Compositions and methods for improving treatment outcomes for patients having hematological malignancies using an expanded stem cell product

Abstract

To provide compositions and methods for improving treatment outcomes for patients having hematological malignancies using an expanded stem cell product.SOLUTION: The present invention relates to methods and compositions for treating a hematological malignancy with an expanded hematopoietic stem cell product in combination with a chemotherapy regimen. The present invention relates to methods and compositions for improving the treatment outcome of a patient having acute myeloid leukemia (AML) or another hematological malignancy. The expanded stem cell product comprises hematopoietic stem cells or hematopoietic stem and progenitor cells derived from multiple donors that are combined (e.g., pooled) without matching (i.e., without regard to) the HLA-type of the cord blood units to each other or to the HLA-type of the patient. The expanded stem cell product can be administered following a chemotherapy regimen, such as an induction regimen, salvage regimen or consolidation regimen of varying intensity.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 849,588, filed May 17, 2019, and U.S. Provisional Application No. 62 / 852,147, filed May 23, 2019, the disclosures of which are incorporated herein by reference in their entireties.

[0002] FIELD OF THE INVENTION The present invention relates to methods and compositions for improving treatment outcomes in patients with acute myeloid leukemia (AML) or another hematologic malignancy. The expanded stem cell product comprises hematopoietic stem cells or hematopoietic stem and progenitor cells from multiple donors combined (e.g., pooled) without matching (i.e., irrespective of) the HLA types of the umbilical cord blood units to each other or to the patient's HLA type. The expanded stem cell product can be administered after a chemotherapy regimen (e.g., induction, salvage, or consolidation regimen of various intensities). [Background technology]

[0003] background Acute myeloid leukemia (AML) is the leading cause of acute leukemia in adults and accounts for the majority of all adult leukemias. Despite extensive research, AML is associated with poor long-term survival; the 5-year overall survival rate is approximately 28.3% for all patients (SEER) and approximately 24% for patients aged 20 years or older. In contrast, for patients younger than 20 years, the 5-year overall survival rate is approximately 67%. Conventional chemotherapy can effectively achieve initial remission of the disease in some AML patients. However, due to the highly heterogeneous nature of the disease, approximately 30% of AML patients do not respond to chemotherapy. It is important to note that chemotherapy does not achieve complete clearance of the disease in most patients, and more than 70% of patients in remission experience AML relapse within two years after initial treatment.

[0004] There is currently no standard treatment regimen for patients with relapsed AML, which is associated with a poor prognosis. Relapsed AML can be caused by a phenomenon called minimal residual disease (MRD), which is mediated by an AML cell population that is resistant to chemotherapy. MRD is proposed to be mediated by a leukemia stem cell (LSC) population because this cell population has the ability to withstand chemotherapy and other treatments. Therefore, the development of treatments that target AML and address MRD to achieve relapse-free clearance of the disease has become an active area of ​​research.

[0005] Allogeneic hematopoietic stem cell transplantation (allo-HSCT) has been investigated as a therapeutic treatment option for AML patients and is associated with higher relapse-free survival rates than conventional chemotherapy. These grafts are commonly derived from bone marrow, peripheral blood, and / or umbilical cord blood, particularly peripheral blood grafts following stem cell mobilization in the donor, for example, by administering GM-CSF. The cells of the graft are a heterogeneous mix of blood and immune cells, including stem cells, red blood cells, white blood cells (including T cells, NK cells, etc.), and platelets. Hematopoietic stem cells constitute a very small proportion of cells in hematopoietic stem cell grafts, generally less than 1% of the total cell population. Donor-derived T cell-mediated anti-leukemia effects contribute to increased patient survival, as autologous and T cell-depleted grafts have been reported to be associated with higher relapse rates. Clinical use of allo-HSCT is limited by the lack of appropriately HLA-matched donors and is associated with toxicity and other related complications. Immune responses have been reported to cause common tissue damage, such as graft-versus-host disease (GVHD).

[0006] Microtransplants (infusion of non-engrafting stem cell grafts) and / or non-engrafting donor lymphocyte infusions have been investigated as potential therapeutic treatments for patients with AML. Unrelated donor-mismatched microtransplants of mobilized PBSCs from a single donor were reported to provide some benefit to patients, but the patient ended up relapsing (Punwani et al., 2018, Leuk. Res. Rep. 9:18-20). HLA-mismatched allogeneic cell therapy has also been investigated for the treatment of AML using mobilization of partially matched peripheral blood cells (Mohrbacher et al., 2014, Blood 124:5944). Five of eight patients were reported to achieve complete remission / complete remission with incomplete hematologic recovery (CR / Cri) lasting 3-10 months or more, but the authors reported that the response did not last as long as expected, despite the partial matching of the graft. Therefore, there remains a need to develop treatments that target AML and other hematological malignancies and achieve relapse-free clearance of the disease. There remains a need in the art to develop less toxic therapies and improve treatment outcomes using existing treatment regimens for patients with AML (including relapsed / refractory AML, newly diagnosed AML, and treatment-related AML) and other hematological malignancies (e.g., myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), and non-Hodgkin's lymphoma (NHL)). [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Punwani et al., 2018, Leuk. Res. Rep. 9:18-20 [Non-patent document 2] Mohrbacher et al., 2014, Blood 124:5944 Summary of the Invention [Means for solving the problem]

[0008] Abstract This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key features of the claimed subject matter, nor is it intended for use as an aid in determining the scope of the claimed subject matter.

[0009] The present invention provides a method for improving treatment outcomes for patients with acute myeloid leukemia (AML) or other hematological malignancies by administering a chemotherapy regimen, or cycles thereof, to a patient in need of treatment and then administering a fixed dose of an expanded stem cell product to the patient, wherein the administering step is performed without matching the HLA type of the expanded stem cell product to the patient's HLA type. The expanded stem cell product is a cell-based product derived from pooled hematopoietic stem cells or hematopoietic stem and progenitor cells of at least two human donors, wherein the donor hematopoietic stem cells or hematopoietic stem and progenitor cells are combined without matching the HLA type of the other donor and without matching the HLA type of the patient. The expanded stem cell product is depleted of T cells and red blood cells.

[0010] Also provided is a method for improving the treatment outcome of a human patient with AML or other hematological malignancies, comprising: (a) selecting an expanded hematopoietic stem cell product for administration to the patient, wherein the selecting step does not take into account (i.e., does not match) the HLA type of the expanded stem cell product or the patient; (b) administering a chemotherapy regimen, or cycles thereof, to the patient; and (c) administering a fixed dose of the selected expanded stem cell product to the patient. The expanded stem cell product is a cell-based product derived from pooled hematopoietic stem cells or hematopoietic stem and progenitor cells of at least two human donors, wherein the donor hematopoietic stem cells or hematopoietic stem and progenitor cells are pooled without matching to the other donor's HLA type and without matching to the patient's HLA type. As described above, the expanded stem cell product is depleted of T cells and red blood cells.

[0011] The present invention further provides a method for treating a patient with AML or other hematological malignancies, comprising administering to the patient a chemotherapy regimen, or cycles thereof, and then administering to the patient a fixed dose of an expanded stem cell product, wherein the administering step is performed without matching the HLA type of the expanded stem cell product to the patient's HLA type. The expanded stem cell product is a cell-based product derived from pooled hematopoietic stem cells or hematopoietic stem and progenitor cells of at least two human donors, wherein the donor's hematopoietic stem cells or hematopoietic stem and progenitor cells are pooled without matching to the other donor's HLA type and without matching to the patient's HLA type. As described above, the expanded stem cell product is depleted of T cells and red blood cells.

[0012] In certain embodiments, the fixed dose of expanded stem cell product comprises about 50 million to about 400 million viable CD34+ cells. In certain embodiments, the fixed dose of expanded stem cell product comprises about 50 million, about 75 million, about 100 million, about 200 million, about 300 million, or about 400 million viable CD34+ cells. In some embodiments, the expanded stem cell product is prepared, cryopreserved, and stored for later use as an "off the shelf" product. The cryopreserved expanded stem cell product is thawed prior to administration to the patient.

[0013] The expanded stem cell product is a pool of at least two expanded hematopoietic stem cell populations and / or at least two expanded hematopoietic stem and progenitor cell populations, where each cell population is derived from a separate donor. In some embodiments, each cell population is obtained from a separate umbilical cord blood or placental blood unit (i.e., from a different human at birth). The HLA types of at least two cell populations in the pool are HLA-matched to each other. Optionally, the expanded stem cell product is a pool of two or more hematopoietic stem cell or stem and progenitor cell populations pooled prior to expansion (which pool is then expanded), or the cell populations are pooled after expansion. Optionally, the expanded stem cell product is a pool of two or more human umbilical cord blood or placental blood stem or stem and progenitor cell populations pooled prior to expansion (which pool is then expanded), or the cell populations are pooled after expansion. In one embodiment, the cell populations in the pool are all derived from umbilical cord and / or placental blood of individuals of the same race (e.g., African American, Caucasian, Asian, Latin American, Native American, Native Australian, Inuit, Pacific Islander) or all derived from umbilical cord and / or placental blood of individuals of the same ethnicity (e.g., Irish, Italian, Indian, Japanese, Chinese, Russian, etc.). In another embodiment, the hematopoietic stem cells or hematopoietic stem and progenitor cells in the pool are combined without regard to either race or ethnicity.

[0014] In yet another embodiment, the method for improving treatment outcomes in patients with AML or another hematological malignancy comprises, prior to the administering step, expanding ex vivo isolated human cord blood stem cells or stem and progenitor cells obtained from the umbilical cord blood and / or placental blood of at least two humans at birth. Preferably, the expanding step comprises contacting the human cord blood stem cells or stem and progenitor cells with an agonist of Notch function. The agonist can be a Delta protein or a Serrate protein, or a fragment of a Delta protein or a Serrate protein, which fragment can bind Notch protein. In another embodiment, the expanding step comprises contacting the hematopoietic stem cells or stem and progenitor cells with a Delta protein or a Serrate protein fragment, which fragment can bind Notch protein. ext-IgG (DXI).

[0015] In another embodiment, a method for improving treatment outcomes for human patients with hematological malignancies comprises: (a) enriching isolated human umbilical cord blood stem or stem and progenitor cells obtained from umbilical cord blood and / or placental blood of at least two humans at birth for hematopoietic stem or hematopoietic stem and progenitor cells to produce a cell population enriched for hematopoietic stem or hematopoietic stem and progenitor cells; (b) expanding the enriched cell population for hematopoietic stem or hematopoietic stem and progenitor cells ex vivo to produce an expanded stem cell product; (c) administering chemotherapy to a subject in need of chemotherapy; and (d) administering a fixed dose of the expanded stem cell product to a human patient in need thereof, wherein the administering step is performed without matching the HLA type of the expanded hematopoietic stem cells or expanded hematopoietic stem and progenitor cells of the expanded stem cell product to the HLA type of the patient, and without matching the HLA types of the expanded hematopoietic stem cells or expanded hematopoietic stem and progenitor cells of the expanded stem cell product to each other. In a preferred embodiment, the expanded hematopoietic stem cells are CD34+ cells. The method may further comprise freezing and storing the expanded stem cell product after step (b), and thawing the expanded stem cell product before step (c). In certain embodiments, the patient is suffering from AML (e.g., newly diagnosed AML, relapsed / refractory AML, or treatment-related AML), or other hematological malignancies (e.g., non-Hodgkin's lymphoma, myelodysplastic syndrome (MDS), or myeloproliferative neoplasm (MPN)).

[0016] definition Although any materials and methods similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. For purposes of the present invention, the following terms are defined below.

[0017] As used herein, an "expanded stem cell product" refers to a cell population enriched for hematopoietic stem or stem / progenitor cells that has been subjected to a technique to expand the hematopoietic stem or stem / progenitor cells of the cell population, which technique has been shown to result in either (i) an increased number of hematopoietic stem or stem / progenitor cells in an aliquot of the so-expanded cells, or (ii) an increased number of severe combined immunodeficiency (SCID) reconstituting cells, as determined by limiting dilution analysis, as indicated by enhanced engraftment in non-obese diabetic / severe combined immunodeficiency (NOD / SCID) mice injected with an aliquot of the so-expanded cells, compared to that observed in an aliquot of cells that has not been subjected to the expansion technique (see U.S. Patent Publication No. 2013 / 0095079; Delaney et al., 2010, Nature Med. 16(2):232-236). Typically, the hematopoietic stem or stem / progenitor cells are CD34+. In some embodiments, the hematopoietic stem cells or hematopoietic stem and progenitor cells are derived from human umbilical cord blood and / or human placental blood. In some embodiments, the expanded stem cell product is prepared using a Notch agonist expansion method. In some embodiments, the expanded stem cell product is prepared using a DXI expansion method. The expanded stem cell product is depleted of T cells and red blood cells.

[0018] As used herein, "chemotherapy regimen" refers to a chemotherapy regimen that defines the drugs to be used, their dosage, frequency and duration of treatment, and other considerations. Such regimens may combine several chemotherapy drugs in combination chemotherapy. Most of the drugs used in chemotherapy are cytostatic or cytotoxic.

[0019] The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description when taken in conjunction with the accompanying drawings. [Brief explanation of the drawings]

[0020] [Figure 1] FIG. 1 illustrates a flow chart showing an exemplary procedure for enriching a population of CD34+ cells and expanding the enriched cell population.

