Kit for treating hematological disease

Abstract

To provide a kit for treating a hematological disease in a subject in need of treatment of the hematological disease.SOLUTION: The kit comprises: (a) obinutuzumab; (b) at least one immunosuppressive agent; (c) an expanded CD3-depleted HLA-haploidentical or HLA-mismatched NK cell fraction which has been expanded by ex-vivo culturing with nutrients, serum, IL-15, and nicotinamide of 1.0 mM to 10 mM; and (d) IL-2.SELECTED DRAWING: None

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 62 / 821,535, filed March 21, 2019, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to methods for expanding natural killer (NK) cells, selection of an expanded NK cell population for transplantation into a subject in need thereof, and therapeutic uses of an ex vivo expanded NK cell fraction suitable for transplantation in a clinical setting for the treatment of hematological malignancies, including in combination with cancer immunotherapy. The present invention also contemplates kits comprising the expanded NK cell fraction. [Background technology]

[0003] Natural killer (NK) cells are lymphoid cells involved in immune responses. These cells have multiple functions, in particular they are important components of the innate immune surveillance system, killing tumor cells, cells undergoing oncogenic transformation, and other abnormal cells in the body. Clinical experience with adoptive immunotherapy using NK cells highlights the need for better methods to effectively and efficiently expand NK cell populations while maintaining and even enhancing their functionality in vivo (killing capacity, trafficking, localization, persistence, and proliferation).

[0004] Unlike T cells, natural killer (NK) cells do not require the presence of specific tumor antigens to kill cancer cells, and target recognition by NK cells is regulated by a balance of activating and inhibitory signals. This ability of NK cells to kill tumor cells without the need for tumor-specific antigen recognition gives them an advantage over T cells, which makes NK cells interesting for study as effectors of immunotherapy. NK cells have attracted considerable attention in recent years as a promising tool for immunotherapy in patients with various refractory hematological and metastatic solid tumors. However, despite the ability of NK cells to kill cancer cells independent of antigen recognition, the full therapeutic potential of NK cell-based immunotherapy has yet to be realized. Results of experimental protocols to date have been mainly limited to partial remissions, with the marginal efficacy being mainly attributed to the relatively low number of NK cells infused, the short in vivo persistence of NK cells, and / or the poor in vivo functionality of NK cells. Therefore, the development of ex vivo NK culture methods that can effectively expand NK cell populations and enhance the functionality of adoptively transferred NK cells in vivo is essential to improve the clinical applicability of NK cell immunotherapy.

[0005] Several methods for in vitro expansion and activation of NK cells have been investigated. These include overnight and long-term culture of enriched NK cells from PBMCs with cytokines, or co-culturing NK cells with support cells such as PBMCs, genetically modified K562 cells (see U.S. Patent Publication No. 2015 / 0224143 to Malmberg et al.), and Epstein-Barr virus transformed lymphoblastoid cell lines (see, e.g., U.S. Patent Publication No. 20150152387 to Lee, et al.). Other methods for NK cell expansion have been reported. Frias et al. (Exp Hematol 2008; 36: 61-68) used NK progenitor cells (CD7 + CD34 - Lin - CD56 -) were grown on a stromal cell layer in serum-free medium and NK differentiation was induced with SCF, IL-7, IL-15, FL and IL-2, resulting in an increase in the number of cytotoxic cultured NK cells. Harada et al. (Exp Hematol. 2004;32:614-21) grew NK cells on cells derived from a Wilms tumor cell line. Waldmann et al. (US Patent Publication No. 20070160578) described the enhancement of proliferation of whole blood, bone marrow or spleen cell-derived NK cells and CD8-T cells in culture using a complex of IL-15 / R ligand activator to suppress unwanted cytokine production. Campana et al. (US Patent Publication No. 20090011498) described the ex vivo culture and activation of NK cells for transplantation in the presence of leukemic cells expressing IL-15 and 4-1BB and lacking or weak expression of MHC-I or II. Childs et al. (U.S. Patent Publication No. 2009 / 0104170) described ex vivo expansion and activation of NK cells by co-culture with irradiated EBV-transformed lymphoblastoid cells in the presence of IL-2. Tsai (U.S. Patent Publication No. 20070048290) used another approach to derive continuous NK cell lines from hematopoietic stem cells by ex vivo culture of immortalized NK progenitor cells with irradiated 3T3-derived OP-9S cells for research and potential therapeutic applications (all of the above references are incorporated herein by reference).

[0006] The therapeutic use of expanded NK cell populations has been the subject of over 40 completed, active, replacement or approved clinical trials (see clinical trials (dot)gov website), investigating the application of expanded NK cells by various protocols for the treatment of various cancerous conditions, including hematological and solid tumors. Expanded NK cell populations have generally been found to maintain cytotoxicity. Adjunctive therapies combining NK cells with chemotherapeutic agents, anti-cancer biologics and other cancer therapies have also been proposed. For example, US 2017 / 0137783 to Li teaches the expansion of chimeric antigen receptor (CAR)-expressing immune cells and their combination with additional therapies for the treatment of cancer. US 2017 / 0137783 to Bedoya et al. teaches the combination of expanded CAR-expressing immune cells with additional therapies, including anti-tumor biologics.

[0007] However, results to date highlight the challenge of designing NK expansion and treatment protocols that are not only safe but also sufficiently effective to target various forms of malignancy.

[0008] The inventors have described efficient ex vivo expansion and enhanced functionality of NK cells cultured with cytokines and the NAD precursor nicotinamide, and reported increased localization and engraftment of expanded NK cells to target organs (e.g., spleen, bone marrow, and peripheral blood) in animal models (see PCT Publication No. WO2011 / 080740 and Frei, et al, Blood, 2011;118:4035). Additional relevant literature includes, inter alia, International Application No. IB2018 / 057475 and Chinese Patent Application No. 201811129572.4. Summary of the Invention

[0009] According to an aspect of some embodiments of the present invention, there is provided a method of treating a hematological disorder in a subject in need thereof, comprising: (a) administering obinutuzumab to a subject; (b) administering to the subject at least one immunosuppressant agent; (c) transplanting the expanded CD3-depleted HLA-half-matched or HLA-mismatched NK cell fraction, expanded by ex vivo culture with nutrients, serum, IL-15, and 1.0 mM to 10 mM nicotinamide, into a subject in need thereof; (d) administering IL-2 to the subject to treat the hematological disorder in the subject. The present invention provides a method comprising:

[0010] According to some embodiments of the invention, the subject and the NK cell fraction are a human subject and a human NK cell fraction.

[0011] According to some embodiments of the invention, the immunosuppressant is a chemotherapeutic immunosuppressant and / or radiation.

[0012] According to some embodiments of the invention, the hematological disorder is a hematological malignancy.

[0013] According to some embodiments of the invention, the hematological disorder is a CD20 positive (CD20+) hematological malignancy. In some embodiments, the hematological disorder is a CD20 positive lymphoid malignancy.

[0014] According to some embodiments of the invention, the hematological disorder is multiple myeloma.

[0015] According to some embodiments of the invention, the multiple myeloma is characterized by at least one of the following: (a) disease relapsed 2–18 months after the initial autologous stem cell transplant; (b) disease relapsed at least 4 months after allogeneic stem cell transplantation without evidence of active graft-versus-host disease (GVHD); (c) relapsed / refractory disease after at least two lines of therapy, including a proteasome inhibitor and an immunomodulatory drug (IMiD); (d) serum IgG, IgA, IgM, or IgD myeloma protein (M protein) ≥ 0.5 g / dL, and (e) Urinary M protein level is 200 mg or more per 24 samples.

[0016] According to some embodiments of the invention, the hematological disease is non-Hodgkin's lymphoma (NHL).

[0017] According to some embodiments of the invention, the NHL is CD20 positive B-cell NHL.

[0018] According to some embodiments of the invention, the NHL is characterized by at least one of the following: (a) relapsed / refractory disease that has failed conventional treatment; (b) disease relapsed at least 60 days after autologous stem cell transplantation; (c) disease relapsed at least 4 months after allogeneic stem cell transplantation without evidence of active graft-versus-host disease, and (d) measurable disease ≥1.5 cm in diameter.

[0019] According to some embodiments of the invention, step (a) is carried out three times.

[0020] According to some embodiments of the invention, step (d) comprises administering a first dose of the expanded CD3-depleted, HLA half-matched or mismatched NK cell fraction, followed two days later by administering a second dose of the expanded CD3-depleted, HLA half-matched or mismatched NK cell fraction.

[0021] According to some embodiments of the invention, step (a) is performed three times: 9-11 days, 3 days, and 11 days after administration of the first dose of the expanded CD3-depleted, HLA half-matched or mismatched NK cell fraction.

[0022] According to some embodiments of the present invention, the NK cell fraction is 1×10 7 pieces / kg~5×10 8 Contains expanded CD3-depleted HLA half-matched or HLA-mismatched NK cells at 100 / kg.

[0023] According to some embodiments of the invention, the sum of the first dose and the second dose is 2×10 7 pieces / kg~2×10 8 Contains expanded CD3-depleted HLA half-matched or HLA-mismatched NK cells at 100 / kg.

[0024] According to some embodiments of the present invention, (a) The first and second doses of the NK cell fraction were each 1×10 7 cells / kg of expanded CD3-depleted HLA-half-matched or mismatched NK cells, with a total dose of expanded CD3-depleted HLA-half-matched or mismatched NK cells of 2 × 10 7 pieces / kg, or (b) The first and second doses of the NK cell fraction were 5×10 7 cells / kg of expanded CD3-depleted HLA half-matched or mismatched NK cells, with a total dose of expanded CD3-depleted HLA half-matched or mismatched NK cells of 1 × 10 8 pieces / kg, or (c) the first and second doses of the NK cell fraction were each 1×10 8 cells / kg of expanded CD3-depleted HLA-half-matched or mismatched NK cells, with a total dose of expanded CD3-depleted HLA-half-matched or mismatched NK cells of 2 × 10 8 Pieces / kg.

[0025] According to some embodiments of the invention, administration of the expanded CD3-depleted HLA-half-matched or HLA-mismatched NK cell fraction to the subject occurs within 1 hour after delivery of the transplant fraction and within 10 hours after final product release of the fraction.

[0026] According to some embodiments of the invention, the expanded CD3-depleted, HLA half-matched or mismatched NK cell fraction is administered to the subject by infusion in no less than 15 minutes and no more than 60 minutes, without the use of a filter or a pump.

[0027] According to some embodiments of the invention, the at least one immunosuppressant comprises cyclophosphamide and / or fludarabine.

[0028] According to some embodiments of the present invention, (i) At least one immunosuppressant was cyclophosphamide (40 mg / kg) and fludarabine (25 mg / m 2 ), (ii) Cyclophosphamide is administered 5 days prior to the infusion of the expanded CD3-depleted, HLA-half-matched or HLA-mismatched NK cells, and fludarabine is administered 5, 4, and 3 days, respectively, prior to the infusion of the expanded CD3-depleted, HLA-half-matched or HLA-mismatched NK cells.

[0029] According to some embodiments, the method of the invention comprises administering to the patient 6×10 6 The method further comprises administering units of IL-2 to the subject, (i) on the day of infusion of expanded CD3-depleted HLA-half-matched or -mismatched NK cells, and (ii) 2 days after infusion of expanded CD3-depleted HLA-half-matched or -mismatched NK cells; and (iii) 4 days after infusion of expanded CD3-depleted HLA half-matched or mismatched NK cells.

[0030] According to some embodiments of the invention, the method further comprises preparing a transplantable NK cell fraction by: (a) obtaining a subject's HLA-haploidentical or HLA-mismatched CD3-depleted NK cell fraction; (b) ex vivo culturing of the CD3-depleted NK cell fraction under conditions that allow cell proliferation, said conditions including supplying nutrients, serum, IL-15 and 1.0 mM to 10 mM nicotinamide; (c) supplementing the CD3-depleted NK cell fraction with fresh nutrients, serum, IL-15 and nicotinamide 8-10 days after step (b) to obtain an expanded CD3-depleted NK cell fraction; (d) recovering the expanded CD3-depleted NK cell fraction 14-16 days after step (b); (e) washing and concentrating the expanded CD3-depleted NK cell fraction of step (d); and obtaining a transplantable NK cell fraction for transplantation into a subject.

[0031] According to some embodiments of the invention, the CD3-depleted NK cell fraction is a human NK cell fraction.

[0032] According to some embodiments of the invention, the CD3-depleted NK cell fraction is obtained by apheresis.

[0033] According to some embodiments of the invention, the ex vivo culture is free of a feeder layer.

[0034] According to some embodiments of the invention, the serum is human serum.

[0035] According to some embodiments of the invention, the conditions allowing cell growth include providing 10% human serum.

[0036] According to some embodiments of the invention, the IL-15 comprises 20 ng / mL of IL-15.

[0037] According to some embodiments of the invention, the nicotinamide comprises 5.0 mM nicotinamide.

[0038] According to some embodiments of the invention, the method includes providing nutrients comprising a minimal essential cell culture medium.

[0039] According to some embodiments of the present invention, the NK cell fraction is derived from an HLA-half-matched or HLA-mismatched donor that meets at least the following criteria: (a) at least two of four class 1 alleles are HLA-matched in intermediate resolution DNA-based class 1 typing of the A and B loci; and (b) Absence of donor-specific anti-HLA antibodies in the recipient (MFI ≦ 1000).

[0040] According to some embodiments of the present invention, the NK cells in step (a) comprise at least 40-90% CD56+ / CD3- cells.

[0041] According to some embodiments of the invention, the harvesting of step (d) comprises harvesting a first portion of the expanded CD3-depleted NK cell fraction 14 days after step (b) and harvesting a second portion of the expanded CD3-depleted NK cell fraction 16 days after step (b).

[0042] According to some embodiments of the invention, the first portion comprises about 50% of the expanded CD3-depleted NK cell fraction and the second portion comprises the remainder of the expanded CD3-depleted NK cell fraction.

[0043] According to some embodiments of the invention, the washed and enriched expanded NK cell fraction produced by step (e) is characterized by the following parameters: (a) at least 70% CD56+ / CD3− cells; (b) survival rate of at least 70%; (c) At the time of infusion, the CD3+ cell count per patient was 5.0 × 10 5 Pieces / weight less than 1kg, (d) endotoxins of 5 EU / kg body weight or less per patient at the time of infusion; and (e) No gram-positive microorganisms.

