Cancer treatment using NK cells and CD38-targeting antibodies

Abstract

The present invention provides, inter alia, methods of treating patients suffering from CD38+ cancers.

Description

[Technical field]

[0001] Priority claim This application claims priority to U.S. Provisional Application No. 63 / 290,351, filed December 16, 2021, and U.S. Provisional Application No. 63 / 172,424, filed April 8, 2021, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Natural killer cells are cytolytic cells of the innate immune system that have the inherent ability to lyse tumor cells and virus-infected cells. Receptor engagement of NK cells promotes effector function by degranulation of cytolytic granules, activation of programmed cell death receptors on the target cell, and secretion of immunoregulatory cytokines. The effector function of natural killer cells is controlled by the balance between receptor-activating and receptor-inhibitory signaling. Classically, NK cells express CD56 + and CD3 - cells, which are CD56 bright CD16 - Cytokine-secreting cells and CD56 dim CD16 + Engagement between CD16 and antibody-opsonized tumor cells is sufficient to induce cytotoxic and cytokine secretion responses by resting NK cells.

[0003] Antibody-dependent cellular cytotoxicity (ADCC) is a core component of the innate immune system in which antibody-coated target cells are killed by cells bearing Fc receptors that recognize the constant region of the bound antibody. ADCC is mediated through the Fc receptor FcγRIII (CD16) expressed on the surface by NK cells. ADCC is recognized as a potent mechanism of action of NK cells, especially in combination with antibodies belonging to the immunoglobulin G1 (IgG1) and IgG3 subclasses.

[0004] Multiple myeloma is a B-cell malignancy characterized by the accumulation and clonal expansion of plasma cells, terminally differentiated, non-dividing cells that survive in the bone marrow for extended periods (months to years) and whose primary function is to produce antigen-specific immunoglobulins. Bone marrow involvement is also a defining feature of multiple myeloma. Signs and symptoms of the disease include anemia, immunoparalysis with increased risk of infection, hypercalcemia, renal insufficiency, fractures and bone pain due to extensive bone damage caused by anemia and / or osteolytic lesions. These symptoms must be present for multiple myeloma to be diagnosed, and recently, predictive biomarkers must be present. Genetic abnormalities commonly found in clonal plasma cells characteristic of multiple myeloma include recurrent translocations involving the heavy chain locus of chromosome 14 (14q32), gains (chromosomes 1q) or losses (chromosomes 1p, 13q, and 17p) of genetic material, and trisomies of odd chromosomes.

[0005] Multiple myeloma diagnoses account for ~2% of all cancers, and ~10% of blood cancers. In 2021, it was estimated that 34,920 new cases of multiple myeloma will be diagnosed, and approximately 12,410 deaths will occur from the disease. The disease is more common in men (58%) than women (42%), and is more likely (2-3 times) to develop in black people than in whites or Asians. In the United States, the average age of diagnosis is 69 years, and the age-adjusted incidence of multiple myeloma has remained relatively stable at approximately 6.6 cases per 100,000.

[0006] Multiple myeloma disease progresses slowly and deliberately. Most patients first develop an asymptomatic underlying condition called monoclonal gammopathy of undetermined significance (MGUS) in their early 50s. The rate at which MGUS progresses to the next most significant condition, smoldering multiple myeloma (SMM), is slow, at about 1% per year. Patients may have MGUS for up to 15 years before progressing to SMM. Progression from SMM to symptomatic myeloma is similarly slow, at about 10% per year for the first 5 years, 3% for the next 5 years, and 1% thereafter. Thus, the risk of progression from MGUS to multiple myeloma is slow, at about 18% over 20 years.

[0007] Newly diagnosed patients are often classified according to their suitability for autologous hematopoietic stem cell transplantation (ASCT). Patients classified as eligible are initiated with induction therapy to reduce the burden of disease, and then undergo ASCT followed by maintenance therapy. Patients who are not amenable to ASCT are treated with alternative drugs such as steroids (e.g., dexamethasone or prednisolone), alkylating agents, and anthracyclines. Although these agents are initially effective, they are prone to induce serious side effects in patients. Along with this, these agents are not curative, and most multiple myeloma patients who undergo such therapy will relapse. Currently, the standard management for multiple myeloma includes a combination of proteasome inhibitors (e.g., bortezomib, carfilzomib), immunomodulators (e.g., lenalidomide), and steroids.

[0008] Targeted therapies such as monoclonal antibody (mAb) therapy have revolutionized cancer treatment. Daratumumab is a mAb approved as monotherapy to treat multiple myeloma in the relapsed setting. Daratumumab has been approved for untreated, relapsed / refractory myeloma in combination with proteasome inhibitors or immunomodulators and corticosteroids. This mAb targets CD38, which is widely expressed on malignant plasma cells. Daratumumab has various mechanisms of action to induce cell death: complement-dependent cytotoxicity, antibody-dependent cellular phagocytosis, apoptosis, and ADCC. Unfortunately, the efficacy of daratumumab can be hindered in multiple myeloma patients by a reduced ADCC response, thus negatively affecting clinical outcomes. One mechanism by which the ADCC response can be reduced is through the loss of NK cells.

[0009] However, NK cells express CD38; if daratumumab binds, NK cells can undergo cytolysis or fratricide. Therefore, NK cells that do not lyse in the presence of daratumumab are needed. Such NK cells should improve the ADCC activity of daratumumab and improve patient outcomes. Despite the recent discovery and development of several anticancer drugs, the poor prognosis of multiple myeloma still requires improved methods and therapeutic agents.

[0010] Allogeneic NK cell therapy has been in clinical use since 2005, but issues with product sourcing, scalability, and dose-specific variation have limited its utility. Combining allogeneic NK cell products with daratumumab can achieve improved efficacy in multiple myeloma patients whose ADCC responses fade over time.

[0011] The present invention solves these and other problems in the art. Summary of the Invention [Problem to be solved by the invention]

[0012] NK cells are immune cells that can engage tumor cells not only through a complex array of their cell surface receptors, but also through ADCC. NK cells can engage antibodies through their surface CD16 receptor to initiate ADCC. NK cells have advantages over other immune cells, such as T cells, used in CAR-T cell therapy and other cell therapies. One exemplary advantage is that NK cells can be used as an allogeneic therapy, where NK cells from a donor can be safely used in one or more patients without HLA matching, gene editing or other genetic manipulation. Allogeneic NK cells with anti-tumor activity can be safely administered to patients without many of the risks associated with T cell therapy, such as severe cytokine release syndrome (CRS) and neurotoxicity or graft-versus-host disease (GvHD).

[0013] Allogeneic NK cells can provide a significant therapeutic option for cancer patients. As one exemplary advantage, NK cells have excellent drug tolerability without the onset of GVHD, neurotoxicity, or CRS associated with other cell-based therapies. As another exemplary advantage, NK cells do not require prior antigen exposure or expression of specific antigens to identify and lyse tumor cells. As another exemplary advantage, NK cells have the unique ability to link innate immunity and mount a multi-clonal adaptive immune response to build long-term anti-cancer immune memory. All of these characteristics contribute to the efficacy of NK cells as a potential cancer treatment option.

[0014] For example, NK cells can recruit and activate other components of the immune system. Activated NK cells secrete cytokines and chemokines, such as interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), and macrophage inflammatory protein 1 (MIP1), which signal and recruit T cells to tumors. NK cells also directly kill tumor cells, thereby exposing tumor antigens for recognition by the adaptive immune system.

[0015] In conjunction with this, by using a diverse cord blood bank as a source of NK cells, cord blood with favorable characteristics (e.g., high affinity CD16 and killer cell immunoglobulin-like receptor (KIR) B-haplotype) can be selected to enhance clinical activity. [Means for solving the problem]

[0016] As described herein, administration of allogeneic NK cells can enhance the ADCC response in patients undergoing, for example, monoclonal antibody therapy.

[0017] Thus, among other things, the present invention provides a method of treating a patient suffering from a CD38+ cancer.

[0018] The present invention provides a method of treating a patient suffering from a CD38+ cancer comprising administering a population of natural killer cells (NK cells) and an antibody that targets human CD38, where the NK cells are allogeneic to the patient, are of the KIR-B haplotype, and are homozygous for a CD16 158V polymorphism for the patient.

[0019] In some embodiments, the cancer is selected from the group consisting of glioma, thyroid cancer, lung cancer, colorectal cancer, head and neck cancer, gastric cancer, liver cancer, pancreatic cancer, renal cancer, urothelial cancer, prostate cancer, testicular cancer, breast cancer, cervical cancer, ovarian cancer, melanoma, lymphoma, and combinations thereof.

[0020] In some embodiments, the cancer is myeloma. In some embodiments, the cancer is multiple myeloma. In some embodiments, the multiple myeloma is high-risk myeloma or lenalidomide-resistant multiple myeloma. In some embodiments, the patient has relapsed after treatment with an anti-CD38 antibody. In some embodiments, the patient has experienced disease progression after treatment with an autologous stem cell transplant or chimeric antigen receptor T-cell therapy (CAR-T).

[0021] In some embodiments, the patient is administered 1×10 NK cells. 8 ~1×10 10 In some embodiments, the patient is administered 1×10 NK cells. 9 ~8×10 9 In some embodiments, the patient is administered 4×10 NK cells. 8 , 1×10 9 , 4×10 9 or 8×10 9 Administer one dose.

[0022] In some embodiments, the antibody is daratumumab, isatuximab or a biosimilar thereof. In some embodiments, the antibody is daratumumab. In some embodiments, the antibody is isatuximab. In some embodiments, the patient undergoes lymphodepleting chemotherapy prior to treatment. In some embodiments, the lymphodepleting chemotherapy is non-myeloablative chemotherapy. In some embodiments, the lymphodepleting chemotherapy includes treatment with one or more of cyclophosphamide and fludarabine. In some embodiments, the lymphodepleting chemotherapy includes treatment with cyclophosphamide and fludarabine. In some embodiments, the cyclophosphamide is administered at a dose of 100-500 mg / m 2 / day. In some embodiments, cyclophosphamide is administered at 250 mg / m 2 / day. In some embodiments, cyclophosphamide is administered at 500 mg / m 2 / day. In some embodiments, fludarabine is administered at 10-50 mg / m 2 / day. In some embodiments, fludarabine is administered at 30 mg / m 2 / day.

[0023] In some embodiments, the method further comprises administering IL-2 to the patient. In some embodiments, IL-2 is administered to the patient at a dose of 1×10 6 IU / m 2 In some embodiments, IL-2 is administered to the patient at 6,000,000 IU. In some embodiments, administration of IL-2 occurs within 1-4 hours of administering the NK cells.

[0024] In some embodiments, the administration of the NK cells and the human CD38-targeting antibody is weekly. In some embodiments, the NK cells and the human CD38-targeting antibody are administered weekly for 4-8 weeks. In some embodiments, the administration of the NK cells is weekly or every other week, and the administration of the human CD38-targeting antibody is every other week or every month. In some embodiments, the administration of the NK cells and the human CD38-targeting antibody is every two weeks.

[0025] In some embodiments, the administration of the NK cells and the antibody comprises four doses at two-week intervals. In some embodiments, the administration of the NK cells and the antibody comprises eight doses at two-week intervals. In some embodiments, the administration of the NK cells and the human CD38-targeting antibody is monthly. In some embodiments, the administration of the NK cells and the antibody comprises eight doses at one-month intervals.

[0026] In some embodiments, the invention involves administering a first course of NK cells and human CD38-targeted antibody weekly, biweekly, or monthly, and a second course of NK cells and human CD38-targeted antibody weekly, biweekly, monthly, or at bimonthly intervals. In some embodiments, the second course of administration continues until the CD38+ cancer progresses, or until administration is discontinued due to patient intolerance to the NK cells, human CD38-targeted antibody, or both, or until the patient exhibits toxicity to the NK cells, human CD38-targeted antibody, or both.

[0027] In some embodiments, the NK cells are not genetically modified. In some embodiments, at least 70% of the NK cells are CD56+ and CD16+. In some embodiments, at least 85% of the NK cells are CD56+ and CD3-. In some embodiments, no more than 1% of the NK cells are CD3+, no more than 1% of the NK cells are CD19+, and no more than 1% of the NK cells are CD14+. In some embodiments, each administration of NK cells is at least 1×10 NK cells. 9 ~5×10 9 In some embodiments, a dose of a CD38-targeting antibody is administered to the patient prior to the first administration of NK cells.

[0028] In some embodiments, the expanded natural killer cells are expanded cord blood natural killer cells.

[0029] In some embodiments, the expanded population of natural killer cells comprises at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% CD16+ cells. In some embodiments, the expanded population of natural killer cells comprises at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% NKG2D+ cells. In some embodiments, the expanded population of natural killer cells comprises at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% NKp46+ cells. In some embodiments, the expanded population of natural killer cells comprises at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% NKp30+ cells. In some embodiments, the expanded population of natural killer cells comprises at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% DNAM-1+ cells. In some embodiments, the expanded population of natural killer cells comprises at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% NKp44+ cells. In some embodiments, the expanded population of natural killer cells comprises less than 20%, e.g., 10% or less, 5% or less, 1% or less, 0.5% or less or 0% CD3+ cells. In some embodiments, the expanded population of natural killer cells comprises less than 20%, e.g., 10% or less, 5% or less, 1% or less, 0.5% or less or 0% CD14+ cells. In some embodiments, the expanded natural killer cell population comprises less than 20%, e.g., 10% or less, 5% or less, 1% or less, 0.5% or less, or 0% CD19+ cells. In some embodiments, the expanded natural killer cell population comprises less than 20%, e.g., 10% or less, 5% or less, 1% or less, 0.5% or less, or 0% CD38+ cells.In some embodiments, the natural killer cells do not comprise the CD16 transgene.

[0030] In some embodiments, the natural killer cells do not express exogenous CD16 protein.In some embodiments, the expanded natural killer cells are not genetically engineered.

[0031] In some embodiments, the expanded natural killer cells are derived from the same cord blood donor.

[0032] In some embodiments, the population of NK cells comprises at least 100 million expanded natural killer cells, e.g., 200 million, 250 million, 300 million, 400 million, 500 million, 600 million, 700 million, 750 million, 800 million, 900 million, 1 billion, 2 billion, 3 billion, 4 billion, 5 billion, 6 billion, 7 billion, 8 billion, 9 billion, 10 billion, 15 billion, 20 billion, 25 billion, 50 billion, 75 billion, 80 billion, 90 billion, 100 billion, 200 billion, 250 billion, 300 billion, 400 billion, 500 billion, 600 billion, 700 billion, 800 billion, 900 billion, 1 trillion, 2 trillion, 3 trillion, 4 trillion, 5 trillion, 6 trillion, 7 trillion, 8 trillion, 9 trillion, or 10 trillion.

[0033] In some embodiments, the population of NK cells is produced by a method comprising the steps of: (a) obtaining seed cells comprising natural killer cells from umbilical cord blood; (b) depleting the seed cells of CD3+ cells; and (c) expanding the natural killer cells by culturing the depleted seed cells with a first plurality of Hut78 cells engineered to express membrane-bound IL-21, a TNFα mutant and a 4-1BBL gene to produce expanded natural killer cells, thereby producing a population of expanded natural killer cells.

[0034] In some embodiments, the population of NK cells is produced by a method comprising the steps of: (a) obtaining seed cells comprising natural killer cells from umbilical cord blood; (b) depleting the seed cells of CD3+ cells; (c) expanding the natural killer cells by culturing the depleted seed cells with a first plurality of Hut78 cells engineered to express membrane-bound IL-21, TNFα mutation and the 4-1BBL gene to produce a master cell bank population of expanded natural killer cells; and (d) expanding the master cell bank population of expanded natural killer cells by culturing with a second plurality of Hut78 cells engineered to express membrane-bound IL-21, TNFα mutation and the 4-1BBL gene to produce expanded natural killer cells; thereby producing a population of expanded natural killer cells.

[0035] In some embodiments, the population of NK cells is produced by a method that, after step (c), additionally comprises the steps of: (i) freezing the master cell bank population of amplified natural killer cells in a plurality of containers; and (ii) thawing the container containing an aliquot of the master cell bank population of amplified natural killer cells, wherein in step (d), expanding the master cell bank population of amplified natural killer cells comprises amplifying the aliquot of the master cell bank population of amplified natural killer cells.

[0036] In some embodiments, the cord blood is from a donor who is KIR-B haplotype and homozygous for the CD16 158V polymorphism.

[0037] In some embodiments, the population of NK cells is produced by a method comprising expanding natural killer cells from umbilical cord blood at least 10,000-fold, e.g., 15,000-fold, 20,000-fold, 25,000-fold, 30,000-fold, 35,000-fold, 40,000-fold, 45,000-fold, 50,000-fold, 55,000-fold, 60,000-fold, 65,000-fold or 70,000-fold.

[0038] In some embodiments, the expanded population of natural killer cells is not enriched or sorted after expansion.

[0039] In some embodiments, the percentage of NK cells expressing CD16 in the expanded population of natural killer cells is the same as or higher than the percentage of natural killer cells in seed cells derived from cord blood. In some embodiments, the percentage of NK cells expressing NKG2D in the expanded population of natural killer cells is the same as or higher than the percentage of natural killer cells in seed cells derived from cord blood. In some embodiments, the percentage of NK cells expressing NKp30 in the expanded population of natural killer cells is the same as or higher than the percentage of natural killer cells in seed cells derived from cord blood. In some embodiments, the percentage of NK cells expressing NKp44 in the expanded population of natural killer cells is the same as or higher than the percentage of natural killer cells in seed cells derived from cord blood. In some embodiments, the percentage of NK cells expressing NKp46 in the expanded population of natural killer cells is the same as or higher than the percentage of natural killer cells in seed cells derived from cord blood. In some embodiments, the percentage of NK cells expressing DNAM-1 in the expanded population of natural killer cells is the same as or higher than the percentage of natural killer cells in seed cells derived from umbilical cord blood.

[0040] In some embodiments, the method additionally comprises administering a steroid to the patient. In some embodiments, the steroid is selected from the group consisting of dexamethasone, methylprednisolone, triamcinolone, prednisolone, prednisone, bethamethasone, and combinations thereof. In some embodiments, the steroid is dexamethasone and / or methylprednisolone. In some embodiments, the steroid is administered within 1-4 hours from the time of administering the NK cells. In some embodiments, administration of the steroid does not reduce or eliminate the ADCC activity of the NK cells.

[0041] In some embodiments, the method comprises administering IL-2 at a dose of 1×10 6 IU / m 2 or 6×10 6 IU. In some embodiments, IL-2 is administered within 1-4 hours of administering the NK cells.

[0042] The present invention also relates to an amplified CD 16+ / CD38 lowCompositions are provided that include a population of NK cells. In some embodiments, the NK cells express CD38 at a lower level than a naturally occurring heterogeneous population of NK cells. In some embodiments, the NK cells exert ADCC activity against CD38+ tumor cells in the presence of a CD38-targeting antibody. In some embodiments, the NK cells exhibit reduced fratricide activity compared to NK cells of a KIR-A haplotype or NK cells lacking CD16 158V polymorphism. In some embodiments, the NK cells exhibit reduced fratricide activity compared to a naturally occurring heterogeneous population of NK cells in the presence of a CD38-targeting antibody. In some embodiments, the NK cells exhibit fratricide in the presence of a CD38-targeting antibody to a level that does not reduce the efficacy of the combination of the NK cells and the CD38-targeting antibody in treating a CD38+ cancer. In some embodiments, the NK cells are CD38+, where the NK cells exert ADCC activity against CD38+ cancer cells in the presence of an anti-CD38 antibody, but do not exhibit substantially ADCC activity against the NK cells in the presence of the antibody.

[0043] The present invention also provides a method for detecting NK cells from a donor homozygous for a KIR-B haplotype or CD16 158V / V genotype, comprising the steps of obtaining a cord blood sample comprising NK cells from the donor; and isolating the NK cells from the donor homozygous for a KIR-B haplotype or CD16 158V / V genotype; and + CD16 including in vitro expansion in the presence of T cell lines + / CD38 low The present invention also provides a method for constructing a population of NK cells, comprising the steps of obtaining a cord blood sample containing NK cells from a donor homozygous for a KIR-B haplotype or CD16 158V / V genotype; 4 3. In Vitro Expansion in the Presence of CD16+ T Cell Lines + / CD38 low Methods for enriching a population of NK cells are provided.

[0044] In some embodiments, the cord blood sample containing NK cells is CD38 highIn some embodiments, the umbilical cord blood sample containing NK cells is a population of CD38 low In some embodiments, the population of CD16 + / CD38 low The population of NK cells is not genetically engineered. + / CD38 low The NK cell population has not been genetically engineered to alter CD38 expression.

[0045] The invention also provides a method of targeting cancer cells comprising administering an antibody comprising an antigen-binding site that independently binds to an NK cell and to a cancer cell; and the NK cell, wherein the NK cell differentially targets the cancer cell bound to the antibody over the NK cell bound to the antibody.

[0046] The invention also provides a method of targeting cancer cells comprising administering NK cells; and an antibody comprising an antigen-binding site that binds independently to NK cells and to cancer cells, wherein the antigen-binding site differentially binds to cancer cells rather than to NK cells.

[0047] The present invention further provides a method for treating a pulmonary artery disease comprising administering a population of NK cells to a patient comprising administering to the patient a population of NK cells ... + wherein the cells are CD38 in the presence of an anti-CD38 antibody. + The cells mediate ADCC against cancer cells, and the cells are capable of isolating other CD38 from the NK cell population in the presence of anti-CD38 antibodies. + 2) administering an anti-CD38 antibody to a subject that does not substantially mediate ADCC against NK cells; and + A method of treating a patient suffering from cancer is provided.

[0048] In some embodiments, the NK cells are allogeneic to the patient, are KIR-B haplotype and homozygous for the CD16 158V polymorphism to the patient.

[0049] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art may also be used. Materials, methods and examples are illustrative only and are not limiting. All publications, patent applications, patents, sequences, database entries and other references mentioned in this application are incorporated herein by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0050] Other features and advantages of the invention will become apparent from the following detailed description and drawings, and from the claims.