[0021] [Figure 2] FIG. 2 shows the subject breakdown during the clinical trial described in Example 3. DETAILED DESCRIPTION OF THE INVENTION

[0022] Detailed Description While exemplary embodiments have been illustrated and described, it will be recognized that various changes can be made therein without departing from the spirit and scope of the invention.

[0023] Hematopoietic stem / progenitor cell transplantation, particularly autologous hematopoietic stem / progenitor cell transplantation, is commonly performed to rescue bone marrow aplasia following high-dose chemotherapy for solid tumors or multiple myeloma. Allogeneic hematopoietic stem cell transplantation has been found to be useful in curing leukemia and other hematopoietic malignancies by eradicating diseased blood and immune systems and restoring blood homeostasis through the infusion of healthy donor hematopoietic stem cell grafts. One of the persistent problems in allogeneic hematopoietic stem cell transplantation is the lack of available allogeneic donors with sufficient HLA antigens and / or alleles compatible with the patient for successful treatment. More recently, methods and compositions have been devised for providing hematopoietic function in immunocompromised human patients by selecting expanded human umbilical cord blood stem / progenitor cells without taking into account the HLA type of the expanded human umbilical cord blood stem / progenitor cell sample or the patient's HLA type. The hematopoietic stem / progenitor cell samples can be used to transiently replace or replenish hematopoietic function or reduce the rate of life-threatening infections in human patients at high risk of morbidity and mortality after hematopoietic stem cell transplantation or high-dose chemotherapy. Unexpectedly, expanded hematopoietic stem cells or hematopoietic stem and progenitor cell products, in which HLA typing is not performed and the cell product does not contain T cells, have been found to be useful in increasing the chances of treating and / or improving the outcomes of human patients with acute myeloid leukemia (AML) or certain other hematologic malignancies.

[0024] The present invention provides methods for treating and improving the treatment outcomes of patients with AML or other hematological malignancies by administering a chemotherapy regimen to the patient followed by administration of a fixed dose of an expanded stem cell product to a patient in need thereof, wherein the administering step is performed without HLA-matching the expanded stem cell product to the patient's HLA type. The expanded stem cell product is a cell-based product comprising hematopoietic stem cells or hematopoietic stem and progenitor cells that are not HLA-matched to each other or to the patient's HLA type. In some embodiments, the expanded stem cell product is a pooled product derived from pooled hematopoietic stem cells or hematopoietic stem and progenitor cells derived from umbilical cord blood or placental blood units from at least two different human donors, comprising hematopoietic stem cells or hematopoietic stem and progenitor cells that are not HLA-matched to each other or to the patient's HLA type. The phrases "without matching the HLA type," "unmatched," and the like mean that no steps have been taken to match any of the HLA antigens or alleles (HLA types) between the patient and the hematopoietic stem cells or hematopoietic stem and progenitor cells in the expanded stem cell product. The expanded stem cell product is selected without matching the HLA type of the patient to whom the expanded stem cell product is administered. Similarly, with respect to the source of the hematopoietic stem cells or hematopoietic stem and progenitor cells from which the expanded stem cell product is derived, e.g., from an umbilical cord blood unit or placental blood unit, the phrase "without matching the HLA type" means that no steps have been taken to match any of the HLA antigens or alleles (HLA types) between the hematopoietic stem cells or hematopoietic stem and progenitor cells in the expanded stem cell product. It should also be noted that the expanded stem cell product is depleted of T cells and red blood cells.

[0025] The expanded stem cell product is typically administered after a chemotherapy regimen. The chemotherapy regimen can be a single-agent or multi-agent regimen. In some embodiments, the chemotherapy regimen is an induction regimen or a consolidation regimen. An induction regimen involves the use of chemotherapy as a primary treatment for patients presenting with advanced cancer for which no alternative treatment exists. A consolidation regimen involves repeated treatment cycles during the immediate post-remission phase, as is typically used in leukemia. In some embodiments, the chemotherapy regimen is a salvage regimen. A salvage regimen involves the use of chemotherapy in patients with a recurrence of a malignant tumor after initial treatment, with the hope of a cure or prolongation of life. In some embodiments, the expanded stem cell product is administered about 12 to about 48 hours after the chemotherapy regimen, or preferably about 24 to 36 hours after the chemotherapy regimen. In some embodiments, the expanded stem cell product is administered about 12 to about 48 hours after each cycle of the chemotherapy regimen, or preferably about 24 to 36 hours after each cycle, where the chemotherapy regimen is administered in more than one cycle.

[0026] In some embodiments, the expanded stem cell product is administered to the patient after the components of the chemotherapy regimen and their active metabolites have cleared from the patient's blood. In some embodiments, the expanded stem cell product is administered to the patient after the components of the induction regimen and their active metabolites have cleared from the patient's blood. In some embodiments, the expanded stem cell product is administered to the patient after the components of the consolidation regimen and their active metabolites have cleared from the patient's blood. In some embodiments, the expanded stem cell product is administered to the patient after the components of the rescue regimen and their active metabolites have cleared from the patient's blood. In some embodiments, if the chemotherapy regimen (e.g., induction regimen, salvage regimen, or consolidation regimen) is administered in more than one cycle, the expanded stem cell product is administered to the patient after the cycle or each cycle after the components of the regimen and their active metabolites have cleared from the patient's blood. As used herein, "after ... regimen and active metabolites thereof have been cleared from the patient's blood" refers to the clearance of components of the regimen (e.g., induction regimen, salvage regimen, or consolidation regimen) and their active metabolites that affect the viability of CD34+ stem cells in the patient's blood, e.g., by reducing the viability of CD34+ stem or progenitor cells by at least 5%, at least 10%, or at least 20%.

[0027] Administration of the expanded stem cell product after each regimen or cycle thereof can improve the treatment outcome of a patient with AML or other hematological malignancy, for example, whether the patient achieves remission (e.g., complete remission (CR) or complete remission with incomplete hematological recovery (CRI)). (Complete Remission without an incomplete hematologic recovery) By improving the chances of achieving In some embodiments, the improved treatment outcome is associated with increased IL-2 levels in the patient after administration of the expanded stem cell product. Increased IL-2 levels are the induction of an increased immune response in the patient. Without intending to be bound by any particular theory, because the expanded stem cell product is derived from unmatched cord blood units from multiple human donors, the expanded hematopoietic stem cell product contains hematopoietic stem cells or hematopoietic stem and progenitor cells with different HLA types and / or alleles. The presence of multiple mismatched HLA types or alleles in the expanded stem cell product after administration to the patient activates and / or augments the patient's immune response, potentially due to an increased antigen load. The resulting activation or stimulation of the patient's immune response may be due, in part, to activation of the patient's own T cells and / or NK cells.

[0028] The expanded stem cell product is not required or expected to engraft and provide a therapeutic benefit to the patient. In some embodiments, the expanded stem cell product does not engraft either transiently or permanently in the patient. In some embodiments, the expanded stem cell product does not engraft transiently in the patient. Engraftment is typically detected as mixed chimerism in the patient, meaning that cells derived from the expanded stem cell product are detected in the patient's blood about 7 to about 14 days after administration of the expanded stem cell product. In some embodiments, the expanded stem cell product does not measurably increase hematopoietic reconstitution, either transiently or long-term. In some embodiments, the expanded stem cell product does not reduce infection rates in the patient.

[0029] Frequent infections are a common complication of chemotherapy regimens used in the treatment of hematological malignancies (e.g., AML) and are a significant cause of treatment failure. Chemotherapeutic agents can also be highly immunosuppressive and / or highly myelosuppressive, which can lead to prolonged neutropenia. Administration of the expanded stem cell product after chemotherapy regimens does not necessarily prevent post-chemotherapy infectious complications or promote transient hematopoietic recovery, but can improve treatment outcomes by inducing a host immune response against leukemia.

[0030] Preparation of expanded stem cell products The expanded stem cell product contains hematopoietic stem cells or stem and progenitor cells and is substantially depleted of T cells and red blood cells, typically containing an enriched number of CD34+ hematopoietic stem cells or stem and progenitor cells. The hematopoietic stem cells or stem and progenitor cells contain multiple HLA types because the hematopoietic stem cells or stem and progenitor cells are not matched to each other or to the patient prior to pooling. As used herein, substantially depleted of T cells refers to less than 1% CD3+ cells, or less than 0.5% CD3+ cells, or less than 0.1% CD3+ cells in the expanded stem cell product.

[0031] In some embodiments, the CD34+ hematopoietic stem cells or hematopoietic stem and progenitor cells are derived from umbilical cord blood or placental blood. Human umbilical cord blood and / or human placental blood are a typical source of umbilical cord blood stem cells. Such blood can be obtained by methods known in the art. For a discussion of collecting umbilical cord blood and placental blood at human birth, see, e.g., U.S. Pat. Nos. 5,004,681 and 7,147,626, and U.S. Patent Publication No. 2013 / 0095079, which are incorporated herein by reference. Collection of umbilical cord blood and / or human placental blood is performed under sterile conditions. Upon collection, umbilical cord or placental blood is mixed with an anticoagulant (e.g., CPD (citrate-phosphate-dextrose), ACD (acid-citrate-dextrose), Alsever's solution (Alsever et al., 1941, NY St. J. Med. 41:126), De Gowin's solution (De Gowin, et al., 1940, J. Am. Med. Ass. 114:850), Edglugate-Mg (Smith, et al., 1959, J. Thorac. Cardiovasc. Surg. 38:573), Rous-Turner solution (Rous and Turner, 1916, J. Exp. Med. 23:219), other glucose mixtures, heparin, ethyl biscoumarate, etc.). See generally, Hurn, 1968, Storage of Blood, Academic Press, New York, pp. 26-160. In one embodiment, ACD may be used.

[0032] Umbilical cord blood may be obtained by direct drainage from the umbilical cord and / or from the delivered placenta by needle aspiration of the umbilical vein and distended veins. Preferably, the collected human umbilical cord blood and / or placental blood is free of contamination, and in particular free of viral contamination. In certain embodiments, the following tests may be performed on the collected blood, either routinely or as clinically indicated:

[0033] Bacterial culture: Established assays can be performed to ensure the absence of microbial contamination (e.g., routine hospital cultures of bacteria under aerobic and anaerobic conditions).

[0034] Diagnostic Screening for Pathogenic Microorganisms: Various diagnostic tests can be used to ensure the absence of specific pathogenic microorganisms. Diagnostic screening for any of the many blood-transmitted pathogens can be performed using standard procedures. As one example, the collected blood sample (or maternal blood sample) can be subjected to diagnostic screening for the presence of viruses. Any of many known assay systems can be used based on the detection of virions, virally encoded proteins, virus-specific nucleic acids, antibodies to viral proteins, etc. The collected blood can also be tested for infectious diseases, including, but not limited to, human immunodeficiency virus-1 or 2 (HIV-1 or HIV-2), human T-cell lymphotropic virus I and II (HTLV-I and HTLV-II), hepatitis B, hepatitis C, cytomegalovirus, syphilis, coronavirus, West Nile virus, etc.

[0035] Preferably, prior to collecting the cord blood, the maternal health history is determined to identify the risk that the cord blood cells may pose to transmitting, for example, a genetic or infectious disease (e.g., cancer, leukemia, immune disorders, neurological disorders, hepatitis, or AIDS). The collected cord blood may be tested for one or more of cell viability, HLA typing, ABO / Rh typing, CD34+ cell count, and total nucleated cell count.

[0036] Once the umbilical cord blood and / or placental blood is collected from a human donor at birth, the blood is processed to produce an enriched population of hematopoietic stem cells or hematopoietic stem and progenitor cells. Preferably, the hematopoietic stem cells or hematopoietic stem and progenitor cells are CD34+ cells or predominantly CD34+ cells. Preferably, the hematopoietic stem or hematopoietic stem and progenitor cell population is substantially depleted of T cells and red blood cells, resulting in a cell population enriched for CD34+ stem cells and / or CD34+ stem and progenitor cells. Enrichment, therefore, refers to a process by which the percentage of hematopoietic stem cells or hematopoietic stem and progenitor cells in the cell population is increased (compared to the percentage in the population prior to the enrichment procedure). Purification is one example of enrichment. In certain embodiments, the increase in the number of CD34+ cells (or other appropriate antigen-positive cells) as a percentage of cells in the expanded stem cell product compared to the population before the enrichment procedure is at least 25-fold, 50-fold, 75-fold, 100-fold, 150-fold, 200-fold, 250-fold, 300-fold, 350-fold, 400-fold, or at least 350-fold, preferably 100-200-fold or 100-400-fold.

[0037] Prior to processing for enrichment, the collected umbilical cord blood and / or placental blood may be fresh or previously cryopreserved. Any suitable cell separation / selection technique known in the art can be used to enrich for hematopoietic stem cells or hematopoietic stem and progenitor cells. Methods relying on differential expression of cell surface markers can be used. For example, cells expressing the cell surface marker CD34 can be positively selected using a monoclonal antibody against CD34, so that cells expressing CD34 are retained and cells that do not express CD34 are not. Furthermore, the separation technique used should maximize the viability of the selected cell population. The particular technique used will depend on the separation efficiency, cytotoxicity of the methodology, ease and speed of execution, and the need for sophisticated equipment and / or technical expertise.