[0044] According to some embodiments of the present invention, the culture in step (b) is carried out in a flask, and 200 to 300 × 10 6 Let us assume that the number of cells is 1.

[0045] According to an aspect of some embodiments of the present invention there is provided a transplantable NK cell fraction prepared according to the methods of the present invention.

[0046] According to some embodiments of the present invention, the transplantable NK cell fraction is characterized by the following parameters: (a) at least 70% CD56+ / CD3− cells; (b) survival rate of at least 70%; (c) At the time of infusion, the CD3+ cell count per patient was 5.0 × 10 5 Pieces / weight less than 1kg, (d) endotoxins of 5 EU / kg body weight or less per patient at the time of infusion; and (e) No gram-positive microorganisms.

[0047] According to some embodiments of the present invention, the transplantable NK cell fraction is provided in fluorinated ethylene propylene (FEP) culture bags.

[0048] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are merely illustrative and are not necessarily intended to be limiting.

[0049] Certain embodiments of the present invention are described herein, by way of example only, with reference to the accompanying drawings, in which it is emphasized that the details shown hereinafter, with particular reference to the drawings, are for the purposes of illustration and for the purpose of detailed description of embodiments of the present invention. Similarly, from viewing the description together with the drawings, it will become apparent to those skilled in the art how embodiments of the present invention may be practiced. [Brief description of the drawings]

[0050] [Figure 1] Figure 1 shows a FACS plot of CD3- / CD56+ NK cells grown for 2 weeks in medium supplemented with exogenous nicotinamide (5 mM) and stained for the cell surface markers CD16 and CD56. Note the high percentage (>75%) of double positive CD16+ / CD56+ cells in the nicotinamide expanded NK population. [Diagram 2]Figure 2, A and B, are histograms showing "cell killing" of CD20+ BL2 cells mediated by the anti-CD20 antibodies obinutuzumab and rituximab by nicotinamide expanded NK cells. Note the superior cell killing function of nicotinamide expanded NK cells and the anti-CD20 monoclonal antibody obinutuzumab. Figure 2, A and B, are two separate experimental groups. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0051] The present invention relates to a method for expanding a natural killer (NK) cell fraction for transplantation into a subject while simultaneously maintaining or enhancing the function of the cells ex vivo and / or in vivo. In one embodiment, ex vivo culture of NK cells with nicotinamide and / or other nicotinamide moieties and NK cell growth factors promotes the production of an NK cell population for use as a therapeutic ex vivo expanded NK cell preparation, comprising an expanded population of functional NK cells with suitable parameters for infusion into a subject (e.g., robust expansion of NK cells with depletion of the CD3+ T cell fraction). In particular in this regard, the present invention can be used to provide a transplantable NK cell fraction and protocols for its use, which can be used for transplantation and infusion of cells for the treatment of cancer and other diseases. Non-limiting applications include combination therapy with anti-cancer antibodies, allogeneic adoptive immunotherapy, and combination with sensitizers and other anti-cancer modalities. In a particular embodiment, the present invention can provide a transplantable NK fraction for use in combination therapy with anti-CD20 monoclonal antibodies.

[0052] The principles and implementations of the present invention may be better understood with reference to the following description.

[0053] Before describing at least one embodiment of the present invention in detail, it should be understood that the invention is not necessarily limited in its application to the details set forth in the following description. The invention is capable of other embodiments and of being practiced or carried out in various ways.

[0054] Natural killer (hereinafter also abbreviated as "NK") cells are lymphoid cells involved in immune responses and exhibit spontaneous non-MHC-restricted cytotoxic activity against tumor cells. Therefore, the development of clinical-grade protocols (e.g., no stromal layer, minimal cytokines) to effectively expand the number and function of viable NK cells ex vivo, as well as the possibility of homing to lymph nodes and homeostatic proliferation in vivo after infusion, could improve the outcome of NK cell-based adoptive immunotherapy for the treatment of cancerous conditions such as solid tumors and hematopoietic malignancies.

[0055] The present invention provides clinically relevant conditions for preparing and characterizing an expanded NK cell fraction suitable for transplantation in a clinical setting based on the culture of NK cells with nicotinamide above a certain concentration, as described in further detail herein. Thus, the present invention, in its embodiments, provides clinically relevant culture conditions for the production of a transplantable NK cell fraction of functionally mature NK cells, without the induction of proliferation of non-NK cells (e.g., CD3+), the transplantable NK fraction and selection criteria thereof, as well as clinical protocols for its use in the treatment of cancerous diseases, particularly hematological malignancies.

[0056] Thus, according to one aspect of an embodiment of the present invention, there is provided a method for preparing a transplantable NK cell fraction for transplantation into a subject in need thereof, comprising the steps of: (a) obtaining a subject's HLA-half-matched or HLA-mismatched CD3-depleted NK cell fraction; (b) ex vivo culturing the CD3-depleted NK cell fraction under conditions that allow cell proliferation, said conditions including supplying nutrients, serum, IL-15, and 1.0 mM to 10 mM nicotinamide; (c) supplementing the CD3-depleted NK cell fraction with fresh nutrients, serum, IL-15 and nicotinamide 8 to 10 days after step (b) to obtain an expanded CD3-depleted NK cell fraction; (d) recovering the expanded CD3-depleted NK cell fraction 14 to 16 days after step (b); (e) washing and concentrating the expanded CD3-depleted NK cell fraction of step (d); wherein said method for obtaining a transplantable NK cell fraction for transplantation into said subject is provided.

[0057] The term "natural killer (NK) cells" as used herein refers to large granular lymphocytes involved in the innate immune response. Functionally, NK cells exhibit cytolytic activity against a variety of targets through exocytosis of cytoplasmic granules that contain various proteins such as perforin and granzyme proteases. Killing is triggered in a contact-dependent, non-phagocytic process that does not require prior sensitization to antigen. Human NK cells are characterized by the presence of cell surface markers CD16 and CD56, and the absence of T cell receptor (CD3). Human bone marrow-derived NK cells are further characterized by a CD2+CD16+CD56+CD3- phenotype, further contain the T cell receptor zeta chain [zeta (ζ)-TCR], and are often characterized by NKp46, NKp30, or NKp44. Non-NK cells, such as NKT cells and CD8NKT, have characteristics and cell surface markers of both T cells and NK cells. In one embodiment, the methods of the invention are used to ex vivo expand mature NK cells from a cell population. As used herein, the term "mature NK cells" is defined as committed NK cells that have characteristic surface markers and NK cell functions, but lack the capacity for further differentiation. As used herein, mature NK cells include those that are resistant to CD56 bright cells (which can proliferate and produce large amounts of cytokines), CD56 dim Cells (showing strong cytotoxicity), CD56 bright CD94 high Cells and CD56 dim CD94 high In other embodiments, NK cells are expanded, including but not limited to, NK precursor cells, or a mixed population of NK precursor cells and mature NK cells. Cell surface expression of CD56, CD3, CD94, and other markers can be confirmed, for example, by FACS analysis or immunohistochemical staining techniques.

[0058] The term "progenitor cell" as used herein refers to an immature cell that can divide and / or differentiate into one or more mature effector cells. Lymphoid progenitor cells include, for example, multipotent hematopoietic stem cells that can give rise to mature cells of the B, T and NK lineages. For the B cell lineage (i.e., the developmental pathway that gives rise to mature B cells), progenitor cells also include pro-B and pre-B cells, which are characterized by immunoglobulin gene rearrangement and expression. For the T and NK cell lineages, progenitor cells include bipotential T / NK progenitor cells from bone marrow [e.g., CD34(+)CD45RA(hi)CD7(+) cells and CD34(+)CD45RA(hi)Lin(-)CD10(+) cells], and progenitor cells within the thymus, including, for example, double negative (for CD4 and CD8) and double positive thymocytes (T cell lineage) and committed NK progenitor cells.

[0059] The NK cells of the present invention can be derived from any source that contains such cells. NK cells are found in many tissues and can be obtained, for example, from lymph nodes, spleen, liver, lung, intestine, decidua, or from iPS cells or embryonic stem cells (ESCs). Umbilical cord blood, peripheral blood, mobilized peripheral blood, and bone marrow, which contain heterogeneous lymphoid cell populations, are usually used to obtain large numbers of NK cells for research and clinical use.

[0060] Clinical experience with NK cell transplantation has shown that allogeneic NK cells can successfully engraft into the host and have a low incidence of graft-versus-host disease (GVHD). When the identity of the transplant candidate (e.g., "subject") is known, parameters such as HLA matching (compatibility) can be ascertained and used as selection criteria.

[0061] Thus, according to certain embodiments, the NK cell fraction is derived from an HLA-half-matched or HLA-mismatched donor. The NK cell donor may be a related or unrelated donor.

[0062] In certain embodiments, the NK cells selected for ex vivo expansion are derived from a donor that is HLA-matched to the subject for at least two of the four HLA class I (medium resolution DNA-based class I typing at HLA-A and HLA-B loci), at least three of the four HLA class I (medium resolution DNA-based class I typing at HLA-A and HLA-B loci), or at least four of the four HLA class I (medium resolution DNA-based class I typing at HLA-A and HLA-B loci). According to certain embodiments, the apheresis unit is derived from a donor that has at least two of the four HLA class I (medium resolution DNA-based class I typing at HLA-A and HLA-B loci) and is free of recipient (host, subject) donor-specific anti-HLA antibodies (mean fluorescence intensity (MFI) ≦1000). The MFI value represents the amount or titer of the antibodies. Class I HLA (or major histocompatibility complex (MHC)) antigens are usually determined on NK cells by microcytotoxicity assays using allogeneic antisera against specific HLA, complement for cytotoxicity, and dyes to identify dead cells. HLA class II is usually determined by mixed lymphocyte reaction (MLR), measuring lymphocyte proliferation after culture of a mixed lymphocyte population. HLA DR antigens can be identified by B cell antisera in microcytotoxicity assays with enriched B cells. Specific monoclonal antibodies can be used instead of antisera.

[0063] Another common method of collecting blood fractions is apheresis, in which a donor's whole blood is separated into blood components (e.g., plasma, white blood cells, and red blood cells), usually by centrifugation, selected components are removed for manipulation (e.g., culturing of the white blood cell fraction), and the remainder is returned to the donor. Apheresis has the advantage of obtaining large amounts of a particular blood fraction (e.g., the white blood cell fraction) without depleting the body fluids (e.g., plasma) or other blood components. Apheresis can be based on continuous flow centrifugation, which requires a small extracorporeal volume, or on intermittent flow centrifugation of blood, which separates the components in multiple cycles, which is usually time-consuming and characterized by a large extracorporeal volume of donor blood. Many suitable apheresis devices are commercially available. Apheresis is usually applied to the separation of blood components from donor peripheral blood.

[0064] Thus, according to one aspect of the present invention, the method comprises culturing a CD3-depleted NK cell fraction, the NK cell fraction being obtained by apheresis. In a particular embodiment, the NK cell fraction is derived from an apheresis unit obtained from a donor using a PCS2 or MCS8150 Haemonetics apheresis machine (Haemonetics, Inc., Boston, Mass.). In one embodiment, the NK cell fraction is derived from an apheresis unit obtained from the peripheral blood of the donor.

[0065] In some embodiments, the NK cells can be cultured from a fresh cell population, while in other embodiments, the NK cells are cultured from a stored cell population (such as cryopreserved or thawed cells) or a pre-cultured cell population.

[0066] Leukocyte fractions, such as "buffy coats" or apheresis units, can be processed to enrich or purify or isolate specific defined cell populations. The terms "purify" and "isolate" do not require absolute purity, but rather are intended as relative terms. Thus, for example, a purified lymphocyte population is one in which a particular cell is more enriched than it is in its source tissue. A substantially pure lymphocyte preparation can be enriched such that the desired cells represent at least 50% of the total cells present in the preparation. In certain embodiments, a substantially pure cell population represents at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% or more of the total cells in the preparation.

[0067] Methods for enriching and isolating lymphocytes are well known in the art and an appropriate method can be selected based on the desired population. For example, one approach enriches the source lymphocytes by removing red blood cells. Red blood cells are separated from lymphocytes and other cells based on density. The lymphocyte-enriched fraction can then be selectively collected. Lymphocytes and their progenitors can also be enriched by centrifugation using a separation medium such as standard lymphocyte separation medium (LSM) available from a variety of commercial sources. Alternatively, lymphocytes / progenitors can be enriched using a variety of affinity-based procedures. Many antibody-mediated affinity preparation methods are known in the art, such as antibody-bound magnetic beads. Lymphocyte enrichment can also be performed using commercially available preparations that negatively select for unwanted cells, such as FICOLL-HYPAQUE™ and other density gradient media formulated for enrichment of total lymphocytes, T cells or NK cells.

[0068] Methods for the selection of NK cells from blood, bone marrow, lymphocyte preparations (e.g., apheresis units) or tissue samples are well known in the art (see, e.g., U.S. Patent No. 5,770,387 to Litwin et al., incorporated herein by reference). The most commonly used protocols are based on the isolation and purification of CD56+ cells, usually followed by fractionation of mononuclear cells followed by depletion of non-NK cells such as CD3+, CD34+, CD133+. A combination of two or more protocols can be used to obtain a more pure NK cell population from non-NK contaminants. The purity of NK cell preparations is crucial for clinical applications, since non-NK cells such as T cells and NKT cells contribute to antigen-specific responses such as GVHD, compromising the potential benefits of NK cell transplantation. Commercially available kits for isolating NK cells include one-step procedures (e.g., CD56 microbeads and CD56+, CD56+CD16+ isolation kit (Miltenyi Biotec, Auburn, Calif.)), multi-step procedures, such as depletion or partial depletion of CD3+, depletion with non-NK cell antibodies (e.g., OKT-3) that recognize and remove T cells, B cells, stem cells, dendritic cells, monocytes, granulocytes, and red blood cells. Thus, in some embodiments, the NK cell population can be selected or enriched for NK cells to provide a CD3-depleted NK cell fraction. In some embodiments, the CD3-depleted fraction comprises CD56+CD16+CD3- cells and / or CD56+CD16-CD3- cells. In certain embodiments, the NK cells selected for culture comprise at least 40% CD56+ / CD3- cells, at least 50% CD56+ / CD3- cells, at least 60% CD56+ / CD3- cells, at least 70% CD56+ / CD3- cells, at least 80% CD56+ / CD3- cells, or at least 90% CD56+ / CD3- cells. In some embodiments, the NK cells selected for culture comprise 40%-90% CD56+ / CD3- cells, 50%-80% CD56+ / CD3- cells, 55-75% CD56+ / CD3- cells, or 60%-70% CD56+ / CD3- cells. In some embodiments, the NK cells selected for culture comprise 40-90% CD56+ / CD3- cells.