[0051] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0052] The novel features of the invention are set forth with particularity in the appended claims. The patent or application file contains one or more drawings executed in color. Copies of this patent or patent application publication containing color drawing(s) will be provided by the Office upon request and payment of the appropriate fee. The features and advantages of the present invention will be better understood with reference to the following detailed description set forth in the accompanying drawings, in which illustrative embodiments in which the principles of the invention are applied are set forth. [Brief description of the drawings]

[0053] [Figure 1] FIG. 1 shows an exemplary embodiment for a method for expanding and stimulating NK cells. [Diagram 2] FIG. 1 shows that in preclinical experiments, umbilical cord blood-derived NK cells (CB-NK) have approximately 10 times higher culture expansion potential than peripheral blood-derived NK cells (PB-NK). [Diagram 3]FIG. 1 shows that expression of tumor-engaging NK-activating immunoreceptors is higher and more consistent in cord blood-derived products compared to peripheral blood sources. [Figure 4] FIG. 1 shows the phenotype of expanded and stimulated NK cell populations. [Diagram 5] FIG. 1 shows the key steps of manufacturing AB-101 drug product as an example of a cord blood-derived NK cell expanded population. [Figure 6] FIG. 1 shows the purity of AB-101 (n=9). [Figure 7] FIG. 1 shows the purity of CD3-depleted cells, MCB and DP produced under GMP conditions. [Figure 8] FIG. 1 shows the expression of NK cell receptors in CD3-depleted cells, MCB and DP produced under GMP conditions. [Figure 9] FIG. 1 shows NK purity (CD56+ / CD3−) as determined by flow cytometry. [Figure 10] FIG. 1 shows CD38+ expression on expanded NK cells from three types of cord blood donors. [Figure 11] FIG. 1 shows the mean fluorescence intensity of CD38+ of CD38+ NK cells from three types of cord blood donors. [Figure 12] (Not stated) [Figure 13] Figure 1 shows cell viability, purity and CD38 expression of AB-101 after thawing. Top left: cell viability of AB-101 after thawing; Top center: purity of AB-101 defined by CD56+CD3-; Top right: CD16 expression is confirmed on CD56+ cells; Bottom left: AB-101 CD56+ cells have low CD38 expression in GMP lot, which remains unchanged after 24 hours of culture; Bottom right: low fluorescence intensity geometric mean of CD38 expression. [Figure 14]Post-thaw PB-NK cell viability, purity and CD38 expression. Top left: Post-thaw PB-NK cell viability; Top center: PB-NK purity defined as CD56+CD3-; Top right: CD16 expression confirmed on CD56+ cells; Bottom left: PB-NK CD56+ cells have high CD38 expression; Bottom right: High fluorescence intensity geometric mean of CD38 expression. [Figure 15] FIG. 1 shows that AB-101 cells do not undergo fratricide when incubated with daratumumab at various concentrations. [Figure 16] Daratumumab induces complement-dependent cytotoxicity (CDC).Top left, top right and bottom left: AB-101 cells do not undergo complement-mediated cytolysis when incubated with various concentrations of daratumumab;bottom right: the control tumor cell line Daudi undergoes CDC lysis in the presence of daratumumab. [Figure 17] FIG. 1 shows the effect of dexamethasone on AB-101 + daratumumab ADCC in the Daudi cell line (average of 3 donors). [Figure 18] FIG. 1 shows the effect of dexamethasone on AB-101 + daratumumab ADCC in the NCI-H929 cell line (average of 3 donors). [Figure 19] FIG. 1 shows the effect of dexamethasone on AB-101 + daratumumab ADCC in RPMI8226 cell line (average of 3 donors). [Figure 20] FIG. 1 shows the effect of dexamethasone on AB-101 + daratumumab ADCC in K562 cell line (average of 3 donors). [Figure 21] FIG. 1 shows that daratumumab induces significant ADCC by AB-101 cells against Daudi. [Figure 22] FIG. 1 shows that daratumumab induces significant ADCC by AB-101 cells against NCI-H929. [Diagram 23] FIG. 1 shows that daratumumab induces significant ADCC by AB-101 cells against RPMI-8226. [Figure 24] FIG. 1 shows that daratumumab induces significant ADCC by AB-101 cells against MM.1R. [Diagram 25] FIG. 1 shows that daratumumab induces significant ADCC by AB-101 cells against MM.1S. [Figure 26] FIG. 1 shows that daratumumab induces significant ADCC by AB-101 cells against K562. [Figure 27] FIG. 1 shows that daratumumab induces AB-101 cells to secrete cytokines. [Figure 28] FIG. 1 shows that daratumumab induces AB-101 cells to secrete cytokines. [Figure 29] FIG. 1 shows that daratumumab induces AB-101 cells to secrete cytokines. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0054] The present invention provides, inter alia, NK cells, eg, expanded and stimulated NK cells, methods for producing NK cells, pharmaceutical compositions comprising NK cells, and methods for treating cancer patients with NK cells.

[0055] I. Expansion and Stimulation of Natural Killer Cells In some embodiments, the NK cells are expanded and stimulated by culture and stimulation with feeder cells.

[0056] NK cells can be expanded and stimulated, for example, as described in US2020 / 0108096 or WO2020 / 101361, both of which are incorporated herein by reference in their entirety. Briefly, the source cells are recombinant HuT-78 (ATCC 61221), engineered to express 4-1BBL, membrane-bound IL-21 and mutant TNFα, as described in US2020 / 0108096. (R) TIB-161TM ) cells.

[0057] Suitable NK cells can also be expanded and stimulated as described herein.

[0058] In some embodiments, the NK cells are expanded and stimulated by a method comprising: (a) providing NK cells, a composition comprising NK cells, e.g., CD3(-) depleted cells; and (b) culturing in medium comprising feeder cells and / or stimulatory factors to produce a population of expanded and stimulated NK cells.

[0059] A. Natural Killer Cell Sources In some embodiments, the NK cell source is selected from the group consisting of peripheral blood, peripheral blood lymphocytes (PBL), peripheral blood mononuclear cells (PBMC), bone marrow, umbilical cord blood (or cord blood), isolated NK cells, NK cells derived from induced pluripotent stem cells, NK cells derived from embryonic stem cells, and combinations thereof.

[0060] In some embodiments, the NK cell source is a single unit of umbilical cord blood.

[0061] In some embodiments, the NK cell source, e.g., a single unit of umbilical cord blood, contains 1×10 total nucleated cells. 7 or about 1 x 10 7 ~1×10 9 or about 1 x 10 9 In some embodiments, the natural killer cell source, e.g., a single unit of umbilical cord blood, comprises 1×10 total nucleated cells. 8 or about 1 x 10 8 ~1.5×10 8 or about 1.5 x 10 8 In some embodiments, the natural killer cell source, e.g., a single unit of umbilical cord blood, comprises 1×10 total nucleated cells. 8In some embodiments, a natural killer cell source, e.g., a single unit of umbilical cord blood, comprises about 1×10 total nucleated cells. 8 In some embodiments, the natural killer cell source, e.g., a single unit of umbilical cord blood, comprises 1×10 total nucleated cells. 9 In some embodiments, a natural killer cell source, e.g., a single unit of umbilical cord blood, comprises about 1×10 total nucleated cells. 9 Includes pieces.

[0062] In some embodiments, the NK cell source, e.g., cord blood unit, comprises about 20% to about 80% CD16+ cells. In some embodiments, the NK cell source, e.g., cord blood unit, comprises about 20% to about 80%, about 20% to 70%, about 20% to 60%, about 20% to 50%, about 20% to 40%, about 20% to 30%, about 30% to 80%, about 30% to 70%, about 30% to 60%, about 30% to 60%, about 30% to 70%, about 30% to 80%, about 30% to 70%, about 30% to 60%, about 30% to 8 ... % to 50% or about 50%, about 30% to 40% or about 40%, about 40% to 80% or about 80%, about 40% to 70% or about 70%, about 40% to 60% or about 60%, about 40% to 50% or about 50%, about 50% to 80% or about 80%, about 50% to 70% or about 70%, about 50% to 60% or about 60%, about 60% to 80% or about 80%, about 60% to 70% or about 70%, or about 70% to 80% or about 80%. In some embodiments, the NK cell source, e.g., umbilical cord blood unit, comprises 80% or less CD16+ cells. Alternatively, some NK cell sources may comprise CD16+ cells at a concentration greater than 80%.

[0063] In some embodiments, the NK cell source, eg, umbilical cord blood unit, comprises no more than 40% MLG2A+ cells, such as no more than 30%, such as no more than 20%, such as no more than 10%, eg, no more than 5%.

[0064] In some embodiments, the NK cell source, e.g., an umbilical cord blood unit, contains no more than 40%, such as no more than 30%, such as no more than 20%, such as no more than 10%, e.g., no more than 5% NKG2C+ cells.

[0065] In some embodiments, the NK cell source, e.g., an umbilical cord blood unit, contains no more than 40%, such as no more than 30%, such as no more than 20%, such as no more than 10%, e.g., no more than 5% NKG2D+ cells.

[0066] In some embodiments, the NK cell source, e.g., an umbilical cord blood unit, contains no more than 40%, such as no more than 30%, such as no more than 20%, such as no more than 10%, e.g., no more than 5% NKp46+ cells.

[0067] In some embodiments, the NK cell source, e.g., an umbilical cord blood unit, contains no more than 40%, such as no more than 30%, such as no more than 20%, such as no more than 10%, e.g., no more than 5% NKp30+ cells.

[0068] In some embodiments, the NK cell source, e.g., an umbilical cord blood unit, contains no more than 40%, such as no more than 30%, such as no more than 20%, such as no more than 10%, e.g., no more than 5% DNAM-1+ cells.

[0069] In some embodiments, the NK cell source, e.g., an umbilical cord blood unit, contains no more than 40%, such as no more than 30%, such as no more than 20%, such as no more than 10%, e.g., no more than 5% NKp44+ cells.

[0070] In some embodiments, the NK cell source, e.g., an umbilical cord blood unit, contains no more than 40%, such as no more than 30%, such as no more than 20%, such as no more than 10%, e.g., no more than 5% CD25+ cells.

[0071] In some embodiments, the NK cell source, e.g., an umbilical cord blood unit, contains no more than 40%, such as no more than 30%, such as no more than 20%, such as no more than 10%, e.g., no more than 5% CD62L+ cells.

[0072] In some embodiments, the NK cell source, e.g., an umbilical cord blood unit, contains no more than 40%, such as no more than 30%, such as no more than 20%, such as no more than 10%, e.g., no more than 5% CD69+ cells.

[0073] In some embodiments, the NK cell source, e.g., an umbilical cord blood unit, contains no more than 40%, such as no more than 30%, such as no more than 20%, such as no more than 10%, e.g., no more than 5% CXCR3+ cells.

[0074] In some embodiments, the NK cell source, e.g., an umbilical cord blood unit, contains no more than 40%, such as no more than 30%, such as no more than 20%, such as no more than 10%, e.g., no more than 5% CD57+ cells.

[0075] In some embodiments, the NK cells in the NK source comprise the KIR B allele of the KIR receptor system. See Hsu et al., "The Killer Cell Immunoglobulin-Like Receptor (KIR) Genomic Region: Gene-Order, Haplotypes and Allelic Polymorphism," Immunological Review 190:40-52 (2002) and Pyo et al., "Different Patterns of Evolution in the Centromeric and Telomeric Regions of Group A and B Haplotypes of the Human Killer Cell Ig-like Receptor Locus," PLoS One 5:e15115 (2010).

[0076] In some embodiments, the NK cells in the NK source contain the 158V / V variant of CD16 (i.e., homozygous CD16 15V polymorphism). See Koene et al., "FcγRIIIa-158V / F Polymorphism Influences the Binding of IgG by Natural Killer Cell FcgammaRIIIa, Independently of the FcgammaRIIIa-48L / R / H Phenotype," Blood 90:1109-14 (1997).

[0077] In some embodiments, the NK cells in the cell source contain both the KIR B allele of the KIR receptor system and the 158V / V variant of CD16.

[0078] In some embodiments, the NK cells in the cell source are not genetically engineered.

[0079] In some embodiments, the NK cells in the cell source do not contain a CD16 transgene.

[0080] In some embodiments, the NK cells in the cell source do not express exogenous CD16 protein.

[0081] In some embodiments, the NK cell source is CD3+ depleted. In some embodiments, the method comprises depleting the NK cell source of CD3+ cells. In some embodiments, depleting the NK cell source of CD3+ cells comprises contacting the NK cell source with a CD3-binding antibody or antigen-binding fragment thereof. In some embodiments, the CD3-binding antibody or antigen-binding fragment thereof is selected from the group consisting of OKT3, UCHT1 and HIT3a, and fragments thereof. In some embodiments, the CD3-binding antibody or antigen-binding fragment thereof is OKT3 or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof is attached to beads, e.g., magnetic beads. In some embodiments, depleting the CD3+ cells in the composition comprises contacting the composition with a CD3-targeting antibody or antigen-binding fragment thereof attached to beads to remove bead-bound CD3+ cells from the composition. Compositions can be CD3 depleted by immunomagnetic selection, for example using the CliniMACS T cell depletion set (LS Depletion set (162-01) Miltenyi Biotec).

[0082] In some embodiments, the NK cell source is enriched for CD56+, for example, by gating on CD56 expression.

[0083] In some embodiments, the NK cell source is CD56+ enriched and CD3+ depleted, for example, by sorting for CD56+CD3- expressing cells.

[0084] In some embodiments, the NK cell source contains both the KIR B allele of the KIR receptor system and the 158V / V variant of CD16, e.g., CD56+ enrichment and CD3+ depletion is achieved by sorting for CD56+CD3- expressing cells.

[0085] B. Feeder cells The present invention discloses feeder cells for expanding NK cells. These feeder cells advantageously allow the expansion of NK cells to numbers suitable for producing the pharmaceutical compositions described herein. In some cases, the feeder cells allow the expansion of NK cells, often with cell expansion on other types of feeder cells or using other methods, without a decrease in CD16 expression. In some cases, the feeder cells allow the expanded NK cells to better tolerate freezing, such that a higher proportion of NK cells remain viable after a freeze / thaw cycle, or the cells remain viable for a longer period during freezing. In some cases, the feeder cells allow the NK cells to maintain high levels of cytotoxicity such as ADCC, prolong survival, increase persistence, and enhance or maintain high levels of CD16. In some cases, the feeder cells allow the NK cells to expand without inducing significant levels of exhaustion or senescence.

[0086] Feeder cells can be used to stimulate and support NK cells to expand more rapidly, for example by providing substrates, growth factors and / or cytokines.

[0087] NK cells can be stimulated using various types of feeder cells, including, but not limited to, peripheral blood mononuclear cells (PBMC), Epstein-Barr virus-transformed B-lymphoblast cells (e.g., EBV-LCL), myeloid leukemia cells (e.g., K562) and CD4+ T cells (e.g., HuT), and their derivatives.

[0088] In some embodiments, the feeder cells are inactivated, for example, by gamma-irradiation or mitomycin-c treatment.

[0089] Feeder cells suitable for use in the methods described herein are described, for example, in US2020 / 0108096, the entire contents of which are incorporated herein by reference.

[0090] In some embodiments, the feeder cells are inactivated CD4+ T cells. In some embodiments, the inactivated CD4+ T cells are HuT-78 cells (ATCC (R) In some embodiments, the HuT-78 derivative is H9 (ATCC 611611), or a mutant or derivative thereof. (R) HTB-176 TM ).

[0091] In some embodiments, the inactivated CD4+ T cells express OX40L. In some embodiments, the inactivated CD4+ T cells are HuT-78 cells that express OX40L (SEQ ID NO: 4), or a variant thereof, or a variant or derivative thereof.

[0092] In some embodiments, the feeder cells are HuT-78 cells engineered to express one or more genes selected from the group consisting of 4-1BBL (UniProtKB P41273, SEQ ID NO:1), membrane-bound IL-21 (SEQ ID NO:2) and mutated TNFα (SEQ ID NO:3) ("eHut-78 cells"), or variants thereof.

[0093] In some embodiments, the inactivated CD4+ T cells are selected from the group consisting of HuT-78 (ATCC 612611) and HuT-78 (ATCC 612611). (R) TIB-161 TM ) cells or mutants or derivatives expressing an OX40L ortholog, or mutants thereof. In some embodiments, the feeder cells are HuT-78 cells engineered to express one or more genes selected from the group consisting of a 4-1BBL ortholog, or mutants thereof, a membrane-bound IL-21 ortholog, or mutants thereof, and a mutant TNFα ortholog, or mutants thereof.

[0094] In some embodiments, the feeder cells are HuT-78 cells that express OX40L (SEQ ID NO: 4) and have been engineered to express 4-1BBL (SEQ ID NO: 1), membrane-bound IL-21 (SEQ ID NO: 2) and mutant TNFα (SEQ ID NO: 3) ("eHut-78 cells"), or variants or derivatives thereof.

[0095] In some embodiments, the feeder cells are expanded, eg, from frozen stocks, prior to culturing with the NK cells, eg, as described in Example 2.

[0096] C. Stimulating factor The NK cells can also be stimulated with one or more other stimulatory factors other than feeder cells, eg, signaling factors, in addition to or instead of feeder cells.

[0097] In some embodiments, the stimulatory factors, e.g., signaling factors, are components of a culture medium, as described herein. In some embodiments, the stimulatory factors, e.g., signaling factors, are supplements to a culture medium, as described herein.

[0098] In some embodiments, the stimulatory factor is a cytokine, hi some embodiments, the cytokine is selected from the group consisting of IL-2, IL-12, IL-15, IL-18, IL-21, IL-23, IL-27, IFN-α, IFN-β, and combinations thereof.

[0099] In some embodiments, the cytokine is IL-2. In some embodiments, the cytokine is a combination of IL-2 and IL-15. In some embodiments, the cytokine is a combination of IL-2, IL-15 and IL-18.

[0100] In some embodiments, the cytokine is a combination of IL-12 and IL-15. In some embodiments, the cytokine is a combination of IL-12 and IL-18. In some embodiments, the cytokine is a combination of IL-15 and IL-18. In some embodiments, the cytokine is a combination of IL-12, IL-15 and IL-18.

[0101] In some embodiments, the cytokines are a combination of IL-2, IL-18 and IL-21.

[0102] D. Culture NK cells can be expanded and stimulated by co-culturing the NK cell source with feeder cells and / or other stimulatory factors. Suitable NK cell sources, feeder cells and stimulatory factors are described herein.

[0103] In some cases, the resulting expanded population of NK cells is enriched and / or sorted after expansion. In some cases, the resulting expanded population of NK cells is not enriched and / or sorted after expansion.

[0104] The present invention also describes compositions comprising the various culture compositions described herein, e.g., NK cells, e.g., compositions comprising an expanded population of cord blood-derived natural killer cells comprising a KIR-B haplotype and homozygous for the CD16 158V polymorphism, and a plurality of engineered HuT78 cells.

[0105] The invention also describes vessels, e.g., vials, cryobags, etc., in which the resulting expanded population of natural killer cells is contained. In some cases, a plurality of vessels contain a portion of the resulting expanded population of natural killer cells, e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 vessels.

[0106] The invention also describes bioreactors containing various culture compositions, e.g., NK cells, as described herein, for example, cultures comprising natural killer cells derived from a natural killer cell source as described herein, and feeder cells, as described herein, for example, for example, as described herein, and the resulting expanded population of natural killer cells.

[0107] 1. Culture Medium The present invention discloses culture media for expanding NK cells. These culture media advantageously allow NK cells to expand to a cell number suitable for producing the pharmaceutical compositions described herein. In some cases, the culture media can expand NK cells without a decrease in CD16 expression, often accompanied by cell expansion on other helper cells or in other media.

[0108] In some embodiments, the culture medium is a basal culture medium, optionally supplemented with additional components, for example, as described herein.

[0109] In some embodiments, the culture medium, e.g., the basal culture medium, is a serum-free culture medium. In some embodiments, the culture medium, e.g., the basal culture medium, is a serum-free culture medium supplemented with human plasma and / or serum.

[0110] Suitable basal culture media include DMEM, RPMI1640, MEM, DMEM / F12, and SCGM (CellGenix (R) , 20802-0500 or 20806-0500), LGM-3 TM (Lonza, CC-3211), TexMACS TM (Miltenyi Biotec, 130-097-196), ALyS TM 505NK-AC (Cell Science and Technology Institute, Inc., 01600P02), ALyS TM 505NK-EX (Cell Science and Technology Institute, Inc., 01400P10), CTS TM AIM-V TM SFM (ThermoFisher Scientific, A3830801), CTS TM OpTmizer TM (ThermoFisher Scientific, A1048501, ABS-001, StemXxVivo), and combinations thereof.

[0111] The culture medium may contain additional components or may be supplemented with additional components such as growth factors, signaling factors, nutrients, antigen binding agents, etc. The culture medium may be supplemented by adding the respective additional component or components to the culture vessel prior to, simultaneously with, or after the medium is added to the culture vessel. The additional component or components may be added together or separately. If added separately, the additional components do not need to be added at the same time.

[0112] In some embodiments, the culture medium comprises plasma, e.g., human plasma. In some embodiments, the culture medium is supplemented with plasma, e.g., human plasma. In some embodiments, the plasma, e.g., human plasma, comprises an anti-coagulant, e.g., trisodium citrate.