[0038] Procedures for separation may include magnetic separation using antibody-coated magnetic beads, affinity chromatography, and "panning" with antibodies attached to a solid matrix (e.g., a plate), or other convenient techniques. Techniques that provide accurate separation / selection include fluorescence-activated cell sorters, which may have various degrees of sophistication (e.g., multiple color channels, low-angle and obtuse-angle light scattering detection channels, impedance channels, etc.).

[0039] The antibodies used in the selection process may be conjugated to markers such as magnetic beads (which allow for direct isolation), biotin (which can be removed with support-bound avidin or streptavidin), fluorescent dyes (which can be used with a fluorescence-activated cell sorter), etc. to allow for ease of isolation of specific cell types. Any technique that is not unduly detrimental to the viability of the remaining cells may be used. Examples include, for example, the FDA-approved CleniMACs® processing system (Miltenyl Biotec BV & Co. KG), Dynabeads, etc. TM CD34 isolation system (Invtrogen Inc.), EasySep TM Examples include the Human CD34 Positive Selection Kit (Stemcell Technologies, Inc.).

[0040] In a preferred embodiment, fresh cord blood units are processed to select (i.e., enrich) CD34+ cells using anti-CD34 antibodies directly or indirectly conjugated to magnetic particles in conjunction with a magnetic cell separator (e.g., the CliniMACS® Cell Separation System (Miltenyi Biotec, Bergisch Gladbach, Germany) that uses nano-sized superparamagnetic particles composed of iron oxide and dextran coupled to specific monoclonal antibodies). The CliniMACS® Cell Separator is a closed, sterile system supplied with a single-use disposable tubing set that can be used to process a single unit of collected cord blood and / or placental blood to enrich for CD34+ cells and can then be discarded.

[0041] In one embodiment, two or more cord blood and / or placental blood units can be pooled prior to enrichment for hematopoietic stem cells or hematopoietic stem and progenitor cells. In another embodiment, individual populations of CD34+ stem cells or CD34+ stem and progenitor cells can be pooled after enrichment for hematopoietic stem cells or hematopoietic stem and progenitor cells. In specific embodiments, the number of pooled cord blood and / or placental blood units or populations of hematopoietic stem cells or hematopoietic stem and progenitor cells is 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, or 40, or at least any of the foregoing numbers. In some embodiments, the pool contains 2-8, 2-10, 4-8, 4-10, 2-20, 4-20, 2-25, or 4-25, and up to 20 or 25 cord blood and / or placental blood units or CD34+ hematopoietic stem and progenitor cell populations. The cord blood and / or placental blood units or hematopoietic stem or stem and progenitor cell populations are pooled without regard to the HLA type of the hematopoietic stem or progenitor cells. In certain embodiments, the cells in the pool are derived from cord blood and / or placental blood of individuals of the same race (e.g., African American, Caucasian, Asian, Hispanic, Native American, Native Australian, Inuit, Pacific Islander) or from individuals of the same ethnicity (e.g., Irish, Italian, Indian, Japanese, Chinese, Russian, etc.). In other embodiments, the cells in the pool are combined without regard to race or ethnicity.

[0042] Optionally, the umbilical cord blood or placental blood may be separated into red and white blood cells prior to enrichment for hematopoietic stem or stem and progenitor cells. Once separation of the red and white blood cells has been performed, the red blood cell fraction may be discarded, and the white blood cell fraction may be processed in a magnetic cell separator as described above to enrich for CD34+ hematopoietic stem or stem and progenitor cells. Separation of the red and white blood cell fractions may be performed by any method known in the art, including centrifugation techniques. Other separation methods that may be used include, for example, the commercially available product FICOLL TM or FICOLL-PAQUE TM or PERCOLL TM (GE Healthcare, Piscataway, New Jersey). TM The plasma is typically placed at the bottom of a conical tube, and whole blood is layered on top. After centrifugation, the following layers are visible in the conical tube, from top to bottom: plasma and other components; a mononuclear cell layer called the buffy coat, which contains peripheral blood mononuclear cells (white blood cells); and FICOLL-PAQUE. TM , as well as red blood cells and granulocytes (which should be present in the form of a pellet). This separation technique allows for the easy collection of peripheral blood mononuclear cells (PBMCs).

[0043] Optionally, prior to CD34+ cell selection, an aliquot of the cord blood or placental blood unit can be checked for total nucleated cell count and / or CD34+ cell content. In a specific embodiment, after the CD34+ cell separation, both CD34+ and CD34- cell fractions are collected. Optionally, DNA can be extracted from a sample of the CD34- cell fraction for initial HLA typing and future chimerism studies, even if HLA matching of the CD34+ cell fraction to the patient or to other cord blood or placental blood units is not performed.

[0044] The CD34+ enriched stem or stem and progenitor cell population can be subsequently treated before expansion, for example, by suspending it in a cell culture medium suitable for storage or transportation. In a preferred embodiment, the cell culture medium is a cell culture medium suitable for maintaining the viability of CD34+ hematopoietic stem or hematopoietic stem and progenitor cells. For example, the cell culture medium may be a medium containing STEMSPAN in the presence of growth factors (e.g., at the following concentrations: 50-300 ng / ml stem cell factor (SCF), 50-300 ng / ml Flt-3 receptor ligand (Flt3L), 50-100 ng / ml thrombopoietin (TPO), 50-100 ng / ml interleukin-6 (IL-6), and 10 ng / ml interleukin-3 (IL-3)). TM Serum Free Expansion Medium or STEMSPAN TM Serum-Free Expansion Medium II (StemCell Technologies, Vancouver, British Columbia) may be used. In more specific embodiments, 300 ng / ml stem cell factor, 300 ng / ml Flt-3 receptor ligand, 100 ng / ml thrombopoietin, 100 ng / ml interleukin-6, and 10 ng / ml interleukin-3, or 50 ng / ml stem cell factor, 50 ng / ml Flt-3 receptor ligand, 50 ng / ml thrombopoietin, 50 ng / ml interleukin-6, and 10 ng / ml interleukin-3 are used. In another preferred embodiment, the cell culture medium is STEMSPAN supplemented with 10 ng / ml recombinant human interleukin-3 (rhIL-3), 50 ng / ml recombinant human interleukin-6 (rhIL-6), 50 ng / ml recombinant human thrombopoietin (rhTPO), 50 ng / ml recombinant human Flt-3 ligand (rhFlt-3L), and 50 ng / ml recombinant human stem cell factor (rhSCF). TM Serum Free Expansion Medium or STEMSPAN TMIn another preferred embodiment, the cell culture medium consists of StemSpan Serum Free Expansion Medium II (SFEM II, StemCell Technologies, Vancouver, British Columbia) supplemented with recombinant human rhSCF, rhFlt-3L, rhTPO, rhIL-6 (each at a final concentration of 50 ng / ml), and rhIL-3 (at a final concentration of 10 ng / ml).

[0045] In a specific embodiment, the cord blood and / or placental blood unit is depleted of red blood cells and the number of CD34+ cells in the red blood cell-depleted fraction is determined. Preferably, the cord blood and / or placental blood sample containing greater than 3.5 million CD34+ cells is subjected to the enrichment method described above.

[0046] After the hematopoietic stem or stem and progenitor cells are isolated according to the enrichment methods described above or other methods known in the art (e.g., from human umbilical cord blood and / or human placental blood collected from humans at birth), the hematopoietic stem or progenitor cells are expanded to increase the number of hematopoietic stem or progenitor cells (e.g., CD34+ cells). Any method known in the art for expanding the number of hematopoietic stem or progenitor cells that results in an expanded (i.e., increased in number) population of hematopoietic stem or progenitor cells can be used. Preferably, the hematopoietic stem or progenitor cells are cultured under cell growth conditions (e.g., that promote mitosis) so that the hematopoietic stem or progenitor cells grow and divide (proliferate) to obtain an expanded population of CD34+ hematopoietic stem or progenitor cells. In one embodiment, individual populations of hematopoietic stem cells or hematopoietic stem and progenitor cells derived from umbilical cord blood and / or placental blood from a single human at birth can be pooled, either before or after expansion, without HLA-matching to other hematopoietic stem cells or hematopoietic stem and progenitor cells. In another embodiment, the hematopoietic stem cells or hematopoietic stem and progenitor cells are expanded before pooling. Preferably, the technique used for expansion has been shown to (i) result in an increased number of hematopoietic stem cells or hematopoietic stem and progenitor cells (e.g., CD34+ cells) in the expanded stem cell product compared to an unexpanded population of hematopoietic stem cells or stem and progenitor cells (wherein the unexpanded and expanded cell populations are derived from different aliquots of the same source of stem cells or stem and progenitor cells, and the expanded cells, but not the unexpanded cells, are subjected to the expansion technique).

[0047] Amplification techniques include, but are not limited to, those described in U.S. Patent No. 7,399,633 B2; U.S. Patent Publication No. 2013 / 0095079; Delaney et al., 2010, Nature Med. 16(2): 232-236; Zhang et al., 2008, Blood 111:3415-3423; or Himburg et al., 2010, Nature Medicine 16(4):475-82 (each of which is incorporated herein by reference), as well as those described below.

[0048] In one embodiment, the hematopoietic stem or stem and progenitor cells are cultured in a culture medium in the presence of a growth factor and exposed to cell growth conditions (e.g., that promote mitosis), resulting in proliferation of the hematopoietic stem or progenitor cells to produce an expanded population of hematopoietic stem or progenitor cells. In a preferred embodiment, the hematopoietic stem or progenitor cells are cultured in the presence of an agonist of Notch function (typically, an immobilized Notch function agonist) in an amount effective to inhibit differentiation and exposed to cell growth conditions (e.g., that promote mitosis), resulting in proliferation of the hematopoietic stem or progenitor cells to produce an expanded population of hematopoietic stem or progenitor cells. In a more preferred embodiment, the hematopoietic stem or stem and progenitor cells are cultured with an agonist of Notch function in an amount effective to inhibit differentiation and in the presence of a growth factor and exposed to cell growth conditions (e.g., promoting mitosis), resulting in proliferation of the hematopoietic stem or progenitor cells to obtain an expanded hematopoietic stem or progenitor cell population. The expanded hematopoietic stem or progenitor cell population thus obtained can be frozen and stored for later use as a "ready-to-use product." Optionally, the Notch pathway agonist is inactivated or removed (e.g., by separation or dilution) from the expanded hematopoietic stem or progenitor cell population prior to transplantation into a patient.

[0049] In specific embodiments, the hematopoietic stem cells or hematopoietic stem and progenitor cells are cultured for 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, or 25 days, or longer; alternatively, preferably, the hematopoietic stem cells or hematopoietic stem and progenitor cells are cultured for at least 10 days or for about 7 to about 14 days.

[0050] Exemplary culture conditions for expanding the above-described hematopoietic stem cells or hematopoietic stem and progenitor cells include culturing in serum-free medium supplemented with the following human growth factors: stem cell factor, Flt-3 receptor ligand, thrombopoietin, interleukin-6, and interleukin-3, and culturing with a fibronectin fragment and the extracellular domain of the Delta protein fused to the Fc domain of human IgG (Delta1). ext-IgGThe method comprises culturing cells for 7 to 14 days in the presence of growth factors. Preferably, the growth factors are present at the following concentrations: 50 to 300 ng / ml stem cell factor, 50 to 300 ng / ml Flt-3 receptor ligand, 50 to 100 ng / ml thrombopoietin, 50 to 100 ng / ml interleukin-6, and 10 ng / ml interleukin-3. In a more specific embodiment, 300 ng / ml stem cell factor, 300 ng / ml Flt-3 receptor ligand, 100 ng / ml thrombopoietin, 100 ng / ml interleukin-6, and 10 ng / ml interleukin-3, or 50 ng / ml stem cell factor, 50 ng / ml Flt-3 receptor ligand, 50 ng / ml thrombopoietin, 50 ng / ml interleukin-6, and 10 ng / ml interleukin-3, are used. In a more preferred embodiment, the cell culture medium is STEMSPAN supplemented with 10 ng / ml recombinant human interleukin-3 (rhIL-3), 50 ng / ml recombinant human interleukin-6 (rhIL-6), 50 ng / ml recombinant human thrombopoietin (rhTPO), 50 ng / ml recombinant human Flt-3 ligand (rhFlt-3L), and 50 ng / ml recombinant human stem cell factor (rhSCF). TM In another more preferred embodiment, the cell culture medium consists of StemSpan Serum Free Expansion Medium II (SFEM II, StemCell Technologies, Vancouver, British Columbia) supplemented with recombinant human rhSCF, rhFlt-3L, rhTPO, rhIL-6 (each at a final concentration of 50 ng / ml), and rhIL-3 (at a final concentration of 10 ng / ml).

[0051] In some embodiments, DXI-mediated expansion is performed as follows: Delta1 ext-IgGIn a specific embodiment, the cell culture dish is immobilized on the surface of a cell culture dish with 2.5 μg / ml Delta1 in phosphate-buffered saline overnight at 4° C. (or at least 2 hours at 37° C.) before adding the hematopoietic stem cells or hematopoietic stem and progenitor cells. ext-IgG and 5 μg / ml RetroNectin® (recombinant human fibronectin fragment (also known as rFN-CH-296)). Preferably, the cell culture medium is STEMSPAN supplemented with 10 ng / ml recombinant human interleukin-3 (rhIL-3), 50 ng / ml recombinant human interleukin-6 (rhIL-6), 50 ng / ml recombinant human thrombopoietin (rhTPO), 50 ng / ml recombinant human Flt-3 ligand (rhFlt-3L), and 50 ng / ml recombinant human stem cell factor (rhSCF). TM Serum-Free Expansion Medium (StemCell Technologies, Vancouver, British Columbia) or StemSpan Serum-Free Expansion Medium supplemented with recombinant human rhSCF, rhFlt-3L, rhTPO, rhIL-6 (each at a final concentration of 50 ng / ml), and rhIL-3 (at a final concentration of 10 ng / ml). Medium II (SFEM II, StemCell Technologies, Vancouver, British Columbia).