[0069] Methods for selecting NK cells according to phenotype include, but are not limited to, immunodetection and FACS analysis. In a particular embodiment, the NK cell fraction is depleted of CD3 cells by immunomagnetic selection, for example using the CliniMACS T cell depletion set (LS depletion set (162-01) Miltenyi Biotec).

[0070] In further embodiments, the CD3-depleted NK cell fraction is treated to remove traces of red blood cells. Thus, in some embodiments, following depletion of CD3 cells, the NK cell fraction is subjected to red blood cell (RBC) lysis prior to culturing. In certain embodiments, red blood cell lysis is performed using ammonium chloride potassium (ACK) buffer (Gibco, Thermo Fischer Scientific).

[0071] NK cells can be cultured ex vivo by short-term or long-term culture. The inventors have demonstrated that culturing NK cells with growth factors and nicotinamide and / or other nicotinamide moieties for as little as seven days or as long as three weeks results in preferential proliferation and / or enhanced functionality of the cultured NK cells compared to cells cultured with cytokines but at less than 0.1 mM nicotinamide and / or other nicotinamide moieties (see WO 2011 / 080740). When preparing a clinically relevant NK cell fraction for transplantation, it is desirable to provide significant ex vivo NK cell expansion and not require long treatment periods, while maintaining the therapeutically beneficial functions of the expanded NK cell fraction.

[0072] Thus, in a particular embodiment, the CD3-depleted NK cell fraction is cultured for a period of 14-16 days.

[0073] According to this aspect of the invention, ex vivo culture of NK cells can be performed by providing conditions for cell expansion to the NK cells ex vivo, culturing the NK cells with a nicotinamide moiety ex vivo, and expanding a population of NK cells ex vivo.

[0074] As used herein, "culturing" includes providing the chemical and physical conditions (e.g., temperature, gases) and growth factors necessary for the maintenance of NK cells. In one embodiment, culturing NK cells includes providing the NK cells with conditions for NK cell proliferation. Examples of chemical conditions that can support NK cell proliferation include, but are not limited to, buffers, nutrients, serum, vitamins, and antibiotics, as well as cytokines and other growth factors that are typically provided in growth (i.e., culture) media. In certain embodiments, the conditions for cell proliferation include nutrients, serum, and cytokines. In one embodiment, the NK media includes a minimum essential medium (MEM) such as MEMα (BI, Bet HaEmek, Israel) and serum. In some embodiments, the serum provides 2-20%, 5-15%, or 5-10% of the media. In certain embodiments, the serum is human serum and provides 10% of the media. In certain embodiments, the media is MEMα with 10% human AB serum (Sigma-Aldrich, St. Louis, MO). Other media suitable for use in the present invention include, but are not limited to, Glasgow Medium (Gibco, Carlsbad, Calif.), RPMI Medium (Sigma-Aldrich, St. Louis, Mo.) or DMEM (Sigma-Aldrich, St. Louis, Mo.). It should be noted that many of the media contain nicotinamide as a vitamin supplement (e.g., MEMα (8.19 μM nicotinamide), RPMI (8.19 μM nicotinamide), DMEM (32.78 μM nicotinamide) and Glasgow Medium (16.39 μM nicotinamide)), but the methods of the present invention involve exogenously added nicotinamide to supplement the nicotinamide and / or nicotinamide moiety contained in the media formulation, or resulting from global adjustment of media component concentrations.

[0075] According to some embodiments of the invention, culturing the NK cells under conditions allowing cell proliferation includes supplying the cells with nutrients, serum, and cytokines. In some embodiments, the at least one growth factor comprises a cytokine and / or a chemokine. Cytokines and other growth factors are typically provided at concentrations ranging from 0.5 to 100 ng / mL, or 1.0 to 80 ng / mL, more typically 5 to 750 ng / mL, and even more typically 5.0 to 50 ng / mL (up to 10 times such concentrations may be contemplated), and are commercially available, for example, from Perpo Tech, Inc. (Rocky Hill, NJ, USA). In one embodiment, the conditions allowing cell proliferation include supplying the cytokine interleukin 15 (IL-15). In a particular embodiment, CD3-depleted NK cells are cultured with 20 ng / mL of IL-15.

[0076] Moreover, in this regard, it will be appreciated that new cytokines are continually being discovered, some of which may find use in the NK cell expansion methods of the present invention. For applications in which cells are introduced (or reintroduced) into a human subject, it is often preferable to use serum-free formulations such as AIM V® Serum-Free Medium or MARROWMAX® Bone Marrow Medium for Lymphocyte Culture. Such media formulations and supplements are available from commercial sources such as Invitrogen (GIBCO) (Carlsbad, Calif.). Cultures may be supplemented with amino acids, antibiotics and / or cytokines to promote optimal viability, proliferation, functionality and / or survival.

[0077] According to one embodiment, the NK cell fraction is cultured with nutrients, serum, cytokines (e.g., IL-15) and nicotinamide and / or nicotinamide moieties. As used herein, the term "nicotinamide moiety" refers to nicotinamide and products derived from nicotinamide, its derivatives, analogs and metabolites, such as NAD, NADH and NADPH, which can effectively and preferentially enhance the proliferation and / or activation of NK cells. Screening of nicotinamide derivatives, analogs and metabolites and evaluation of their effect on ex vivo NK culture proliferation can be performed by addition to maintained NK cultures as described below, or by addition to functional assays such as killing assays and motility assays, or by automated screening protocols designed for high throughput assays as known in the art.

[0078] As used herein, the phrase "nicotinamide analog" refers to any molecule known to behave similarly to nicotinamide in the above assays or similar assays. Representative examples of nicotinamide analogs include, but are not limited to, benzamide, nicotinethioamide (the thiol analog of nicotinamide), nicotinic acid, and α-amino-3-indolepropionic acid.

[0079] The phrase "nicotinamide derivative" further refers to any structural derivative of nicotinamide itself or a nicotinamide analogue. Examples of such derivatives include, but are not limited to, substituted benzamides, substituted nicotinamides and nicotinethioamides, and N-substituted nicotinamides and nicotinethioamides, 3-acetylpyridine, and sodium nicotinate. In one particular embodiment of the present invention, the nicotinamide moiety is nicotinamide.

[0080] Concentrations of nicotinamide or nicotinamide moieties suitable for use in some embodiments of the invention typically range from about 0.5 mM to about 50 mM, about 1.0 mM to about 25 mM, about 1.0 mM to about 25 mM, about 2.5 mM to about 10 mM, about 5.0 mM to about 10 mM. Exemplary effective concentrations of nicotinamide can be about 0.5 to about 15 mM, 1.0 to 10.0 mM, typically 2.5 or 5.0 mM, based on the effect of such concentrations of nicotinamide on proliferation and NK cell function. According to some embodiments of the invention, nicotinamide is provided in a concentration range (mM) of about 0.5, about 0.75, about 1.0, about 1.25, about 1.5, about 1.75, about 2.0, about 2.25, about 2.5, about 2.75, about 3.0, about 3.25, about 3.5, about 3.75, about 4.0, about 4.25, about 4.5, about 4.75, about 5.0, about 5.25, about 5.5, about 5.75, about 6.0, about 6.25, about 6.5, about 6.75, about 7.0, about 7.25, about 7.5, about 7.75, about 8.0, about 8.25, about 8.5, about 8.75, about 9.0, about 9.25, about 9.5, about 9.75, about 10.0, about 11.0, about 12.0, about 13.0, about 14.0, about 15.0, about 16.0, about 17.0, about 18.0 and about 20.0 mM. All effective intermediate concentrations are contemplated. In certain embodiments, the growth-enabling conditions include 1.0-10.0 mM nicotinamide. In yet other embodiments, the growth-enabling conditions include 5.0 mM nicotinamide.

[0081] A suitable concentration of nicotinamide and / or nicotinamide moiety can be determined according to any assay of NK proliferation and / or activity, e.g., cell culture or function. A suitable concentration of nicotinamide is one whose use in culture "enhances" or results in an increase in the proliferation and / or function of NK cells in culture, when compared to a "control" culture with less than 0.1 mM nicotinamide, tested with the same NK cell source (e.g., umbilical cord blood, bone marrow or peripheral blood preparation) in the same assay and under similar culture conditions (duration of exposure to nicotinamide, time of exposure to nicotinamide).

[0082] According to some studies, ex vivo expansion of purified NK cells by culture with nutrients, serum, cytokines and nicotinamide does not require medium supplementation or manipulation during the culture period, while other studies recommend medium supplementation ("feeding") at various intervals during NK cell culture. In certain embodiments of the invention, the NK cell fraction is "feeded" during the culture period. Thus, in certain embodiments, preparation of a transplantable NK cell fraction for transplantation includes supplementing the CD3-depleted NK cell fraction with fresh nutrients, serum, IL-15 and nicotinamide 8-10 days after the initiation of ex vivo culture (step (b)). In some embodiments, supplementation occurs 8-9 days after the initiation of ex vivo culture, 9-10 days after the initiation of ex vivo culture, or 8-10 days after the initiation of culture of CD3-depleted NK cells. In some embodiments, supplementation (or "feeding") involves removing about 30-80%, about 40-70%, or about 45-55% of the medium of the NK cell fraction culture and replacing it with new medium (the amount of which is similar (e.g., equal) to the amount of medium removed) having the same composition and the same levels of nutrients, serum, cytokines (e.g., IL-15), and nicotinamide as the removed medium. In some embodiments, supplementation (or "feeding") involves removing about 50% of the medium of the NK cell fraction culture and replacing the removed medium with new medium (the amount of which is similar (e.g., equal) to the amount of medium removed) having the same composition and the same levels of nutrients, serum, cytokines (e.g., IL-15), and nicotinamide as the removed medium. In other embodiments, the amount of medium after feeding reaches about twice the original amount of medium at the initiation ("seeding") of the NK cell culture.

[0083] The NK cell population can be cultured using a variety of methods and devices. The choice of culture device is usually based on the scale and purpose of the culture. For scale-up of cell culture, it is preferable to use dedicated devices. Devices for large-scale clinical grade NK cell production are detailed, for example, in Spanholtz et al. (PLoS ONE 2010;5:e9221) and Sutlu et al. (Cytotherapy 2010, Early Online 1-12). In some embodiments, the culture of the NK cell fraction (e.g., step (b) and / or step (c) of the method) is carried out at 100-4000×10 in flasks. 6 In a specific embodiment, the culture of the NK cell fraction (e.g., initiation of ex vivo culture and / or "feeding") is performed at a cell density of 200-300 × 10 cells / flask. 6 The cell density is 100 cells / flask. In one embodiment, the flask is a flask containing a gas-permeable membrane, such as a G-Rex culture device (G-Rex 100M or closed system G-Rex MCS (WolfWilson, St. Paul, Minnesota)).

[0084] It will be appreciated that the cell density in the culture flask increases with the proliferation of the cells during the culture period. Thus, in some embodiments, during the course of proliferation in culture, the NK cell culture of the NK cell fraction is at a density of 100-4000×10 6 Cells / flask, 100-4000 x 10 6 Cells / flask, 100-4000 x 10 6 Cells / flask, 100-4000 x 10 6 Cells / flask, 200-3000 x 10 6 Cells / flask, 300-2000 x 10 6 Cells / flask, 400-1000 x 10 6 Cells / flask, 250-800 x 10 6 Cells / flask, 100-600 x 10 6 Cells / flask, or 150-500 x 10 6In a specific embodiment, the NK cells of the NK cell fraction are cultured at a cell density of 100-3000×10 cells / flask over the culture period in the flask. 6 The cell density is determined as: cells / flask.

[0085] The culture of NK cells can be performed with or without feeder cells or a feeder layer. Ex vivo culture without a feeder layer is highly advantageous for clinical application of cultured cells (including NK cells). Thus, according to one embodiment, the culture of the NK cell population is performed without a feeder layer or feeder cells.

[0086] In certain embodiments, CD3-depleted NK cells are harvested from the culture 14-16 days after initiation of the NK cell culture (step (b)). Cell harvesting can be performed manually by detaching attached cells (e.g., by "scraping" the surface of the culture vessel) or by a cell harvester designed to efficiently wash the cells from the culture vessel and automatically collect the cells. In certain embodiments, the expanded CD3-depleted NK cell fraction is harvested from the culture vessel by a cell harvester (e.g., a G-Rex MCS harvester (WolfWilson, St. Paul, MN)).

[0087] In some embodiments, harvesting the expanded NK cell fraction from the culture removes most or almost all of the cells from the culture vessel. In other embodiments, harvesting can be done in two or more steps, and unharvested cells can remain in the culture until harvested at a later time. In one embodiment, harvesting the expanded CD3-depleted NK cell fraction is done in two steps, harvesting a first portion of the expanded CD3-depleted NK cell fraction followed by harvesting a second portion of the expanded CD3-depleted NK cell fraction. The harvesting of the two portions can be done with an interval of several hours, days, or more between harvesting the first and second portions. The two harvested portions may contain approximately equal proportions of the culture (e.g., equal amounts of cultured NK cells), or one portion may contain a greater proportion of cultured NK cells than the other. In one embodiment, harvesting includes harvesting a first portion of the expanded CD3-depleted NK cells about 14 days after step (b) (the initiation of the culture) and harvesting a second portion of the expanded CD3-depleted NK cell fraction about 2 days thereafter. In a specific embodiment, the first portion is harvested 14 days after the initiation of the ex vivo culture, and the second portion is harvested 16 days after the initiation of the ex vivo culture.

[0088] In one embodiment, the first and second portions are approximately equal, i.e., the first (recovery) portion comprises about 50% of the expanded CD3-depleted NK cell fraction and the second (recovery) portion comprises the remainder of the expanded CD3-depleted NK cell fraction.

[0089] To prepare an expanded CD3-depleted NK cell fraction for transplantation, the harvested cells must be flushed of media, vital parameters assessed, and concentrations adjusted to be appropriate for infusing volumes over a clinically relevant period of time.