[0113] In some embodiments, the medium comprises and / or is supplemented with plasma, e.g., human plasma, at 0.5% or about 0.5%-10% or about 10% v / v. In some embodiments, the medium comprises and / or is supplemented with 0.5% or about 0.5%-9% or about 9%, 0.5% or about 0.5%-8% or about 8%, 0.5% or about 0.5%-7% or about 7%, 0.5% or about 0.5%-6% or about 6%, 0.5% or about 0.5%-5% or about 5%, 0.5% or about 0.5%-4% or about 4%, 0.5% or about 0.5%-3% or about 3%, 0.5% or about 0.5%-2% or about 2%, 0.5% or about 0.5% to 1% or about 1%, 1% or about 1% to 10% or about 10%, 1% or about 1% to 9% or about 9%, 1% or about 1% to 8% or about 8%, 1% or about 1% to 7% or about 7%, 1% or about 1% to 6% or about 6%, 1% or about 1% to 5% or about 5%, 1% or about 1% to 4% or about 4%, 1% or about 1% to 3% or about 3%, 1% or about 1% to 2% or about 2%, 2% or about 2% to 10% or about 10%, 2% or about 2% to 9% or about 9%, 2% or about 2% to 8% or about 8%, 2% or about 2% to 7% or about 7%, 2% or about 2% to 6% or about 6%, 2% or about 2% to 5% or about 5%, 2% or about 2% to 4% or about 4%, 2% or about 2% to 3% or about 3%, 3% or about 3% to 10% or about 10%, 3% or about 3% to 9% or about 9%, 3% or about 3% to 8% or about 8%, 3% or about 3% to 7% or about 7%, 3% or about 3% to 6% or about 6%, 3% or about 3% to 5% or about 5%, 3% or about 3% to 4% or about 4%, 4% or about 4% to 10% or about 10%, 4% or about 4% to 9% or about 9%, 4% or about 4% to 8% or about 8%, 4% or about 4% to 7% or about 7%, 4% or about 4% to 6% or about 6%, 4% or about 4% to 5% or about 5%, 5% or about 5% to 10% or about 10%, 5% or about 5% to 9% or about 9%, 4% or about 4% to 8% or about 8%, 5% or about 5% to 7% or about 7%, 5% or about 5% to 6% or The culture medium is supplemented with plasma, e.g., human plasma, at about 6%, 6% or about 6%-10% or about 10%, 6% or about 6%-9% or about 9%, 6% or about 6%-8% or about 8%, 6% or about 6%-7% or about 7%, 7% or about 7%-10% or about 10%, 7% or about 7%-9% or about 9%, 7% or about 7%-8% or about 8%, 8% or about 8%-10% or about 10%, 8% or about 8%-9% or about 9%, or 9% or about 9%-10% or about 10% v / v. In some embodiments, the culture medium comprises and / or is supplemented with human plasma at 0.8%-1.2% v / v. In some embodiments, the culture medium comprises and / or is supplemented with human plasma at 1.0% v / v. In some embodiments, the culture medium comprises and / or is supplemented with human plasma at about 1.Contains and / or supplemented with 0% v / v.

[0114] In some embodiments, the culture medium comprises serum, e.g., human serum. In some embodiments, the culture medium is supplemented with serum, e.g., human serum. In some embodiments, the serum is inactivated, e.g., heat inactivated. In some embodiments, the serum is filtered, e.g., sterile-filtered.

[0115] In some embodiments, the culture medium comprises glutamine. In some embodiments, the culture medium is supplemented with glutamine. In some embodiments, the culture medium comprises and / or is supplemented with 2.0 or about 2.0 to 6.0 or about 6.0 mM glutamine. In some embodiments, the culture medium comprises and / or is supplemented with 2.0 or about 2.0 to 5.5 or about 5.5, 2.0 or about 2.0 to 5.0 or about 5.0, 2.0 or about 2.0 to 4.5 or about 4.5, 2.0 or about 2.0 to 4.0 or about 4.0, 2.0 or about 2.0 to 3.5 or about 3.5, 2.0 or about 2.0 to 3.0 or about 3.0, 2.0 or about 2.0 to 2.5 or about 2.5, 2.5 or about 2.5 to 6.0 or about 6.0 ... or about 2.5 to 5.5 or about 5.5, 2.5 or about 2.5 to 5.0 or about 5.0, 2.5 or about 2.5 to 4.5 or about 4.5, 2.5 or about 2.5 to 4.0 or about 4.0, 2.5 or about 2.5 to 3.5 or about 3.5, 2.5 or about 2.5 to 3.0 or about 3.0, 3.0 or about 3.0 to 6.0 or about 6.0, 3.0 or about 3.0 to 5.5 or about 5.5, 3.0 or about 3.0 to 5.0 or about 5.0, 3.0 or about 3.0 to 4.5 or about 4.5, 3.0 or about 3.0-4.0 or about 4.0, 3.0 or about 3.0-3.5 or about 3.5, 3.5 or about 3.5-6.0 or about 6.0, 3.5 or about 3.5-5.5 or about 5.5, 3.5 or about 3.5-5.0 or about 5.0, 3.5 or about 3.5-4.5 or about 4.5, 3.5 or about 3.5-4.0 or about 4.0, 4.0 or about 4.0-6.0 or about 6.0, 4.0 or about 4.0-5.5 or In some embodiments, the culture medium comprises and / or is supplemented with about 5.5, 4.0 or about 4.0-5.0 or about 5.0, 4.0 or about 4.0-4.5 or about 4.5, 4.5 or about 4.5-6.0 or about 6.0, 4.5 or about 4.5-5.5 or about 5.5, 4.5 or about 4.5-5.0 or about 5.0, 5.0 or about 5.0-6.0 or about 6.0, 5.0 or about 5.0-5.5 or about 5.5, or 5.5 or about 5.5-6.0 or about 6.0 mM glutamine. In some embodiments, the culture medium comprises and / or is supplemented with 3.2 mM to 4.8 mM glutamine.In some embodiments, the culture medium comprises and / or is supplemented with 4.0 mM glutamine. In some embodiments, the culture medium comprises and / or is supplemented with about 4.0 mM glutamine.

[0116] In some embodiments, the culture medium comprises one or more cytokines. In some embodiments, the culture medium is supplemented with one or more cytokines.

[0117] In some embodiments, the cytokine is selected from IL-2, IL-12, IL-15, IL-18, and combinations thereof.

[0118] In some embodiments, the culture medium comprises and / or is supplemented with IL-2. In some embodiments, the culture medium comprises and / or is supplemented with IL-2 at 150 or about 150-2,500 or about 2,500 IU / ml. In some embodiments, the culture medium comprises and / or is supplemented with IL-2 at 200 or about 200-2,250 or about 2,250, 200 or about 200-2,000 or about 2,000, 200 or about 200-1,750 or about 1,750, 200 or about 200-1,500 or about 1,500, 200 or about 200-1,250 or about 1,250, 200 or about 200-1,000 or about 1,000, 200 or about 200-750 or about 750, 200 or about 200-500 or about 500, 200 or about 200-250 or about 250, 250 or about 250-2,500 or about 2,500, 250 or about 250-2,250 or about 2,250, 250 or about 250-2,000 or about 2,000, 250 or about 250-1,750 or about 1,750, 250 or about 250-1,500 or about 1,500, 250 or about 250-1,250 or about 1,250, 250 or about 250-1,000 or about 1,000, 250 or about 250-7 50 or about 750, 250 or about 250-500 or about 500, 500 or about 500-2,500 or about 2,500, 500 or about 500-2,250 or about 2,250, 500 or about 500-2,000 or about 2,000, 500 or about 500-1,750 or about 1,750, 500 or about 500-1,500 or about 1,500, 500 or about 500-1,250 or about 1,250, 500 or about 500-1,000 or about 1,000, 500 or is about 500 to 750 or about 750, 750 or about 750 to 2,250 or about 2,250, 750 or about 750 to 2,000 or about 2,000, 750 or about 750 to 1,750 or about 1,750, 750 or about 750 to 1,500 or about 1,500, 750 or about 750 to 1,250 or about 1,250, 750 or about 750 to 1,000 or about 1,000, 1,000 or about 1,000 to 2,500 or about 2,500, 1,000 or about 1,000 to 2,250 or about 2,250, 1,000 or about 1,000 to 2,000 or about 2,000, 1,000 or about 1,000 to 1,750 or about 1,750, 1,000 or about 1,000 to 1,500 or about 1,500, 1,000 or about 1,000 to 1,250 or about 1,250, 1,250 or about 1,250 to 2,50 0 or about 2,500, 1,250 or about 1,250 to 2,250 or about 2,250, 1,250 or about 1,250 to 2,000 or about 2,000, 1,250 or about 1,250 to 1,750 or about 1,750, 1,250 or about 1,250 to 1,500 or about 1,500, 1,500 or about 1,500 to 2,500, is about 2,500, 1,500 or about 1,500 to 2,250 or about 2,250, 1,500 or about 1,500 to 2,000 or about 2,000, 1,500 or about 1,500 to 1,750 or about 1,750, 1,750 or about 1,750 to 2,500 or about 2,500, 1,750 or about 1,750 to 2,250 or about 2,250, 1,750 or about 1,750 to 2,000 or about 2,000, 2,000 or about 2,000 to 2,500 or about 2,500, 2,000 or about 2,000 to 2,250 or about 2,250, or 2,250 or about 2,250 to 2,500 or about 2,500 IU / ml.

[0119] In some embodiments, the culture medium comprises and / or is supplemented with IL-2 at 64 μg / l to 96 μg / l. In some embodiments, the culture medium comprises and / or is supplemented with IL-2 at 80 μg / l (about 1,333 IU / ml). In some embodiments, the culture medium comprises and / or is supplemented with IL-2 at about 80 μg / l.

[0120] In some embodiments, the culture medium comprises and / or is supplemented with a combination of IL-2 and IL-15. In some embodiments, the culture medium comprises and / or is supplemented with a combination of IL-2, IL-15 and IL-18. In some embodiments, the culture medium comprises and / or is supplemented with a combination of IL-2, IL-18 and IL-21.

[0121] In some embodiments, the culture medium comprises and / or is supplemented with glucose. In some embodiments, the culture medium comprises and / or is supplemented with 0.5 or about 0.5-3.5 g / l or about 3.5 g / l of glucose. In some embodiments, the culture medium comprises and / or is supplemented with 0.5 or about 0.5-3.0 or about 3.0, 0.5 or about 0.5-2.5 or about 2.5, 0.5 or about 0.5-2.0 or about 2.0, 0.5 or about 0.5-1.5 or about 1.5, 0.5 or about 0.5-1.0 or about 1.0, 1.0 or about 1.0-3.0 or about 3.0, 1.0 or about 1.0-2.5 or about 2.5, 1.0 or about 1.0-2.0 or about In some embodiments, the culture medium comprises and / or is supplemented with 2.0, 1.0 or about 1.0-1.5 or about 1.5, 1.5 or about 1.5-3.0 or about 3.0, 1.5 or about 1.5-2.5 or about 2.5, 1.5 or about 1.5-2.0 or about 2.0, 2.0 or about 2.0-3.0 or about 3.0, 2.0 or about 2.0-2.5 or about 2.5, or 2.5 or about 2.5-3.0 or about 3.0 g / l of glucose. In some embodiments, the culture medium comprises and / or is supplemented with 1.6-2.4 g / l of glucose. In some embodiments, the culture medium comprises and / or is supplemented with 2.0 g / l of glucose. In some embodiments, the culture medium comprises about 2.0 g / l of glucose.

[0122] In some embodiments, the culture medium comprises and / or is supplemented with sodium pyruvate. In some embodiments, the culture medium comprises and / or is supplemented with 0.1 or about 0.1-2.0 or about 2.0 mM sodium pyruvate. In some embodiments, the culture medium comprises and / or is supplemented with 0.1 or about 0.1-1.8 or about 1.8, 0.1 or about 0.1-1.6 or about 1.6, 0.1 or about 0.1-1.4 or about 1.4, 0.1 or about 0.1-1.2 or about 1.2, 0.1 or about 0.1-1.0 or about 1.0, 0.1 or about 0.1-0.8 or about 0.8, 0.1 or about 0.1-0.6 or about 0.6, 0.1 or about 0.1-0.4 or about 0.4, 0.1 or about 0.1-0.2 or about 0.2, 0. 2 or about 0.2 to 2.0 or about 2.0, 0.2 or about 0.2 to 1.8 or about 1.8, 0.2 or about 0.2 to 1.6 or about 1.6, 0.2 or about 0.2 to 1.4 or about 1.4, 0.2 or about 0.2 to 1.2 or about 1.2, 0.2 or about 0.2 to 1.0 or about 1.0, 0.2 or about 0.2 to 0.8 or about 0.8, 0.2 or about 0.2 to 0.6 or about 0.6, 0.2 or about 0.2 to 0.4 or about 0.4, 0.4 or about 0.4 to 2.0 or about 2.0, 0 0.4 or about 0.4-1.8 or about 1.8, 0.4 or about 0.4-1.6 or about 1.6, 0.4 or about 0.4-1.4 or about 1.4, 0.4 or about 0.4-1.2 or about 1.2, 0.4 or about 0.4-1.0 or about 1.0, 0.4 or about 0.4-0.8 or about 0.8, 0.4 or about 0.4-0.6 or about 0.6, 0.6 or about 0.6-2.0 or about 2.0, 0.6 or about 0.6-1.8 or about 1.8, 0.6 or about 0.6-1.6 or about 1.6 , 0.6 or about 0.6-1.4 or about 1.4, 0.6 or about 0.6-1.2 or about 1.2, 0.6 or about 0.6-1.0 or about 1.0, 0.6 or about 0.6-0.8 or about 0.8, 0.8 or about 0.8-2.0 or about 2.0, 0.8 or about 0.8-1.8 or about 1.8, 0.8 or about 0.8-1.6 or about 1.6, 0.8 or about 0.8-1.4 or about 1.4, 0.8 or about 0.8-1.4 or about 1.4, 0.8 or about 0.8-1.2 or about 1.2, 0.8 or about 0.8-1.0 or about 1.0, 1.0 or about 1.0-2.0 or about 2.0, 1.0 or about 1.0-1.8 or about 1.8, 1.0 or about 1.0-1.6 or about 1.6, 1.0 or about 1.0-1.4 or about 1.4, 1.0 or about 1.0-1.2 or about 1.2, 1.2 or about 1.2-2.0 or about 2.0, 1.2 or about 1.2-1.8 or about 1.8, 1.2 or about 1.2-1.6 or or about 1.6, 1.2 or about 1.2-1.4 or about 1.4, 1.4 or about 1.4-2.0 or about 2.0, 1.4 or about 1.4-1.8 or about 1.8, 1.4 or about 1.4-1.6 or about 1.6, 1.6 or about 1.6-2.0 or about 2.0, 1.6 or about 1.6-1.8 or about 1.8, or 1.8 or about 1.8-2.0 or about 2.0 mM sodium pyruvate. In some embodiments, the culture medium comprises 0.8-1.2 mM sodium pyruvate. In some embodiments, the culture medium comprises 1.0 mM sodium pyruvate. In some embodiments, the culture medium comprises about 1.0 mM sodium pyruvate.

[0123] In some embodiments, the culture medium comprises and / or is supplemented with sodium bicarbonate. In some embodiments, the culture medium comprises and / or is supplemented with 0.5 or about 0.5-3.5 or about 3.5 g / l of sodium bicarbonate. In some embodiments, the culture medium comprises and / or is supplemented with 0.5 or about 0.5-3.0 or about 3.0, 0.5 or about 0.5-2.5 or about 2.5, 0.5 or about 0.5-2.0 or about 2.0, 0.5 or about 0.5-1.5 or about 1.5, 0.5 or about 0.5-1.0 or about 1.0, 1.0 or about 1.0-3.0 or about 3.0, 1.0 or about 1.0-2.5 or about 2.5, 1.0 or about 1.0-2.0 or about 2. In some embodiments, the culture medium comprises and / or is supplemented with 0, 1.0 or about 1.0-1.5 or about 1.5, 1.5 or about 1.5-3.0 or about 3.0, 1.5 or about 1.5-2.5 or about 2.5, 1.5 or about 1.5-2.0 or about 2.0, 2.0 or about 2.0-3.0 or about 3.0, 2.0 or about 2.0-2.5 or about 2.5, or 2.5 or about 2.5-3.0 or about 3.0 g / l of sodium bicarbonate. In some embodiments, the culture medium comprises and / or is supplemented with 1.6-2.4 g / l of sodium bicarbonate. In some embodiments, the culture medium comprises and / or is supplemented with 2.0 g / l of sodium bicarbonate. In some embodiments, the culture medium comprises about 2.0 g / l of sodium bicarbonate.

[0124] In some embodiments, the culture medium comprises and / or is supplemented with albumin, e.g., human albumin, e.g., a human albumin solution described herein. In some embodiments, the culture medium comprises and / or is supplemented with a 20% albumin solution, e.g., 0.5% or about 0.5% to 3.5% or about 3.5% v / v of a 20% human albumin solution. In some embodiments, the culture medium comprises a 20% albumin solution, e.g., a 20% human albumin solution, at 0.5 or about 0.5% to 3.0 or about 3.0%, 0.5% or about 0.5% to 2.5 or about 2.5%, 0.5% or about 0.5% to 2.0 or about 2.0%, 0.5% or about 0.5% to 1.5 or about 1.5%, 0.5% or about 0.5% to 1.0 or about 1.0%, 1.0 or about 1.0% to 3.0 or about 3.0%, 1.0 or about 1.0% to 2.5 or about 2.5%, 1.0 or about 1.0% to 2.5 or about 2.5%, 1.0 or about 1.0% to 3.0 or about 3.0 ...%, 1.0 or about 1.0% to 3.0%, 1. In some embodiments, the culture medium comprises and / or is supplemented with a 20% albumin solution, e.g., 1.0 or about 1.0% to 2.0 or about 2.0%, 1.0 or about 1.0% to 1.5 or about 1.5%, 1.5 or about 1.5% to 3.0 or about 3.0%, 1.5 or about 1.5% to 2.5 or about 2.5%, 1.5 or about 1.5% to 2.0 or about 2.0%, 2.0% or about 2.0% to 3.0 or about 3.0%, 2.0% or about 2.0% to 2.5 or about 2.5%, or 2.5% or about 2.5% to 3.0 or about 3.0% v / v. In some embodiments, the culture medium comprises and / or is supplemented with a 20% albumin solution, e.g., 1.6% to 2.4% v / v of a 20% human albumin solution. In some embodiments, the culture medium comprises and / or is supplemented with a 20% albumin solution, e.g., a 20% human albumin solution at 2.0% v / v. In some embodiments, the culture medium comprises a 20% albumin solution, e.g., a 20% human albumin solution at about 2.0% v / v.

[0125] In some embodiments, the culture medium comprises and / or is supplemented with 2 or about 2-6 or about 6 g / l of albumin, e.g., human albumin. In some embodiments, the culture medium comprises and / or is supplemented with 2 or about 2-5.5 or about 5.5, 2 or about 2-5.0 or about 5.0, 2 or about 2-4.5 or about 4.5, 2 or about 2-4 or about 4, 2 or about 2-3.5 or about 3.5, 2 or about 2-3 or about 3, 2 or about 2-2.5 or about 2.5, 2.5 or about 2.5-6 or about 6, 2.5 or about 2.5 to 5.5 or about 5.5, 2.5 or about 2.5 to 5.5 or about 5.5, 2.5 or about 2.5 to 5.0 or about 5.0, 2.5 or about 2.5 to 4.5 or about 4.5, 2.5 or about 2.5 to 4.0 or about 4.0, 2.5 or about 2.5 to 3.5 or about 3.5, 2.5 or about 2.5 to 3.0 or about 3.0, 3 or about 3 to 6 or about 6, 3 or about 3 to 5.5 or about 5.5, 3 or about 3-5 or about 5, 3 or about 3-4.5 or about 4.5, 3 or about 3-4 or about 4, 3 or about 3-3.5 or about 3.5, 3.5 or about 3.5-6 or about 6, 3.5 or about 3.5-5.5 or about 5.5, 3.5 or about 3.5-5 or about 5, 3.5 or about 3.5-4.5 or about 4.5, 3.5 or about 3.5-4 or about 4, 4 or about 4-6 or about 6, 4 or or about 4-5.5 or about 5.5, 4 or about 4-5 or about 5, 4 or about 4-4.5 or about 4.5, 4.5 or about 4.5-6 or about 6, 4.5 or about 4.5-5.5 or about 5.5, 4.5 or about 4.5-5 or about 5, 5 or about 5-6 or about 6, 5 or about 5-5.5 or about 5.5, or 5.5 or about 5.5-6 or about 6 g / l. In some embodiments, the culture medium comprises and / or is supplemented with 3.2-4.8 g / l of albumin, e.g., human albumin. In some embodiments, the culture medium comprises 4 g / l of albumin, e.g., human albumin. In some embodiments, the culture medium comprises about 4 g / l of albumin, e.g., human albumin.

[0126] In some embodiments, the culture medium is supplemented with Poloxamer 188. In some embodiments, the culture medium comprises and / or is supplemented with 0.1 or about 0.1-2.0 or about 2.0 g / l of Poloxamer 188. In some embodiments, the culture medium comprises and / or is supplemented with 0.1 or about 0.1-1.8 or about 1.8, 0.1 or about 0.1-1.6 or about 1.6, 0.1 or about 0.1-1.4 or about 1.4, 0.1 or about 0.1-1.2 or about 1.2, 0.1 or about 0.1-1.0 or about 1.0, 0.1 or about 0.1-0.8 or about 0.8, 0.1 or about 0.1-0.6 or about 0.6, 0.1 or about 0.1-0.4 or about 0.4, 0.1 or about 0.1-0.2 or about 0.2, 0.2 or about 0.2-2.0 or about 2.0, 0.2 or about 0.2-1.8 or about 1.8, 0.2 or about 0.2-1.6 or about 1.6, 0.2 or about 0.2-1.4 or about 1.4, 0.2 or about 0.2-1.2 or about 1.2, 0.2 or about 0.2-1.0 or about 1.0, 0.2 or about 0.2-0.8 or about 0.8, 0.2 or about 0.2-0.6 or about 0.6, 0.2 or about 0.2-0.4 or about 0.4, 0.4 or about 0.4-2.0 or is about 2.0, 0.4 or about 0.4 to 1.8 or about 1.8, 0.4 or about 0.4 to 1.6 or about 1.6, 0.4 or about 0.4 to 1.4 or about 1.4, 0.4 or about 0.4 to 1.2 or about 1.2, 0.4 or about 0.4 to 1.0 or about 1.0, 0.4 or about 0.4 to 0.8 or about 0.8, 0.4 or about 0.4 to 0.6 or about 0.6, 0.6 or about 0.6 to 2.0 or about 2.0, 0.6 or about 0.6 to 1.8 or about 1.8, 0.6 or about 0.6 to 1.6 or about 1.6, 0.6 or about 0.6-1.4 or about 1.4, 0.6 or about 0.6-1.2 or about 1.2, 0.6 or about 0.6-1.0 or about 1.0, 0.6 or about 0.6-0.8 or about 0.8, 0.8 or about 0.8-2.0 or about 2.0, 0.8 or about 0.8-1.8 or about 1.8, 0.8 or about 0.8-1.6 or about 1.6, 0.8 or about 0.8-1.4 or about 1.4, 0.8 or about 0.8-1.4 or about 1.4, 0.8 or about 0.8-1.2 or about 1.2, 0.8 or about 0.8-1.0 or about 1.0, 1.0 or about 1.0-2.0 or about 2.0, 1.0 or about 1.0-1.8 or about 1.8, 1.0 or about 1.0-1.6 or about 1.6, 1.0 or about 1.0-1.4 or about 1.4, 1.0 or about 1.0-1.2 or about 1.2, 1.2 or about 1.2-2.0 or about 2.0, 1.2 or about 1.2-1.8 or about 1.8, 1.2 or about 1.2- In some embodiments, the culture medium comprises and / or is supplemented with 1.6 or about 1.6, 1.2 or about 1.2 to 1.4 or about 1.4, 1.4 or about 1.4 to 2.0 or about 2.0, 1.4 or about 1.4 to 1.8 or about 1.8, 1.4 or about 1.4 to 1.6 or about 1.6, 1.6 or about 1.6 to 2.0 or about 2.0, 1.6 or about 1.6 to 1.8 or about 1.8, or 1.8 or about 1.8 to 2.0 or about 2.0 g / l of Poloxamer 188. In some embodiments, the culture medium comprises 0.8 to 1.2 g / l of Poloxamer 188. In some embodiments, the culture medium comprises 1.0 g / l of Poloxamer 188. In some embodiments, the culture medium comprises about 1.0 g / l of Poloxamer 188.