[0052] Other exemplary culture conditions for expanding hematopoietic stem or stem / progenitor cells are provided in Zhang et al., 2008, Blood 111:3415-3423 (incorporated herein by reference). In a specific embodiment, the hematopoietic stem or stem / progenitor cells can be cultured in serum-free medium supplemented with heparin, stem cell factor, thrombopoietin, insulin-like growth factor-2 (IGF-2), fibroblast growth factor-1 (FGF-1), and Angptl3 or Angptl5. In a specific embodiment, the medium is supplemented with 10 μg / ml heparin, 10 ng / ml stem cell factor, 20 ng / ml thrombopoietin, 20 ng / ml IGF-2, 10 ng / ml FGF-1, and 100 ng / ml Angptl3 or Angptl5, and the cells are cultured for approximately 19 to 23 days. In another specific embodiment, the hematopoietic stem cells or hematopoietic stem and progenitor cells can be expanded by culturing the cells for about 11 to 19 days in serum-free medium supplemented with 10 μg / ml heparin, 10 ng / ml stem cell factor, 20 ng / ml thrombopoietin, 10 ng / ml FGF-1, and 100 ng / ml Angptl 5. In another specific embodiment, the hematopoietic stem cells or stem and progenitor cells can be expanded by culturing the cells for about 10 days in serum-free medium supplemented with 50 ng / ml stem cell factor, 10 ng / ml thrombopoietin, 50 ng / ml Flt-3 receptor ligand, and 100 ng / ml insulin-like growth factor binding protein-2 (IGFBP2) or 500 ng / ml Angptl 5. In yet another embodiment, the hematopoietic stem cells or hematopoietic stem and progenitor cells can be expanded by culturing the cells for about 11 days in serum-free medium supplemented with 10 μg / ml heparin, 10 ng / ml stem cell factor, 20 ng / ml thrombopoietin, 10 ng / ml FGF-1, 500 ng / ml Angptl5, and 500 ng / ml IGFBP2 (see Zhang et al., 2008, Blood 111:3415-3423, incorporated herein by reference).

[0053] Further exemplary culture conditions for expanding the hematopoietic stem or stem and progenitor cells are set forth in Himburg et al., 2010, Nature Medicine 16(4):475-482 (incorporated herein by reference). In a specific embodiment, the hematopoietic stem or stem and progenitor cells can be cultured in a liquid suspension culture supplemented with thrombopoietin, stem cell factor, Flt-3 receptor ligand, and pleiotrophin. In a specific embodiment, the liquid suspension culture is supplemented with 20 ng / ml thrombopoietin, 125 ng / ml stem cell factor, 50 ng / ml Flt-3 receptor ligand, and 10, 100, 500, or 1000 ng / ml pleiotrophin, and the hematopoietic stem or progenitor cells are cultured for approximately 7 days.

[0054] After expansion of the hematopoietic stem cells or hematopoietic stem and progenitor cells, the total number of cells and viable CD34+ cells is determined. For example, on day 14 during expansion, a sample can be taken to determine the total viable nucleated cell count. Furthermore, the total number of CD34+ cells can be determined by multiparameter flow cytometry, and thus the percentage of CD34+ cells in the sample. Preferably, cultures that do not result in at least a 10-fold increase in the absolute number of CD34+ cells are discontinued. Similarly, before cryopreservation or after thawing, an aliquot of the expanded hematopoietic stem or hematopoietic stem and progenitor cell population can be taken for determination of the percentage of total nucleated cells and viable CD34+ cells to calculate the total viable CD34+ cell count in the expanded population. In a preferred embodiment, populations containing fewer than 50 million viable CD34+ cells can be discarded.

[0055] In a specific embodiment, the number of live total CD34+ (or other antigen-positive) cells can be considered a potency assay for releasing a final product for therapeutic use. Viability can be determined by any method known in the art, such as trypan blue exclusion or 7-amino-actinomycin D (7-AAD) exclusion. Preferably, the total nucleated cell count (TNC) and other data are used to calculate the potency of the product. The percentage of live CD34+ cells can be assessed by flow cytometry and the use of a dye that is excluded by live cells. Percentage of live CD34+ cells = the number of CD34+ cells that exclude 7-AAD (or other appropriate dye) in an aliquot of a sample divided by the TNC (both live and non-viable) of that aliquot. The live CD34+ cells in the sample can be calculated as follows: Live CD34+ cells = TNC of sample × % of live CD34+ cells in sample. The proportional increase in viable CD34+ cells during enrichment or expansion can be calculated as follows: total viable CD34+ cells after culture / total viable CD34+ cells before culture.

[0056] In some embodiments, the hematopoietic stem or progenitor cells are expanded by culturing the cells in the presence of an agonist of Notch function and one or more of a growth factor or cytokine for a predetermined period of time, as described above. An agonist of Notch function (also referred to as a Notch agonist) is an agent that promotes, i.e., causes or increases, the activation of Notch pathway function. As used herein, "Notch function" means a function mediated by the Notch signaling (signal transduction) pathway, including, but not limited to, nuclear translocation of the intracellular domain of Notch, nuclear translocation of RBP-Jκ or its Drosophila homolog Suppressor of Hairless; activation of bHLH genes in the Enhancer of Split complex, e.g., Mastermind; activation of the HES-1 gene or the KBF2 (also known as CBF1) gene; inhibition of Drosophila neuroblast separation; and binding of Notch to Delta, Jagged / Serrate, Fringe, Deltex, or RBP-Jκ / Suppressor of Hairless, or homologs or analogs thereof. Generally, for a discussion of the Notch signaling pathway and its effects upon activation, see the review article by Kopan et al., 2009, Cell 137:216-233; see also Jarriault et al., 1998, Mol. Cell. Biol. 18:7423-7431 (both of which are incorporated by reference in their entireties).

[0057] Notch activation is achieved by exposing cells to a Notch agonist. The agonist of Notch function can be, but is not limited to, a soluble molecule, a molecule recombinantly expressed on the cell surface, a molecule on the cell monolayer to which the hematopoietic stem or hematopoietic stem and progenitor cells are exposed, or a molecule immobilized on a solid phase. Exemplary Notch agonists are the extracellular binding ligands Delta and Serrate, which bind to the extracellular domain of Notch and activate Notch signaling, or fragments of Delta or Serrate that bind to the extracellular domain of Notch and activate Notch signaling. The nucleic acid and amino acid sequences of Delta and Serrate have been isolated from several species (including humans) and are known in the art, and are disclosed in International Patent Publication No. WO 2014 / 023900. 93 / 12141, WO 96 / 27610, WO 97 / 01571, and Gray et al., 1999, Am. J. Path. 154:785-794. In a preferred embodiment, the Notch agonist is a Delta or Serrate protein (Delta and Serrate, respectively) consisting of the extracellular domain of the Delta or Serrate protein fused to a myc epitope tag. ext-myc or Serrate ext-myc ), or Delta or Serrate proteins consisting of the extracellular domains of Delta or Serrate proteins fused to the Fc portion of IgG (Delta and Serrate, respectively). ext-IgG or Serrate ext-IgG) is an immobilized fragment. Notch agonists include, but are not limited to, Notch proteins and their analogs and derivatives (including fragments); proteins that are other elements of the Notch pathway and their analogs and derivatives (including fragments); antibodies against them, and fragments or other derivatives of such antibodies containing their binding regions; nucleic acids encoding the above proteins, derivatives, or analogs; and proteins and their analogs and derivatives that bind to or otherwise interact with Notch proteins or other proteins in the Notch pathway, thereby promoting Notch pathway activity. Such agonists include, but are not limited to, Notch proteins and derivatives thereof containing intracellular domains, Notch nucleic acids encoding the above, and proteins containing the Notch-interacting domain of a Notch ligand (e.g., the extracellular domain of Delta or Serrate). Other agonists include, but are not limited to, RBPJκ / Suppressor of Hairless or Deltex. Fringe, for example, can be used in conjunction with Delta protein to enhance Notch activity. These proteins, fragments, and derivatives thereof can be recombinantly expressed, isolated, or chemically synthesized.

[0058] In another specific embodiment, the Notch agonist is a cell recombinantly expressing a protein or fragment or derivative thereof that agonizes Notch, wherein the cell expresses the Notch agonist in such a manner that it is available (e.g., it is secreted, expressed on the cell surface, etc.) to hematopoietic stem or stem and progenitor cells in which Notch signaling is to be activated.

[0059] In yet another specific embodiment, Notch agonist is the peptide mimic or peptide analogue or organic molecule that binds to the member of Notch signal transduction pathway.This agonist can be identified by the binding assay selected from those known in the art (for example, the cell aggregation assay described in Rebay et al., 1991, Cell 67:687-699 and International Patent Publication No. WO 92 / 19734 (both are incorporated herein by reference)).

[0060] In a preferred embodiment, the agonist is a protein consisting of at least a fragment of a protein encoded by a Notch-interacting gene that mediates binding to the Notch protein or a fragment of Notch (wherein the fragment of Notch includes the region of Notch responsible for binding to the agonist protein, e.g., Notch epidermal growth factor required repeats 11 and 12). As used herein, Notch-interacting gene refers to the Notch gene, Delta gene, Serrate gene, RBPJκ gene, Suppressor of Hairless gene, and Deltex gene, as well as other members of the Delta / Serrate or Deltex family. These genes can be identified by sequence homology or genetic interaction, and more generally, members of the "Notch cascade" or "Notch group" of genes, identified by molecular interaction (e.g., in vitro binding, or genetic interaction (phenotypically, e.g., as demonstrated in Drosophila)). Exemplary fragments of Notch-binding proteins that contain the region responsible for binding to Notch are described in US Pat. Nos. 5,648,464; 5,849,869; and 5,856,441, which are incorporated herein by reference.

[0061] The Notch agonists utilized by the methods described herein can be obtained commercially, produced by recombinant expression, or chemically synthesized.

[0062] In a specific embodiment, the exposure of the cells to the Notch agonist is not by incubation with other cells recombinantly expressing a Notch ligand on their cell surface (although in other embodiments this method may be used), but rather by exposure to a cell-free Notch ligand, e.g., by incubation with a cell-free ligand of Notch, which ligand is immobilized on a solid surface (e.g., immobilized on the surface of a tissue culture substrate, dish, flask, bottle, bag, etc.).

[0063] In a specific embodiment, Notch activity is promoted by the binding of a Notch ligand (e.g., Delta, Serrate) to the extracellular portion of the Notch receptor. Notch signaling appears to be triggered by physical interaction between the extracellular domain of Notch and its ligand, which is either membrane-bound on adjacent cells or immobilized on a solid surface. Full-length ligands are agonists of Notch, since their expression on one cell triggers pathway activation in neighboring cells that express the Notch receptor. Soluble truncated Delta or Serrate molecules containing the extracellular domain of the protein or its Notch-binding portion and immobilized on a solid surface (e.g., tissue culture plate) are particularly preferred Notch pathway agonists. Such soluble proteins can be immobilized on a solid surface by antibodies or interacting proteins (e.g., antibodies against an epitope tag with which Delta or Serrate is expressed as a fusion protein (e.g., a myc epitope tag recognized by antibody 9E10)) or proteins that interact with an epitope tag with which Delta or Serrate is expressed as a fusion protein (e.g., an immunoglobulin epitope tag bound by protein A).

[0064] In another specific embodiment, and as described in U.S. Pat. No. 5,780,300 (Artavanis-Tsakonas et al.), Notch agonists include agents that promote or activate cellular processes that mediate the maturation or processing steps required for activation of Notch or members of the Notch signaling pathway (e.g., furin-like convertases required for Notch processing), Kuzbanian (a disintegrin-metalloproteinase (ADAM) thought to be required for activation of the Notch pathway upstream of or in parallel with Notch) (Schlondorff and Blobel, 1999, J. Cell Sci. 112:3603-3617), or more generally, cellular trafficking and processing proteins (e.g., the rab family of GTPases required for movement between cellular compartments) (for a review on Rab GTPases, see Olkkonen and Stenmark, 1997, Int. Rev. Cytol. 176:1-85). The agonist can be any molecule that increases the activity of one of the above processes (e.g., a nucleic acid encoding a furin, Kuzbanian, or rab protein, or a fragment, derivative, or dominant-active mutant thereof, or a peptidomimetic, peptide analog, or organic molecule that binds to or activates the function of the above protein).

[0065] U.S. Patent No. 5,780,300 (incorporated herein by reference) further discloses classes of Notch agonist molecules (and methods for identifying them) that can be used to activate the Notch pathway, e.g., molecules that induce dissociation of Notch ankyrin repeats from RBP-Jκ, thereby promoting translocation of RBP-Jκ from the cytoplasm to the nucleus.