[0090] After collection, the expanded CD3-depleted NK cell fraction can be washed manually until it is free of medium, or, preferably for clinical applications, using an automated device that employs a closed system. The washed cells can be reconstituted with an infusion fluid (e.g., an exemplary infusion fluid includes 8% w / v HSA and 6.8% w / v dextran-40). In some embodiments, the reconstitution is performed in a closed system. In some embodiments, the infusion fluid is screened for suitability for use in the methods and compositions of the invention. Exemplary criteria for selecting an appropriate infusion fluid include safety testing, including the absence of bacterial, yeast, or mold growth, endotoxin content less than 0.5 Eu / mL, and a clear, non-foreign appearance.

[0091] The term "multiplication" or "expansion" as used herein refers to growth, e.g., cell growth, and an increase in cell number. As used herein, proliferation and expansion refer to an increase in the number of NK cells that occurs during an incubation period. The in vitro or in vivo proliferation of cells exhibiting a NK cell phenotype is a known phenomenon, e.g., following stimulation with IL-2, Epstein-Barr virus transformed lymphoblastoid lines, etc.

[0092] Assays for cell proliferation known in the art include, but are not limited to, clonogenic assays (cells are plated and grown at low density and colonies are counted), mechanical assays [flow cytometry (e.g., FACS™), propidium iodide] (cell number is measured mechanically), metabolic assays (e.g., tetrazolium salt (e.g., XTT, MTT, etc.) uptake) (measures the number of viable cells), and direct proliferation assays (e.g., bromodeoxyuridine, thymidine uptake) (measures DNA synthesis of the growing population). In one embodiment, cell proliferation of NK cell populations cultured with an effective concentration of nicotinamide and / or other nicotinamide moieties according to the invention is measured at a predetermined time after seeding the NK cells in culture (e.g., about 10 hours, 12 hours, about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 2 months or more) and confirmed by FACS analysis using anti-CD56 and anti-CD3 markers to identify and quantify the CD56+CD3- NK cell fraction of the population. NK cell proliferation can be expressed as a fold increase (e.g., expansion or fold expansion) of NK cells compared to the original NK cell fraction prior to culture. In some embodiments, for populations of NK cells exposed to an effective concentration of nicotinamide according to the present invention, after about 5 days, about 7 days, about 12 days, about 14 days, about 16 days, about 18 days, about 21 days, about 25 days, about 30 days or more of culture, the NK cell population increases at least 2-fold, at least 10-fold, at least 20-fold, at least 40-fold, at least 50-fold, at least 75-fold, at least 100-fold, at least 150-fold, at least 250-fold, at least 500-fold or more. In other embodiments, the expansion of a NK cell population exposed to an effective concentration of nicotinamide and determined by FACS™ is at least about 1.2-fold, about 1.3-fold, about 1.5-fold, about 1.75-fold, about 2-fold, about 2.25-fold, about 2.5-fold, about 2.75-fold, about 3.0-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or about 10-fold compared to NK cells cultured under identical conditions at less than 0.1 mM nicotinamide and / or other nicotinamide moieties.

[0093] The term "function" or "NK cell function" as used herein means any biological function attributable to NK cells. Non-limiting examples of NK cell functions include, for example, cytotoxicity, induction of apoptosis, cell motility, directed migration, cytokine and other cell signaling responses, cytokine / chemokine production and secretion, expression of activating and inhibitory cell surface molecules in vitro, cell homing and engraftment (in vivo retention) in a transplant host, and modification of a disease or disease process in vivo. In some embodiments, NK cell functions enhanced by exposure to nicotinamide and / or other nicotinamide moieties include at least one of increased expression of the CD62L surface marker, increased migratory response, and higher cytotoxic activity of NK cells, as well as increased homing and in vivo retention of infused NK cells.

[0094] Assays for adhesion and migration molecules such as CD62L, CXCR-4, CD49e, etc., important for cell homing / engraftment and retention in transplantation, are well known in the art. CD62L expression on cells can be assayed, for example, by flow cytometry, immunodetection, quantitative cDNA amplification, hybridization, etc. In one embodiment, detection of CD62L expression on different populations of NK cells is performed by exposing the cells to a fluorescently labeled specific anti-human CD62L monoclonal antibody (e.g., CD62L PE, Cat. No. 304806, BioLegend, San Diego, CA, USA) and sorting the cells by fluorescence activated cell sorting (FACS).

[0095] Assays for cell migration are well known in the art. Cell migration can be assayed, for example, by transmigration assays or gap closure assays. In transmigration assays, such as the two-chamber technique, cells are separated from the stimulus by a barrier (e.g., a filter) and cell migration is detected by counting the loss of cells from the origin, the accumulation of cells across the barrier, or both at specific intervals. In gap closure assays, cells are placed around a visible gap (notched agar plate, periphery, etc.) and incubated with the stimulus. Closure of the gap between cells by cell movement in response to the stimulus is visualized using cytometry, immunodetection, microscopy / morphometry, etc. In one embodiment, the migration capacity of different populations of NK cells is confirmed by the "Transwell" (trademark) transmigration assay in response to SDF (250 ng / mL).

[0096] Assays for homing and in vivo retention of injected or transplanted cells are well known in the art. As used herein, the term "homing" refers to the ability of injected or transplanted cells to reach and survive in a target organ of the host. For example, an NK cell target organ can be lymphoid tissue, a hepatic cell target organ can be the liver parenchyma, an alveolar cell target organ can be the lung parenchyma, etc. As used herein, the term "in vivo retention" (also known as "engraftment") refers to the ability of injected or transplanted cells to proliferate and remain viable in the target organ. Animal models for assaying homing and in vivo retention of transplanted NK cells include immune-deficient small mammals (e.g., SCID mice and IL2Rγ mice). null Examples of suitable models include, but are not limited to, human NK cell models (e.g., mice). The SCID-Hu mouse model employs CB-17 scid / scid (SCID) mice engrafted with human fetal thymus and liver tissue or fetal BM tissue, providing a suitable model for evaluating the retention and therapeutic potential of engrafted human NK cells. Homing and in vivo retention of engrafted cells can also be evaluated in human host subjects. In one embodiment, homing and in vivo retention assays are performed using, for example, approximately 15×10 NK cells cultured with an effective concentration of nicotinamide according to the present invention. 4pieces, approximately 15×10 5 pieces, approximately 15×10 6 pieces, approximately 15×10 7 Irradiated NOD / SCID mice injected with 10 or more human NK cells are sacrificed at a predetermined time after injection (e.g., about 5 hours, about 10 hours, about 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 2 months, 3 months, 4 months or more after injection). Upon sacrifice of the mice, samples of spleen, bone marrow, peripheral blood and other organs are assessed by FACS for the presence of human NK cells (CD56+CD45+) using human-specific antibodies. In vivo retention is expressed as the percentage of cells in the organ that display the donor phenotype (e.g., CD45 on human cells).

[0097] Assays for cytotoxicity ("cell killing") are well known in the art. Examples of target cells suitable for use in redirected killing assays include cancer cell lines, primary cancer cells, solid tumor cells, leukemia cells, or virus-infected cells. In particular, K562, BL-2, colo250, and primary leukemia cells can be used, although any of a number of other cell types can be used and are well known in the art (see, for example, Sivori et al. (1997) J. Exp. Med. 186: 1129-1136; Vitale et al. (1998) J. Exp. Med. 187: 2065-2072; Pessino et al. (1998) J. Exp. Med. 188: 953-960; Neri et al. (2001) Clin. Diag. Lab. Immun. 8:1131-1135). Cell killing is assessed by cell viability assays (eg, dye exclusion, chromium release, CFSE), metabolic assays (eg, tetrazolium salts) and direct observation methods.

[0098] Once the expanded CD3-depleted NK cell fraction has been washed and concentrated, the expanded fraction can be evaluated for suitability for use in transplantation. Typical criteria for selecting an appropriate transplantable NK cell fraction include the percentage of CD56+ / CD3- cells, cell viability, size of the CD3+ cell fraction, presence of endotoxins, microbial contamination, etc. It should be noted that the CD56+, CD3+ and CD56+ / CD3- cell content of the expanded NK cell fraction is important for successful engraftment of transplanted NK cells and is therefore an important criterion for proceeding with ex vivo expansion. Thus, in certain embodiments, the washed and concentrated expanded NK cell fraction produced in step (e) of the method of the invention is characterized by about 60% to about 90% CD56+ / CD3- cells, about 68% to about 85% CD56+ / CD3- cells, about 72% to about 82% CD56+ / CD3- cells, and about 76 to 79% CD56+ / CD3- cells. In one embodiment, the washed and enriched expanded NK cell fraction produced in step (e) of the method of the invention is characterized by at least 60%, at least 64%, at least 70%, at least 74%, at least 80% or at least 85% CD56+ / CD3- cells. In a further embodiment, the washed and enriched expanded NK cell fraction produced in step (e) of the method of the invention is characterized by at least 70% CD56+ / CD3- cells. Identification of NK cell phenotype by CD56 and CD3 cell markers is described above.

[0099] The presence of allogeneic T (CD3+) cells in the cell fraction intended for transplantation is problematic as it significantly increases the risk of GVHD. Thus, an important parameter for the suitability of the transplantable expanded NK cell fraction is the amount or percentage of CD3+ cells. Thus, in a specific embodiment, the washed and enriched expanded NK cell fraction produced by the method of the present invention has a CD3+ cell count of 1.0×10 per patient. 5 ~1.0×10 6 In a further embodiment, the washed and enriched expanded NK cell fraction produced by the methods of the invention is characterized by a CD3+ cell count of 7.0×10 5cells / kg body weight, CD3+ cell count per patient is 6.5×10 5 cells / kg body weight, CD3+ cell count per patient is 6.0×10 5 cells / kg body weight, CD3+ cell count per patient is 5.5×10 5 cells / kg body weight, CD3+ cell count per patient is 5.0×10 5 <1 kg body weight, CD3+ cell count per patient 4.5 × 10 5 cells / kg body weight, CD3+ cell count per patient is 4.0×10 5 <1 kg body weight, CD3+ cell count 3.5 × 10 per patient 5 < 1 kg body weight or CD3+ cell count 3.0 × 10 5 In one embodiment, the washed and enriched expanded NK cell fraction produced by the methods of the invention is characterized by a CD3+ cell count of less than 7.0×10 5 The fraction, percentage or content of CD3+ cells in the washed and enriched expanded NK cell fraction produced by the method of the present invention, expressed per Kg of patient body weight, is characterized by less than 100 CD3+ cells / Kg body weight. It should be noted that the calculation of the CD3+ fraction, percentage or content of the washed and enriched expanded NK cell fraction produced by the method of the present invention, expressed per Kg of patient body weight, is based on the total amount of CD3+ cells transferred (e.g., infused) to the patient (i.e., subject). The fraction, percentage or content of CD3+ cells in the washed and enriched expanded NK cell fraction produced by step (e) of the method of the present invention can also be expressed as a ratio of CD56+ / CD3- cells to CD3+ cells, or as a volume fraction (e.g., CD3+ cells / mL) or weight fraction (CD3+ cells / 100 g) of the washed and enriched expanded NK cell fraction produced by the method of the present invention. The identification of CD3+ cell markers is described above.

[0100] Sterility and safety of the expanded CD3-depleted NK cell fraction for transplantation is ensured, inter alia, by monitoring endotoxin content and the presence of bacterial, fungal, viral and mycoplasma contamination. In some embodiments, the expanded NK cell fraction selected for transplantation has an endotoxin content of 5 Eu / mL or less after washing and concentration. In some embodiments, the expanded NK cell fraction for transplantation is characterized as being free of microorganisms (e.g., gram-positive microorganisms) after washing and concentration.

[0101] In some embodiments, the expanded NK cell fraction suitable for transplantation is characterized by a viability of about 50% to about 85%. In some embodiments, an expanded NK cell fraction is selected that has a viability of about 55%, about 60%, about 63%, about 65%, about 68%, about 70%, about 75%, about 78%, about 80%, about 82%, about 83%, about 84% to about 85% or more. In further embodiments, the NK cell fraction selected for ex vivo expansion is at least 70% viable cells. In further embodiments, the expanded NK cell fraction suitable for transplantation is characterized by at least 70% viable cells after washing and concentration. In further embodiments, the expanded NK cell fraction suitable for transplantation is at least 85% viable cells.

[0102] The term "viability" as used herein refers to the distinction between viable and non-viable cells. Cell viability may be determined by morphological changes, or by changes in membrane permeability and / or physiological state inferred from the exclusion of certain dyes or the uptake and retention of other dyes. Assessment of cell viability is well known in the art and includes, but is not limited to, assays (e.g., dye exclusion, chromium release), metabolic assays (e.g., tetrazolium salts), and direct observation methods (Coder, D., Current Protocols in Cytometry, 1997, John Wiley and Sons, Inc., Unit 9.2, 9.2.1-9.2.14).

[0103] In some embodiments, the parameters of CD56+ / CD3- cell fraction, CD3+ cell fraction, viability, endotoxin and microbial content are monitored in samples taken before NK cell culture, during NK cell culture, after collection of the first and / or second portion, and / or after washing and enrichment of the expanded NK cell fraction. In some embodiments, collection of samples from any apheresis unit is performed prior to processing (100×10 6 (cells), post-column (CD3 depletion) pre-culture sample (10 × 10 6 (cells), post-expansion before washing (10 mL sample), and the washed and concentrated final expanded NK cell product on the day of first infusion (day 0) (10 × 10 6 (cells) and the washed and concentrated final expanded NK cell product (10 × 10 6 The method may be performed using one or more cells (individuals) or any combination thereof.

[0104] Thus, according to certain embodiments, the washed and enriched expanded NK cell fraction produced by the methods of the present invention is characterized by the following parameters: (a) at least 70% CD56+ / CD3− cells; (b) survival rate of at least 70%; (c) At the time of infusion, the CD3+ cell count per patient was 5.0 × 10 5 Pieces / weight less than 1kg, (d) endotoxins of 5 EU / kg body weight or less per patient at the time of infusion; and (e) No gram-positive microorganisms.

[0105] An expanded CD3-depleted NK cell fraction that meets the above criteria can be used for transplantation into a subject (e.g., a patient) in need thereof. As described in this section and in the sections that follow, any of the methods for ex vivo expansion (culture), selection and preparation of an NK cell fraction for transplantation described above, and each of the embodiments thereof, can be used alone or in various combinations to affect the method of transplanting the expanded NK cell fraction.