[0127] In some embodiments, the culture medium contains and / or is supplemented with one or more antibiotic agents.

[0128] An exemplary first culture medium is shown in Table 1.

[0129] [Table 1]

[0130] Exemplary second culture media are referred to in Table 2.

[0131] [Table 2]

[0132] 2.CD3 binding antibody In some embodiments, the culture medium comprises and / or is supplemented with a CD3 binding antibody or antigen-binding fragment thereof. In some embodiments, the CD3 binding antibody or antigen-binding fragment thereof is selected from the group consisting of OKT3, UCHT1 and HIT3a, or a variant thereof. In some embodiments, the CD3 binding antibody or antigen-binding fragment thereof is OKT3 or an antigen-binding fragment thereof.

[0133] In some embodiments, the CD3 binding antibody or antigen-binding fragment thereof and the feeder cells are added to the culture vessel prior to the addition of the NK cells and / or culture medium.

[0134] In some embodiments, the culture medium comprises and / or is supplemented with OKT3 at 5 or about 5 ng / ml to 15 or about 15 ng / ml. In some embodiments, the culture medium comprises and / or is supplemented with OKT3 at 5 or about 5 to 12.5 or about 12.5, 5 or about 5 to 10 or about 10, 5 or about 5 to 7.5 or about 7.5, 7.5 or about 7.5 to 15 or about 15, 7.5 or about 7.5 to 12.5 or about 12.5, 7.5 or about 7.5 to 10 or about 10, 10 or about 10 to 15 or about 15, 10 or about 10 to 12.5 or about 12.5, or 12.5 or about 12.5 to 15 or about 15 ng / ml. In some embodiments, the culture medium comprises and / or is supplemented with OKT3 at 10 ng / ml. In some embodiments, the culture medium contains and / or is supplemented with OKT3 at about 10 ng / ml.

[0135] 3.Culture container A variety of vessels are contemplated within the teachings of the present invention. In some embodiments, the culture vessel is selected from the group consisting of a flask, a bottle, a dish, a multi-well plate, a roller bottle, a bag, and a bioreactor.

[0136] In some embodiments, the culture vessel is treated to be hydrophilic. In some embodiments, the culture vessel is treated to promote attachment and / or proliferation. In some embodiments, the surface of the culture vessel is coated with serum, collagen, laminin, gelatin, poly-L-lysine, fibronectin, extracellular matrix proteins, and combinations thereof.

[0137] In some embodiments, different types of culture vessels are used for different culture stages.

[0138] In some embodiments, the culture vessel has a volume of 100 ml or about 100 ml to 1,000 l or about 1,000 l. In some embodiments, the culture vessel has a volume of 125 ml or about 125 ml, 250 ml or about 250 ml, 500 ml or about 500 ml, 1 l or about 1 l, 5 l or about 5 l, 10 l or about 10 l, or 20 l or about 20 l.

[0139] In some embodiments, the culture vessel is a bioreactor.

[0140] In some embodiments, the bioreactor is a rocking bed (wave motion) bioreactor. In some embodiments, the bioreactor is a stirred tank bioreactor. In some embodiments, the bioreactor is a rotating wall vessel. In some embodiments, the bioreactor is a perfusion bioreactor. In some embodiments, the bioreactor is an isolation / expansion automated system. In some embodiments, the bioreactor is an automated or semi-automated bioreactor. In some embodiments, the bioreactor is a disposable bag bioreactor.

[0141] In some embodiments, the bioreactor has a volume of about 100 ml to about 1,000 liters. In some embodiments, the bioreactor has a volume of about 10 liters to about 1,000 liters. In some embodiments, the bioreactor has a volume of about 100 liters to about 900 liters. In some embodiments, the bioreactor has a volume of about 10 liters to about 800 liters. In some embodiments, the bioreactor may be about 10 L to about 700 L, about 10 L to about 600 L, about 10 L to about 500 L, about 10 L to about 400 L, about 10 L to about 300 L, about 10 L to about 200 L, about 10 L to about 100 L, about 10 L to about 90 L, about 10 L to about 80 L, about 10 L to about 70 L, about 10 L to about 60 L, about 10 L to about 50 L, about 10 L to about 40 L, about 10 L to about 30 L, about 10 L to about 20 L, about 10 L to about 25 L, about 10 L to about 30 L, about 10 L to about 40 L, about 10 L to about 50 L, about 10 L to about 40 L, about 10 L to about 30 L, about 10 L to about 20 L, about 10 L to about 25 L, about 10 L to about 3 ... 0l, about 20l to about 1,000l, about 20l to about 900l, about 20l to about 800l, about 20l to about 700l, about 20l to about 600l, about 20l to about 500l, about 20l to about 400l, about 20l to about 300l, approximately 20l to approximately 200l, approximately 20l to approximately 100l, approximately 20l to approximately 90l, approximately 20l to approximately 80l, approximately 20l to approximately 70l, approximately 20l to approximately 60l, approximately 20l to approximately 50l, approximately 20l to approximately 40l, approximately 20l to 30l, 30l to 1,000l, 30l to 900l, 30l to 800l, 30l to 700l, 30l to 600l, 30l to 500l, 30l to 400l , about 30l to about 300l, about 30l to about 200l, about 30l to about 100l, about 30l to about 90l, about 30l to about 80l, about 30l to about 70l, about 30l to about 60l, about 30l to about 50l, about 30l ~40l, 40l~1,000l, 40l~900l, 40l~800l, 40l~700l, 40l~600l, 40l~500l, 40l~400l, 40 l ~ about 300l, about 40l - about 200l, about 40l - about 100l, about 40l - about 90l, about 40l - about 80l, about 40l - about 70l, about 40l - about 60l, about 40l - about 50l, about 50l - about 1,000l, about 50l to about 900l, about 50l to about 800l, about 50l to about 700l, about 50l to about 600l, about 50l to about 500l, about 50l to about 400l, about 50l to about 300l, about 50l to about 200l, about 50l to about 100l, about 50l to about 90l, about 50l to about 80l, about 50l to about 70l, about 50l to about 60l, about 60l to about 1,000l, about 60l to about 900l, about 60l to about 800l, about 60l to about 700l, about 60l to about 600l, about 60l to about 500l, about 60l to about 400l, about 60l to about 300l, about 60l to about 200l, about 60l to about 100L, about 60l to about 90l, about 60l to about 80l, about 60l to about 70l, about 70l to about 1,000l, about 70l to about 900l, about 70l to about 800l, about 70l to about 700l, about 70l to about 600l, about 70l to about 500l, about 70l to about 400l, about 70l to about 300l, about 70l to about 200l, about 70l to about 100l, about 70l to about 90l, about 70l to about 80l, about 80l to about 1,000l, about 80l to about 900l, about 80l to about 800l, about 80l to about 700l, about 80l to about 600l, about 80l to about 500l, about 80l to about 400l, about 80l to about 300l, about 80l to about 200l, about 80l to about 100l, about 80l to about 90l, about 90l to about 1,000l, about 90l to about 900l, about 90l to about 800l, about 90l to about 700l, about 90l to about 600l, about 90l to about 500l, about 90l to about 400l, about 90l to about 300l, about 90l to about 200l, about 90l to about 100l, about 100l to about 1,000l, about 100l to about 900l, about 100l to about 800l, about 100l to about 700l, about 100l to about 600l, about 100l to about 500l, about 100l to about 400l, about 100l to about 300l, about 100l to about 200l, about 200l to about 1,000l, about 200l to about 900l, about 200l to about 800l, about 200l to about 700l, about 200l to about 600l, about 200l to about 500l, about 200l to about 400l, about 200l to about 300l, about 300l to about 1,000l, about 300l to about 900l, about 300l to about 800l, about 300l to about 700l, about 300l to about 600l, about 300l to about 500l, about 300l to about 400l, about 400l to about 1,000l, about 400l to about 900l, about 400l to about 800l, about 400l to about 700l, about 400l to about 600l, about 400l to about 500l, about 500l ~ approx. 1,000l, approx. 500l ~ approx. 900l, approx. 500l ~ approx. 800l, approx. 500l ~ approx. 700l, approx. 500l ~ approx. 600l, approx. 600l The bioreactor has a volume of about 1,000 L, about 600 L to about 900 L, about 600 L to about 800 L, about 600 L to about 700 L, about 700 L to about 1,000 L, about 700 L to about 900 L, about 700 L to about 800 L, about 800 L to about 1,000 L, about 800 L to about 900 L, or about 900 L to about 1,000 L. In some embodiments, the bioreactor has a volume of about 50 L.

[0142] In some embodiments, the bioreactor has a volume of 100 ml to 1,000 liters. In some embodiments, the bioreactor has a volume of 10 liters to 1,000 liters. In some embodiments, the bioreactor has a volume of 100 liters to 900 liters. In some embodiments, the bioreactor has a volume of 10 liters to 800 liters. In some embodiments, the bioreactor is configured to have a volume of 10 l to 700 l, 10 l to 600 l, 10 l to 500 l, 10 l to 400 l, 10 l to 300 l, 10 l to 200 l, 10 l to 100 l, 10 l to 90 l, 10 l to 80 l, 10 l to 70 l, 10 l to 60 l, 10 l to 50 l, 10 l to 40 l, 10 l to 30 l, 10 l to 20 l, 20 l to 1,000 l, 20 l to 900 l, 20 l to 800 l, 20 l to 700 l, 20 l to 600 l, 20 l to 500 l , 20l~400l, 20l~300l, 20l~200l, 20l~100l, 20l~90l, 20l~80l, 20l~70l, 20l~60l, 20l~50l, 20l~40l, 20l~30l, 30l~1,000 l, 30l~900l, 30l~800l, 30l~700l, 30l~600l, 30l~500l, 30l~400l, 30l~300l, 30l~200l, 30l~100l, 30l~90l, 30l~80l, 30l ~70l, 30l~60l, 30l~50l, 30l~40l, 40l~1,000l, 40l~900l, 40l~800l, 40l~700l, 40l~600l, 40l~500l, 40l~400l, 40l~300 l, 40l~200l, 40l~100l, 40l~90l, 40l~80l, 40l~70l, 40l~60l, 40l~50l, 50l~1,000l, 50l~900l, 50l~800l, 50l~700l, 50l~ 600l, 50l~500l, 50l~400l, 50l~300l, 50l~200l, 50l~100l, 50l~90l, 50l~80l, 50l~70l, 50l~60l, 60l~1,000l, 60l~900l, 60l~800l, 60l~700l, 60l~600l, 60l~500l, 60l~400l, 60l~300l, 60l~200l, 60l~100L, 60l~90l, 60l~80l, 60l~70l, 70l~1,000l, 70l~900l, 70l~800l, 70l~700l, 70l~600l, 70l~500l, 70l~400l, 70l~300l, 70l~200l, 70l~100l, 70l~90l, 70 l~80l, 80l~1,000l, 80l~900l, 80l~800l, 80l~700l, 80l~600l, 80l~500l, 80l~400l, 80l~300l, 80l~200l, 80l~100l , 80l~90l, 90l~1,000l, 90l~900l, 90l~800l, 90l~700l, 90l~600l, 90l~500l, 90l~400l, 90l~300l, 90l~200l, 90l~ 100l, 100l~1,000l, 100l~900l, 100l~800l, 100l~700l, 100l~600l, 100l~500l, 100l~400l, 100l~300l, 100l~200l, 200l~1,000l, 200l~900l, 200l~800l, 200l~700l, 200l~600l, 200l~500l, 200l~400l, 200l~300l, 300l~1,000l, 30 0l~900l, 300l~800l, 300l~700l, 300l~600l, 300l~500l, 300l~400l, 400l~1,000l, 400l~900l, 400l~800l, 400l~70 In some embodiments, the bioreactor has a volume of 0 liters, 400 liters to 600 liters, 400 liters to 500 liters, 500 liters to 1,000 liters, 500 liters to 900 liters, 500 liters to 800 liters, 500 liters to 700 liters, 500 liters to 600 liters, 600 liters to 1,000 liters, 600 liters to 900 liters, 600 liters to 800 liters, 600 liters to 700 liters, 700 liters to 1,000 liters, 700 liters to 900 liters, 700 liters to 800 liters, 800 liters to 1,000 liters, 800 liters to 900 liters, or 900 liters to 1,000 liters. In some embodiments, the bioreactor has a volume of 50 liters.

[0143] 4. Cell Expansion and Stimulation In some embodiments, a natural killer cell source, for example, a single unit of umbilical cord blood, is co-cultured with feeder cells to produce expanded and stimulated NK cells.

[0144] In some embodiments, the co-culture is performed in a culture medium described herein, for example, Culture Medium Example #1 (Table 1) or Culture Medium Example #2 (Table 2).

[0145] In some embodiments, the natural killer cell source, e.g., umbilical cord blood single unit, is cultured at 1×10 total nucleated cells prior to expansion. 7 or about 1 x 10 7 ~1×10 9 or about 1 x 10 9 In some embodiments, the natural killer cell source, e.g., a single unit of umbilical cord blood, contains 1×10 total nucleated cells prior to expansion. 8 or about 1 x 10 8 ~1.5×10 8 or about 1.5 x 10 8 In some embodiments, the natural killer cell source, e.g., a single unit of umbilical cord blood, contains 1×10 total nucleated cells prior to expansion. 8 In some embodiments, the natural killer cell source, e.g., a single unit of umbilical cord blood, contains about 1×10 total nucleated cells prior to expansion. 8 In some embodiments, the natural killer cell source, e.g., a single unit of umbilical cord blood, contains 1×10 total nucleated cells prior to expansion. 9 In some embodiments, the natural killer cell source, e.g., a single unit of umbilical cord blood, contains about 1×10 total nucleated cells prior to expansion. 9 Includes pieces.

[0146] In some embodiments, the natural killer cell source, e.g., cells derived from a co-culture of a single cord blood unit with feeder cells, are harvested and frozen, e.g., harvested and frozen in a cryopreservation composition as described herein. In some embodiments, the frozen cells derived from the co-culture are an infusion-ready drug product. In some embodiments, the frozen cells derived from the co-culture are used as a master cell bank (MCB) for producing an infusion-ready drug product, e.g., by one or more additional co-culture steps as described herein. Thus, for example, the natural killer cell source can be expanded and stimulated as described herein to produce expanded and stimulated NK cells suitable for use in an infusion-ready drug product without making any intermediate product. The natural killer cell source can also be expanded and stimulated as described herein to produce an intermediate product, e.g., a primary master cell bank (MCB). The primary MCB can be used to produce expanded and stimulated NK cells suitable for use in an infusion-ready drug product, or alternatively, to produce other intermediate products, e.g., a secondary MCB. The secondary MCB can be used to produce expanded and stimulated NK cells suitable for use in injectable, finished pharmaceutical products, or alternatively, to produce other intermediate products, such as the tertiary MCB.

[0147] In some embodiments, the ratio of feeder cells:natural killer cell source or MCB cells inoculated into the co-culture is 1:1 or about 1:1 to 4:1 or about 4:1. In some embodiments, the ratio of feeder cells:natural killer cell source or MCB cells is 1:1 or about 1:1 to 3.5:1 or about 3.5:1, 1:1 or about 1:1 to 3:1 or about 3:1, 1:1 or about 1:1 to 2.5:1 or about 2.5:1, 1:1 or about 1:1 to 2:1 or about 2:1, 1:1 or about 1:1 to 1.5 or about 1.5:1, 1.5:1 or about 1.5:1 to 4:1 or about 4:1, 1.5:1 or about 1.5:1 to 3.5:1 or about 3.5:1, 1.5:1 or about 1.5:1 to 3:1 or about 3:1, 1.5:1 or about 1.5:1 to 2.5:1 or about 2.5:1, 2:1 or about 1.5:1 to 2:1 or about 2:1, 2:1 or about 2:1 to 4:1 or about 4:1, 2:1 or about 2:1 to 3.5:1 or about 3.5:1, 2:1 or about 2:1 to 3:1 or about 3:1, 2:1 or about 2:1 to 2.5:1 or about 2.5:1, 2.5:1 or about 2.5:1 to 4:1 or about 4:1, 2.5:1 or about 2.5:1 to 3.5:1 or about 3.5:1, 2.5:1 or about 2.5:1 to 3:1 or about 3:1, 3:1 or about 3:1 to 4:1 or about 4:1, 3:1 or about 3:1 to 3.5:1 or about 3.5:1, or 3.5:1 or about 3.5:1 to 4:1 or about 4:1. In some embodiments, the ratio of feeder cells to natural killer cell source or MCB cells inoculated into the co-culture is 2.5: 1. In some embodiments, the ratio of feeder cells to natural killer cell source or MCB cells inoculated into the co-culture is about 2.5: 1.

[0148] In some embodiments, the co-culture is performed in a disposable culture bag, for example, a 1 liter disposable culture bag. In some embodiments, the co-culture is performed in a bioreactor, for example, a 50 liter bioreactor. In some embodiments, after the primary inoculation, culture medium is added to the co-culture.

[0149] In some embodiments, the co-culture is carried out for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, or longer. In some embodiments, the co-culture is carried out for up to 16 days.

[0150] In some embodiments, the co-culture is performed at or about 37°C.

[0151] In some embodiments, the co-cultivation is carried out at or about pH 7.9.

[0152] In some embodiments, the co-cultivation is carried out at a dissolved oxygen (DO) level of 50% or greater.

[0153] In some embodiments, exemplary culture medium #1 (Table 1) is used to produce MCB and culture medium #2 (Table 2) is used to produce cells suitable for injectable-finished drug product.

[0154] In some embodiments, a source of natural killer cells, e.g., umbilical cord blood single units in co-culture with feeder cells, e.g., MCB cells or infusion-ready pharmaceutical cells, 50×10 8 or about 50 x 10 8 ~50×10 12 or about 50 x 10 12 In some embodiments, such expansion produces 50×10 cells, e.g., MCB cells or injectable finished drug product cells. 8 or about 50 x 10 8 ~25×10 10 or about 25 x 10 10 , 10×10 8 or about 10 x 10 8 ~1×10 10 or about 1 x 10 10 , 50×10 8 or about 50 x 10 8 ~75×10 9 or about 75 x 10 9 , 50×108 or about 50 x 10 8 ~50×10 9 or about 50 x 10 9 , 50×10 8 or about 50 x 10 8 ~25×10 9 or about 25 x 10 9 , 50×10 8 or about 50 x 10 8 ~1×10 9 or about 1 x 10 9 , 50×10 8 or about 50 x 10 8 ~75×10 8 or about 75 x 10 8 , 75×10 8 or about 75 x 10 8 ~50×10 10 or about 50 x 10 10 , 75×10 8 or about 75 x 10 8 ~25×10 10 or about 25 x 10 10 , 75×10 8 or about 75 x 10 8 ~1×10 10 or about 1 x 10 10 , 75×10 8 or about 75 x 10 8 ~75×10 9 or about 75 x 10 9 , 75×10 8 or about 75 x 10 8 ~50×10 9 or about 50 x 10 9 , 75×10 8 or about 75 x 10 8 ~25×10 9 or about 25 x 10 9 , 75×10 8 or about 75 x 10 8 ~1×10 9 or about 1 x 10 9 , 1×10 9 or about 1 x 10 9 ~50×10 10 or about 50 x 10 10 , 1×10 9 or about 1 x 109 ~25×10 10 or about 25 x 10 10 , 1×10 9 or about 1 x 10 9 ~1×10 10 or about 1 x 10 10 , 1×10 9 or about 1 x 10 9 ~75×10 9 or about 75 x 10 9 , 1×10 9 or about 1 x 10 9 ~50×10 9 or about 50 x 10 9 , 1×10 9 or about 1 x 10 9 ~25×10 9 or about 25 x 10 9 , 25×10 9 or about 25 x 10 9 ~50×10 10 or about 50 x 10 10 , 25×10 9 or about 25 x 10 9 ~25×10 10 or about 25 x 10 10 , 25×10 9 or about 25 x 10 9 ~1×10 10 or about 1 x 10 10 , 25×10 9 or about 25 x 10 9 ~75×10 9 or about 75 x 10 9 , 25×10 9 or about 25 x 10 9 ~50×10 9 or about 50 x 10 9 , 50×10 9 or about 50 x 10 9 ~50×10 10 or about 50 x 10 10 , 50×10 9 or about 50 x 10 9 ~25×10 10 or about 25 x 10 10 , 50×10 9 or about 50 x 10 9 ~1×10 10or about 1 x 10 10 , 50×10 9 or about 50 x 10 9 ~75×10 9 or about 75 x 10 9 , 75×10 9 or about 75 x 10 9 ~50×10 10 or about 50 x 10 10 , 75×10 9 or about 75 x 10 9 ~25×10 10 or about 25 x 10 10 , 75×10 9 or about 75 x 10 9 ~1×10 10 or about 1 x 10 10 , 1×10 10 or about 1 x 10 10 ~50×10 10 or about 50 x 10 10 , 1×10 10 or about 1 x 10 10 ~25×10 10 or about 25 x 10 10 , or 25 × 10 10 or about 25 x 10 10 ~50×10 10 or about 50 x 10 10 Pieces are produced.