[0066] In some preferred embodiments, the DXI expression method is used. The Notch agonist is expressed as an immobilized fragment of Delta (Delta), consisting of the extracellular domain of the protein fused to the Fc portion of IgG, as described in U.S. Patent No. 7,399,633. ext-IgG or DXI), or immobilized Notch-1 or Notch-2 specific antibodies as described in U.S. Patent No. 10,208,286 (both of which are incorporated herein by reference). ext-IgG In a specific embodiment, the cell culture dish is immobilized with 2.5 μg / ml Delta1 in phosphate-buffered saline overnight at 4° C. (or at least 2 hours at 37° C.) before adding the hematopoietic stem cells or hematopoietic stem and progenitor cells. ext-IgG and 5 μg / ml RetroNectin® (recombinant human fibronectin fragment (also known as rFN-CH-296)). Preferably, the cell culture medium is StemSpan supplemented with 10 ng / ml recombinant human interleukin-3 (rhIL-3), 50 ng / ml recombinant human interleukin-6 (rhIL-6), 50 ng / ml recombinant human thrombopoietin (rhTPO), 50 ng / ml recombinant human Flt-3 ligand (rhFlt-3L), and 50 ng / ml recombinant human stem cell factor (rhSCF). TM The hematopoietic stem cells or stem / progenitor cells are cultured in Serum-Free Expansion Medium (StemCell Technologies, Vancouver, British Columbia) or StemSpan Serum-Free Expansion Medium II (SFEM II, StemCell Technologies, Vancouver, British Columbia) supplemented with recombinant human rhSCF, rhFlt-3L, rhTPO, rhIL-6 (final concentration 50 ng / ml each), and rhIL-3 (final concentration 10 ng / ml).

[0067] Once the expanded hematopoietic stem cells or stem and progenitor cells are obtained to form an expanded stem cell product, the expanded hematopoietic stem or stem and progenitor cell population can be collected and cryopreserved, for example, to prepare a "ready-to-use" product. In one embodiment, the expanded hematopoietic stem or stem and progenitor cell population can be divided into one or more bags (or units) and frozen. In another embodiment, two or more expanded hematopoietic stem or stem and progenitor cell populations can be pooled and divided into separate aliquots, with each aliquot frozen. In a preferred embodiment, about 50 million to about 400 million CD34+ cells are frozen in one bag (or unit) of expanded stem cell product. In another preferred embodiment, about 100 million to about 300 million CD34+ cells are frozen in one bag (or unit) of expanded stem cell product. In other preferred embodiments, approximately 100 million, 200 million, 300 million, or 400 million CD34+ cells are frozen in one bag (or unit) of expanded stem cell product.

[0068] In a preferred embodiment, the expanded stem product is fresh, i.e., it has not been previously frozen prior to expansion or cryopreservation. The terms "frozen / freezing" and "cryopreserved / cryopreserving" are used interchangeably in this application. Cryopreservation can be by any method known in the art for freezing cells in a viable form. Freezing cells is typically destructive. Upon cooling, intracellular water freezes. Damage then occurs due to osmotic effects on cell membranes, cell dehydration, solute concentration, and ice crystal formation. As ice forms outside the cells, available water is removed from solution and pulled from the cells, causing osmotic dehydration and increased solute concentration, which ultimately destroys the cells. For a discussion, see Mazur, P., 1977, Cryobiology 14:251-272.

[0069] These damaging effects can be avoided by (a) the use of cryoprotectants, (b) controlling the rate of freezing, and (c) storage at temperatures low enough to minimize degradative reactions.

[0070] Cryoprotectants that may be used include, but are not limited to, dimethyl sulfoxide (DMSO) (Lovelock and Bishop, 1959, Nature 183:1394-1395; Ashwood-Smith, 1961, Nature 190:1204-1205), glycerol, polyvinylpyrrolidone (Rinfret, 1960, Ann. NY Acad. Sci. 85:576), polyethylene glycol (Sloviter and Ravdin, 1962, Nature 196:548), albumin, dextran, sucrose, ethylene glycol, i-erythritol, D-ribitol, D-mannitol (Rowe et al., 1962, Fed. Proc. 21:157), D-sorbitol, i-inositol, D-lactose, choline chloride (Bender et al., 1960, J. Appl. Physiol. 15:520), amino acids (Phan The Tran and Bender, 1960, Exp. Cell Res. 20:651), methanol, acetamide, glycerol monoacetate (Lovelock, 1954, Biochem. J. 56:265), inorganic salts (Phan The Tran and Bender, 1960, Proc. Soc. Exp. Biol. Med. 104:388; Phan The Tran and Bender, 1961, in Radiobiology, Proceedings of the Third Australian Conference on Radiobiology, ed. Ilbery, Butterworth, London, p. 59), and CryoStor® CS10 (BioLife Solutions Inc., Bothell, WA). In a preferred embodiment, DMSO is used. The liquid is non-toxic to cells at low concentrations. The addition of plasma (e.g., to a concentration of about 20-25%) can enhance the protective effect of DMSO. After adding DMSO, the cells should be kept at 0°C until frozen, because DMSO concentrations of about 1% are toxic at temperatures above 4°C.

[0071] A controlled, slow cooling rate can be important. Different cryoprotectants (Rapatz et al., 1968, Cryobiology 5(1):18-25) and different cell types have different optimal cooling rates (see, e.g., Rowe and Rinfret, 1962, Blood 20:636; Rowe, 1966, Cryobiology 3(1):12-18; Lewis, et al., 1967, Transfusion 7(1):17-32; and Mazur, 1970, Science 168:939-949 for the effect of cooling rate on the survival of bone marrow stem cells and their transplantability). The heat of the fusion phase, in which water turns to ice, must be minimal. The cooling procedure can be performed, for example, by using a programmable freezing device or a methanol bath procedure.

[0072] Programmable freezing devices allow for the determination of optimal cooling rates and facilitate standard, reproducible cooling. Programmable rate-controlled freezers (e.g., Cryomed or Planar) allow for the adjustment of freezing regimens to the desired cooling curve. For example, for bone marrow cells in 10% DMSO and 20% plasma, the optimal rate is 1°C to 3°C per minute from 0°C to -80°C. This cooling rate can be used in preferred embodiments. The container holding the cells must be stable at ultralow temperatures and allow rapid heat transfer for effective control of both freezing and thawing. Sealed plastic vials (e.g., Nunc, Wheaton cryosols) or glass ampoules can be used for many small volumes (1–2 ml), while larger volumes (100–200 ml) can be frozen in polyolefin bags (e.g., Delmed) held between metal plates for better heat transfer during cooling. Bags of bone marrow cells were successfully frozen by placing them in a -80°C freezer, which fortunately gives a cooling rate of approximately 3°C / min).

[0073] In an alternative embodiment, a methanol bath cooling method can be used. The methanol bath method is well suited for routine cryopreservation of large numbers of small items on a large scale. The method does not require manual control of the freezing rate or a recorder to monitor that rate. In a preferred embodiment, DMSO-treated cells are pre-chilled on ice and transferred to a tray containing chilled methanol, which is then placed in a mechanical refrigerator (e.g., Harris or Revco) at -80°C. Thermocouple measurements of the methanol bath and sample indicate a desired cooling rate of 1°C to 3°C / min. After at least 2 hours, the specimens have reached a temperature of -80°C and can be placed directly into liquid nitrogen (-196°C) for permanent storage.

[0074] After complete freezing, the expanded stem cell product can be rapidly transferred to a long-term freezing storage container. In a preferred embodiment, samples can be cryogenically stored in liquid nitrogen (-196°C) or its vapor (-165°C). Such storage is greatly facilitated by the availability of highly efficient liquid nitrogen coolers similar to large Thermos containers with extremely low vacuum and internal super-insulation, so that heat leakage and nitrogen loss are minimized.

[0075] Suitable racking systems are commercially available and can be used for cataloguing, storing, and retrieving individual specimens.

[0076] The considerations and procedures for the manipulation, cryopreservation, and long-term storage of hematopoietic stem cells (especially those derived from bone marrow or peripheral blood) described above are largely applicable to expanded hematopoietic stem cells or stem and progenitor cells. Such considerations can be found, for example, in the following references (incorporated herein by reference): Gorin, 1986, Clinics in Haematology 15(1):19-48; Bone-Marrow Conservation, Culture and Transplantation, Proceedings of a Panel, Moscow, July 22-26, 1968, International Atomic Energy Agency, Vienna, pp. 107-186.

[0077] Other methods of cryopreservation of living cells, or modifications thereof, are available and are contemplated for use (e.g., cold metal-mirror techniques; Livesey and Linner, 1987, Nature 327:255; Linner et al., 1986, J. Histochem. Cytochem. 34(9):1123-1135; see also U.S. Pat. No. 4,199,022 (Senkan et al.), U.S. Pat. No. 3,753,357 (Schwartz), U.S. Pat. No. 4,559,298 (Fahy)).

[0078] Cryogenically stored or frozen cells are preferably thawed quickly (e.g., in a water bath maintained at 37°C to 41°C) and cooled rapidly upon thawing. In a specific embodiment, the vial containing the frozen cells can be immersed up to its neck in a warm water bath; gentle rotation ensures mixing of the cell suspension as it melts and increases heat transfer from the warm water to the internal ice mass. Once the ice has completely melted, the vial can be immediately placed back into ice.

[0079] In one embodiment of the present invention, the expanded stem cell product can be thawed, or a portion thereof can be infused into a human patient in need thereof (e.g., with AML or other hematological malignancies). Several procedures for processing thawed cells are available and can be used if deemed desirable.

[0080] It may be desirable to treat the cells to prevent clumping upon thawing. A variety of procedures can be used to prevent clumping, including, but not limited to, the addition of DNase (Spitzer et al., 1980, Cancer 45:3075-3085), low molecular weight dextran and citrate, hydroxyethyl starch (Stiff et al., 1983, Cryobiology 20:17-24), etc., before and / or after freezing.

[0081] If the cryoprotectant is toxic in humans, it should be removed before the thawed expanded stem cell product is used therapeutically.In one embodiment, when DMSO is used as the cryoprotectant, it is preferable to omit this step to avoid cell loss.However, if removal of the cryoprotectant is desired, it is preferably achieved during thawing.

[0082] One way to remove the cryoprotectant is by dilution to a slight concentration. This can be achieved by adding medium, followed by one or more centrifugation cycles, if necessary, to pellet the cells, removing the supernatant, and resuspending the cells. For example, the intracellular DMSO in the thawed cells can be reduced to a level (less than 1%) that does not adversely affect the recovered cells. This is preferably done slowly to minimize the potentially damaging osmotic gradient that occurs during DMSO removal.

[0083] After removal of the cryoprotectant, cell counting (e.g., by using a hemocytometer) and viability testing (e.g., by trypan blue exclusion; Kuchler, 1977, Biochemical Methods in Cell Culture and Virology, Dowden, Hutchinson & Ross, Stroudsburg, Pa., pp. 18-19; 1964, Methods in Medical Research, Eisen et al., eds., Vol. 10, Year Book Medical Publishers, Inc., Chicago, pp. 39-47) can be performed to confirm cell viability. The percentage of live antigen (e.g., CD34)-positive cells can be determined by calculating the number of antigen-positive cells that exclude 7-AAD (or other suitable dye excluded by live cells) in an aliquot of cells, divided by the total number of nucleated cells (TNC) (both live and non-live cells) in the aliquot of cells. The number of live antigen-positive cells can then be determined by multiplying the percentage of live antigen-positive cells by TNC.

[0084] Before cryopreservation and / or after thawing, the total number of nucleated cells, or in a specific embodiment, the total number of CD34+ cells, can be determined. For example, a total nucleated cell count can be performed using a hemocytometer and trypan blue exclusion. Highly cellular specimens can be diluted to a concentration range appropriate for manual counting. The final cell count of the product is calibrated at any dilution factor. Total nucleated cell count = viable nucleated cells / mL x product volume (in mL). The number of CD34+ positive cells in a sample can be determined, for example, by flow cytometry using an anti-CD34 monoclonal antibody conjugated to a fluorescent dye.

[0085] In certain embodiments, the identity and purity of the starting hematopoietic stem or stem and progenitor cell population, umbilical cord blood and / or placental blood, or expanded stem cell product before cryopreservation, or the expanded stem cell product after thawing, can be subjected to multiparameter flow cytometry immunophenotyping, which provides the percentage of viable, antigen-positive cells present in the sample. Each sample can be tested using a panel of monoclonal antibodies directly conjugated to fluorochromes for one or more of the following cell phenotypes: 1. CD34+ HPC 2. T cells (including both CD3+, CD4+ and CD8+ subsets) 3. B cells (CD19+ or CD20+) 4. NK cells (CD56+) 5. Monocytes (CD14+) 6. Bone marrow monocytes (CD15+) 7. Megakaryocytes (CD41+) 8. Dendritic cells (lineage negative / HLA-DRbright and CD123bright, or lineage negative / HLA-DRbright and CD11cbright).

[0086] Treatment method According to the present invention, methods are provided for improving the treatment outcomes of patients with AML or other hematological malignancies. Methods for treating patients with AML or other hematological malignancies are also provided. The patient is treated by administering a chemotherapy regimen, or cycles thereof, followed by administering a fixed dose of an expanded stem cell product to the patient, wherein the administering step is performed without matching the HLA type of the expanded stem cell product to the HLA type of the patient. The expanded stem cell product is a pooled product derived from hematopoietic stem cells or hematopoietic stem and progenitor cells of at least two or at least four human donors, without matching the HLA types of the donors to each other and to the HLA type of the patient. The phrase "without matching the HLA type" means that no steps have been taken to match either HLA antigens or alleles between the patients and / or between the donors contributing to the expanded stem cell product (or the hematopoietic stem cells or hematopoietic stem and progenitor cells in the expanded stem cell product).