[0106] Thus, in some embodiments, a transplantable NK cell fraction is provided that is prepared according to any of the methods for preparing a transplantable NK cell fraction described herein. In certain embodiments, the transplantable NK cell fraction is characterized by the following parameters: (a) at least 70% CD56+ / CD3− cells; (b) survival rate of at least 70%; (c) At the time of infusion, the CD3+ cell count per patient was 5.0 × 10 5 Pieces / weight less than 1kg, (d) endotoxins of 5 EU / kg body weight or less per patient at the time of infusion; and (e) No gram-positive microorganisms.

[0107] In some embodiments, after washing and concentration, the transplantable NK cell fraction is transferred to a container (e.g., for transport to a transplant (infusion) site). In some embodiments, the container is a culture bag. Culture bags constructed of inert materials with high gas permeability, low water loss, flexibility, and high light transmittance are preferred. In certain embodiments, the transplantable expanded NK cell fraction is provided in a fluorinated ethylene propylene (FEP) culture bag.

[0108] In other embodiments, a transplantable human NK cell fraction is provided that is characterized by the following parameters: (a) at least 70% CD56+ / CD3− cells; (b) survival rate of at least 70%; (c) At the time of infusion, the CD3+ cell count per patient was 5.0 × 10 5 Pieces / weight less than 1kg, (d) endotoxins of 5 EU / kg body weight or less per patient at the time of infusion; and (e) No gram-positive microorganisms.

[0109] The expanded NK cell fraction of the present invention can be used for transplantation into a subject in need thereof.

[0110] The term "transplantation" as used herein means administration of cells expected to have a therapeutic effect to a subject, preferably a subject in need thereof, for example, as a treatment for a patient against a disease or condition, in the context of cell therapy, adoptive transfer, cellular immunotherapy, etc. Such cell therapy involves the introduction of a therapeutic cell fraction into the subject's body via a connection to a blood vessel, and therefore "transplantation" and "administration" of NK cells as used herein are synonymous with "infusion." Typically, the therapeutic cell fraction is intravenously infused into the subject, for example, via a central venous catheter (e.g., a Hickman catheter). The infusion rate of the therapeutic cell fraction into the subject can be controlled by a pump or fed by gravity without assistance, and adjusted by the height difference between the cell fraction and the inlet catheter. In some embodiments, the expanded NK cell fraction is transplanted (infused, administered) intravenously by gravity feed, without the use of pump(s) and / or filters.

[0111] In some embodiments, the subject in need of a transplant suffers from a hematological disorder. In some embodiments, the subject suffers from a hematological malignancy. In some embodiments, the hematological disorder is a CD20 positive (CD20+) hematological disorder. In some embodiments, the subject in need of a transplant suffers from a CD20 positive lymphoid malignancy. In certain embodiments, hematological malignancies suitable for treatment with the expanded NK cell fraction or methods described herein are multiple myeloma and non-Hodgkin's lymphoma.

[0112] Accordingly, in some embodiments, there is provided a method of treating a hematological disorder in a subject in need thereof, comprising: (a) administering an anti-cancer monoclonal antibody to a subject; (b) administering to the subject at least one immunosuppressant agent; (c) transplanting the expanded CD3-depleted HLA-half-matched or HLA-mismatched NK cell fraction, expanded by ex vivo culture with nutrients, serum, IL-15, and 1.0 mM to 10 mM nicotinamide, into a subject in need thereof; (d) administering IL-2 to the subject to treat the hematological disorder in the subject. A method is provided that includes:

[0113] In certain embodiments, the anti-cancer monoclonal antibody is an anti-CD20 monoclonal antibody, specifically the anti-cancer monoclonal antibody obinutuzumab (e.g., Gazyva®).

[0114] As used herein, a "subject" or "patient" can be any mammal, e.g., a human, a primate, a mouse, a rat, a dog, a cat, a cow, a horse, a pig, a sheep, a goat, a camel. In certain embodiments, the subject is a human. In further embodiments, the subject is a human and the NK cell fraction is a human NK cell fraction.

[0115] As used herein, a "subject in need of an NK cell fraction" is a subject in need of transplantation, transfusion, infusion or implantation of an NK cell fraction of the present invention to treat or ameliorate a disease, disorder or condition. In one embodiment, the subject has (has been diagnosed with) or is afflicted with a hematological disorder. In some embodiments, the hematological disorder is a cell proliferative disorder. In other embodiments, the hematological disorder is a hematological tumor.

[0116] As used herein, the term "risk of" or "probability of" refers to the likelihood of an event occurring. In some embodiments, the risk or probability of an event in an individual (e.g., engraftment or non-engraftment of an NK cell fraction, non-relapse mortality, etc.) refers to a risk calculated from comparative data of a treated group and a similar untreated group. In some embodiments, an increase or decrease in risk or probability reflects the difference between a treated group and a control group with respect to the outcome under consideration. In some embodiments, an increase or decrease in risk or probability of a particular event or condition is only relative and is not expressed in a numerical value.

[0117] The term "cell proliferative disorder" as used herein refers to a condition in which uncontrolled or abnormal growth of cells or both can lead to the development of an undesirable condition or disease (whether cancerous or not). Examples of cell proliferative disorders of the present invention include various conditions in which cell division is deregulated. The term "rapidly dividing cells" as used herein is defined as any cell that divides at a rate that exceeds that expected or observed between adjacent or juxtaposed cells in the same tissue. Cell proliferative disorders include precancer or precancerous conditions. Cell proliferative disorders include cancer. In certain embodiments, the methods provided herein are used to treat or alleviate symptoms of cancer. The term "cancer" encompasses solid tumors, as well as hematological tumors and / or malignancies. In certain embodiments, the hematological malignancy is non-Hodgkin's lymphoma (NHL) or multiple myeloma (MM).

[0118] In some embodiments, the methods, compositions, and kits of the present invention can be used to treat subjects of all ages, hi certain embodiments, the subject or patient is greater than 18 years old and less than 70 years old.

[0119] In some embodiments, the subject in need of an NK cell fraction may have multiple myeloma. In further embodiments, the multiple myeloma (MM) is characterized by at least one of the following criteria: (a) disease relapsed 2-18 months after initial autologous stem cell transplant, (b) disease relapsed at least 4 months after allogeneic stem cell transplant with no evidence of active graft-versus-host disease (GVHD), (c) relapsed / refractory disease after at least two lines of therapy including a proteasome inhibitor and an immunomodulatory drug (IMiD), (d) serum IgG, IgA, IgM or IgD myeloma protein (M protein) ≥ 0.5 g / dL, and (e) urinary M protein ≥ 200 mg / 24 collections. In some embodiments, the multiple myeloma is also characterized by serum IgE myeloma protein (M protein) ≥ 0.5 g / dL and has undergone plasma exchange ≥ 4 weeks prior to initiation of NK therapy. In some embodiments, a subject in need of an NK cell fraction has multiple myeloma characterized by two or more of the criteria described herein.

[0120] A subject in need of an NK cell fraction may have non-Hodgkin's lymphoma (NHL). In some embodiments, the non-Hodgkin's lymphoma is a CD20 positive B-cell NHL where CD20 expression has been confirmed by flow cytometry or immunohistochemistry. In further embodiments, the NHL is characterized by at least one of the following features: (a) relapsed / refractory disease that has failed conventional therapy, (b) disease that has relapsed at least 60 days after autologous stem cell transplant, (c) disease that has relapsed at least 4 months after allogeneic stem cell transplant without evidence of active graft-versus-host disease, and (d) measurable disease 1.5 cm or greater in diameter. In some embodiments, a subject in need of an NK cell fraction has an NHL that is characterized by two or more of the criteria described herein.

[0121] In some embodiments, subjects in need of the NK cell fraction may be further defined according to the following criteria: a Karnofsky performance score of at least 60% and adequate organ function, as defined below. a. Cardiac function: left ventricular ejection fraction (LVEF) ≥ 40% by echocardiogram, radionuclide scan, or cardiac MRI; b. Pulmonary function: Oxygen saturation at least 90% on room air; pulmonary function tests showing FVC and FEV1 ≥ 50% predicted for age; cDLCO ≥ 50% predicted; c. Renal function: Creatinine clearance test (Cockcroft-Gault formula) 40mL / min or more, or creatinine 1.5mg / dL or less, d. Liver function: Total serum bilirubin is generally less than 1.5 times the upper limit of the normal range, and liver transaminases (ALT and AST) are generally less than 3 times the upper limit of the normal range. e. Blood: total white blood cell (WBC) count ≥ 3000 / μL, absolute neutrophil count (ANC) ≥ 1000 / μL, platelet count ≥ 75000 / μL, and hemoglobin ≥ 8.0 g / dL (may be waived when abnormalities are due to disease-related bone marrow involvement), and f.Calcium (for multiple myeloma patients only): Corrected calcium level less than 11.5 mg / dL within 2 weeks prior to treatment enrollment.

[0122] In some embodiments, eligible subjects are required to discontinue prednisone or other immunosuppressive agents for at least 3 days prior to NAM-NK cell infusion (excluding pre-regimen premedication). Sexually active women of childbearing potential and men with partners of childbearing potential may be asked to agree to use effective contraception during treatment and for 4 months after completing treatment.

[0123] In some embodiments, a subject may be excluded as a candidate for treatment for any of the following reasons: 1. High titer donor-specific anti-HLA antibodies (MFI>1000). 2. Active, untreated CNS involvement. 3. Chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL) or high-grade lymphoma (Burkitt's lymphoma / lymphoblastic lymphoma). 4. Pregnant or breastfeeding. 5. For subjects with multiple myeloma: Females of childbearing potential must have a negative serum or urine pregnancy test (minimum sensitivity 25 IU / L or equivalent units of HCG) within 14 days of initiating treatment (24 hours prior to initiating anticancer antibody administration). 6. Marked baseline prolongation of the QT / QTc interval (e.g., display of a QTc interval greater than 500 ms). 7. New York Heart Association Functional Classification Class II or higher (Appendix III) or severe cardiac arrhythmias likely to increase the risk of cardiac complications from cytokine therapy (e.g., ventricular tachycardia, frequent ectopic ventricular activation, or supraventricular tachyarrhythmias requiring chronic treatment). 8. Active autoimmune disease requiring immunosuppressive therapy. 9. History of severe asthma currently receiving chronic medication (history of mild asthma requiring only inhaled steroids is eligible). 10. New or progressive pulmonary infiltrates on screening chest x-ray or chest CT scan [unless study by a pulmonologist is permitted. Infiltrates due to infection must be stable / improving (with associated clinical improvement) after 1 week of appropriate treatment (4 weeks if fungal infection is presumed or proven)]. 11. Active, uncontrolled bacterial, fungal, or viral infections – All prior infections must have resolved with optimal treatment. 12. Known hypersensitivity to any of the therapeutic agents used in the methods of the invention. 13. MM patients only: prior radiation therapy within 2 weeks, surgery within 4 weeks, or chemotherapy within 3 weeks (within 6 weeks for melphalan or monoclonal antibodies) prior to administration of the NK cell fraction of the present invention. 14. Administration of an investigational drug within 14 days prior to initiation of treatment with NK cell fraction.

[0124] In some embodiments, NK cell donors (e.g., apheresis candidates identified as HLA half-matched or HLA mismatched, related or unrelated) are selected according to the following criteria. 1. HLA-half-matched or HLA-mismatched related donor / recipient combinations based on a minimum of intermediate resolution DNA-based class I typing at the A and B loci (at least two of four class I alleles) and the absence of recipient anti-HLA antibodies (with MFI≦1000) to the selected donor. 2. 12-70 years: Priority is given to age (<35 years), followed by HLA matching (half-matched donor, and if none is available, a fully mismatched donor). 3.Weight at least 40 kilograms. 4. General health status as assessed by a health care provider. 5. Adequate organ function defined as follows: Blood: hemoglobin, WBC, platelets within 10% of the upper and lower limits of the test normal range (hemoglobin values ​​are gender-specific), Liver: ALT less than 2x upper limit of normal, and Renal: serum creatinine less than 1.8 mg / dL. 6. Completion of donor infectious disease testing panel (including CMV antibody, Hepatitis B surface antigen, Hepatitis B core antibody, Hepatitis C antibody, HIV PCR, HIV1 / 2 antibody, HTLVA1 / 2 antibody, rapid plasma repeat (RPR) treponemes, Trypanosoma cruzi (T. Cruzi), HCV by NAT, HIV by NAT, and WNV (West Nile Virus) per NAT or current panel); must be negative for HIV and active Hepatitis B. 7. Not pregnant: Women of childbearing potential must have a negative pregnancy test within 7 days of apheresis. 8. Able and willing to undergo apheresis. 9. Voluntary written consent (using an assent form for donors under 18 years of age).

[0125] In some embodiments, the subject in need undergoes myeloablative therapy or conditioning treatment. In certain embodiments, the subject undergoes myeloablative therapy or conditioning treatment prior to, concurrent with, and after transplantation or administration of the compositions of the invention. Myeloablative therapy or conditioning treatment can include total body irradiation (TBI), immunotherapy, and chemotherapy and / or immunosuppressive therapy.

[0126] In some embodiments, implantation or administration of a composition of the invention may be provided as an adjunct to, or in combination with, other therapeutic procedures or compositions.

[0127] Combination therapy In some embodiments, a subject in need thereof is treated with a combination of an expanded CD3-depleted NK cell fraction as described herein and an additional cancer therapy. In some embodiments, the additional cancer therapy comprises a cytotoxic agent and / or a non-cytotoxic agent. "Cytotoxic agent" refers to a substance that inhibits or prevents the function of a cell and / or causes the destruction of a cell. This term includes radioisotopes (e.g., 131 I, 125 I, 90 Y and 186Re), chemotherapeutic agents, and toxins (enzymatically active toxins of bacterial, fungal, plant or animal origin or synthetic toxins, or fragments thereof). Non-cytotoxic agents refer to substances that do not inhibit or prevent cellular function and / or cause destruction of cells. "Non-cytotoxic agents" may also include those that can be activated to become cytotoxic. Non-cytotoxic agents may also include beads, liposomes, matrices, or particles (see, for example, U.S. Patent Publication Nos. 2003 / 0028071 and 2003 / 0032995, which are incorporated herein by reference). Such agents may be bound, coupled, linked, or associated with the expanded CD3-depleted NK cell fraction compositions described herein.