[0155] In some embodiments, the amplification produces 60 or about 60-100 or about 100 vials containing 600 million or about 600 million to 1 billion or about 1 billion cells, e.g., MCB cells or injectable-finished drug product cells, respectively. In some embodiments, the amplification produces 80 or about 80 vials containing or consisting of 800 million or about 800 million cells, e.g., MCB cells or injectable-finished drug product cells, respectively.

[0156] In some embodiments, the expansion increases the number of cells, e.g., MCB cells, by 100-fold or about 100-fold to 500-fold or about 500-fold relative to the number of cells, e.g., NK cells, in a natural killer cell source. In some embodiments, the expansion is achieved by increasing the number of cells, e.g., the number of MCB cells, relative to the number of cells in a natural killer cell source, e.g., NK cells, by 100-fold or about 100-fold to 500-fold or about 500-fold, 100-fold or about 100-fold to 400-fold or about 400-fold, 100-fold or about 100-fold to 300-fold or about 300-fold, 100-fold or about 100-fold to 200-fold or about 200-fold, 200-fold or about 200-fold to 450-fold or about 450-fold, 200-fold or about 200-fold to 400-fold or about 400-fold, 100-fold or about 100-fold to 350-fold or about 350-fold, 200-fold or about 200-fold to 300-fold or about 300-fold, 200-fold or about 200-fold to 250-fold or about 250-fold, 250-fold or about 250-fold, 50-500-fold or about 500-fold, 250-fold or about 250-fold or about 450-fold, 200-fold or about 200-fold or about 400-fold, 250-fold or about 250-fold or about 350-fold, 250-fold or about 250-fold or about 300-fold, 300-fold or about 300-fold or about 500-fold, 300-fold or about Increase by 300-fold to 450-fold or about 450-fold, 300-fold or about 300-fold to 400-fold or about 400-fold, 300-fold or about 300-fold to 350-fold or about 350-fold, 350-fold or about 350-fold to 500-fold or about 500-fold, 350-fold or about 350-fold to 450-fold or about 450-fold, or 350-fold or about 350-fold to 400-fold or about 400-fold.

[0157] In some embodiments, the expansion increases the number of cells, e.g., MCB cells, by 100-fold or about 100-fold to 70,000-fold or about 70,000-fold relative to the number of cells, e.g., NK cells, in the natural killer cell source. In some embodiments, the expansion increases the number of cells, e.g., MCB cells, by at least 10,000-fold, e.g., 15,000-fold, 20,000-fold, 25,000-fold, 30,000-fold, 35,000-fold, 40,000-fold, 45,000-fold, 50,000-fold, 55,000-fold, 60,000-fold, 65,000-fold, or 70,000-fold relative to the number of cells, e.g., NK cells, in the natural killer cell source.

[0158] In some embodiments, co-culture of MCB cells with feeder cells produces cells, e.g., 500 million or about 500 million to 1.5 billion or about 1.5 billion NK cells suitable for use in the MCB and / or injectable finished pharmaceutical product. In some embodiments, co-culture of MCB cells with feeder cells produces cells, e.g., 500 million or about 500 million to 1.5 billion or about 1.5 billion, 500 million or about 500 million to 1.25 billion or about 1.25 billion, 500 million or about 500 million to 1 billion or about 1 billion, 500 million or about 500 million to 750 million or about 750 million, 750 million or about 750 million to 1.5 billion or about 1.5 billion, 500 million or about 500 million to 1.25 billion or about 1.25 billion, 750 million or about 750 million to 1 billion or about 1 billion, 1 billion or about 1 billion to 1.5 billion or about 1.5 billion, 1 billion or about 1 billion to 1.25 billion or about 1.25 billion, or 1.25 billion to 1.5 billion or about 1.5 billion NK cells suitable for use in the MCB and / or injectable-finished pharmaceutical product.

[0159] In some embodiments, co-culture of MCB cells with feeder cells produces 50 or about 50 to 150 or about 150 vials of cells, e.g., injectable-finished drug product cells, containing at or about 750 million to at or about 750 million to at or about 1.25 billion cells, e.g., MCB and / or NK cells suitable for use in an injectable-finished drug product, respectively. In some embodiments, co-culture of MCB cells with feeder cells produces 100 or about 100 vials containing or consisting of at or about 1 billion cells, e.g., MCB and / or NK cells suitable for use in an injectable-finished drug product, respectively.

[0160] In some embodiments, the expansion increases the number of cells suitable for use in the MCB and / or injectable-finished drug product, e.g., NK cell numbers, by 100-fold or about 100-fold to 500-fold or about 500-fold compared to the initial MCB cell numbers. In some embodiments, the expansion involves increasing the cell numbers, e.g., increasing the cell numbers of the MCB and / or NK cells suitable for use in an injectable finished drug product by 100-fold or about 100-fold to 500-fold or about 500-fold, 100-fold or about 100-fold to 400-fold or about 400-fold, 100-fold or about 100-fold to 300-fold or about 300-fold, 100-fold or about 100-fold to 200-fold or about 200-fold, 200-fold or about 200-fold to 450-fold or about 450-fold, 200-fold or about 200-fold to 400-fold or about 400-fold, 100-fold or about 100-fold to 350-fold or about 350-fold, 200-fold or about 200-fold to 300-fold or about 300-fold, 200-fold or about 200-fold to 250-fold or about 250-fold, 250-fold or about 250-fold to about 500-fold or about 500-fold, 250-fold or about 250-fold to about 450-fold or about 450-fold, 200-fold or about 200-fold to about 400-fold or about 400-fold, 250-fold or about 250-fold to about 350-fold or about 350-fold, 250-fold or about 250-fold to about 300-fold or about 300-fold, 300-fold or about 300-fold to about 500-fold or about 500-fold, 300-fold or about 300-fold to 450-fold or about 450-fold, 300-fold or about 300-fold to 400-fold or about 400-fold, 300-fold or about 300-fold to 350-fold or about 350-fold, 350-fold or about 350-fold to 500-fold or about 500-fold, 350-fold or about 350-fold to 450-fold or about 450-fold, 350-fold or about 350-fold to 400-fold or about 400-fold.

[0161] In some embodiments, the expansion increases the number of cells suitable for use in the MCB and / or injectable-finished drug product, e.g., NK cell number, by at least 10,000-fold, e.g., 15,000-fold, 20,000-fold, 25,000-fold, 30,000-fold, 35,000-fold, 40,000-fold, 45,000-fold, 50,000-fold, 55,000-fold, 60,000-fold, 65,000-fold, or 70,000-fold, compared to the initial MCB cell number.

[0162] In embodiments in which cells are engineered during expansion and stimulation as described herein, not all expanded and stimulated cells will necessarily be successfully engineered, e.g., successfully transduced, e.g., successfully transduced with a vector comprising a heterologous protein, e.g., a heterologous protein comprising a CAR and / or IL-15, as described herein. Thus, the methods described herein can additionally include sorting engineered cells, e.g., engineered cells as described herein, from non-engineered cells.

[0163] In some embodiments, engineered cells, e.g., transduced cells, are separated from non-engineered cells, e.g., non-transduced cells, using a reagent specific for an antigen of the engineered cells, e.g., an antibody that targets an antigen of the engineered cells but not the non-engineered cells. In some embodiments, the antigen of the engineered cells is a component of a CAR, e.g., a component of a CAR described herein.

[0164] Systems for antigen-based cell separation are commercially available, e.g., CliniMACS (R) There is a classification system (Miltenyi Biotec).

[0165] In some embodiments, engineered cells, eg, transduced cells, are sorted from non-engineered cells, eg, non-transduced cells, using flow cytometry measurements.

[0166] In some embodiments, the sorted engineered cells are used as an MCB. In some embodiments, the sorted engineered cells are used as components of an injectable finished pharmaceutical product.

[0167] In some embodiments, engineered cells, e.g., transduced cells, are sorted from non-engineered cells, e.g., non-transduced cells, using microfluidic cell sorting methods, such as those referenced in Dalili et al., "A Review of Sorting, Separation and Isolation of Cells and Microbeads for Biomedical Applications: Microfluidic Approaches," Analyst 144:87 (2019).

[0168] In some embodiments, 1% or about 1% to 99% or about 99% of the expanded and stimulated cells are successfully engineered, e.g., successfully transduced, e.g., successfully transduced with a vector comprising a heterologous protein, e.g., a heterologous protein comprising a CAR and / or IL-15, as described herein. In some embodiments, 1% or about 1% to 90% or about 90%, 1% or about 1% to 80% or about 80%, 1% or about 1% to 70% or about 70%, 1% or about 1% to 60% or about 60%, 1% or about 1% to 50% or about 50%, 1% or about 1% to 40% or about 40%, 1% or about 1% to 30% or about 30%, 1% or about 1% to 20% or about 20%, 1% or about 1% to 10% or about 10%, 1% or about About 1% to 5% or about 5%, 5% or about 5% to 99% or about 99%, 5% or about 5% to 90% or about 90%, 5% or about 5% to 80% or about 80%, 5% or about 5% to 70% or about 70%, 5% or about 5% to 60% or about 60%, 5% or about 5% to 50% or about 50%, 5% or about 5% to 40% or about 40%, 5% or about 5% to 30% or about 30%, 5% or about 5% to 20% or about 20%, 5% or about 5% to 10% or about 1 0%, 10% or about 10% to 99% or about 99%, 10% or about 10% to 90% or about 90%, 10% or about 10% to 80% or about 80%, 10% or about 10% to 70% or about 70%, 10% or about 10% to 60% or about 60%, 10% or about 10% to 50% or about 50%, 10% or about 10% to 40% or about 40%, 10% or about 10% to 30% or about 30%, 10% or about 10% to 20% or about 20%, 20% or about 20 % to 99% or about 99%, 20% or about 20% to 90% or about 90%, 20% or about 20% to 80% or about 80%, 20% or about 20% to 70% or about 70%, 20% or about 20% to 60% or about 60%, 20% or about 20% to 50% or about 50%, 20% or about 20% to 40% or about 40%, 20% or about 20% to 30% or about 30%, 30% or about 30% to 99% or about 99%, 30% or about 30% to 90% or about 90%,30% or about 30% to 80% or about 80%, 30% or about 30% to 70% or about 70%, 30% or about 30% to 60% or about 60%, 30% or about 30% to 50% or about 50%, 30% or about 30% to 40% or about 40%, 40% or about 40% to 99% or about 99%, 40% or about 40% to 90% or about 90%, 40% or about 40% to 80% or about 8 0%, 40% or about 40%-70% or about 70%, 40% or about 40%-70% or about 70%, 40% or about 40%-60% or about 60%, 40% or about 40%-50% or about 50%, 50% or about 50%-99% or about 99%, 50% or about 50%-90% or about 90%, 50% or about 50%-80% or about 80%, 50% or about 50%-70% or About 70%, 50% or about 50%-60% or about 60%, 60% or about 60%-99% or about 99%, 60% or about 60%-90% or about 90%, 60% or about 60%-80% or about 80%, 60% or about 60%-70% or about 70%, 70% or about 70%-99% or about 99%, 70% or about 70%-90% or about 90%, 70% or about 70%-80% or about 80%, 80% or about 80%-99% or about 99%, 80% or about 80%-90% or about 90% or 90% or about 90%-99% or about 99% are successfully engineered, e.g., successfully transduced, e.g., successfully transduced with a vector comprising a heterologous protein, e.g., a heterologous protein comprising CAR and / or IL-15, as described herein.

[0169] In some embodiments, frozen cells of the primary or secondary MCB are thawed and cultured. In some embodiments, a single frozen cell vial of the primary or secondary MCB, e.g., a single vial containing 800 million or about 800 million cells of each of the primary or secondary MCB, is thawed and cultured. In some embodiments, the frozen primary or secondary MCB cells are cultured with additional feeder cells to produce cells suitable for use as a secondary or tertiary MCB or in an injectable-finished drug product. In some embodiments, cells are harvested and frozen from a co-culture of the primary or secondary MCB.

[0170] In some embodiments, cells are harvested from a co-culture of a natural killer cell source, primary MCB, or secondary MCB, and frozen in a cryopreservation composition, e.g., a cryopreservation composition described herein. In some embodiments, the cells are washed after harvesting. Thus, the present invention provides pharmaceutical compositions and cryopreservation compositions, e.g., cryopreservation compositions described herein, comprising activated and stimulated NK cells, e.g., activated and stimulated NK cells produced by the methods described herein, e.g., harvested and washed activated and stimulated NK cells produced by the methods described herein.

[0171] In some embodiments, the cells are mixed with a cryopreservation composition prior to freezing, e.g., as described herein. In some embodiments, the cells are frozen in a cryobag. In some embodiments, the cells are frozen in a cryovial.

[0172] In some embodiments, the method additionally comprises isolating NK cells from the expanded and stimulated population of NK cells.

[0173] An example of a process for expanding and stimulating NK cells is shown in FIG.

[0174] 5.Operation In some embodiments, the method additionally includes engineering the NK cells to express a heterologous protein, e.g., a heterologous protein, e.g., a heterologous protein described herein, e.g., a heterologous protein including CAR and / or IL-15.

[0175] In some embodiments, engineering the NK cells to express a heterologous protein described herein comprises transforming, e.g., stably transforming, the NK cells with a vector comprising a polynucleic acid encoding a heterologous protein described herein. Suitable vectors are described herein.

[0176] In some embodiments, engineering NK cells to express a heterologous protein as described herein includes introducing the heterologous protein by gene editing (e.g., zinc finger nuclease (ZFN) gene editing, ARCUS gene editing, CRISPR-Cas9 gene editing, or megaTAL gene editing) in combination with adeno-associated virus (AAV) technology.

[0177] In some embodiments, the NK cells are engineered to express a heterologous protein as described herein, for example, during or after culturing the composition in medium containing feeder cells.

[0178] In some embodiments, the methods additionally include engineering the NK cells, for example, to express, overexpress, knock out, or knock down a gene or gene product.

[0179] In some embodiments, the natural killer cells are not genetically engineered.

[0180] A. Characterization of expanded and stimulated NK cells For example, after ex vivo expansion and stimulation as described herein, the expanded and stimulated population of NK cells not only has a number / density (e.g., as described above) that does not naturally occur in the human body, but also has phenotypic characteristics (e.g., gene expression and / or surface protein expression) that differ from the starting source material or other naturally occurring populations of NK cells.

[0181] In some cases, the starting NK cell source is a sample derived from only one individual, e.g., a single umbilical cord blood unit without in vitro expansion. Thus, in some cases, the expanded and stimulated NK cells share a common lineage, i.e., they are all generated from the expansion of the starting NK cell source, and thus share a genotype due to the clonal expansion of a cell population that is itself derived from a single organism. However, these cells may not naturally occur at the densities achieved by in vitro expansion, and differences in phenotypic characteristics exist from the starting NK cell source.

[0182] In some cases, the population of expanded and stimulated NK cells includes at least 100 million, e.g., 200 million, 250 million, 300 million, 400 million, 500 million, 600 million, 700 million, 750 million, 800 million, 900 million, 1 billion, 2 billion, 3 billion, 4 billion, 5 billion, 6 billion, 7 billion, 8 billion, 9 billion, 10 billion, 15 billion, 20 billion, 25 billion, 50 billion, 75 billion, 80 billion, 90 billion, 100 billion, 200 billion, 250 billion, 300 billion, 400 billion, 500 billion, 600 billion, 700 billion, 800 billion, 900 billion, 1 trillion, 2 trillion, 3 trillion, 4 trillion, 5 trillion, 6 trillion, 7 trillion, 8 trillion, 9 trillion, or 10 trillion expanded natural killer cells.

[0183] In some embodiments, the expanded and stimulated NK cells comprise at least 80%, e.g., at least 90%, at least 95%, at least 99% or 100% CD56+CD3- cells.

[0184] In some embodiments, the expanded and stimulated NK cells are not genetically engineered.

[0185] In some embodiments, the expanded and stimulated NK cells do not comprise the CD16 transgene.

[0186] In some embodiments, the expanded and stimulated NK cells do not express exogenous CD16 protein.

[0187] The expanded and stimulated NK cells are characterized, for example, by surface expression of one or more of CD16, CD56, CD3, CD38, CD14, CD19, NKG2D, NKp46, NKp30, DNAM-1, and NKp44.

[0188] The expression levels of surface proteins referenced herein are optionally achieved without positive selection for the particular proteins referenced. For example, optionally, a NK cell source, e.g., a single umbilical cord blood unit, contains both the KIR B allele of the KIR receptor system and the 158V / V variant of CD16, and is CD56+ enriched and CD3(+) depleted, e.g., by gating on CD56+CD3- expression, but does not select for other surface protein expression during expansion and stimulation.

[0189] In some embodiments, the expanded and stimulated NK cells, e.g., expanded and stimulated NK cells derived from the expansion and stimulation of a single cord blood unit, e.g., as described above, comprise at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% NKG2D+ cells.

[0190] In some embodiments, the expanded and stimulated NK cells, e.g., expanded and stimulated NK cells derived from the expansion and stimulation of a single cord blood unit, e.g., as described above, comprise at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% NKp46+ cells.

[0191] In some embodiments, the expanded and stimulated NK cells, e.g., expanded and stimulated NK cells derived from the expansion and stimulation of a single cord blood unit, e.g., as described above, comprise at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% NKp30+ cells.

[0192] In some embodiments, the expanded and stimulated NK cells, e.g., expanded and stimulated NK cells derived from the expansion and stimulation of a single umbilical cord blood unit, as described above, comprise at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% DNAM-1+ cells.

[0193] In some embodiments, the expanded and stimulated NK cells, e.g., expanded and stimulated NK cells derived from the expansion and stimulation of a single cord blood unit, e.g., as described above, comprise at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% NKp44+ cells.

[0194] In some embodiments, the expanded and stimulated NK cells, e.g., expanded and stimulated NK cells derived from the expansion and stimulation of a single cord blood unit, e.g., as described above, comprise at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% CD94+(KLRD1) cells.

[0195] In some embodiments, the expanded and stimulated NK cells, e.g., expanded and stimulated NK cells derived from the expansion and stimulation of a single cord blood unit, e.g., as described above, comprise 20% or less, e.g., 10% or less, 5% or less, 1% or less, or 0% CD3+ cells.

[0196] In some embodiments, the expanded and stimulated NK cells, e.g., expanded and stimulated NK cells derived from the expansion and stimulation of a single cord blood unit, e.g., as described above, comprise 20% or less, e.g., 10% or less, 5% or less, 1% or less, or 0% CD14+ cells.

[0197] In some embodiments, the expanded and stimulated NK cells, e.g., expanded and stimulated NK cells derived from the expansion and stimulation of a single cord blood unit, e.g., as described above, comprise 20% or less, e.g., 10% or less, 5% or less, 1% or less, or 0% CD19+ cells.

[0198] In some embodiments, the expanded and stimulated NK cells, e.g., expanded and stimulated NK cells derived from the expansion and stimulation of a single cord blood unit, e.g., as described above, comprise 20% or less, e.g., 10% or less, 5% or less, 1% or less, or 0% CXCR+ cells.

[0199] In some embodiments, the expanded and stimulated NK cells, e.g., expanded and stimulated NK cells derived from the expansion and stimulation of a single cord blood unit, e.g., as described above, comprise 20% or less, e.g., 10% or less, 5% or less, 1% or less, or 0% CD122+(IL2RB) cells.

[0200] As described herein, the inventors have surprisingly found that NK cells expanded and stimulated by the methods described herein express high levels of CD16 due to the expansion and stimulation steps, resulting in a cell population with high CD16 expression. The high expression of CD16 obviates the need to engineer the expanded cells to express CD16, which is important for initiating ADCC, which is a surprising and unexpected advantage of the expansion and stimulation methods described herein. Thus, in some embodiments, the expanded and stimulated NK cells, e.g., expanded and stimulated NK cells derived from the expansion and stimulation of a single cord blood unit, as described above, comprise 50% or more, e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% CD16+ NK cells.

[0201] In some embodiments, the expanded and stimulated NK cells, e.g., as described above, e.g., expanded and stimulated NK cells derived from the expansion and stimulation of a single cord blood unit, contain both the KIR B allele of the KIR receptor system and the 158V / V variant of CD16 and comprise 50% or more, e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% CD16+ NK cells.

[0202] In some embodiments, the percentage of expanded and stimulated NK cells that express CD16, e.g., derived from the expansion and stimulation of a single cord blood unit, as described above, is the same as or higher than the percentage of natural killer cells in seed cells derived from cord blood.

[0203] In some embodiments, the percentage of expanded and stimulated NK cells that express NKG2D, e.g., derived from the expansion and stimulation of a single cord blood unit, e.g., as described above, is the same as or higher than the percentage of natural killer cells in seed cells derived from cord blood.

[0204] In some embodiments, the percentage of expanded and stimulated NK cells that express NKp30, e.g., derived from the expansion and stimulation of a single cord blood unit, e.g., as described above, is the same as or higher than the percentage of natural killer cells in seed cells derived from cord blood.

[0205] In some embodiments, the percentage of expanded and stimulated NK cells that express DNAM-1, e.g., the percentage of expanded and stimulated NK cells derived from the expansion and stimulation of a single umbilical cord blood unit as described above, is the same as or higher than the percentage of natural killer cells in seed cells derived from umbilical cord blood.

[0206] In some embodiments, the percentage of expanded and stimulated NK cells that express NKp44, e.g., derived from the expansion and stimulation of a single cord blood unit, e.g., as described above, is the same as or higher than the percentage of natural killer cells in seed cells derived from cord blood.