[0087] In some embodiments, the fixed dose of expanded stem cell product may be administered after a chemotherapy regimen or cycle thereof (e.g., induction regimen). The fixed dose of expanded stem cell product may also be administered after a consolidation regimen or cycle thereof. The fixed dose of expanded stem cell product may also be administered after a salvage regimen or cycle thereof. In some embodiments, the fixed dose of expanded stem cell product may be administered after a second induction regimen or cycle thereof, or after a second cycle of induction regimen, if desired or necessary. In some embodiments, the fixed dose of expanded stem cell product may be administered after a second consolidation regimen or cycle thereof, or after a second cycle of consolidation regimen, if desired or necessary. In some embodiments, the fixed dose of expanded stem cell product may be administered after a second salvage regimen or cycle thereof, or after a second cycle of salvage regimen, if desired or necessary.

[0088] As discussed above, the expanded stem cell product is typically administered after the last dose of the regimen has been administered, or for regimens having more than one cycle, after the last dose of each cycle of the regimen has been administered. In some embodiments, the expanded stem cell product is administered about 12 to about 48 hours after completion of the regimen, or preferably about 24 to about 36 hours after completion of the regimen.

[0089] In some embodiments, the expanded stem cell product is administered to the patient after the components of the chemotherapy regimen and their active metabolites have cleared from the patient's blood. In some embodiments, the expanded stem cell product is administered to the patient after the components of the induction regimen and their active metabolites have cleared from the patient's blood. In some embodiments, the expanded stem cell product is administered to the patient after the components of the consolidation regimen and their active metabolites have cleared from the patient's blood. In some embodiments, the expanded stem cell product is administered to the patient after the components of the rescue regimen and their active metabolites have cleared from the patient's blood. In some embodiments, if the chemotherapy regimen (e.g., induction regimen, salvage regimen, or consolidation regimen) is administered in more than one cycle, the expanded stem cell product is administered to the patient after each cycle after the components of the regimen and its active metabolites have cleared from the patient's blood. As used herein, "...after the regimen and its active metabolites have cleared from the patient's blood" refers to the clearance of components of the regimen (e.g., a chemotherapy regimen, induction regimen, salvage regimen, or consolidation regimen) and the active metabolites of those components that affect the viability of CD34+ stem cells in the patient's blood, e.g., by reducing the viability of CD34+ stem or progenitor cells by at least 5%, at least 10%, or at least 20%.

[0090] In some embodiments, the chemotherapy regimen is an induction regimen. In some embodiments, the induction regimen is the administration of cytarabine and an anthracycline (e.g., daunorubicin or idarubicin). In some embodiments, the chemotherapy regimen is a "7+3" regimen of cytarabine and daunorubicin or idarubicin. The combination of cytarabine (Cytosar-U®) given over about 4 to about 7 days and an anthracycline drug (e.g., daunorubicin (Cerubidine®) or idarubicin (Idamycin®)) given for about 3 days is most frequently used. Patients may also receive hydroxyurea (Droxia®, Hydrea®) to help reduce white blood cell counts.

[0091] In some embodiments, for somewhat older adults, decitabine (Dacogen) TM ), azacitidine (Vidaza®), and low-dose cytarabine may alternatively be used in induction regimens.

[0092] In some embodiments, the induction regimen is GCLAC (G-CSF-clofarabine-high-dose cytarabine).

[0093] In some embodiments, the chemotherapy regimen is an intensive regimen. In some embodiments, the intensive regimen is the administration of high-dose cytarabine. In some embodiments, the intensive regimen is medium-dose cytarabine. In some embodiments, 2-4 cycles (rounds) of high-dose or medium-dose cytarabine are administered. The expanded stem cell product can be administered after each cycle.

[0094] In some embodiments, the chemotherapy regimen is a salvage regimen. In some embodiments, the salvage regimen is administration of cladribine, high-dose cytarabine, and G-CSF (CLAG). In some embodiments, the salvage regimen is a combination of etoposide, cytarabine, and mitoxantrone (MEC). The expanded stem cell product may be administered after each cycle.

[0095] In other embodiments, the chemotherapy regimen may be: 7+3 (7 days of Ara-C (cytarabine) plus 3 days of anthracycline antibiotic (either daunorubicin (DA or DAC variant) or idarubicin (IA or IAC variant)); 5+2 (5 days of Ara-C (cytarabine) plus 2 days of idarubicin (IA or IAC variant); BACOD (bleomycin, doxorubicin, cyclophosphamide, vincristine, dexamethasone); CBV (cyclophosphamide, BCNU (carmustine), VP-16 (etoposide)); CHOEP (cyclophosphamide, hydroxydaunorubicin (doxorubicin), etoposide, vincristine (Oncovin®), prednisone); CEPP (cyclophosphamide, etoposide, procarbazine, prednisone); CHOP (cyclophosphamide, hydroxydaunorubicin (doxorubicin), vincristine, and prednisone); CHOP-R or R-CHOP (CHOP plus rituximab); CVAD and Hyper-CVAD (cyclophosphamide, vincristine, doxorubicin, and dexamethasone); DA or DAC (3 days of daunorubicin plus 7 days of ara-C (cytarabine), a modification of the 7+3 regimen); DAT (daunorubicin, cytarabine (ara-C), and thioguanine); DHAP (dexamethasone, cytarabine (ara-C), and a platinum agent); DHAP-R or R-DHAP (dexamethasone, cytarabine (ara-C), and a platinum agent plus rituximab); DICE (dexamethasone, ifosfamide, cisplatin, etoposide (VP-16)); EPOCH (etoposide, prednisone, vincristine, cyclophosphamide, hydroxydaunorubicin); EPOCH-R or R-EPOCH (etoposide, prednisone, vincristine, cyclophosphamide, hydroxydaunorubicin + rituximab); ESHAP (etoposide, methylprednisolone, cytarabine (ara-C) + platinum drug); FCM or FMC (fludarabine, cyclophosphamide, mitoxantrone); FCM-R or R-FCM or R-FMC or FMC-R (fludarabine, cyclophosphamide, mitoxantrone + rituximab); FCR (fludarabine, cyclophosphamide, rituximab); FM (fludarabine and mitoxantrone); FM-R or R-FM or RFM or FMR (fludarabine, mitoxantrone, and rituximab); FLAG (fludarabine, cytarabine, and G-CSF); FLAG-Ida or FLAG-IDA or IDA-FLAG or Ida-FLAG (fludarabine, cytarabine, idarubicin, and G-CSF); FLAG-Mito or FLAG-MITO or Mito-FLAG or MITO-FLAG or FLANG (mitoxantrone, fludarabine, cytarabine, and G-CSF); FLAMSA (fludarabine, cytarabine, and amsacrine); FLAMSA-BU or FLAMSA-Bu (fludarabine, cytarabine, and amsacrine and busulfan); FLAMSA-MEL or FLAMSA-Mel (fludarabine, cytarabine, and amsacrine and melphalan); GDP (gemcitabine, dexamethasone, cisplatin); GemOx or GEMOX (gemcitabine, oxaliplatin); GemOx-R or GEMOX-R or R-GemOx or R-GEMOX (gemcitabine, oxaliplatin, rituximab); GCLAC (G-CSF, clofarabine, high-dose cytarabine); IA or IAC (idarubicin x 3 days + Ara-C (cytarabine) x 7 days); ICE (ifosfamide, carboplatin, etoposide (VP-16)); ICE-R or R-ICE or RICE (ICE + rituximab);m-BACOD (methotrexate, bleomycin, doxorubicin (Adriamycin®), cyclophosphamide, vincristine, dexamethasone); MACOP-B (methotrexate, leucovorin (folinic acid), doxorubicin, cyclophosphamide, vincristine, prednisone, bleomycin); MINE (mesna, ifosfamide, novantrone, etoposide); MINE-R or R-MINE (mesna, ifosfamide, novantrone, etoposide, and rituximab); ProMACE-MOPP (methotrexate, doxorubicin, cyclophosphamide, etoposide, and MOPP); R-Benda (rituximab and bendamustine); R-DHAP or DHAP-R (rituximab and DHAP); R-FCM or FCM-R (rituximab and FCM); R-ICE, ICE-R, or RICE (rituximab plus ICE); or TAD (thioguanine-cytarabine (ara-C)-daunorubicin).

[0096] In some embodiments, administration of the expanded stem cell product may improve the treatment outcome of patients with AML by improving the patient's chance of achieving remission. In some embodiments, administration of the expanded stem cell product may improve the treatment outcome of patients with AML by improving the patient's chance of achieving complete remission (CR), e.g., morphologic, cytogenetic, or molecular CR, or complete remission with incomplete recovery of blood counts (CRi). In some embodiments, the improved treatment outcome is other than a morphologic, leukemia-free state, partial remission, or disease stabilization. In some embodiments, the improved treatment outcome is associated with increased IL-2 levels in the patient after administration of the expanded stem cell product.

[0097] In some embodiments, the patient with AML is between about 20 and about 60 years of age. In some embodiments, the patient with AML is under 20 years of age. In some embodiments, the patient with AML is older than 60 years of age or older than 70 years of age. In some embodiments, the patient with AML is older than 60 years of age or older than 70 years of age and is undergoing a reduced-intensity chemotherapy regimen.

[0098] The expanded stem cell product is administered as a fixed dose to a human patient with AML or other hematological malignancies in need thereof to improve the patient's treatment outcome. Preferably, the expanded stem cell product is administered by infusion (e.g., intravenous infusion). Other suitable methods of administering the expanded stem cell product are encompassed by the present invention. The expanded stem cell product may be administered by any convenient route, for example, by bolus injection, and may be administered together with other biologically active agents.

[0099] The fixed dose of the expanded stem cell product administered is effective in treating a particular disorder or condition, such as AML or other hematologic malignancies, such as myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), and non-Hodgkin's lymphoma (NHL). In some embodiments, the patient has AML (e.g., relapsed / refractory AML, newly diagnosed AML, or treatment-related AML). In some embodiments, the patient has a myelodysplastic syndrome (MDS), such as MDS with multilineage dysplasia (MDS-MLD); MDS with single lineage dysplasia (MDS-SLD); MDS with ringed sideroblasts (MDS-RS); MDS with excess blasts (MDS-EB); MDS with isolated deletion (5q), or unclassifiable MDS (MDS-U). In some embodiments, the patient has a myeloproliferative neoplasm (MPN), such as chronic myeloid leukemia, polycythemia vera (p. vera), primary myelofibrosis, essential thrombocythemia, chronic neutrophilic leukemia, or chronic eosinophilic leukemia.

[0100] In a specific embodiment, a suitable fixed dose of the expanded stem cell product for administration is approximately 50 million, 75 million, 100 million, 200 million, 300 million, or 400 million CD34+ cells per dose, which may be administered to a patient once, twice, three times, or more times, as often as necessary, with intervals. If the expanded stem cell product is a frozen or cryogenically frozen product, the number of CD34+ cells refers to the number of cells prior to freezing or cryopreservation. In a specific embodiment, a patient receives a single fixed dose of the expanded stem cell product per regimen or, if applicable (e.g., for a multi-cycle regimen), per cycle of the regimen. In a specific embodiment, a patient receives a fixed dose of the expanded stem cell product per cycle of the regimen, with administration occurring after completion of the cycle. In a specific embodiment, a patient receives a fixed dose of the expanded stem cell product per regimen, with administration occurring after completion of the regimen.

[0101] Pharmaceutical Compositions The expanded stem cell product can be administered to a patient as a pharmaceutical (therapeutic) composition comprising a fixed dose (which is a therapeutically effective amount of the expanded stem cell product), wherein the administration is performed without matching the HLA type of the expanded stem cell product to the patient or to the hematopoietic stem cells or hematopoietic stem and progenitor cells in the expanded stem cell product relative to each other.

[0102] The present invention provides pharmaceutical compositions. Such compositions comprise a fixed dose of the expanded stem cell product, which is a therapeutically effective amount, and a pharmaceutically acceptable carrier or excipient. Such carriers can be, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The carriers and compositions are preferably sterile. The formulation should be adapted to the mode of administration. The pharmaceutical compositions are acceptable for therapeutic use in humans. The compositions can also contain a pH buffering agent, if desired.

[0103] In a preferred embodiment, the composition is formulated according to conventional procedures as the pharmaceutical composition that is suitable for intravenous administration of stem cells to humans.Typically, the composition for intravenous administration is a solution in sterile isotonic aqueous buffer solution.If necessary, the composition can also contain solubilizing agent and local anesthetic (such as lidocaine) to relieve pain at injection site.

[0104] The present invention also provides pharmaceutical packs or kits containing one or more containers or bags filled with one or more doses of the expanded stem cell product and a diluent (e.g., sterile isotonic aqueous buffer). If desired, a notice in the form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceutical or biological products may be associated with such containers or bags, indicating approval by the agency of manufacture, use, or sale for human administration.