[0128] In some embodiments, known cancer therapeutics are administered together with the compositions described herein. In some cases, a subject in need of treatment is treated with the expanded CD3-depleted NK cell fraction described herein together with one or more additional agents that target cancer cells. Highly suitable agents include agents that promote DNA damage in cancer cells, e.g., double-strand breaks in cellular DNA. Any form of DNA damaging agent known to those skilled in the art can be used. DNA damage can typically be created by radiation therapy and / or chemotherapy. DNA damaging agents are also referred to as genotoxic agents. As used herein, "together" shall mean that the expanded CD3-depleted NK cell fraction is administered to a subject simultaneously (contemporaneously or at a separate but close interval) with one or more additional therapies, or administered before or after administration of one or more additional therapies.

[0129] Examples of radiation therapy include, but are not limited to, external radiation therapy and internal radiation therapy (also called brachytherapy). Energy sources for external radiation therapy include x-rays, gamma rays, and particle beams, while energy sources for internal radiation therapy include radioactive iodine (iodine 125 or iodine 131 ),strontium 89and radioactive isotopes of phosphorus, palladium, cesium, indium, phosphate, or cobalt. Methods for administering radiation therapy are well known to those skilled in the art.

[0130] Particularly useful DNA damaging chemotherapeutic agents include, but are not limited to, busulfan (Myelan), carboplatin (Paraplatin), carmustine (Carmustme) (BCNU), chlorambucil (Leukeran), cisplatin (Pratomol), cyclophosphamide (Cytoxan, Neosar), dacarbazume (DTIC-Dome), ifosfamide (Ifex), lomustme (CCNU), mechlorethamine (nitrogen mustard, Mustargen), melphalan (Alkeran), and procarbazine (Matulane).

[0131] Several other chemotherapeutic agents, either individually or in combination, can be used in the methods described herein, including methotrexate, vincristine, adriamycin, cisplatin, non-sugar-containing chloroethylnitrosourea, 5-fluorouracil, mitomycin C, bleomycin, doxorubicin, dacarbazine, taxol, flagellin, meglamin GLA, valrubicin, carmstein, and polyferposan, MMI270, BAY 12-9566, RAS farnesyltransferase inhibitors, farnesyltransferase inhibitors, MMP, MTA / LY231514, LY264618 / romotexol, Gramorec, CI-994, TNP-470, hicamucine / topotecan, PKC412, valspodar / PSC833, novantrone / mitroxantrone, metholet / suramin, batimastat, E7070, BCH-4556, CS-682, 9-AC, AG3340, AG3433, Incel / VX-710, VX-853, ZD0101, ISI641, ODN 698, TA 2516 / marimistat, BB2516 / marimistat, CDP 845, D2163, PD183805, DX8951f, Lemonal DP 2202, FK 317, Picibanil / OK-432, AD 32 / Valrubicin, Methastron / Strontium derivative, Temodar / Temozolomide, Everset / Liposomal doxorubicin, Eutaxan / Paclitaxel, Taxol / Paclitaxel, Xeloda (Xeload) / Capecitabine, Furtulon / Doxifluridine, Cyclopax / Oral paclitaxel, Oral taxoid, SPU-077 / Cisplatin, HMR 1275 / Flavopiridol, CP-358 (774) / EGFR, CP-609(754) / RAS oncogene inhibitors, BMS-182751 / oral platinum, UFT (tegafur / uracil), Ergamisole / levamisole, Eniluracil / 776C85 / 5FU enhancer, Campto / levamisole, Camptosar / irinotecan, Tumodex / latitrexed, Lostatin / cladribine, Pachyex / paclitaxel, Doxil / liposomal doxorubicin, Kaelix / liposomal doxorubicin, Fludara / fludarabine, Farmarubicin / epurubicin, DepoCyt, ZD1839, LU 79553 / bisnaphthalimide, LU 103793 / dolastein, Kaetix / liposomal doxorubicin, Gemzar / gemcitabine, ZD 0473 / anormed, YM 116, iodine seeds, CDK4 and CDK2 inhibitors, PARP inhibitors, D4809 / dextrose, Ifes / Mesnex / ifosamide, Bumon / teniposide, Paraplatin / carboplatin, Platinol / cisplatin, Bepesid / etoposide, ZD9331, texotere / docetaxel, prodrugs of guanine arabinoside, taxane analogs, nitrosoureas, alkylating agents such as melpheran and cyclophosphamide, aminoglutethimide, asparaginase, busulfan, carboplatin, chloromubucil, cisplatin, cytarabine HCl, dactinomycin, danrubicin HCl, estramustine sodium phosphate, etoposide (VP16-213), floxuridine, fluorouracil (5-FU), flutamide, hydroxyurea (hydroxycarbamide), isofamide, interferon alpha-2a, alpha-2b, leuprolide acetate (LHRH-releasing factor analog), lomustine (CCNU), mechloroethamine HCl (NaCl), These include, but are not limited to, 2'-deoxy-2'-nitrogen mustard, mercaptopurine, mesna, mitotane (o.p'-DDD), mitoxantrone HCl, octreotide, plicamycin, procarbazine HCl, streptozocin, tamoxifen citrate, thioguanine, thiotepa, vinblastine sulfate, amsacrine (m-AMSA), azacitidine, erythropoietin, hexamethylmelamine (HMM), interleukin 2, mitoguazone (methyl GAG), methylglyoxal bisguanylhydrazone (MGBG), pentostatin (2' deoxyformycin), semustine (methyl CCNU), teniposide (VM-26), and vindesine sulfate.

[0132] Additionally, the following agents may be useful in the present invention:alkylating agents such as carboplatin and cisplatin, nitrogen mustard alkylating agents, nitrosourea alkylating agents such as carmustine (BCNU), antimetabolites such as methotrexate, folinic acid, purine analog antimetabolites, pyrimidine analog antimetabolites such as mercaptopurine, fluorouracil, and gemcitabine (Gemzar®), hormonal antineoplastic agents such as goserelin, leuprolide, and tamoxifen, natural antineoplastic agents such as aldesleukin, interleukin-2, docetaxel, etoposide (VP-16), interferon alpha, paclitaxel (Taxol®), and tretinoin (ATRA), antibody-based natural antineoplastic agents such as bleomycin, ductomycin, daunorubicin, doxorubicin, daunomycin, and the mitomycins, including mitomycin C, and vinblastine. vinca alkaloid antineoplastic agents such as vincristine, vindesine, hydroxyurea, acetone, adriamycin, ifosamide, enocitabine, epitiostanol, aclarubicin, ancitabine, nimustine, procarbazine hydrochloride, carboquone, carboplatin, carmofur, chromomycin A3, antitumor polysaccharides, antitumor platelet factors, cyclophosphamide (Cytoxan®), sizofiran, cytalbine (cytosine arabinoside), dacarbazine, thymosin, thiotepa, tegafur, dolastatin, dolastatin analogs such as auristatin, CPT-11 (irinotecan), mitoxantrone, vinorelbine, teniposide, aminopterin, carbomycin, esperamicin (see, for example, U.S. Pat. No. 4,675,187, which is incorporated herein by reference), neocarzinostatin, OK 432, bleomycin, furtulon, bromodeoxyuridine (broxundine), busulfan, honban, peplomycin, bestatin (Ubenimex®), interferon-0, mepitiostane, mitobromthol, melphalan, laminin peptides, lentinan, Coriolus versicolor extract, tegafur / uracil, estramustine (estrogen / mechlorethamine), thalidomide and lenalidomide (Revulmid®).

[0133] Other suitable chemotherapeutic agents include proteasome inhibitors. Proteasome inhibitors block the activity of the proteasome, a cellular complex that breaks down proteins, especially short-lived proteins involved in cell maintenance, growth, division, and cell death. Examples of proteasome inhibitors include bortezomib (Velcade®), lactacystin (AG Scientific, Inc., San Diego, Calif.), MG132 (Biomol International, Plymouth Meeting, Pa.), PS-519, eponemycin, epoxomicin, aclacinomycin A, dipeptide benzamide, CVT-63417, and vinylsulfone tripeptide proteasome inhibitors.

[0134] In some embodiments, the methods described herein are combined with one or more other cancer treatments, including cancer immunotherapy. Cancer immunotherapy is the use of the immune system to reject cancer. The main premise is to stimulate the subject's immune system to attack the tumor cells responsible for the disease. This can be either by immunizing the subject, in which case the subject's own immune system will recognize the tumor cells as targets to be destroyed, or by administering a therapeutic agent (e.g., an antibody) as a drug, in which case the subject's immune system will be recruited by the therapeutic agent to destroy the tumor cells. Cancer immunotherapy includes antibody-based therapy and cytokine-based therapy.

[0135] Cytokine-based cancer therapy utilizes one or more cytokines to regulate a subject's immune response. Non-limiting examples of cytokines useful in cancer treatment include interferon alpha (IFN-alpha), interleukin 2 (IL-2), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin 12 (IL-12).

[0136] To facilitate tumor targeting and antibody-dependent cellular cytotoxicity (ADCC), in some embodiments, disease-specific monoclonal antibodies can be administered to a subject in need thereof in conjunction with (e.g., prior to, simultaneously with, or after) administration of an expanded CD3-depleted NK cell fraction as described herein.

[0137] A non-limiting list of monoclonal antibodies suitable for use with the methods and expanded CD3-depleted NK cell fractions and compositions of the present invention, their cancer cell targets, and the specific diseases for which they are currently approved for use are provided in Table 1 below.

[0138] [Table 1-1]

[0139] [Table 1-2]

[0140] Thus, in some embodiments, when the hematological malignancy is multiple myeloma, one or more MM-specific monoclonal antibodies (such as elotuzumab) are administered to a subject in need thereof. An example of a dosage of elotuzumab useful in the methods of the present invention is 10 mg / kg body weight per subject. When the hematological malignancy is NHL, one or more NHL-specific monoclonal antibodies (such as rituximab) are administered to a subject in need thereof. An example of a dosage of rituximab useful in the methods of the present invention is 375 mg / m2 per subject. 2 It is.

[0141] In some particular embodiments where the hematological malignancy is a B cell malignancy (e.g., lymphoma, leukemia), the B cell specific monoclonal antibody is an anti-CD20 monoclonal antibody. In particular embodiments, the anti-CD20 monoclonal antibody is obinutuzumab. Although dosages and administration schedules often vary depending on the disease being treated, the severity, patient characteristics observed by the treating physician, and response to treatment (see "dosage" and "obinutuzumab" at the "drugs(dot)com" website for current practice), an exemplary dose of obinutuzumab can be 100-1000 mg per infusion. An exemplary obinutuzumab dosing regimen, for example, for CLL, can be: Cycle 1, 100 mg IV over 4 hours at 25 mg / hour on day 1 with no increase in infusion rate, transitioning to 900 mg IV on day 2, 1000 mg IV on days 8 and 15, and 1000 mg IV through cycles 2-6 for six 28-day treatment cycles.

[0142] An exemplary dosing regimen for follicular lymphoma is also based on six 28-day treatment cycles, with a total dose of 1000 mg IV. Specific information regarding premedications and adjuvants for antibody therapy with obinutuzumab is available from the manufacturer's specifications and on the "drugs(dot)com" website under "dosage" and "obinutuzumab." In certain embodiments, combination therapy with obinutuzumab is provided for subjects with relapsed / refractory lymphoma.

[0143] Thus, in some aspects of the invention, there is provided a method of treating a hematological disorder in a human subject in need thereof, comprising administering obinutuzumab to the subject, administering at least one immunosuppressant to the subject, and transplanting an expanded CD3-depleted, HLA-half-matched or HLA-mismatched NK cell fraction into a subject in need thereof, the expanded CD3-depleted, HLA-half-matched or HLA-mismatched NK cell fraction having been expanded by ex vivo culture with nutrients, serum, IL-15, and nicotinamide, particularly 1.0 mM to 10 mM nicotinamide, according to the methods of the invention, thereby providing a method of treating a hematological disorder in a subject. In further particular embodiments, the method further comprises administering IL-2 to the subject.

[0144] In another specific embodiment, the method of the present invention comprises steps of preparing a transplantable NK cell fraction for use in combination therapy with an anti-CD20 monoclonal antibody, the steps comprising obtaining an HLA half-matched or HLA mismatched CD3-depleted NK cell fraction, ex vivo culturing the CD3-depleted NK cell fraction under conditions allowing cell proliferation, specifically under conditions providing nutrients, serum, IL-15 and 1.0 mM to 10 mM nicotinamide, adding fresh nutrients, serum, IL-15 and nicotinamide to the CD3-depleted NK cell fraction after 8 to 10 days of ex vivo culture to produce an expanded CD3-depleted NK cell fraction, recovering the expanded CD3-depleted NK cell fraction after 14 to 16 days of ex vivo culture, and washing and concentrating the expanded CD3-depleted NK cell fraction to obtain transplantable NK cells for transplantation into a subject.

[0145] In certain embodiments, the disease-specific monoclonal antibody treatment involves three administrations of monoclonal antibody, i.e., the first administration 10 days prior to administration (infusion, transplantation) of the NK cell fraction, the second administration 3 days prior to administration (infusion, transplantation) of the NK cell fraction, and the third (final) administration 11 days after administration (infusion, transplantation) of the NK cell fraction, but in some embodiments, approximately one week after the final (second) administration (infusion, transplantation) of the NK cell fraction. In certain embodiments, the disease-specific monoclonal antibody is administered 9-11 days prior to, 3 days prior to, and 11 days after the first administration of the expanded CD3-depleted HLA half-matched or mismatched NK cell fraction.

[0146] Standard guidelines are followed for monitoring of infusions, response to monoclonal antibody administration, and toxicity. Elotuzumab is usually administered with a premedication regimen including dexamethasone, an H1 blocker such as diphenylhydramine, an H2 blocker such as ranitidine, and acetaminophen prior to the start of the infusion.