[0207] In some embodiments, the percentage of expanded and stimulated NK cells that express NKp46, e.g., derived from the expansion and stimulation of a single cord blood unit, e.g., as described above, is the same as or higher than the percentage of natural killer cells in seed cells derived from cord blood.

[0208] As described herein, the inventors have surprisingly found that NK cells expanded and stimulated by the methods described herein express low levels of CD38. CD38 is an effective target in certain cancer therapies (e.g., multiple myeloma and acute myeloid leukemia). See, for example, Jiao et al., "CD38: Targeted Therapy in Multiple Myeloma and Therapeutic Potential for Solid Cancerrs," Expert Opinion on Investigational Drugs 29(11):1295-1308 (2020). However, when administering an anti-CD38 antibody such as daratumumab together with NK cells, there is a risk of increased fratricide, since NK cells naturally express CD38. However, NK cells expanded and stimulated by the methods described herein express low levels of CD38, thus eliminating the expected fratricide. While other groups have relied on engineering methods such as genome editing to reduce CD38 expression (see, e.g., Gurney et al., "CD38 Knockout Natural Killer Cells Expressing an Affinity Optimized CD38 Chimeric Antigen Receptor Successfully Target Acute Myeloid Leukemia with Reduced Effector Cell Fratricide," Haematologica doi:10.3324 / haematol.2020.271908 (2020)), NK cells expanded and stimulated by the methods described herein express low levels of CD38 without the need for genetic engineering, providing a surprising and unexpected advantage for using NK cells expanded and stimulated as described herein in combination with, for example, a CD38 antibody such as daratumumab to treat CD38+ cancers.

[0209] Thus, in some embodiments, the expanded and stimulated NK cells, e.g., expanded and stimulated NK cells derived from the expansion and stimulation of a single cord blood unit, as described above, comprise 80% or less CD38+ cells, e.g., 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, or 20% or less CD38+ cells.

[0210] In some embodiments, the expanded and stimulated NK cells, e.g., expanded and stimulated NK cells derived from the expansion and stimulation of a single cord blood unit, e.g., as described above, contain both the KIR B allele of the KIR receptor system and the 158V / V variant of CD16 and comprise 80% or less CD38+ cells, e.g., 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, or 20% or less CD38+ cells.

[0211] In some embodiments, the expanded and stimulated NK cells, e.g., derived from the expansion and stimulation of a single cord blood unit, e.g., as described above, comprise both the KIR B allele of the KIR receptor system and the 158V / V variant of CD16, and comprise 80% or less, e.g., 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less or 20% or less, CD38+ cells, and 50% or more, e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% CD16+ NK cells.

[0212] In some embodiments, the expanded and stimulated NK cells, e.g., expanded and stimulated NK cells derived from the expansion and stimulation of a single cord blood unit, e.g., as described above, comprise both the KIR B allele of the KIR receptor system and the 158V / V variant of CD16, and include: i) 50% or more, e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% CD16+ NK cells; and / or ii) 80% or less CD38+ cells, e.g., 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less or 20% or less CD38+ ... i) at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% NKG2D+ cells; and / or iv) at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% NKp46+ cells; and / or v) at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95% , at least 99% or 100% NKp30+ cells; and / or vi) at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% DNAM-1+ cells; and / or vii) at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% NKp44+ cells; and / or viii) at least 60% , e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% CD94+(KLRD1) cells; and / or ix) 20% or less, e.g., 10% or less, 5% or less, 1% or less or 0% CD3+ cells; and / or x) 20% or less, e.g., 10% or less, 5% or less, 1% or less or 0% CD14+ cells; and / or xi) 20% or less, e.g., 10% or less, 5% or less, 1% or less or 0% CD19+ cells;and / or xii) 20% or less, e.g., 10% or less, 5% or less, 1% or less, or 0% CXCR+ cells; and / or xiii) 20% or less, e.g., 10% or less, 5% or less, 1% or less, or 0% CD122+ (IL2RB) cells;

[0213] In some embodiments, a residual signature of the feeder cells may be detected, for example, by the presence of residual cells (e.g., by detecting cells expressing certain surface proteins) or by residual nucleic acids and / or proteins expressed by the feeder cells, but which do not persist in the expanded and stimulated NK cells.

[0214] For example, in some cases, the methods described herein include expanding and stimulating natural killer cells using engineered feeder cells, e.g., the aforementioned eHuT-78 feeder cells, engineered to express sequences not expressed by cells in a natural killer cell source, such as natural killer cells. For example, the engineered feeder cells can be engineered to express one or more genes selected from the group consisting of 4-1BBL (UniProtKB P41273, SEQ ID NO: 1), membrane-bound IL-21 (SEQ ID NO: 2) and mutant TNFα (SEQ ID NO: 3) ("eHut-78 cells"), or variants thereof.

[0215] Although these feeder cells may not persist in the expanded and stimulated NK cells, the expanded and stimulated NK cells may retain detectable residual amounts of cells, proteins and / or nucleic acids derived from the feeder cells. Thus, the presence of such residues in the expanded and stimulated NK cells can be detected, for example, by detecting the cells themselves (e.g., by flow cytometric measurements), proteins expressed by these cells, and / or nucleic acids expressed by these cells.

[0216] Thus, the present invention also describes a population of expanded and stimulated NK cells that contain residual feeder cells (viable or dead) or cellular impurities of the residual feeder cells (e.g., proteins or parts thereof, and / or genetic material such as nucleic acids or parts thereof) of the residual feeder cells. In some cases, the expanded and stimulated NK cells contain residual feeder cells, e.g., eHuT-78 feeder cells, at a level greater than 0% and less than or equal to 0.3%.

[0217] In some cases, the expanded and stimulated NK cells contain residual feeder cells encoding, for example, residual 4-1BBL (UniProtKB P41273, SEQ ID NO: 1), membrane-bound IL-21 (SEQ ID NO: 2), and / or mutant TNFα (SEQ ID NO: 3), or portions thereof. In some cases, the membrane-bound IL-21 contains the CD8 transmembrane domain.

[0218] In some cases, the expanded and stimulated NK cells comprise a residual % of feeder cells greater than 0% and less than or equal to 0.2%, e.g., as measured by the relative proportion of feeder cell-specific proteins or nucleic acid sequences (i.e., proteins or nucleic acid sequences not expressed by natural killer cells) in the sample, e.g., by qPCR, e.g., as described herein.

[0219] In some embodiments, the remaining feeder cells are CD4(+) T cells. In some embodiments, the remaining feeder cells are engineered CD4(+) T cells. In some embodiments, the remaining feeder cells are engineered to express one or more genes selected from the group consisting of 4-1BBL (UniProtKB P41273, SEQ ID NO: 1), membrane-bound IL-21 (SEQ ID NO: 2) and mutated TNFα (SEQ ID NO: 3) ("eHut-78 cells"), or variants thereof. Thus, in some cases, the feeder cell specific protein is 4-1BBL (UniProtKB P41273, SEQ ID NO: 1), membrane-bound IL-21 (SEQ ID NO: 2) and / or mutated TNFα (SEQ ID NO: 3). Thus, the feeder cell specific nucleic acid is a nucleic acid encoding 4-1BBL (UniProtKB P41273, SEQ ID NO: 1), membrane-bound IL-21 (SEQ ID NO: 2) and / or mutated TNFα (SEQ ID NO: 3), or a portion thereof. In some cases, the membrane-bound IL-21 comprises a CD8 transmembrane domain.

[0220] A wide variety of different methods can be used to analyze and detect the presence of nucleic acid or protein gene products in biological samples. As used herein, "detection" can refer to a method used to discover, confirm or verify the coexistence or presence of a compound and / or substance (e.g., a cell, a protein and / or a nucleic acid). In some embodiments, the detection method can be used to detect proteins. In some embodiments, detection can include chemiluminescent or fluorescent techniques. In some embodiments, detection can include immunologically-based methods (e.g., quantitative enzyme-linked immunosorbent assay (ELISA), Western blotting or dot blotting) that use antibodies to specifically react with the whole protein or specific epitopes of the protein. In some embodiments, detection can include immunoprecipitation of the protein (Jungblut et al., J Biotechnol. 31; 41(2-3): 111-20 (1995); Franco et al., Eur J Morphol. 39(1): 3-25 (2001)). In some embodiments, the detection method can be used to detect nucleic acids (e.g., DNA and / or RNA). In some embodiments, detection can include Northern blot analysis, nuclease protection assay (NPA), in situ hybridization or reverse transcription-polymerase chain reaction (RT-PCR) (Raj et al., Nat. Methods 5, 877-879 (2008); Jin et al., J Clin Lab Anal. 11(1):2-9 (1997); Ahmed, J Environ Sci Health C Environ Carcinog Ecotoxicol Rev. 20(2):77-116 (2002)).

[0221] For this reason, the present invention also describes methods for detecting a population of expanded and stimulated NK cells co-cultured with engineered feeder cells, e.g., eHuT-78 feeder cells as described herein, e.g., a population of expanded and stimulated NK cells using the methods described herein.

[0222] II. Manipulation of Natural Killer Cells In some embodiments, natural killer cells are engineered to produce CAR-NK and / or NK expressing IL-15.

[0223] In some embodiments, the natural killer cells are manipulated, e.g., transduced, during expansion and stimulation, e.g., during expansion and stimulation as described herein. In some embodiments, the natural killer cells are manipulated during expansion and stimulation, e.g., during production of an MCB as described herein. In some embodiments, the natural killer cells are manipulated during expansion and stimulation, e.g., during production of NK cells suitable for use in an injectable-finished pharmaceutical product and / or during production of an MCB as described above. Thus, in some embodiments, the NK cells are host cells, and the invention provides NK host cells that express a heterologous protein, e.g., a heterologous protein as described above.

[0224] In some embodiments, the natural killer cells are manipulated prior to expansion and stimulation. In some embodiments, the natural killer cells are manipulated after expansion and stimulation.

[0225] In some embodiments, the NK cells are engineered by transduction with a vector. Suitable vectors are described herein, such as lentiviral vectors, e.g., lentiviral vectors comprising a heterologous protein, e.g., a heterologous protein described herein. In some embodiments, the NK cells are transduced during production of the first MCB, as described herein.

[0226] In some embodiments, the NK cells are transduced at a multiplicity of infection of 1 or about 1 to 40 or about 40 viral particles / cell. In some embodiments, the NK cells are transduced at a multiplicity of infection of 1 or about 1, 5 or about 5, 10 or about 10, 15 or about 15, 20 or about 20, 25 or about 25, 30 or about 30, 35 or about 35, or 40 or about 40 viral particles / cell.

[0227] A. Chimeric Antigen Receptor In some embodiments, the heterologous protein is a fusion protein, e.g., a fusion protein comprising a chimeric antigen receptor (CAR), is introduced into the NK cells, e.g., during the expansion and stimulation process.

[0228] In some embodiments, the CAR comprises one or more of the following: a signal sequence, an extracellular domain, a hinge, a transmembrane domain, and one or more intracellular signaling domain sequences. In some embodiments, the CAR additionally comprises a spacer sequence.

[0229] In some embodiments, a CAR comprises (from N-terminus to C-terminus) a signal sequence, an extracellular domain, a hinge, a spacer, a transmembrane domain, a first signaling domain sequence, a second signaling domain sequence, and a third signaling domain sequence.

[0230] In some embodiments, a CAR comprises (from N-terminus to C-terminus) a signal sequence, an extracellular domain, a hinge, a transmembrane domain, a first signaling domain sequence, a second signaling domain sequence, and a third signaling domain sequence.

[0231] In some embodiments, the extracellular domain comprises an antibody or an antigen-binding portion thereof.

[0232] In some embodiments, the one or more intracellular signaling domain sequences are CD28 intracellular signaling sequences. In some embodiments, the CD28 intracellular signaling sequence comprises or consists of SEQ ID NO:5.

[0233] In some embodiments, the one or more intracellular signaling domain sequences are OX40L signaling sequences. In some embodiments, the OX40L signaling sequence comprises or consists of SEQ ID NO:8.

[0234] In some embodiments, the one or more intracellular signaling domain sequences are CD3ζ intracellular signaling domain sequences. In some embodiments, the CD3ζ intracellular signaling sequence comprises or consists of SEQ ID NO:11.

[0235] In some embodiments, the CAR comprises a CD28 intracellular signaling sequence (SEQ ID NO:5), an OX40L intracellular signaling sequence (SEQ ID NO:8), and a CD3 zeta intracellular signaling sequence (SEQ ID NO:11).

[0236] In some embodiments, the CAR comprises an intracellular signaling domain that comprises or is made up of SEQ ID NO:19.

[0237] In some embodiments, the CAR does not comprise an OX40L intracellular signaling domain sequence.

[0238] In some embodiments, the CAR comprises the CD28 intracellular signaling sequence (SEQ ID NO:5) and the CD3ζ intracellular signaling sequence (SEQ ID NO:11), but does not comprise the OX40L intracellular signaling domain sequence.

[0239] B.IL-15 In some embodiments, NK cells are engineered to express IL-15, e.g., human IL-15 (UniProtKB#P40933; NCBI Gene ID#3600), e.g., soluble human IL-15 or an ortholog thereof, or a mutant of any of the foregoing. In some embodiments, IL-15 is expressed as part of a fusion protein that additionally includes a cleavage site. In some embodiments, IL-15 is expressed as part of a polyprotein that includes a T2A ribosomal skip sequence site (sometimes referred to as a self-cleavage site).

[0240] In some embodiments, the IL-15 comprises or consists of SEQ ID NO:16.

[0241] In some embodiments, the T2A truncation comprises or consists of SEQ ID NO:14.

[0242] In some embodiments, IL-15 is expressed as part of a fusion protein that includes a CAR, e.g., a CAR described herein.

[0243] In some embodiments, the fusion protein comprises (N-terminal to C-terminal) CAR, a cleavage site and IL-15.

[0244] In some embodiments, the fusion protein comprises SEQ ID NO:20.

[0245] C. Inhibitory receptors In some embodiments, the NK cells are engineered to alter, eg, reduce, the expression of one or more inhibitory receptor genes.

[0246] In some embodiments, the inhibitory receptor gene is an HLA-specific inhibitory receptor. In some embodiments, the inhibitory receptor gene is an HLA-non-specific inhibitory receptor.

[0247] In some embodiments, the inhibitory receptor gene is selected from the group consisting of KIR, CD94 / NKG2A, LILRB1, PD-1, IRp60, Siglec-7, LAIR-1, and combinations thereof.

[0248] D. Polynucleic acids, vectors and host cells The present invention also provides polynucleic acids encoding the fusion proteins or portions thereof, e.g., polynucleotide sequences encoding the polypeptides described herein, as set out in the sequence tables provided herein.

[0249] Additionally, the present invention provides vectors comprising the polynucleic acids, and cells, such as NK cells, comprising such vectors.

[0250] In some embodiments, the vector is a lentiviral vector. See, e.g., Milone et al., "Clinical Use of Lentiviral Vectors", Leukemia 32:1529-41 (2018). In some embodiments, the vector is a retroviral vector. In some embodiments, the vector is a gamma retroviral vector. In some embodiments, the vector is a non-viral vector, such as a piggyback non-viral vector (PB transposon, e.g., Wu et al., "piggyback is a Flexible and Highly Active Transposon as Compared to Sleeping Beauty, Tol2, and Mos1 in Mammalian Cells", PNAS 103(41):15008-13 (2006)), a Sleeping Beauty non-viral vector (SB transposon, see, e.g., Hudecek et al., "Going Non-Viral: the Sleeping Beauty Transposon System Breaks on Through to the Clinical Side", Critical Reviews in Biochemistry and Molecular Biology 52(4):355-380 (2017)), or an mRNA vector.

[0251] III. Freezing storage A. Freeze preservation composition The present invention provides cryopreservation compositions, e.g., cryopreservation compositions suitable for intravenous administration, e.g., for intravenous administration of NK cells, e.g., NK cells described herein. In some embodiments, a pharmaceutical composition comprises the cryopreservation composition and cells, e.g., NK cells described herein.

[0252] 1. Albumin

[0253] In some embodiments, the cryopreservation composition comprises an albumin protein, such as a human albumin protein (UniProtKB Accession P0278, SEQ ID NO:21), or a variant thereof. In some embodiments, the cryopreservation composition comprises an albumin protein, such as an orthologue of a human albumin protein, or a variant thereof. In some embodiments, the cryopreservation composition comprises an albumin protein, such as a biologically active portion of human albumin, or a variant thereof.

[0254] In some embodiments, albumin, for example human albumin, is provided as a solution, which is also referred to herein as albumin solution or human albumin solution.Thus, in some embodiments, the cryopreservation composition is or comprises an albumin solution, for example a human albumin solution.In some embodiments, the albumin solution is a serum-free albumin solution.

[0255] In some embodiments, the albumin solution is suitable for intravenous use.

[0256] In some embodiments, the albumin solution comprises 40 or about 40-200 or about 200 g / l of albumin. In some embodiments, the albumin solution comprises 40 or about 40-50 or about 50 g / l of albumin, e.g., human albumin. In some embodiments, the albumin solution comprises about 200 g / l of albumin, e.g., human albumin. In some embodiments, the albumin solution comprises 200 g / l of albumin, e.g., human albumin.

[0257] In some embodiments, the albumin solution comprises a protein composition that is 95% or more albumin protein, e.g., human albumin protein. In some embodiments, 96%, 97%, 98%, or 99% or more of the protein is albumin, e.g., human albumin.

[0258] In some embodiments, the albumin solution additionally comprises sodium. In some embodiments, the albumin solution comprises 100 or about 100-200 or about 200 mmol of sodium. In some embodiments, the albumin solution comprises 130 or about 130-160 or about 160 mmol of sodium.

[0259] In some embodiments, the albumin solution additionally comprises potassium. In some embodiments, the albumin solution additionally comprises up to 3 mmol of potassium. In some embodiments, the albumin solution additionally comprises up to 2 mmol of potassium.

[0260] In some embodiments, the albumin solution additionally comprises one or more stabilizers. In some embodiments, the stabilizers are selected from the group consisting of sodium caprylate, caprylic acid, (2S)-2-acetamido-3-(1H-indol-3-yl)propanoic acid (also referred to herein as: acetyltryptophan, N-acetyl-L-tryptophan and acetyl-L-tryptophan), 2-acetamido-3-(1H-indol-3-yl)propanoic acid (also referred to herein as: N-acetyltryptophan, DL-acetyltryptophan and N-acetyl-DL-tryptophan). In some embodiments, the solution comprises less than 0.1 mmol of one or more stabilizers per gram of protein in the solution. In some embodiments, the solution comprises 0.05 or about 0.05 to 0.1 or about 0.1, e.g., 0.064 or about 0.064 to 0.096 or about 0.096 mmol of stabilizers per gram of protein in the solution. In some embodiments, the solution comprises less than 0.1 mmol of total stabilizing agent per gram of protein in the solution, in some embodiments, the solution comprises 0.05 or about 0.05 to 0.1 or about 0.1, e.g., 0.064 or about 0.064 to 0.096 or about 0.096 mmol of total stabilizing agent per gram of protein in the solution.

[0261] In some embodiments, the albumin solution is comprised of a protein composition of 95% or more albumin protein in water, one or more stabilizers selected from the group consisting of sodium, potassium and sodium caprylate, caprylic acid, (2S)-2-acetamido-3-(1H-indol-3-yl)propanoic acid (also referred to herein as: acetyltryptophan, N-acetyl-L-tryptophan and acetyl-L-tryptophan), 2-acetamido-3-(1H-indol-3-yl)propanoic acid (also referred to herein as: N-acetyltryptophan, DL-acetyltryptophan and N-acetyl-DL-tryptophan).

[0262] In some embodiments, the cryopreservation composition comprises an albumin solution, such as an albumin solution described herein, at or about 10% v / v to 50% or about 50% v / v. In some embodiments, the cryopreservation composition comprises an albumin solution, such as an albumin solution described herein, at or about 10% v / v to 50% or about 50%, at or about 10% v / v to 45%, at or about 10% v / v to 40%, at or about 10% v / v to 35%, at or about 35%, at or about 10% v / v to 30%, at or about 10% v / v to 25%, at or about 10% v / v to 20%, at or about 10% v / v to 15%, at or about 15% v / v to 50%, at or about 10% v / v to 50%, at or about 10% v / v to 50%, at or about 10% v / v to 45%, at or about 10% v / v to 40%, at or about 10% v / v to 35%, at or about 10% v / v to 30%, at or about 10% v / v to 25%, at or about 10% v / v to 20%, at or about 10% v / v to 5 ...45%, at or about 10% v / v to 40%, at or about 10% v / v to 40%, at or about 10% v / v to 35%, at or about 10% v / v to 30%, at or about 10% v / v to 25%, at or about 10% v / v to 20%, at 0% or about 50%, 15% or about 15% to 45% or about 45%, 15% or about 15% to 40% or about 40%, 15% or about 15% to 35% or about 35%, 15% or about 15% to 30% or about 30%, 15% or about 15% to 25% or about 25%, 15% or about 15% to 20% or about 20%, 20% or about 20% to 50% or about 50%, 20% or about 20% to 45% or about 45%, 20% or about 20% to 40% or about 40%, 0% or about 20%-35% or about 35%, 20% or about 20%-30% or about 30%, 20% or about 20%-25% or about 25%, 25% or about 25%-50% or about 50%, 25% or about 25%-45% or about 45%, 25% or about 25%-40% or about 40%, 25% or about 25%-35% or about 35%, 25% or about 25%-30% or about 30%, 30% or about 30%-50% or about 50%, 30% or about 30%-4 The albumin solution may comprise 5% or about 45%, 30% or about 30%-40% or about 40%, 30% or about 30%-35% or about 35%, 35% or about 35%-50% or about 50%, 35% or about 35%-45% or about 45%, 35% or about 35%-40% or about 40%, 40% or about 40%-50% or about 50%, 40% or about 40%-45% or about 45%, or 45% or about 45%-50% or about 50% v / v of the albumin solution described herein.In some embodiments, the cryopreservation composition comprises about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% v / v of an albumin solution described herein.In some embodiments, the cryopreservation composition comprises 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% v / v of an albumin solution described herein.