[0105] In some embodiments, the present invention provides for the use of a fixed dose of an expanded stem cell product to improve treatment outcomes in patients with AML or other hematological malignancies, the expanded stem cell product comprising a pool of expanded hematopoietic stem cells or stem and progenitor cells from multiple donors, wherein the hematopoietic stem cells or stem and progenitor cells are not HLA-matched to each other or to the patient. In some embodiments, the expanded hematopoietic stem or stem and progenitor cells are CD34+. In some embodiments, a suitable fixed dose of the expanded stem cell product is approximately 50 million, 75 million, 100 million, 200 million, 300 million, or 400 million CD34+ cells. [Example]

[0106] Example 1: Generation of a human expanded stem cell product from a human umbilical cord blood unit The following sections describe the generation and storage of expanded stem cell products, as shown as a flow chart in FIG.

[0107] Umbilical cord blood / placental blood units were collected from human donors at birth. The collected blood was then mixed with an anticoagulant to prevent clotting. The blood was stored in quarantine at 4°C in a monitored refrigerator. The received units were evaluated to determine which units should be processed for expansion. The following information was collected for the units: date of receipt, age of the unit (in hours), gestational age of the donor (in weeks), sex of the donor, and volume of the unit. Additionally, total nucleated cell count and total CD34+ cell count for each unit were determined, and percent CD34+ cells were calculated. If the unit had fewer than 3.5 million CD34+ cells, the unit was discarded. If a unit was selected for expansion, it was removed from quarantine and assigned a unique lot number identifier, which was retained throughout the manufacturing process.

[0108] Prior to the start of planned expansion, tissue culture vessels were first inoculated with 2.5 μg / ml Delta1 in phosphate-buffered saline (PBS) overnight at 4°C or at 37°C for a minimum of 2 hours. ext-IgGand 5 μg / ml RetroNectin® (recombinant human fibronectin fragment) (Clontech Laboratories, Inc., Madison, WI). The flasks were then washed with PBS and then blocked with PBS-2% human serum albumin (HSA). Fresh cord blood units were processed to select for CD34+ cells using the CliniMACS® Plus Cell Separation System. Prior to CD34+ cell selection, an aliquot of the fresh cord blood unit was checked for total cell count and CD34+ cell content. Both CD34+ and CD34- cell fractions were collected after processing. After enrichment, the percentage of CD34+ cells increased 88- to 400-fold compared to the percentage of CD34+ cells in the sample before enrichment. The enriched CD34+ cell fraction was resuspended in final culture medium. This medium was prepared using STEMSPAN supplemented with rhIL-3 (10 ng / ml), rhIL-6 (50 ng / ml), rhTPO (50 ng / ml), rhFlt-3L (50 ng / ml), and rhSCF (50 ng / ml). TM It consists of Serum Free Expansion Medium II (StemCell Technologies, Vancouver, British Columbia).

[0109] Multiple donor CD34+ enriched cells were cultured at ≤1.8 x 10 4 Total nucleated cells / cm 2 The CD34+ enriched cells were added to specifically labeled and prepared tissue culture vessels at a concentration of 1000µg / ml per vessel surface area and then placed in an individually monitored and alarm-equipped incubator dedicated to that product lot. The CD34+ enriched cells were not HLA-matched to each other. After approximately 2 to 4 days of culture, 50% of the original volume of fresh culture medium (as described above) was added to the vessel. The cell culture vessels were periodically (every 1 to 3 days) removed from the incubator and examined under an inverted microscope for signs of cell growth and contamination. On approximately days 5 to 8, the vessels were gently agitated to mix the cells, and a 1 ml sample was removed for process testing. Cell samples were counted and phenotyped for expression of CD34, CD7, CD14, CD15, and CD56. Throughout the culture period, cells were cultured at a cell density of ≥8 x 10 5 When expanded to cells / ml, they were transferred to additional flasks as needed. Fresh medium was added the day before cells were harvested for cryopreservation.

[0110] On day 14, the expanded stem cell population was harvested for cryopreservation. The container was vortexed and the entire contents transferred to a sterile 500 ml centrifuge tube. The harvested cells were centrifuged, then washed once by centrifugation in phosphate-buffered saline (PBS) and resuspended in a cryopreservation solution containing human serum albumin (HAS), a sterile, pyrogen-free, isotonic solution of balanced electrolytes in water (Normosol-R®; Hospira, Lake Forrest, IL), and dimethyl sulfoxide (DMSO), or in CryoStor® CS10 cryopreservation medium containing 10% DMSO. Samples were taken to complete release testing. The expanded stem cell product was frozen in a controlled-rate freezer and transferred for storage in a vapor-phase liquid nitrogen (LN2) freezer.

[0111] At the end of the culture period, the resulting cell population was heterogeneous and consisted of CD34+ stem and progenitor cells and more mature myeloid and lymphoid precursors, as evidenced by flow cytometry analysis for the presence of CD34, CD7, CD14, CD15, and CD56 antigens. There was a significant expansion of CD34+ cell and total cell numbers during the culture period (ranging from approximately 100-fold to approximately 400-fold expansion of CD34+ cells and 617-fold to 3337-fold expansion of total cell numbers (N = 9 individual cord blood units, processed according to the final expansion procedure described above)). There was an essentially complete lack of T cells, as determined by immunophenotyping. Functionally, these cells are capable of multilineage human hematopoietic engraftment in a NOD / SCID mouse model as previously described (see U.S. Patent Publication No. 2013 / 0095079).

[0112] Example 2: Generation of a human expanded stem cell product from frozen human umbilical cord blood units Expanded stem cell products were prepared containing all cell progeny generated from enriched CD34+ cells selected from pooled human cord blood units (pools of 4-20 individual units). The pooled human cord blood units were transfected with the Notch ligand Delta1 as follows: ext-IgG (DXI) and recombinant cytokines.

[0113] Cord blood units containing approximately 2 to 20 million cells were selected for use. The cord blood units were thawed, subsequently centrifuged to remove cryoprotectant, resuspended in selection buffer, and pooled into a single container. The selection buffer was typically PBS with 1 mM EDTA and other components. The cord blood units were typically thawed in pairs. The cells were washed twice in selection buffer. The cells were pooled without regard to HLA antigens or alleles (i.e., unmatched). The pooled cord blood units were preincubated with paramagnetic beads and then processed by CliniMACS to isolate CD34+ cells using a single-use tubing set. After selection, the cells were centrifuged, and the collected CD34+ cells were suspended in cell culture medium (StemSpan Serum Free Expansion Medium II (SFEMII) medium supplemented with five recombinant human cytokines: IL-3 (10 ng / ml), and IL-6, TPO, SCF, and Flt-3L (50 ng / ml each)). The enriched CD34+ cells were then sampled to determine the viable cell yield and percentage of CD34+ cells in the composition. CD34+ cells were plated into coated flasks at the appropriate target seeding density using StemSpan SFEMII medium supplemented with five recombinant human cytokines (IL-3, IL-6, TPO, SCF, Flt-3L). Before use, the flasks were coated with recombinant protein DXI (2.5 micrograms / ml) and RetroNectin® recombinant human fibronectin fragment (rFN-CH-206) (5 micrograms / ml); unbound protein was washed from the flasks before use. The flasks were fed with fresh SFEMII medium and cytokines as needed. When the cells reached a sufficient cell number, they were harvested, pooled, and passaged into larger vessels at the appropriate target seeding density using the same SFEMII medium and five cytokines. The vessels were also pre-coated overnight with DXI and RetroNectin® recombinant human fibronectin fragment (rFN-CH-206) as described above. The vessels were monitored for cell density and viability and fed with up to the full volume of fresh SFEMII medium and cytokines as needed. When the cells reached the desired cell density, they were harvested by slight agitation, concentrated by centrifugation, the medium was removed, and the cells were resuspended in wash buffer. The viable CD34+ cell count was determined. After washing and harvesting, the cell pellet was resuspended in a balanced electrolyte solution with albumin. The final stem cell product typically contained approximately 50 to 100 million cells / ml.

[0114] The final cell product was then added to cryoprotective medium, which was then aseptically filled into labeled CryoStore bags, cryopreserved in a controlled-rate freezer, and stored in a vapor-phase liquid nitrogen (LN2) freezer at <-150°C. The bags were filled with approximately 50 million to 400 million CD34+ cells in a volume of approximately 20 ml / bag. The cryoprotective medium was either Normosol-R or CyroStor®, as described. CS10 contained approximately 4% human serum albumin (HSA) and 10% dimethyl sulfoxide (DMSO).

[0115] Example 3: Treatment of patients with AML with expanded stem cell products Patients with acute myeloid leukemia receiving intensive myelosuppressive chemotherapy regimens are at risk for life-threatening infections that impact overall treatment outcomes. The use of a non-HLA-matched pooled umbilical cord blood-derived ex vivo expanded CD34+ stem cell product (dilanubicel or NLA101) on the rate of serious bacterial or fungal infections was investigated in phase I and phase II trials. Dilanubicel was administered with induction and consolidation chemotherapy. In this international, phase II, randomized, open-label trial, 146 of the planned 220 subjects were enrolled in one of four treatment arms: standard of care (SOC) alone or SOC plus low-, intermediate-, or high-dose dilanubicel (100 × 10 6 , 300×10 6 , or 800×10 6CD34+ cells). Up to three doses of dilanubicel could be given with each round of chemotherapy, and subjects were followed until 84 or 30 days after the last dose of chemotherapy or dilanubicel. When the study was discontinued, no specific effect on infection rates was observed, and surprisingly, dilanubicel-treated subjects experienced a higher complete remission (CR) rate compared with patients in the control arm who received chemotherapy alone. Furthermore, treatment with dilanubicel was associated with a transient, dose-dependent increase in serum interleukin-2 (IL-2) levels. There were no Data Safety Monitoring Board-related safety concerns, although a small number of unexpected serious adverse events (SAEs) were observed. However, neither graft-versus-host disease nor cytokine release syndrome was observed.

[0116] material and method Study Design: This study was a phase 2, open-label, multicenter, randomized, controlled, dose-ranging study of the safety and efficacy of dilanubicel to reduce infection rates associated with chemotherapy-induced neutropenia in adult subjects with AML. The study was conducted at 36 sites in the United States (US), South Korea (SK), and Australia (AU). After enrollment, subjects were randomized 1:1:1:1 to either the control arm (standard of care (SOC) chemotherapy) or one of three investigational arms (SOC chemotherapy plus low-, intermediate-, or high-dose dilanubicel). Randomization was based on region (US). The data were stratified by age (vs. SK / AU).

[0117] Subjects randomized to the investigational arm were eligible to receive a single, fixed-assigned dose of dilanubicel after the first cycle of chemotherapy, and up to two additional doses (1 infusion / cycle) after subsequent chemotherapy cycles. Subjects randomized to the SOC arm were treated similarly, but without dilanubicel infusions, for up to three chemotherapy cycles. All subjects were followed for 84 days after randomization, or 30 days after the last dilanubicel infusion, or for the SOC arm, 30 days after the last chemotherapy infusion, whichever was longer. The study was halted after enrollment of 146 subjects (66%) following completion of an unplanned interim analysis.

[0118] The protocol and its amendments were approved by relevant institutional review boards and ethics committees, and required written informed consent prior to any study procedures. Safety was overseen by an independent Data Safety Monitoring Board (DSMB).

[0119] Patients: Eligible subjects must have untreated newly diagnosed or secondary AML and be scheduled to receive at least two cycles of chemotherapy with curative intent according to local institutional standards. Induction chemotherapy was required to include an anthracycline and cytarabine backbone and be predicted to result in moderate to severe myelosuppression. Subjects were also required to have a Karnofsky score ≥ 50 or an Eastern Cooperative Oncology Group (ECOG) performance status of 0, 1, or 2, adequate renal, hepatic, pulmonary, and cardiac function, and no evidence of active, uncontrolled infection at screening. Concurrent use of granulocyte transfusions, immunotherapy, or other study drugs was excluded.

[0120] Test Treatment: Dilanubicel is an ex vivo expanded hematopoietic stem and progenitor cell (HSPC) product derived from pooled, unmatched cord blood-derived CD34+ cells as described above. CD34+ cells were isolated from eligible, screened cord blood donors and cultured for 16 days in the presence of immobilized Notch ligand and recombinant cytokines (generally as described above). The expanded stem cell product was cryopreserved until infusion and delivered to the test site at fixed doses of approximately 100 million (low dose), 300 million (mid dose), and 800 million (high dose) CD34+ cells / bag in a volume of approximately 20 ml. Dilanubicel was given intravenously over 5-10 minutes approximately 24-36 hours after the last dose of chemotherapy for a given cycle. Oral acetaminophen and an intravenous antihistamine were administered immediately prior to administration.

[0121] Endpoints and Statistical Analysis: The primary endpoint of the study was the incidence of serious adverse events (Common Terminology Criteria) over the course of the 84-day study period. The incidence of bacterial or fungal infections (CTCAE Grade 3 or higher) was assessed by counting the number of unique Grade ≥ 3 infections within a subject and normalizing by study days. The normalized infection rates were regressed against treatment arm (SOC as reference) and region using negative binomial regression to compare infection rates between treatment arms. Study days from study day 1 were used as an offset variable to account for differential follow-up due to death or loss to follow-up. Event rate ratios and 95% confidence intervals were calculated as measures of association strength and precision, respectively.