[0147] In some embodiments, the subject in need undergoes a conditioning regimen of immunosuppressive therapy prior to administration (infusion, transplantation) of the NK cell fraction. Suitable immunosuppressants include, but are not limited to, alkylating agents, purine analogs, antimetabolites, and the like. Some immunosuppressants are also considered chemotherapeutic immunosuppressants. In certain embodiments, the immunosuppressive therapy comprises administration of cyclophosphamide and fludarabine. An example dosage of cyclophosphamide useful in the methods of the present invention is 40 mg / kg body weight per subject (patient), and an example dosage of fludarabine useful in the methods of the present invention is 25 mg / m2 per subject (patient). 2 In a specific embodiment, administration of cyclophosphamide is performed 5 days prior to administration (transplantation, infusion) of the expanded CD3-depleted HLA-half-matched or HLA-mismatched NK cells, and administration of fludarabine is performed 5 days, 4 days, and 3 days, respectively, prior to administration (transplantation, infusion) of the expanded CD3-depleted HLA-half-matched or HLA-mismatched NK cells. Alternatively, administration of fludarabine and cyclophosphamide can be timed so that the final administration of the immunosuppressant is completed 2 days or 3 days prior to the start of administration of the NK cell fraction.

[0148] According to the methods of the invention, in some embodiments, the NK cell fraction is administered twice to a subject in need thereof, in certain embodiments, the administration of the NK cell fraction comprises administering a first dose of the expanded CD3-depleted, HLA-half-matched or HLA-mismatched NK cell fraction, followed two days later by administering a second dose of the expanded CD3-depleted, HLA-half-matched or HLA-mismatched NK cell fraction.

[0149] In some embodiments, the NK cell fraction for administration to a subject (patient) is 1×10 7 pieces / kg~5×10 8 pieces / kg, 2×10 7 pieces / kg~2×10 8 pieces / kg, 5×10 7 pieces / kg~1×10 8 Pieces / kg or 2 x 10 7 pieces / kg~5×10 7 In some embodiments, the sum of the first dose and the second dose of NK cell fraction comprises 2×10 cells / kg of expanded CD3-depleted HLA-half-matched or HLA-mismatched NK cells. 7 pieces / kg~2×10 8 In some embodiments, the first and second doses of the NK cell fraction each comprise 1×10 cells / kg of expanded CD3-depleted HLA half-matched or HLA-mismatched NK cells. 7 cells / kg of expanded CD3-depleted HLA-half-matched or mismatched NK cells, with a total dose of expanded CD3-depleted HLA-half-matched or mismatched NK cells of 2 × 10 7 In another embodiment, the first and second doses of the NK cell fraction are each 5×10 7 cells / kg of expanded CD3-depleted HLA half-matched or mismatched NK cells, with a total dose of expanded CD3-depleted HLA half-matched or mismatched NK cells of 1 × 10 8 In yet another embodiment, the first and second doses of the NK cell fraction are each 1×10 8 cells / kg of expanded CD3-depleted HLA-half-matched or mismatched NK cells, with a total dose of expanded CD3-depleted HLA-half-matched or mismatched NK cells of 2 × 10 8 Pieces / kg.

[0150] Administration of the NK cell fraction is typically performed as a treatment for hospitalized patients. Administration of the NK cell fraction described herein is performed by infusion, and in certain embodiments, the NK cell fraction is infused into the subject (patient) within 1 hour of arrival of the transplantable NK cell fraction and within 10 hours of final product release of the washed and enriched expanded CD3-depleted NK cell fraction. In certain embodiments, the washed and enriched expanded CD3-depleted NK cell fraction is kept at room temperature until administration and is not refrigerated prior to use.

[0151] Thus, in some embodiments, the expanded CD3-depleted, HLA-half-matched or HLA-mismatched NK cell fraction is administered to the subject within 1 hour of providing the NK cell fraction for transplant and within 10 hours of final product release of the NK cell fraction. In some embodiments, the expanded CD3-depleted, HLA-half-matched or mismatched NK cell fraction is administered to the subject by intravenous infusion, without the use of a filter or pump, over a period of no less than 15 minutes and no more than 60 minutes per infusion.

[0152] In some embodiments, a subject in need thereof receives a supportive regimen of interleukin 2 (IL-2) following administration of the NK cell fraction.

[0153] In some embodiments, subcutaneous (SC) administration of IL-2 is at a dose of 6 MU (for patients weighing less than 45 kilograms, the IL-2 dose is 3 MU / m 2 ), on the day of the first NK cell fraction administration (transplant, infusion), on the day of the second NK cell fraction administration (transplant, infusion), and two days after the second NK cell fraction administration (transplant, infusion). In some embodiments, IL-2 is administered on the day of NAM-NK cell infusion, no later than 4 hours after NAM-NK cell infusion. In certain embodiments, the first two IL-2 administrations are administered as part of the hospitalization for the NK cell infusion. The third IL-2 administration can be administered in an outpatient setting. Thus, in certain embodiments, IL-2 is administered after infusion of expanded CD3-depleted NK cells, at a dose of 6×10 6 units of IL-2, (i) on the day of infusion of the expanded CD3-depleted HLA-half-matched or mismatched NK cells; and (ii) 2 days after infusion of the expanded CD3-depleted HLA-half-matched or mismatched NK cells; and (iii) 4 days after infusion of the expanded CD3-depleted, HLA-half-matched or -mismatched NK cells.

[0154] Additionally, if a patient experiences grade 2 or greater IL-2 infusion-related toxicity after the first or second dose, administration of IL-2 can be delayed for up to 48 hours. If toxicity resolves to grade 1 or better within 48 hours, IL-2 can be administered for all planned doses, but the administration of remaining doses should be separated by at least 24 hours.

[0155] In some embodiments, subjects may receive any or all of the following: infusion support (e.g., diphenylhydramine or dexchlorpheniramine, hydrocortisone and acetaminophen), supportive cytokines (e.g., G-CSF), blood products as needed, antiviral, antibacterial, PCP and / or fungal prophylaxis, CMV, EBV and HHV6 monitoring, and IV immune globulin as needed.

[0156] In some embodiments, the subject receives any or all of the additional treatments for the hematological disorder, which can be a treatment selected from the group consisting of immunosuppressive treatment, chemotherapy, and radiation therapy.

[0157] Accordingly, in some embodiments, there is provided a method of treating a hematological disorder in a subject in need thereof, comprising: (i) obtaining a HLA-half-matched or HLA-mismatched CD3-depleted NK cell fraction for a subject; (ii) ex vivo culturing of the CD3-depleted NK cell fraction under conditions that allow cell proliferation, the conditions including supplying nutrients, serum, IL-15, and 1.0 mM to 10 mM nicotinamide; (iii) supplementing the CD3-depleted NK cell fraction with fresh nutrients, serum, IL-15 and nicotinamide 8-10 days after step (ii) to obtain a proliferated CD3-depleted NK cell fraction; (iv) recovering the expanded CD3-depleted NK cell fraction 14 to 16 days after step (ii); (v) washing and concentrating the expanded CD3-depleted NK cell fraction of step (iv) to obtain a transplantable NK cell fraction for transplantation into the subject; (vi) administering an anti-cancer monoclonal antibody to a subject; (vii) administering to the subject at least one immunosuppressant agent; (viii) transplanting the expanded CD3-depleted, HLA-half-matched or mismatched NK cell fraction of (v) into a subject in need thereof; (ix) administering IL-2 to the subject to treat the blood disorder in the subject; A method is provided that includes:

[0158] In some embodiments, the NK cell fraction infusion solution is stored in a bag at 8-20° C. until its use (e.g., transplantation, infusion). In some specific embodiments, prior to transplantation (administration, infusion) of the NK cell fraction, a safety assessment of the subject in need thereof is performed on the day of NK cell transplantation, which typically includes a physical exam, CBC, blood chemistry (e.g., at least serum creatinine, total bilirubin, alkaline phosphatase, AST, ALT, and magnesium), vital signs: weight, temperature, blood pressure, pulse and respiratory rate, and administration of concomitant medications (including RBC and platelet transfusions).

[0159] Infusion of the expanded NK cell fraction into a subject in need thereof is typically performed by infusion via the patient's central venous catheter, subject to the limitations of individual site practice.

[0160] The method for treating blood diseases of the present invention can be used to treat blood tumors, including but not limited to MM and NHL. As used herein, the term "treatment of blood disease" or "treatment of blood tumor" refers to alleviating symptoms or signs of blood disease. In some embodiments, the treatment of blood disease or blood tumor is evaluated according to, but not limited to, suppression of symptoms over time, improvement of clinical parameters, reduction in hospitalization, and reduction in risk of recurrence or death.

[0161] In some embodiments, infusion of an expanded NK cell fraction as described herein increases the likelihood of successful in vivo expansion of the infused NK cells as compared to infusion of NK cells that have not been cultured and / or administered according to the methods described herein, in some embodiments, successful in vivo expansion is measured at days 7 and 14 after infusion.

[0162] In other embodiments, infusion of an expanded NK cell fraction as described herein enhances NK cell function in the peripheral blood of a subject as compared to infusion of NK cells that have not been cultured and / or administered according to the methods described herein, in some embodiments, NK cell function is measured 7 and 14 days after infusion.

[0163] According to some embodiments of the methods of the invention, infusion of an expanded NK cell fraction as described herein increases the probability of a favorable disease response infusion of the NK cell fraction, compared to infusion of NK cells that have not been cultured and / or administered according to the methods described herein. In some embodiments, NK cell function is measured 28 days after infusion and one year after infusion. In a particular embodiment, the hematological malignancy is NHL, and the disease response criteria for NHL are assessed according to the International Working Group Response Criteria for NHL (see Cheson, et al, J Clin Oncol 2014;32:3059-68 for details). In a further particular embodiment, the hematological malignancy is MM, and the disease response criteria for MM are assessed according to the following criteria:

[0164] Uniform response criteria for plasma cell leukemia Stringent complete response (sCR): In addition to the CR (defined below), an sCR requires all of the following: Absence of malignant plasma cells in bone marrow as determined by flow cytometry Absence of malignant plasma cells in peripheral blood as determined by flow cytometry Normal free light chain ratio (FLC)

[0165] Complete remission (CR): CR requires all of the following: Fewer than 5% plasma cells in bone marrow aspirate No plasma cells in peripheral blood Absence of the original monoclonal paraprotein in serum and urine as determined by routine electrophoresis and immunofixation No extramedullary disease

[0166] Very good partial response (VGPR) VGPR requires all of the following: Fewer than 5% plasma cells in bone marrow aspirate No plasma cells in peripheral blood - Suppression of serum monoclonal paraproteins by 90% or more and paraproteins of 100mg / 24 hours 2 less than No extramedullary disease

[0167] Partial response (PR) Partial remission requires all of the following: - Plasma cells in bone marrow aspirate are 5%-25% -1% to 5% of plasma cells in peripheral blood 200 mg / 24 hours with 50% or more suppression of serum monoclonal paraproteins and 90% or more suppression of urinary monoclonal paraproteins in 24 hours 3 less than -50% or greater reduction in size of extramedullary lesions

[0168] Steady State (SD) Patients who do not meet the criteria for sCR, CR, VGPR, PR, or progressive disease (defined below) are considered to be in stable disease (SD). If serum and urinary M protein are not measurable, a normal serum κ / λ FLC ratio is also required. If serum and urinary M protein cannot be measured, a 90% or greater reduction in the difference between involved and uninvolved FLC levels is required in place of M protein. If serum and urinary M protein cannot be measured, a 50% or greater reduction in the difference between involved and uninvolved FLC levels is required in place of M protein.

[0169] progressive disease Progression from CR or sCR requires one or more of the following: Increase in plasma cells in bone marrow aspirate of more than 25% or absolute increase of 10% or more Absolute increase in peripheral blood plasma cells >5% Increase in serum monoclonal paraprotein levels by more than 25% with an absolute increase of 5 g / L or more Increase in 24-hour urinary protein electrophoresis of more than 25% with an absolute increase of at least 200 mg / 24 hours Hypercalcemia -Clear increase in lytic bone lesions A clear increase in the size or number of extramedullary lesions

[0170] In some embodiments, the products, compositions or kits of the invention further comprise instructions for administering the expanded NK cell fraction suitable for transplant to a subject in need thereof.

[0171] In some embodiments of the products, compositions, or kits of the invention, an expanded NK cell fraction suitable for transplantation into a subject in need thereof has a total viable NK cell count of at least 7×10 8 In some embodiments, an expanded NK cell fraction suitable for transplantation into a subject in need thereof has a total viable NK cell count of at least 8×10 8 Pieces, at least 10 x 10 8Pieces, at least 15 x 10 8 Pieces, at least 20 x 10 8 or at least 25 x 10 8 There are 10.

[0172] The selected cell populations of the present invention may be provided with the culture medium in which they are contained, may be isolated from the culture medium, or may be combined with a pharma- ceutically acceptable carrier and additional agents that may promote cell engraftment and / or organ function (e.g., immunosuppressants, antibiotics, growth factors). Thus, the cell populations of the present invention may be administered in a pharma- ceutically acceptable carrier or diluent, such as sterile saline or aqueous buffer. The use of such carriers and diluents is well known in the art.

[0173] The compositions of the invention may, if desired, be provided in a pack or dispenser device, such as an FDA approved kit or article of manufacture, which may contain one or more unit dosage forms containing the active ingredient (e.g., cells). The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser device may also be accompanied by a notice in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals, which notice reflects approval by the agency of the form of the composition for human or animal administration. Such notice may include, for example, labeling approved by the U.S. Food and Drug Administration for prescription drugs or an approved product insert. Compositions comprising the formulations of the invention formulated in a pharma- ceutically acceptable carrier, as described in further detail above, may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.

[0174] The cells prepared according to the method of the present invention may be administered to a subject as they are, or may be administered to a subject as a pharmaceutical composition in which the cells are mixed with a suitable carrier or excipient.

[0175] As used herein, a "pharmaceutical composition" refers to a preparation of one or more active ingredients described herein that includes other chemical components, such as physiologically suitable carriers or excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

[0176] Hereinafter, the terms "physiologically acceptable carrier" and "pharmaceutical acceptable carrier" (which may be used interchangeably) refer to a carrier or diluent that does not cause significant irritation to an organism and does not abolish the biological activity and properties of the administered compound. These terms include adjuvants.

[0177] As used herein, the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Techniques for drug formulation and administration can be found in the latest edition of "Remington's Pharmaceutical Sciences" (Mack Publishing Co., Easton, Pa.), which is incorporated herein by reference.

[0178] Thus, pharmaceutical compositions for use in accordance with the present invention can be formulated in a conventional manner using one or more physiologically acceptable carriers, including excipients and auxiliaries, which facilitate processing of the active ingredient into a pharma- ceutically usable preparation. The appropriate formulation will depend on the chosen route of administration.

[0179] For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.