[0263] In some embodiments, the cryopreservation composition comprises albumin, e.g., human albumin, at 20 or about 20-100 or about 100 g / l. In some embodiments, the cryopreservation composition comprises albumin, e.g., human albumin, at 20 or about 20-100 or about 100, 20 or about 20-90 or about 90, 20 or about 20-80 or about 80, 20 or about 20-70 or about 70, 20 or about 20-60 or about 60, 20 or about 20-50 or about 50, 20 or about 20-40 or about 40, 20 or about 20-30 or about 30, 30 or about 30-10 0 or about 100, 30 or about 30-90 or about 90, 30 or about 30-80 or about 80, 30 or about 30-70 or about 70, 30 or about 30-60 or about 60, 30 or about 30-50 or about 50, 30 or about 30-40 or about 40, 40 or about 40-100 or about 100, 40 or about 40-90 or about 90, 40 or about 40-80 or about 80, 40 or About 40-70 or about 70, 40 or about 40-60 or about 60, 40 or about 40-50 or about 50, 50 or about 50-100 or about 100, 50 or about 50-90 or about 90, 50 or about 50-80 or about 80, 50 or about 50-70 or about 70, 50 or about 50-60 or about 60, 60 or about 60-100 or about 100, 60 or about 60-90 or about 90 , 60 or about 60-80 or about 80, 60 or about 60-70 or about 70, 70 or about 70-100 or about 100, 70 or about 70-90 or about 90, 70 or about 70-80 or about 80, 80 or about 80-100 or about 100, 80 or about 80-90 or about 90, or 90 or about 90-100 or about 100 g / l of albumin, e.g., human albumin.

[0264] In some embodiments, the cryopreservation composition comprises albumin, e.g., human albumin, at 20 g / l. In some embodiments, the cryopreservation composition comprises albumin, e.g., human albumin, at 40 g / l. In some embodiments, the cryopreservation composition comprises albumin, e.g., human albumin, at 70 g / l. In some embodiments, the cryopreservation composition comprises albumin, e.g., human albumin, at 100 g / l.

[0265] In some embodiments, the cryopreservation composition comprises albumin, e.g., human albumin, at about 20 g / l. In some embodiments, the cryopreservation composition comprises albumin, e.g., human albumin, at about 40 g / l. In some embodiments, the cryopreservation composition comprises albumin, e.g., human albumin, at about 70 g / l. In some embodiments, the cryopreservation composition comprises albumin, e.g., human albumin, at about 100 g / l.

[0266] In some embodiments, the cryopreservation composition additionally comprises a stabilizing agent, for example, an albumin stabilizing agent. In some embodiments, the stabilizing agent is selected from the group consisting of sodium caprylate, caprylic acid, (2S)-2-acetamido-3-(1H-indol-3-yl)propanoic acid (also referred to herein as: acetyltryptophan, N-acetyl-L-tryptophan, and acetyl-L-tryptophan), and 2-acetamido-3-(1H-indol-3-yl)propanoic acid (also referred to herein as: N-acetyltryptophan, DL-acetyltryptophan, and N-acetyl-DL-tryptophan). In some embodiments, the cryopreservation composition comprises less than .1 mmol of each of one or more stabilizing agents per gram of protein, for example, albumin protein, in the composition. In some embodiments, the cryopreservation composition comprises 0.05 or about 0.05-0.1 or about 0.1, e.g., 0.064 or about 0.064-0.096 or about 0.096 mmol of stabilizer per gram of protein, e.g., albumin protein, in the composition. In some embodiments, the cryopreservation composition comprises less than 0.1 mmol of total stabilizer per gram of protein, e.g., albumin protein, in the cryopreservation composition. In some embodiments, the cryopreservation composition comprises 0.05 or about 0.05-0.1 or about 0.1, e.g., 0.064 or about 0.064-0.096 or about 0.096 mmol of total stabilizer per gram of protein, e.g., albumin protein, in the cryopreservation composition.

[0267] 2. Dextran In some embodiments, the cryopreservation composition comprises dextran or a derivative thereof.

[0268] Dextran is an anhydroglucose polymer ((C6H 10 O5) nDextran fractions are provided with molecular weights ranging from about 1,000 daltons to about 2,000,000 daltons. This is designated by a number (Dextran X), e.g., Dextran 1, Dextran 10, Dextran 40, Dextran 70, etc., where X is the average molecular weight divided by 1,000 daltons. Thus, for example, Dextran 40 has an average molecular weight of at or about 40,000 daltons.

[0269] In some embodiments, the average molecular weight of the dextran is at or about 1,000 Daltons to 2,000 or about 2,000,000 Daltons. In some embodiments, the average molecular weight of the dextran is at or about 40,000 Daltons. In some embodiments, the average molecular weight of the dextran is at or about 70,000 Daltons.

[0270] In some embodiments, the dextran is selected from the group consisting of dextran 40, dextran 70, and combinations thereof. In some embodiments, the dextran is dextran 40.

[0271] In some embodiments, the dextran, e.g., Dextran 40, is provided in solution, also referred to herein as a dextran solution or a Dextran 40 solution. Thus, in some embodiments, the composition comprises a dextran solution, e.g., a Dextran 40 solution.

[0272] In some embodiments, the dextran solution is suitable for intravenous use.

[0273] In some embodiments, the dextran solution comprises about 5% to about 50% w / w of dextran, for example, dextran 40. In some embodiments, the dextran solution comprises 5% or about 5% to 50% or about 50%, 5% or about 5% to 45% or about 45%, 5% or about 5% to 40% or about 40%, 5% or about 5% to 35% or about 35%, 5% or about 5% to 30% or about 30%, 5% or about 5% to 25% or about 25%, 5% or about 5% to 20% or about 20%, 5% or about 5% to 15% or about 15%, 5% or about 5% to 10% or about 10%, 10% or about 10% to 50% or about 5 ... 0% to 45% or about 45%, 10% or about 10% to 40% or about 40%, 10% or about 10% to 35% or about 35%, 10% or about 10% to 30% or about 30%, 10% or about 10% to 25% or about 25%, 10% or about 10% to 20% or about 20%, 10% or about 10% to 15 or about 15%, 15% or about 15% to 50% or about 50%, 15% or about 15% to 45% or about 45%, 15% or about 15% to 40% or about 40%, 15% or about 15% to 35% or about 35% , 15% or about 15% to 30% or about 30%, 15% or about 15% to 25% or about 25%, 15% or about 15% to 20% or about 20%, 20% or about 20% to 50% or about 50%, 20% or about 20% to 45% or about 45%, 20% or about 20% to 40% or about 40%, 20% or about 20% to 35% or about 35%, 20% or about 20% to 30% or about 30%, 20% or about 20% to 25% or about 25%, 25% or about 25% to 50% or about 50%, 25% or about 25% ~45% or about 45%, 25% or about 25% to 40% or about 40%, 25% or about 25% to 35% or about 35%, 25% or about 25% to 30% or about 30%, 30% or about 30% to 50% or about 50%, 30% or about 30% to 45% or about 45%, 30% or about 30% to 40% or about 40%, 30% or about 30% to 35% or about 35%, 35% or about 35% to 50% or about 50%, 35% or about 35% to 45% or about 45%, 35% or about 35% to 40% or about 40%,40% or about 40%-50% or about 50%, 40% or about 40%-45% or about 45%, or 45% or about 45%-50% or about 50% w / w dextran, e.g., dextran 40. In some embodiments, the dextran solution comprises 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% w / w dextran, e.g., dextran 40. In some embodiments, the dextran solution comprises about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% w / w dextran, e.g., dextran 40.

[0274] In some embodiments, the dextran solution comprises dextran, for example, dextran 40, at or about 25 g / l to 200 or about 200 g / l. In some embodiments, the dextran solution comprises dextran, e.g., dextran 40, 35 or about 35-200 or about 200, 25 or about 25-175 or about 175, 25 or about 25-150 or about 150, 25 or about 25-125 or about 125, 25 or about 25-100 or about 100, 25 or about 25-75 or about 75, 25 or about 25-50 or about 50, 50 or about 50-200 or about 200, 50 or about 50-175 or about 175, 50 or about 50-150 or about 150, 50 or about 50-125 or about 125, 50 or about 50-100 or about 100, 50 or about 50-75 or about 75, 75 or about 75-200 or about 200, 75 or about 75-175 or about 175, 75 or about 75-150 or about 150, 75 or about 75-125 or about 125, 75 or about 75-100 or about 100, 100 or about 100-200 or about 200, 100 or about 100-175 or about 175, 100 or about 100-150 or about 150, 100 or The dextran solution comprises about 100-125 or about 125, 125 or about 125-200 or about 200, 125 or about 125-175 or about 175, 125 or about 125-150 or about 150, 150 or about 150-200 or about 200, 150 or about 150-175 or about 175, or 175 or about 175-200 or about 200 g / l. In some embodiments, the dextran solution comprises 25, 50, 75, 100, 125, 150, 175 or 200 g / l of dextran, e.g., Dextran 40. In some embodiments, the dextran solution comprises 100 g / l of dextran, e.g., Dextran 40. In some embodiments, the dextran solution comprises about 25, about 50, about 75, about 100, about 125, about 150, about 175, or about 200 g / l of dextran, e.g., Dextran 40. In some embodiments, the dextran solution comprises about 100 g / l of dextran, e.g., Dextran 40.

[0275] In some embodiments, the dextran solution additionally comprises glucose (also referred to as dextrose). In some embodiments, the dextran solution comprises 10 or about 10 g / l to 100 or about 100 g / l of glucose. In some embodiments, the dextran solution comprises 10 or about 10 to 100 or about 100, 10 or about 10 to 90 or about 90, 10 or about 10 to 80 or about 80, 10 or about 10 to 70 or about 70, 10 or about 10 to 60 or about 60, 10 or about 10 to 50 or about 50, 10 or about 10 to 40 or about 40, 10 or about 10 to 30 or about 30, 10 or about 10 to 20 or about 20, 20 or about 20 to 100 or about 100, 20 or about 20 ~90 or about 90, 20 or about 20-80 or about 80, 20 or about 20-70 or about 70, 20 or about 20-60 or about 60, 20 or about 20-50 or about 50, 20 or about 20-40 or about 40, 20 or about 20-30 or about 30, 30 or about 30-100 or about 100, 30 or about 30-90 or about 90, 30 or about 30-80 or about 80, 30 or about 30-70 or about 70, 30 or about 30-60 or about 60, 30 or about 30-50 or about 50, 30 or about 30-40 or about 40, 40 or about 40-100 or about 100, 40 or about 40-90 or about 90, 40 or about 40-80 or about 80, 40 or about 40-70 or about 70, 40 or about 40-60 or about 60, 40 or about 40-50 or about 50, 50 or about 50-100 or about 100, 50 or about 50-90 or about 90, 50 or about 50-80 or about 80, 50 or about 50-70 or about 70, 50 or about 50-60 or about 60, 60 or about 60-100 or about 100, 60 or about 60-90 or about 90, 60 or about 60-80 or about 80, 60 or about 60-70 or about 70, 70 or about 70-100 or about 100, 70 or about 70-90 or about 90, 70 or about 70-80 or about 80, 80 or about 80-90 or about 90, 80 or about 80-100 or about 100, 80 or about 80-90 or about 90, or 90 or about 90-100 or about 100 g / l.In some embodiments, the dextran solution comprises 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 g / l of glucose. In some embodiments, the dextran solution comprises 50 g / l of glucose. In some embodiments, the dextran solution comprises about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90 or about 100 g / l of glucose. In some embodiments, the dextran solution comprises 50 g / l of glucose.

[0276] In some embodiments, the dextran solution is composed of dextran, eg, dextran 40, and glucose in water.

[0277] In some embodiments, the cryopreservation composition comprises 10% or about 10% v / v to 50% or about 50% v / v of a dextran solution described herein. In some embodiments, the cryopreservation composition comprises 10% or about 10% to 50% of a dextran solution, e.g., 10% or about 10% to 45% or about 45%, 10% or about 10% to 40% or about 40%, 10% or about 10% to 35% or about 35%, 10% or about 10% to 30% or about 30%, 10% or about 10% to 25% or about 25%, 10% or about 10% to 20% or about 20%, 10% or about 10% to 15% or 20% of a dextran solution, e.g., a dextran solution described herein. or about 15%, 15% or about 15% to 50% or about 50%, 15% or about 15% to 45% or about 45%, 15% or about 15% to 40% or about 40%, 15% or about 15% to 35% or about 35%, 15% or about 15% to 30% or about 30%, 15% or about 15% to 25% or about 25%, 15% or about 15% to 20% or about 20%, 20% or about 20% to 50% or about 50%, 20% or about 20% to 45% or about 45%, 20% or is about 20% to 40% or about 40%, 20% or about 20% to 35% or about 35%, 20% or about 20% to 30% or about 30%, 20% or about 20% to 25% or about 25%, 25% or about 25% to 50% or about 50%, 25% or about 25% to 45% or about 45%, 25% or about 25% to 40% or about 40%, 25% or about 25% to 35% or about 35%, 25% or about 25% to 30% or about 30%, 30% or about 30% to 50% or About 50%, 30% or about 30%-45% or about 45%, 30% or about 30%-40% or about 40%, 30% or about 30%-35% or about 35%, 35% or about 35%-50% or about 50%, 35% or about 35%-45% or about 45%, 35% or about 35%-40% or about 40%, 40% or about 40%-50% or about 50%, 40% or about 40%-45% or about 45%, or 45% or about 45%-50% or about 50% v / v. In some embodiments, the cryopreservation composition comprises a dextran solution, e.g., a dextran solution described herein, at 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% v / v.In some embodiments, the cryopreservation composition comprises a dextran solution, such as a dextran solution described herein, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% v / v.

[0278] In some embodiments, the cryopreservation composition comprises 10 or about 10-50 or about 50 g / l of dextran, e.g., dextran 40. In some embodiments, the cryopreservation composition comprises 10 or about 10-50 or about 50, 10 or about 10-45 or about 45, 10 or about 10-40 or about 40, 10 or about 10-35 or about 35, 10 or about 10-30 or about 30, 10 or about 10-25 or about 25, 10 or about 10-20 or about 20, 10 or about 10 to 15 or about 15, 15 or about 15 to 50 or about 50, 15 or about 15 to 45 or about 45, 15 or about 15 to 40 or about 40, 15 or about 15 to 35 or about 35, 15 or about 15 to 30 or about 30, 15 or about 15 to 25 or about 25, 15 or about 15 to 20 or about 20, 20 or about 20 to 50 or about 50, 20 or about 20 to 4 5 or about 45, 20 or about 20-40 or about 40, 20 or about 20-30 or about 30, 20 or about 20-25 or about 25, 25 or about 25-50 or about 50, 25 or about 25-45 or about 45, 25 or about 25-40 or about 40, 25 or about 25-35 or about 35, 25 or about 25-30 or about 30, 30 or about 30-50 or about 50, In some embodiments, the cryopreservation composition comprises 10, 15, 20, 25, 30, 30, 35, 40, 50, or about 50 g / l of dextran, e.g., dextran 40. In some embodiments, the cryopreservation composition comprises about 10, about 15, about 20, about 25, about 30, about 30, about 35, about 40, about 45, or about 50 g / l of dextran, e.g., dextran 40.

[0279] 3. Glucose In some embodiments, the cryopreservation composition comprises glucose.

[0280] In some embodiments, the cryopreservation composition as described herein comprises a dextran solution containing glucose.

[0281] In some embodiments, the cryopreservation composition comprises a dextran solution that does not contain glucose. In some embodiments, for example, when the dextran solution does not contain glucose, glucose is added separately to the cryopreservation composition.

[0282] In some embodiments, the cryopreservation composition comprises 5 or about 5-25 or about 25 g / l of glucose. In some embodiments, the cryopreservation composition comprises 5 or about 5-25 or about 25, 5 or about 5-20 or about 20, 5 or about 5-15 or about 15, 5 or about 5-10 or about 10, 10 or about 10-25 or about 25, 10 or about 10-20 or about 20, 10 or about 10-15 or about 15, 15 or about 15-25 or about 25, 15 or about 15-20 or about 20, or 20 or about 20-25 or about 25 g / l of glucose. In some embodiments, the cryopreservation composition comprises 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5 or 25 g / l of glucose. In some embodiments, the cryopreservation composition comprises 12.5 g / l of glucose. In some embodiments, the cryopreservation composition comprises about 5, about 7.5, about 10, about 12.5, about 15, about 17.5, about 20, about 22.5, or about 25 g / l of glucose. In some embodiments, the cryopreservation composition comprises about 12.5 g / l of glucose.

[0283] In some embodiments, the cryopreservation composition comprises glucose at less than 2.75% w / v. In some embodiments, the cryopreservation composition comprises glucose at less than 27.5 g / l. In some embodiments, the cryopreservation composition comprises glucose at less than 2% w / v. In some embodiments, the cryopreservation composition comprises glucose at less than 1.5% w / v. In some embodiments, the cryopreservation composition comprises about 1.25% w / v or less.

[0284] 4. Dimethyl sulfoxide In some embodiments, the cryopreservation composition comprises dimethylsulfoxide (DMSO, also known as methylsulfoxide and methylsulfinylmethane).

[0285] In some embodiments, DMSO is provided as a solution, also referred to herein as a DMSO solution. Thus, in some embodiments, the cryopreservation composition comprises a DMSO solution.

[0286] In some embodiments, the DMSO solution is suitable for intravenous use.

[0287] In some embodiments, the DMSO solution comprises 1.1 g / ml DMSO. In some embodiments, the DMSO solution comprises about 1.1 g / ml DMSO.

[0288] In some embodiments, the cryopreservation composition comprises a DMSO solution at 1% or about 1% to 10% or about 10% v / v. In some embodiments, the cryopreservation composition comprises a DMSO solution at 1% or about 1% to 10% or about 10%, 1% or about 1% to 9% or about 9%, 1% or about 1% to 8% or about 8%, 1% or about 1% to 7% or about 7%, 1% or about 1% to 6% or about 6%, 1% or about 1% to 5% or about 5%, 1% or about 1% to 4% or about 4%, 1% or about 1% to 3% or about 3%, 1% or about 1% to 2% or about 2%, 2% or about 2% to 10% or about 10%, 2% or about 2% to 10% or about 10%, 3% or about 3%, 4% or about 4% or about 4%, 5% or about 5%, 6% or about 6% or about 6%, 7% or about 7%, 8% or about 8% or about 8%, 9% or about 9% or about 9%, 1% or about 1% to 5% or about 5%, 1% or about 1% to 4% or about 4%, 1% or about 1% to 3% or about 3%, 1% or about 1% to 2% or about 2%, 2% or about 2% to 10% or about 10%, 3% or about 3%, 4% or about 4% or about 4%, 5% or about 5%, 6% or about 6% or about 6%, 7% or about 7%, 8% or about 8% or about 9%, 9% or about 9%, 10% or about 10%, 10% or about 10%, 10% or % or about 2%-9% or about 9%, 8% or about 8%, 2% or about 2%-7% or about 7%, 2% or about 2%-6% or about 6%, 2% or about 2%-5% or about 5%, 2% or about 2%-4% or about 4%, 2% or about 2%-3% or about 3%, 3% or about 3%-10% or about 10%, 3% or about 3%-9% or about 9%, 3% or about 3%-8% or about 8%, 3% or about 3%-7% or about 7%, 3% or about 3%-6% or about 6%, 3% or about 3% to 5% or about 5%, 3% or about 3% to 4% or about 4%, 4% or about 4% to 10% or about 10%, 4% or about 4% to 9% or about 9%, 4% or about 4% to 8% or about 8%, 4% or about 4% to 7% or about 7%, 4% or about 4% to 6% or about 6%, 4% or about 4% to 5% or about 5%, 5% or about 5% to 10% or about 10%, 5% or about 5% to 9% or about 9%, 5% or about 5% to 8% or about 8%, 5% or about 5% to 7% or about 7%, 5% or about 5% to 6% or about 6%, 6% or about 6% to 10% or about 10%, 6% or about 6% to 9% or about 9%, 6% or about 6% to 8% or about 8%, 6% or about 6% to 7% or about 7%, 7% or about 7% to 10% or about 10%, 7% or about 7% to 9% or about 9%, 7% or about 7% to 8% or about 8%, 8% or about 8% to 10% or about 10%, 8% or about 8% to 9% or about 9%, or 9% or about 9% to 10% or about 10% v / v. In some embodiments, the cryopreservation composition comprises 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% v / v of a DMSO solution.In some embodiments, the cryopreservation composition comprises a 5% DMSO solution. In some embodiments, the cryopreservation composition comprises about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% v / v DMSO solution. In some embodiments, the cryopreservation composition comprises about 5% DMSO solution.

[0289] In some embodiments, the cryopreservation composition comprises 11 or about 11-110 or about 110 g / l DMSO. In some embodiments, the cryopreservation composition comprises 11 or about 11-110 or about 110, 11 or about 11-99 or about 99, 11 or about 11-88 or about 88, 11 or about 11-77 or about 77, 11 or about 11-66 or about 66, 11 or about 11-55 or about 55, 11 or about 11-44 or about 44, 11 or about 11-33 or about 33, 11 or about 11-22 or about 22, 22 or about 22-110 or about 110, 22 or about 22 to 99 or about 99, 22 or about 22 to 88 or about 88, 22 or about 22 to 77 or about 77, 22 or about 22 to 77 or about 77, 22 or about 22 to 66 or about 66, 22 or about 22 to 55 or about 55, 22 or about 22 to 44 or about 44, 22 or about 22 to 33 or about 33, 33 or about 33 to 110 or about 110, 33 or about 33 to 99 or about 99, 33 or about 33 to 88 or about 88, 33 or about 33 to 77 or about 77, or about 33 to 66 or about 66, 33 or about 33 to 55 or about 55, 33 or about 33 to 44 or about 44, 44 or about 44 to 110 or about 110, 44 or about 44 to 99 or about 99, 44 or about 44 to 88 or about 88, 44 or about 44 to 77 or about 77, 44 or about 44 to 66 or about 66, 44 or about 44 to 55 or about 55, 55 or about 55 to 110 or about 110, 55 or about 55 to 99 or about 99, 55 or about 55 to 88 or about 8 In some embodiments, the cryopreservation composition comprises 11, 22, 33, 44, 55, 66, 77, 88, 99, or 110 g / l of DMSO.In some embodiments, the cryopreservation composition comprises 55 g / l DMSO. In some embodiments, the cryopreservation composition comprises about 11, about 22, about 33, about 44, about 55, about 66, about 77, about 88, about 99, or about 110 g / l DMSO. In some embodiments, the cryopreservation composition comprises about 55 g / l DMSO.