[0122] Key secondary endpoints included best overall treatment response, filgrastim use, and incidence and duration of febrile neutropenia, and safety. Treatment response was defined as complete remission (CR) or complete remission with incomplete recovery of counts (CRi) according to the Revised International Working Group criteria, and treatment arms were compared using the Cochran-Mantel-Hansel test and stratified by region.

[0123] result Of the 162 subjects screened for this study before the enrollment deadline, 146 were enrolled and randomized to receive study treatment: 37 in the low-dose arm, 38 in the intermediate-dose arm, 35 in the high-dose arm, and 36 in the SOC arm. At the time of study halt, 18 subjects (48.6%) in the low-dose arm, 17 subjects (44.7%) in the intermediate-dose arm, and 10 subjects (28.6%) in the high-dose arm, compared with 6 subjects (16.7%) in the SOC arm, had completed the study according to protocol. A summary of subject breakdown is provided in Figure 2. The number of subjects treated with dilanubicel was 33 (89.2%) in the low-dose arm, 34 (89.5%) in the intermediate-dose arm, and 34 (97.1%) in the high-dose arm. The median number of dilanubicel doses received per subject was 2 across all 3 arms.

[0124] The overall median age of randomized subjects was 60 years (range, 19-77). Subjects were evenly split between men (75 subjects; 51.4%) and women (71 subjects, 48.6%). The majority of subjects were Caucasian (110 subjects; 75.3%). Most underlying disease characteristics were relatively balanced between groups, although a higher percentage of subjects in the SOC arm had an intermediate (unfavorable) risk of AML.

[0125] The total number of grade ≥3 bacterial or fungal infections occurring during the study period was 23 in the low-dose arm, 22 in the intermediate-dose arm, and 25 in the high-dose arm, and 20 in the SOC arm. All dilanubicel vs. SOC tests were not statistically significant (p=0.9604). The incidence rates and associated 95% CIs for each treatment arm relative to the SOC arm were 0.88 (0.44, 1.77; p=0.7291) for the low dose, 0.93 (0.46, 1.88; p=0.8471) for the intermediate dose, and 1.05 (0.53, 2.09; p=0.8868) for the high dose.

[0126] Table 1 summarizes the best overall response rates of complete remission (CR) (including morphologic CR, cytogenetic CR, molecular CR, or CRi) versus non-CR (all other non-CR response assessments) by treatment arm over the course of the study. Each treatment arm had a numerically advantageous CR rate compared with the SOC arm, which was statistically significant in the intermediate-dose arm (p=0.0024) and in all treatment groups (p=0.0086). This observation was unexpected, especially in light of a previous study using single-donor unmatched expanded cord blood products (Delaney et al., 2016, Lancet 3(7):PE330-339). While CR rate was not the primary endpoint of the previous study, no increase in CR rate was reported. [Table 1] Note: CR = morphologic CR, cytogenetic CR, molecular CR, or CR with incomplete recovery of blood counts (CRi); non-CR = morphologic leukemia-free status, partial remission / response, initial evaluation, treatment failure p-values ​​from CMH stratified by region

[0127] Overall, dilanubicel was generally well tolerated, with a dose-dependent increase in related events, although the overall incidence of safety events in the high-dose arm was only moderately higher than in the SOC arm. The most common adverse events assessed as related to dilanubicel were fever / febrile neutropenia, infusion reactions, and inflammatory signs and symptoms. The incidence of death in the study was not elevated in any of the expanded stem cell products compared with the SOC arm. The DSMB monitored safety throughout the study and raised no safety concerns. The numerically favorable CR rates in each treatment arm compared with the SOC arm were unexpected.

[0128] Example 4: Treatment of patients with hematological malignancies with expanded stem cell products Patients with hematological malignancies and receiving intensive chemotherapy regimens were randomized to receive standard of care (SOC) plus low-dose, intermediate-dose, or high-dose dilanubicel (100 × 10 6 , 300×10 6 , or 800×10 6 Patients are treated with CD34+ cells. Dilanubicel is administered after each cycle of chemotherapy. Patients are followed according to standard practice, and the best overall response rate of complete remission (CR) (including morphologic CR, cytogenetic CR, molecular CR, or CRi) vs. non-CR (all other non-CR response assessments) is assessed over the course of the study.

[0129] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

[0130] Various publications, including patents, patent application publications, and scientific literature, are cited herein, the disclosures of which are incorporated by reference in their entireties for all purposes. The embodiments of the invention in which exhaustive features or advantages are claimed are defined as follows: The present invention provides, for example, the following items. (Item 1) 1. A method for improving treatment outcomes in a human patient with acute myeloid leukemia (AML), the method comprising: administering an induction chemotherapy regimen to said patient; administering to the patient, after administration of the induction regimen, a fixed dose of a CD34+ enriched, T cell depleted, expanded stem cell product; wherein the expanded stem cell product comprises hematopoietic stem cells or hematopoietic stem and progenitor cells derived from cord blood units of at least two different human donors, wherein the cord blood units are HLA-matched to the donors. and selecting the antibody without matching the HLA type of the human patient; monitoring the patient's condition to determine whether the patient has achieved remission; and if the patient has not achieved remission, administering a second induction chemotherapy regimen followed by administering a second fixed dose of the expanded stem cell product to the patient; A method that encompasses (Item 2) 13. The method of claim 1, further comprising administering an intensive chemotherapy regimen to the patient achieving remission, followed by administration of a fixed dose of the expanded stem cell product. (Item 3) 3. The method of any one of items 1 or 2, wherein each dose of the expanded stem cell product is administered about 12 to 48 hours, or preferably about 24 to 36 hours, after the induction chemotherapy regimen. (Item 4) The method of claim 1, wherein each dose of the expanded stem cell product is administered to the patient after the components of the induction chemotherapy regimen and their active metabolites have cleared from the patient's blood. (Item 5) 3. The method of claim 2, wherein the expanded stem cell product dose is administered about 12 to 48 hours, or preferably about 24 to 36 hours, after the intensive chemotherapy regimen. (Item 6) 3. The method of claim 2, wherein the expanded stem cell product dose is administered after the components of the intensive chemotherapy regimen and their active metabolites have cleared from the patient's blood. (Item 7) 2. The method of item 1, wherein the induction chemotherapy regimen comprises administration of a combination of cytarabine and an anthracycline. (Item 8) 8. The method of claim 7, wherein the anthracycline is daunorubicin or idarubicin. (Item 9) 8. The method of item 7, wherein the induction regimen is a 7+3 regimen. (Item 10) 8. The method of item 7, wherein the induction chemotherapy regimen comprises administration of cytarabine and daunorubicin. (Item 11) 3. The method of item 2, wherein the intensive chemotherapy regimen comprises administration of medium-dose or high-dose cytarabine. (Item 12) 10. The method of claim 1, further comprising administering a salvage chemotherapy regimen. (Item 13) 13. The method of item 12, wherein the salvage chemotherapy regimen comprises cladribine-high-dose cytarabine-G-CSF (CLAG) or etoposide-cytarabine-mitoxantrone (MEC). (Item 14) The method of any of the preceding items, wherein each fixed dose of the expanded stem cell product comprises about 50 million CD34+ cells to about 400 million CD34+ cells. (Item 15) The method of any of the preceding items, wherein each fixed dose of the expanded stem cell product comprises about 100 million to about 300 million CD34+ cells. (Item 16) a. each fixed dose of the expanded stem cell product is the same; b. each fixed dose of the expanded stem cell product administered after the induction chemotherapy regimen is the same; or c. each fixed dose of the expanded stem cell product administered after the induction chemotherapy regimen is different from the fixed dose administered after the consolidation chemotherapy regimen; 10. The method according to any of the preceding items. (Item 17) The method of any of the preceding items, wherein the expanded stem cell product further comprises a cryoprotectant. (Item 18) The method of any of the preceding items, wherein the expanded stem cell product is produced by a process comprising enriching for CD34+ human umbilical cord blood stem and progenitor cells and expanding the CD34+ enriched human umbilical cord blood stem and progenitor cells with a Notch agonist. (Item 19) The Notch agonist consists of the extracellular domain of Delta fused to the Fc portion of IgG (Delta ext-IgG Item 19. The method according to Item 18, wherein (Item 20) The method of any of the preceding items, wherein the expanded stem cell product is derived from cord blood units from at least four different human donors, at least six different human donors, or at least eight different human donors. (Item 21) The method of any of the preceding items, wherein the expanded stem cell product does not transiently engraft in the patient 14 days after administration. (Item 22) The method of any of the preceding items, wherein the monitoring step comprises determining whether the patient has <5% bone marrow blasts by morphology. (Item 23) The method of any of the preceding items, wherein after the induction regimen and before the consolidation regimen, the patient does not receive an umbilical cord blood unit that is at least partially matched to the patient's HLA type. (Item 24) 24. The method of claim 23, wherein the at least partially matched cord blood unit is an autologous graft, a haploidentical graft, a matched related donor graft, a matched unrelated donor graft, or a mismatched unrelated donor graft. (Item 25) 1. A method for improving treatment outcomes in a human patient having a hematological malignancy, said method comprising: administering a chemotherapy regimen to said patient; administering to the patient a fixed dose of an expanded stem cell product after administration of the chemotherapy regimen; wherein the expanded stem cell product comprises hematopoietic stem cells or hematopoietic stem and progenitor cells of at least two different human donors, and wherein the hematopoietic stem cells or hematopoietic stem and progenitor cells are selected without matching to the donor's HLA type and without matching to the human patient's HLA type; monitoring the patient's condition to determine whether the patient has achieved remission; and If the patient has not achieved remission, optionally administering a second chemotherapy regimen followed by administering another fixed dose of the expanded stem cell product to the patient; A method that encompasses (Item 26) 26. The method of claim 25, wherein each dose of the expanded stem cell product is administered about 12 to 48 hours, or preferably about 24 to 36 hours, after the chemotherapy regimen. (Item 27) 26. The method of claim 25, wherein each dose of the expanded stem cell product is administered to the patient after components of the chemotherapy regimen and their active metabolites have cleared from the patient's blood. (Item 28) 29. The method of any one of items 25 to 28, wherein the hematological malignancy is selected from acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), non-Hodgkin's lymphoma (NHL) and myeloproliferative neoplasm (MPN). (Item 29) 29. The method of item 28, wherein the AML is newly diagnosed acute myeloid leukemia (AML), relapsed / refractory AML, or treatment-related AML. (Item 30) 29. The method of item 28, wherein the MDS is selected from MDS with multilineage dysplasia (MDS-MLD); MDS with single lineage dysplasia (MDS-SLD); MDS with ringed sideroblasts (MDS-RS); MDS with excess blasts (MDS-EB); MDS with isolated deletion (5q); and unclassifiable MDS (MDS-U). (Item 31) 29. The method of claim 28, wherein the MPN is selected from chronic myeloid leukemia, polycythemia vera, primary myelofibrosis, essential thrombocythemia, chronic neutrophilic leukemia, or chronic eosinophilic leukemia. (Item 32) 32. The method of any one of items 25 to 31, wherein the expanded stem cell product comprises hematopoietic stem cells or hematopoietic stem and progenitor cells derived from cord blood units of at least two different human donors. (Item 33) 33. The method of any one of items 25 to 32, wherein the chemotherapy regimen is selected from an induction regimen, a salvage regimen, and a consolidation regimen. (Item 34) 34. The method of any one of items 25 to 33, wherein the chemotherapy regimen is an induction regimen comprising administration of cytarabine and an anthracycline. (Item 35) 35. The method of claim 34, wherein the anthracycline is daunorubicin or idarubicin. (Item 36) 35. The method of item 34, wherein the induction regimen is a 7+3 regimen. (Item 37) 34. The method of any one of items 25 to 33, wherein the chemotherapy regimen is an intensive regimen comprising administration of medium-dose or high-dose cytarabine. (Item 38) 38. The method of claim 37, wherein the chemotherapy regimen is a salvage regimen. (Item 39) 34. The method of any one of items 25 to 33, wherein the salvage regimen is cladribine-high-dose cytarabine-G-CSF (CLAG) or etoposide-cytarabine-mitoxantrone (MEC). (Item 40) 40. The method of any one of items 25 to 39, wherein each fixed dose of the expanded stem cell product comprises about 50 million CD34+ cells to about 400 million CD34+ cells. (Item 41) 41. The method of any one of items 25 to 40, wherein each fixed dose of the expanded stem cell product comprises about 100 million to about 300 million CD34+ cells. (Item 42) 42. The method of any one of items 25 to 41, wherein each fixed dose of the expanded stem cell product is the same. (Item 43) 43. The method of any one of items 25 to 42, wherein the expanded stem cell product further comprises a cryoprotectant. (Item 44) 44. The method of any one of paragraphs 25 to 43, wherein the expanded stem cell product is produced by a process comprising enriching for CD34+ human cord blood stem and progenitor cells and expanding the CD34+ enriched human cord blood stem and progenitor cells with a Notch agonist. (Item 45) The Notch agonist consists of the extracellular domain of Delta fused to the Fc portion of IgG (Delta ext-IgG Item 45. The method according to Item 44, wherein (Item 46) The method of any of the preceding items, wherein the expanded stem cell product is derived from cord blood units from at least four different human donors, at least six different human donors, or at least eight different human donors. (Item 47) 47. The method of any one of items 25-46, wherein the expanded stem cell product does not transiently engraft in the patient as determined 14 days after administration.

Claims

[Claim 1] The invention described in the specification.

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