figures 1 and 2.
• the rate of platelet engraftment is shown in Figures 3 and 4.

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