[0180] Pharmaceutical compositions suitable for use in the context of the present invention include compositions containing an effective amount of the active ingredient to achieve its intended purpose. More specifically, a "therapeutically effective amount" refers to an amount of the active ingredient (e.g., expanded CD3-depleted NK cells) effective to prevent, reduce or ameliorate symptoms of a disorder (e.g., leukemia, multiple myeloma) or prolong the survival of a subject being treated.

[0181] Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0182] Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures, or in experimental animals. Data obtained from such in vitro, cell culture assays, and animal studies can be used in formulating a range of dosages for use in humans. Dosages may vary depending on the dosage form employed and the route of administration utilized. The exact formulation, route of administration, and dosage can be selected by the individual physician in view of the patient's condition (see, for example, Fingl, E. et al. (1975), "The Pharmacological Basis of Therapeutics," Ch. 1, p.1.).

[0183] Depending on the severity and responsiveness of the condition being treated, single or multiple administrations may be employed. The amount of a composition administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

[0184] As used herein, "about" refers to ±10%.

[0185] The terms "comprises," "comprising," "includes," "including," "having" and conjugations thereof mean "including but not limited to."

[0186] The term "consisting of" means "including and limited to."

[0187] The term "consisting essentially of" means that a composition, method, or structure may include additional ingredients, steps, and / or moieties, provided that the additional ingredients, steps, and / or moieties do not materially alter the basic and novel characteristics of the claimed composition, method, or structure.

[0188] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, "a compound" or "at least one compound" includes a plurality of compounds, and may also include mixtures thereof.

[0189] Throughout this application, various embodiments of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and is not an inflexible limitation of the scope of the invention. Thus, the description of a range should be considered to specifically disclose all of the possible subranges and individual numerical values ​​within that range. For example, description of a range such as 1 to 6 specifically discloses not only subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., but also individual numerical values ​​within that range, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the magnitude of the range.

[0190] Whenever a numerical range is given herein, it is intended to include any recited number (fractional or integer) within the range given. The phrases "range between" a first indicated number and a second indicated number and "range from" a first indicated number to a second indicated number are used interchangeably herein and are intended to include the first indicated number and the second indicated number, and all fractional and integer numbers therebetween.

[0191] As used herein, the term "method" means manner, means, techniques, and procedures for accomplishing a given task, including, but not limited to, those known to practitioners in the fields of chemistry, pharmacology, biology, biochemistry, and medicine, or those that can be readily developed by practitioners from known manners, means, techniques, and procedures.

[0192] It will be understood that certain features of the invention that are, for clarity, described in the context of separate embodiments, may also be provided in a single embodiment in any combination of those features. Conversely, several features of the invention that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or with respect to other described embodiments as appropriate. Certain features described in the context of various embodiments should not be construed as essential to that embodiment, unless the particular embodiment is inoperable without that element.

[0193] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples. EXAMPLES

[0194] Reference is now made to the following examples which, together with the above descriptions, further illustrate some embodiments of the invention and are not intended to limit the invention.

[0195] The nomenclature used herein and the laboratory procedures used in the present invention generally include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are fully explained in the literature, e.g., "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, RM, ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York. (1998), U.S. Patent Nos. 4,666,828, 4,683,202, 4,801,531, 5,192,659, and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, JE, ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, NY (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan JE, ed. (1994); Stites et al.(eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", WH Freeman and Co., New York (1980). Available immunoassays have been described extensively in the patent and scientific literature, e.g., in U.S. Pat. Nos. 3,791,932, 3,839,153, 3,850,752, 3,850,578, 3,853,987, 3,867,517, 3,879,262, 3,901,654, 3,935,074, 3,984,533, 3,996,345, 4,034,074, 4,098,876, 4,879,219, 5,011,771, and 5,281,521; "Oligonucleotide Synthesis" Gait, MJ, ed. (1984); "Nucleic Acid Hybridization" Hames, BD, and Higgins SJ, eds. (1985), "Transcription and Translation" Hames, BD, and Higgins SJ, eds. (1984), "Animal Cell Culture" Freshney, RI, ed. (1986), "Immobilized Cells and Enzymes" IRL Press, (1986), "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press, "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990), Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996). All of the above references are hereby incorporated by reference as if fully set forth herein. Other general references are provided throughout this specification. The procedures described therein are believed to be well known in the art and are provided for the convenience of the reader. All of the information contained therein is hereby incorporated by reference.

[0196] Materials and Experimental Methods Blood cell samples and T cell depletion Blood cells were collected by apheresis from healthy donors on day 0. Red blood cells (RBCs) were lysed by washing with ACK buffer (Gibco, Dublin, Ireland). CD3+ cells were depleted using CliniMACS and CD3 reagent (Miltenyi 273-01) (Miltenyi Biotec, Gladbach, Germany) according to the manufacturer's recommendations.

[0197] Ex vivo culture: CD3+ depleted NK cells were seeded at 280 × 10 in MEMa w / nucleosides (HyClone, South Logan, UT) containing gentamicin (Octapharma, Lachen, Switzerland), 2 mM L-glutamine (Biologica Industries, Inc., West Sacramento, CA), 10% AB human serum (Gemini Bio Products, Inc., West Sacramento, CA), 5 mM nicotinamide, and 20 ng / mL IL-15 (Miltenyi, Gladbach, Germany). 6Cells were seeded into G-REX100MCS cell culture flasks (Wilson Wolf, St. Paul, MN) containing 800 mL of medium and incubated at 5% CO2 and 37°C in a humidified incubator. On day 8, the cell population was split by shaking the flask and transferring half the volume to a fresh G-REX100MCS cell culture flask (Wilson Wolf). 400 mL of freshly prepared medium was added to each G-REX100MCS culture flask. On day 14, cells were harvested and washed with 0.5% HAS (human serum albumin) (Octapharma) in phosphate saline (PBS) (Biological Industries, Israel). At harvest, the cell suspension was >90% CD56+ (clone B159, BD, San Jose, CA) cells as determined by FCS Canto II (BD, San Jose, CA).

[0198] Target cells: The BL2 cell line was derived from a 7 year old male patient with Burkitt's lymphoma and is CD20+. For further details of the BL2 cell line, see cellosaurus(slash)CVCL 1966 on the expasy(dot)org website.

[0199] BL2 cells were cultured in a 5% CO2, 37°C incubator in the following medium: RPMI1640 (Biological Industries), 10% FBS, gentamicin (Octapharma), and L-glutamine (BI) in T-flasks. Cells were passaged twice a week to a total concentration of ∼1 × 10 6 The cells were cultured at 100 x g / mL.

[0200] Antibody-dependent cytotoxicity (ADCC) assay: The harvested expanded NK cells (effector cells) were incubated at a 1:1 ratio with BL2 cells (target cells) previously labeled with violet CFSE (Invitrogen, Thermo Fisher, Waltham, MA) as recommended by the manufacturer. Co-incubation of NK and BL2 cells in the presence or absence of anti-CD20 antibodies lasted for 3 h in a 37°C incubator with 5% CO2. Evaluation of target cell killing by staining with propidium iodide (PI) (Sigma) was performed immediately before validation with an FCS Canto II (BD Biosciences). FACS data analysis was performed with FACS DIVA software (BD Biosciences). BL2 cells lysed by NK cells were expressed as the percentage of double positive (PI+ / CFSE+) BL2 cells over the total number of BL2 CFSE+ cells.

[0201] result Example I: Nicotinamide enhances Fc receptor (CD16) expression by NK cells Recognition of antibody-coated cells by the NK cell surface Fc receptor (FCgammaRIII), also known as CD16, leads to direct cell killing and cytokine production by peripheral blood NK cells. FACS analysis of surface CD16 expression by NK cells cultured with added exogenous nicotinamide (5 mM) showed abundant CD16 expression in the CD3- / CD56+ NK cell population (see Figure 1, CD56+ / CD16+ fraction ≥75%).

[0202] Example II: Anti-CD20 antibody-dependent cellular cytotoxicity (ADCC) in nicotinamide-expanded NK cells CD20 is a B-cell surface tumor marker that is gaining clinical relevance in the immunotherapy of hematological cancers (e.g., lymphomas and leukemias) and B-cell autoimmune diseases. Several CD20-targeting monoclonal antibodies have been approved for clinical use.

[0203] The inventors have previously shown (see WO 2011 / 080740) that incubation with nicotinamide enhances NK cell proliferation in culture, enhances NK cell motility, and enhances migratory (CXCR4), adhesion (CD49e), and trafficking (CD62L) receptor expression by cultured cord blood-derived NK cells.

[0204] To assess the efficiency of the CD20-mediated "cell killing" function of nicotinamide-expanded NK cells, nicotinamide-expanded NK cells were incubated with BL2 (Burkitt's lymphoma cells, CD20+) and the anti-CD20 monoclonal antibodies rituximab and obinutuzumab in an ADCC assay.

[0205] Although both anti-CD20 antibodies mediated NK killing of BL2 target cells, the combination of nicotinamide-expanded NK cells with the anti-CD20 monoclonal antibody obinutuzumab proved to be far superior to its combination with rituximab. As shown in Figure 2A and B, over the entire range of antibody concentrations tested (double digits, 0.5-50.0 ng / mL), the presence of either anti-CD20 monoclonal antibody significantly enhanced NK cell cytotoxicity (cytolysis measured by FACS analysis based on PI staining). However, at all concentrations, CD20-mediated NK cytotoxicity of BL2 cells was greater with obinutuzumab, up to three-fold greater than with rituximab.

[0206] All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference in their entirety to the same extent as if each such publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Furthermore, citation or identification of any reference in this application should not be construed as an admission that such reference is prior art to this application. The use of section headings should not necessarily be construed as limiting. Additionally, all documents relating to the prior application of this application are hereby incorporated by reference in their entirety.

Claims

1. 1. A kit for treating a hematological disorder in a subject in need thereof, comprising: (a) obinutuzumab, (b) at least one immunosuppressant; (c) a fraction of HLA half-matched or HLA mismatched expanded CD3-depleted NK cells expanded in ex vivo culture with nutrients, serum, IL-15 and 1.0 mM to 10 mM nicotinamide; a fraction of expanded CD3-depleted NK cells that are HLA half-matched or HLA mismatched; (d) IL-2 and Kit including:

2. The kit of claim 1 , wherein the immunosuppressant is a chemotherapeutic immunosuppressant.

3. 1. A kit for treating a hematological disorder in a subject in need thereof, comprising: (a) obinutuzumab, (b) a fraction of HLA half-matched or HLA mismatched expanded CD3-depleted NK cells expanded in ex vivo culture with nutrients, serum, IL-15 and 1.0 mM to 10 mM nicotinamide; a fraction of HLA half-matched or HLA mismatched expanded CD3-depleted NK cells; (c) IL-2 and wherein the subject is an immunosuppressed subject that has undergone radiation therapy.

4. The kit according to any one of claims 1 to 3, wherein the blood disease is a blood tumor, optionally a CD20-positive lymphoid tumor.

5. The kit according to any one of claims 1 to 3, wherein the blood disease is multiple myeloma.

6. The kit according to any one of claims 1 to 3, wherein the hematological disease is non-Hodgkin's lymphoma (NHL), optionally CD20 positive B-cell NHL.

7. The kit of any one of claims 1 to 6, wherein the expanded CD3-depleted HLA half-matched or mismatched NK cell fraction is provided in two doses for administration on consecutive days.

8. The NK cell fraction was 1×10 ７ pieces / kg ~ 5×10 ８ The kit according to any one of claims 1 to 6, comprising cells / kg of expanded CD3-depleted HLA half-matched or HLA-mismatched NK cells.

9. (a) the first dose and the second dose of the NK cell fraction are each 1×10 ７ The total dose of expanded CD3-depleted HLA half-matched or mismatched NK cells was 2×10 ７ pieces / kg, or (b) the first dose and the second dose of the NK cell fraction are each 5×10 ７ The total dose of expanded CD3-depleted HLA half-matched or mismatched NK cells is 1×10 ８ pieces / kg, or (c) the first dose and the second dose of the NK cell fraction are each 1×10 ８ The total dose of expanded CD3-depleted HLA half-matched or mismatched NK cells was 2×10 ８ The kit according to claim 7, wherein the concentration is 1 / kg.

10. The at least one immunosuppressant comprises cyclophosphamide and / or fludarabine, optionally in combination with cyclophosphamide (40 mg / kg) and fludarabine (25 mg / kg). ２ 2. The kit of claim 1 , comprising both

11. The IL-2 was administered at 6×10 ６ 10. The kit of claim 1 comprising three unit doses.

12. The kit according to any one of claims 1 to 11, wherein the HLA half-matched or HLA mismatched expanded CD3-depleted NK cell fraction is prepared by the steps of: (a) culturing ex vivo a fraction of HLA-half-matched or HLA-mismatched CD3-depleted NK cells for said subject under conditions allowing cell proliferation, said conditions including the provision of nutrients, serum, IL-15 and 1.0 mM to 10 mM nicotinamide; (b) supplementing the CD3-depleted NK cell fraction with fresh nutrients, serum, IL-15 and nicotinamide 8-10 days after step (a) to obtain an expanded CD3-depleted NK cell fraction; (c) recovering the expanded CD3-depleted NK cell fraction 14 to 16 days after step (a); (d) washing and concentrating the expanded CD3-depleted NK cell fraction of step (c); and preparing a transplantable NK cell fraction.

13. 13. The kit of claim 12, wherein the CD3-depleted NK cell fraction is obtained by apheresis, and / or the serum is 10% human serum, and / or the IL-15 comprises 20 ng / mL IL-15.

14. 14. The kit of claim 12 or 13, wherein the nicotinamide comprises 5.0 mM nicotinamide.

15. The kit according to claim 12, wherein the NK cells in step (a) comprise at least 40-90% CD56+ / CD3- cells.

16. The kit according to any one of claims 1 to 15, wherein the expanded CD3-depleted HLA half-matched or HLA-mismatched NK cell fraction is characterized by the following parameters: (a) at least 70% CD56+ / CD3− cells; (b) a survival rate of at least 70%; (c) CD3+ cell count per patient at the time of infusion was 5.0×10 ５ Pieces / Weight less than 1kg, (d) endotoxin levels per patient at the time of infusion of 5 EU / kg body weight or less; and (e) No gram-positive microorganisms.

17. The kit of any one of claims 1 to 16, wherein the expanded CD3-depleted HLA half-matched or HLA-mismatched NK cell fraction is provided in fluorinated ethylene propylene (FEP) culture bags.

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