[0290] 5. Buffers In some embodiments, the cryopreserved composition comprises a buffer solution, for example a buffer solution suitable for intravenous administration.

[0291] Buffer solutions include, but are not limited to, phosphate buffered saline (PBS), Ringer's solution, Tyrode's buffer, Hank's balanced salt solution, Earle's balanced salt solution, saline and Tris.

[0292] In some embodiments, the buffer solution is phosphate buffered saline (PBS).

[0293] 6. Exemplary Cryopreservation Compositions In some embodiments, the cryopreservation composition comprises or is composed of the following components: 1) albumin, e.g., human albumin, 2) dextran, e.g., dextran 40, 3) DMSO, and 4) a buffer solution. In some embodiments, the cryopreservation composition additionally comprises glucose. In some embodiments, the cryopreservation composition is composed of 1) albumin, e.g., human albumin, 2) dextran, e.g., dextran 40, 3) glucose, 4) DMSO, and 5) a buffer solution.

[0294] In some embodiments, the cryopreservation composition comprises 1) an albumin solution described herein, 2) a dextran solution described herein, 3) a DMSO solution described herein, and 4) a buffer solution.

[0295] In some embodiments, the cryopreservation composition is comprised of 1) an albumin solution as described herein, 2) a dextran solution as described herein, 3) a DMSO solution as described herein, and 4) a buffer solution.

[0296] In some embodiments, the cryopreservation composition does not include cell culture medium.

[0297] In one embodiment, the cryopreservation composition comprises approximately 40 mg / ml human albumin, 25 mg / ml dextran 40, 12.5 mg / ml glucose and 55 mg / ml DMSO.

[0298] In one embodiment, the cryopreservation composition comprises or consists of approximately 40 mg / ml human albumin, 25 mg / ml dextran 40, 12.5 mg / ml glucose, 55 mg / ml DMSO, and 0.5 mg / ml 100% phosphate buffered saline (PBS) in water.

[0299] In one embodiment, the cryopreservation composition comprises approximately 32 mg / ml human albumin, 25 mg / ml dextran 40, 12.5 mg / ml glucose and 55 mg / ml DMSO.

[0300] In one embodiment, the cryopreservation composition comprises or consists of approximately 32 mg / ml human albumin, 25 mg / ml dextran 40, 12.5 mg / ml glucose, 55 mg / ml DMSO, and 0.54 mg / ml 100% phosphate buffered saline (PBS) in water.

[0301] Examples of cryopreservation compositions are shown in Table 3.

[0302] [Table 3]

[0303] [Table 4]

[0304] [Table 5]

[0305] B. Freezing preservation method The cryopreservation compositions described herein can be used to cryopreserve cells, e.g., therapeutic cells, e.g., natural killer (NK) cells, e.g., NK cells, as described herein.

[0306] In some embodiments, the cell is an animal cell, hi some embodiments, the cell is a human cell.

[0307] In some embodiments, the cell is an immune cell, hi some embodiments, the immune cell is selected from a basophil, an eosinophil, a neutrophil, a mast cell, a monocyte, a macrophage, a neutrophil, a dendritic cell, a natural killer cell, a B cell, a T cell, and combinations thereof.

[0308] In some embodiments, the immune cells are natural killer (NK) cells. In some embodiments, the natural killer cells are expanded and stimulated by the methods described herein.

[0309] In some embodiments, cryopreservation of cells includes mixing the cells with a cryopreservation composition described herein or a component thereof to produce a composition, e.g., a pharmaceutical composition, and freezing the mixture.

[0310] In some embodiments, cryopreservation of cells includes mixing a composition comprising cells with a cryopreservation composition described herein or a component thereof to produce a composition, e.g., a pharmaceutical composition; and freezing the mixture. In some embodiments, the composition comprising cells includes cells and a buffer. Suitable buffers are described herein.

[0311] In some embodiments, cryopreservation of cells comprises mixing a composition comprising cells and a buffer, e.g., PBS, with a composition comprising albumin, dextran and DMSO, e.g., as described herein; freezing the mixture.

[0312] In some embodiments, cryopreservation of cells includes mixing a composition comprising cells and a buffer, e.g., PBS, 1:1 with a composition comprising 40 mg / ml albumin, e.g., human albumin, 25 mg / ml dextran, e.g., dextran 40, 12.5 mg / ml glucose, and 55 mg / ml DMSO.

[0313] In some embodiments, a composition comprising cells and a buffer, e.g., PBS, comprises 2×10 cells. 7 or about 2 x 10 7 ~2×10 9 or about 2 x 10 9 In some embodiments, a composition comprising cells and a buffer, e.g., PBS, contains 2×10 cells / ml. 8 In some embodiments, the composition comprising cells and a buffer, e.g., PBS, comprises about 2×10 cells / ml. 8 Contains cells / ml.

[0314] In some embodiments, cell cryopreservation includes mixing cells, a buffer, eg, PBS, albumin, eg, human albumin, dextran, eg, dextran 40, and DMSO; freezing the mixture.

[0315] In some embodiments, the mixture comprises 1×10 7 or about 1 x 10 7 ~1×10 9 or about 1 x 10 9 In some embodiments, the mixture comprises 1×10 8 In some embodiments, the mixture comprises about 1×10 cells / ml. 8 Contains cells / ml.

[0316] Suitable albumin, dextran and DMSO ranges are described above.

[0317] In some embodiments, the composition is frozen at -135°C or below.

[0318] In some embodiments, the composition freezes at a controlled rate.

[0319] IV. Antibodies CD20-targeting antibodies suitable for use in the methods described herein include, but are not limited to, the antibodies set forth in Table 6 and combinations thereof.

[0320] [Table 6]

[0321] In some embodiments, the CD38-targeting antibody is selected from the group consisting of daratumumab (or a biosimilar thereof), isatuximab (or a biosimilar thereof), and combinations thereof.

[0322] In some embodiments, the antibody is administered intravenously or subcutaneously.

[0323] V. Pharmaceutical Compositions The present invention provides pharmaceutical compositions comprising the natural killer cells described herein, and dosage units of the pharmaceutical compositions described herein.

[0324] In some cases, the dosage unit comprises between 100 million and 1.5 billion cells, e.g., 100 million, 200 million, 300 million, 400 million, 500 million, 600 million, 700 million, 800 million, 900 million, 1 billion, 1.1 billion, 1.2 billion, 1.3 billion, 1.4 billion, or 1.5 billion cells.

[0325] Pharmaceutical compositions typically include a pharma- ceutically acceptable carrier. As used herein, the term "pharma- ceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., suitable for pharmaceutical administration.

[0326] In some embodiments, the pharmaceutical composition comprises: a) a natural killer cell described herein; and b) a cryopreserved composition.

[0327] Suitable cryopreservation compositions are described herein.

[0328] In some embodiments, the composition is frozen. In some embodiments, the composition is frozen for at least 3 months, e.g., at least 6 months, at least 9 months, at least 12 months, at least 15 months, at least 18 months, at least 24 months, or at least 36 months.

[0329] In some embodiments, at least 60%, eg, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% of the natural killer cells survive post-thawing.

[0330] In some embodiments, the pharmaceutical composition comprises: a) a cryopreservation composition described herein; and b) therapeutic cells.

[0331] In some embodiments, the therapeutic cell is an animal cell, hi some embodiments, the therapeutic cell is a human cell.

[0332] In some embodiments, the therapeutic cell is an immune cell, hi some embodiments, the immune cell is selected from a basophil, an eosinophil, a neutrophil, a mast cell, a monocyte, a macrophage, a neutrophil, a dendritic cell, a natural killer cell, a B cell, a T cell, and combinations thereof.

[0333] In some embodiments, the immune cells are natural killer (NK) cells. In some embodiments, the natural killer cells are expanded and stimulated by the methods described herein.

[0334] In some embodiments, the pharmaceutical composition additionally comprises c) a buffer solution. Suitable buffer solutions are, for example, as described herein for the cryopreserved composition.

[0335] In some embodiments, the pharmaceutical composition comprises 1×10 cells. 7 cells / ml or approximately 1 x 10 7 ~1×10 9 cells / ml or approximately 1 x 10 9 In some embodiments, the pharmaceutical composition contains 1×10 cells / ml. 8 In some embodiments, the pharmaceutical composition contains about 1×10 cells / ml. 8 Contains cells / ml.

[0336] In some embodiments, the pharmaceutical composition additionally comprises an antibody or antigen-binding fragment thereof, eg, an antibody described herein.

[0337] Pharmaceutical compositions are typically formulated for the intended route of administration, including parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.

[0338] Methods for formulating suitable pharmaceutical compositions are known in the art, see, for example, Remington: The Science and Practice of Pharmacy, 21st ed., 2005; and the books in the series Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY). For example, the solution or suspension used for parenteral, intradermal or subcutaneous application can contain the following components: sterile diluent, for example, water for injection, saline solution, fixed oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvent; antibacterial agent, for example, benzyl alcohol or methyl paraben; antioxidant, for example, ascorbic acid or sodium bisulfite; chelating agent, for example, ethylenediaminetetraacetic acid; buffer, for example, acetate, citrate or phosphate, and tonicity adjuster, for example, sodium chloride or dextrose. pH can be titrated with acid or base, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0339] Pharmaceutical compositions suitable for injectable use can include sterile acids and sterile aqueous solutions (where water soluble) or dispersions for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL TM(BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and fluid enough for easy syringability. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. The prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to add isotonic agents, for example, sugars, polyalcohols, for example, mannitol, sorbitol, sodium chloride to the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0340] Sterile injectable solution can be prepared by adding active compound in the required amount to a suitable solvent with one or a combination of the above-listed ingredients, and then sterilizing by filtration as required.Generally, dispersion is prepared by adding active compound to a sterile vehicle that contains a basic dispersion medium and other necessary ingredients from the above-listed ones.For the case of sterile acid solution for preparing sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying, which obtains a powder of active ingredient and any additional desired ingredient from the previously sterilized-filtered solution.

[0341] VI. Treatment method The NK cells described herein have utility in treating cancer or other proliferative disorders.

[0342] Thus, the invention also provides a method of treating a patient suffering from a disorder, e.g., a disorder associated with cancer, e.g., a CD38+ cancer, comprising administering an NK cell, e.g., an NK cell described herein, and a CD38-targeting antibody, e.g., an antibody described herein.

[0343] The invention also provides methods for preventing, reducing and / or inhibiting the recurrence, growth, proliferation, migration and / or metastasis of a cancer cell or cancer cell population in an individual in need thereof comprising administering an NK cell, e.g., an NK cell described herein, and a CD38-targeting antibody, e.g., an antibody described herein.

[0344] Additionally, the present invention provides methods of enhancing, improving and / or enhancing a response to anti-cancer therapy in an individual in need thereof comprising administering NK cells, e.g., NK cells described herein, and a CD38-targeting antibody, e.g., an antibody described herein.

[0345] The present invention also provides a method of inducing an immune system in an individual in need thereof, comprising administering NK cells, e.g., NK cells described herein, and a CD38-targeting antibody, e.g., an antibody described herein.

[0346] The methods described herein include methods of treating disorders associated with abnormal apoptotic or differentiation processes, e.g., cell proliferation or differentiation disorders, e.g., cancers such as solid tumors and hematopoietic cancers. Generally, the methods include administering a therapeutically effective amount of a therapeutic agent as described herein to an individual in need thereof or determined to be in need of treatment. In some embodiments, the methods include administering a therapeutic agent comprising NK cells, e.g., NK cells as described herein, and a CD38-targeting antibody, e.g., an antibody as described herein, in a therapeutically effective amount.

[0347] As used herein, the terms "treatment", "treat" and "treating" refer to the regression, alleviation, delayed onset or inhibition of progression of disorders associated with abnormal apoptotic or differentiation processes. For example, treatment can achieve a reduction in tumor size or growth rate. The administration of a compound described herein in a therapeutically effective amount to treat a condition associated with abnormal apoptotic or differentiation processes should achieve, among other things, a reduction in tumor size or growth rate, a reduction in the risk or frequency of recurrence, a delay in recurrence, a reduction in metastasis, an increase in survival and / or a reduction in morbidity and mortality. In some embodiments, the therapeutic agent can be administered after one or more symptoms have appeared. In other embodiments, the therapeutic agent can be administered in the absence of symptoms. For example, the therapeutic agent can be administered to a susceptible individual before symptoms have begun (e.g., in light of a history of symptoms and / or in light of genetic or other susceptibility factors). The therapeutic agent can also continue to be administered after symptoms have resolved, for example, to prevent or delay recurrence.

[0348] As used herein, the term "inhibit", in relation to cancer and / or cancer cell proliferation, means inhibiting the growth, division, maturation or viability of cancer cells, either individually or collectively with other cancer cells, and / or inducing the death of cancer cells, by cytotoxicity, nutrient depletion or induction of apoptosis.

[0349] As used herein, "delaying" the progression of a disease or disorder, or one or more symptoms thereof, means delaying, preventing, slowing, retarding, stabilizing and / or postponing the onset of the disease, disorder or symptoms thereof. The delay may be of varying length of time depending on the individual's history of the disease and / or treatment. As will be appreciated by those skilled in the art, a sufficient or significant delay encompasses prevention, in that the disease, disorder or symptoms thereof do not in fact occur in the individual. For example, a method of "delaying" the onset of cancer is one that reduces the probability of the onset of the disease for a given time range and / or reduces the extent of the disease for a given time range, compared to not utilizing the method. Such comparisons can be based on clinical trials using a statistically significant number of individuals.

[0350] As used herein, "prevention" or "preventing" refers to the use of preventing a disease or disorder from initiating so that clinical symptoms of the disease do not occur. Thus, "prevention" refers to administering a therapy (e.g., administration of a therapeutic agent) to an individual before symptoms of the disease are detectable in the individual and / or before a particular stage of the disease is reached (e.g., administration of a therapeutic agent to a patient whose cancer has not yet metastasized). The individual may be at risk of developing a disease or disorder or at risk of disease progression, e.g., at risk of cancer metastasis, e.g., an individual having one or more risk factors known to be associated with the onset or initiation of a disease or disorder. For example, the individual may carry a mutation associated with the onset or progression of cancer. Furthermore, it is understood that prevention does not achieve a complete prevention against the onset of a disease or disorder. In some cases, prevention includes a reduction in the risk of developing a disease or disorder. A reduction in risk may not achieve a complete elimination of the risk of developing a disease or disorder.

[0351] By "increased" or "enhanced" (e.g., in relation to anti-tumor response, metastasis of cancer cells) is meant an increase of 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold or higher (e.g., 100 fold, 500 fold, 1000 fold) over the amounts or levels referred to herein (including all integers and decimal values ​​greater than 1 between the referred integers, e.g., 2.1, 2.2, 2.3, 2.4, etc.). This can also encompass an increase of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 500% or at least 1000% relative to the amounts or levels referred to herein.

[0352] A "decreased" or "reduced" or "lesser" amount (e.g., with respect to tumor size, cancer cell proliferation or growth) refers to a 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold or higher (e.g., 100 fold, 500 fold, 1000 fold) decrease relative to the amount or level referenced herein (including all integers and decimal values ​​greater than 1 between the referenced integers, e.g., 1.5, 1.6, 1.7, 1.8, etc.). This can also encompass a reduction of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 500% or at least 1000% relative to the amounts or levels referred to herein.

[0353] A. Disability The disclosed method and the prepared composition have application for targeting a number of disorders, such as cell proliferation disorders.In the present invention, the advantage of such an approach is that allogeneic cells are used in combination with the administration of exogenous antibody to target the specific proliferative cells targeted by the exogenous antibody.By using such an approach and pharmaceutical composition of the present invention, unlike existing therapies such as chemotherapy or radiation therapy, it is possible to specifically target cells that show potentially harmful proliferative activity without administering systemic drugs or toxins that indiscriminately affect proliferative cells.

[0354] Examples of cell proliferative and / or differentiative disorders include cancer, e.g., carcinoma, sarcoma, metastatic disorders, or hematopoietic neoplastic disorders, e.g., leukemia, etc. Metastatic tumors can originate from a variety of primary tumor types, including, but not limited to, prostate, colon, lung, breast, and hepatic origin.

[0355] As used herein, the terms "cancer," "hyperproliferative," and "neoplastic" refer to cells having the capacity for autonomous growth, i.e., an abnormal state or pathology characterized by rapidly proliferating cell growth. Hyperproliferative and neoplastic disease states can be classified as pathological, i.e., characterized or diseased by a disease state, or non-pathological, i.e., not a normal state but not associated with a disease state. The term is meant to encompass all types of cancerous growth or oncogenic processes, metastatic tissues, or malignantly transformed cells, tissues, or organs, regardless of histopathological type or stage of invasion. "Pathological hyperproliferative" cells occur in disease states characterized by malignant tumor growth. Examples for non-pathological hyperproliferative cells include cell proliferation associated with wound healing.

[0356] The term "cancer" or "neoplasm" encompasses malignancies of various organ systems, such as those occurring in the lung, breast, thyroid, lymphatic system, gastrointestinal and genitourinary tract, as well as malignancies such as adenocarcinomas, e.g., most colon cancers, renal cell carcinoma, prostate cancer and / or testicular tumors, non-small cell lung carcinoma, small intestine cancer and esophageal cancer.

[0357] The term "carcinoma" is understood in the art and refers to malignant tumors of epithelial or endocrine tissues, such as respiratory system carcinoma, gastrointestinal system carcinoma, genitourinary system carcinoma, testicular carcinoma, breast carcinoma, prostate carcinoma, endocrine system carcinoma, and melanoma. In some embodiments, the disease is renal cancer or melanoma. Examples of carcinomas include those arising from tissues of the cervix, lung, prostate, breast, head and neck, colon, and ovary. The term also encompasses carcinosarcoma, including, for example, malignant tumors composed of carcinoma and sarcoma tissue. "Adenocarcinoma" refers to a carcinoma derived from glandular tissue or whose tumor cells originate from recognizable glandular structures.

[0358] The term "sarcoma" is art recognized and refers to malignant tumors of mesenchymal origin.

[0359] Additional examples for proliferative disorders include hematopoietic neoplastic disorders. As used herein, the term "hematopoietic neoplastic disorder" includes diseases involving hyperplastic / neoplastic cells of hematopoietic origin, e.g., originating from the myeloid, lymphoid or erythroid lineages or precursor cells thereof. Preferably, the diseases arise from poorly differentiated acute leukemias, e.g., erythroblastic leukemia and acute megakaryoblastic leukemia. Examples of additional myeloid disorders include, but are not limited to, acute promyelocytic leukemia (APML), acute myeloid leukemia (AML) and chronic myeloid leukemia (CML) (Vaickus, L. (1991) Crit Rev. in Oncol. / Hemotol. 11:267-97); lymphoid malignancies include, but are not limited to, ALL, including B-lineage acute lymphoblastic leukemia (ALL) and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphoblastic leukemia (PLL), hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM). Additional forms of malignant lymphoma include, but are not limited to, non-Hodgkin's lymphoma and its variants, peripheral T-cell lymphoma, adult T-cell leukemia / lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease, and Reed-Sternberg disease.

[0360] In some embodiments, the cancer is selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, Kaposi's sarcoma, AIDS-related lymphoma, primary CNS lymphoma, anal cancer, appendix cancer, astrocytoma, typical teratoma / rhabdoid tumor, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, bronchial tumor, Burkitt's lymphoma, carcinoid, cardiac tumor, medulloblastoma, germ cell tumor, primary CNS lymphoma, cervical cancer, bile duct cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloproliferative neoplasm, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, ductal carcinoma in situ, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, Ewing's sarcoma. , extracranial germ cell tumors, extragonadal germ cell tumors, eye cancer (e.g., intraocular melanoma or retinoblastoma), fallopian tube cancer, fibrous histiocytoma of bone, osteosarcoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), germ cell tumors, gestational trophoblastic disease, hairy cell leukemia, head and neck cancer, cardiac tumors, hepatocellular carcinoma, histiocytosis, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumors, Pancreatic neuroendocrine tumors, renal (renal cell) carcinoma, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer, lung cancer (e.g., non-small cell lung cancer, small cell lung carcinoma, pleuropulmonary blastoma, and tracheobronchial tumors), lymphoma, male breast cancer, malignant fibrous histiocytoma of bone, melanoma, Merkel cell carcinoma, mesothelioma, metastatic carcinoma, metastatic squamous cell carcinoma, midline carcinoma tract carcinoma, oral cavity cancer, multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm, myeloproliferative neoplasm, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's lymphoma, oral cavity cancer, lip and oral cavity cancer, oropharyngeal cancer, osteosarcoma, malignant fibrous histiocytoma, ovarian cancer, pancreatic cancer, pancreatic neuroendocrine tumor, papillomatosis, paraganglioma, paranasal sinus and nasal cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary gland tumor, plasma cell neoplasm, multiple myeloma, pleuropulmonary blastoma, pregnancy and breast cancercancer), primary malignant lymphoma of the central nervous system, primary peritoneal cancer, prostate cancer, rectal cancer, recurrent cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcomas (e.g. childhood rhabdomyosarcoma, childhood vascular tumors, Ewing's sarcoma, Kaposi's sarcoma, osteosarcoma, soft tissue sarcoma, uterine sarcoma), Sezary syndrome, skin cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, cervical squamous cell carcinoma, gastric cancer, T-cell lymphoma, testicular cancer, laryngeal cancer, nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer, thymoma and thymic carcinoma, thyroid cancer, tracheobronchial tumors, transitional cell carcinoma of the renal pelvis and ureter, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vascular tumors, vulvar cancer, and Wilms' tumor.

[0361] In some embodiments, the cancer is a solid tumor.

[0362] In some embodiments, the cancer is metastatic.

[0363] In some embodiments, the cancer is a CD38+ cancer.

[0364] In some embodiments, the CD38+ cancer is selected from the group consisting of glioma, thyroid cancer, lung cancer, colorectal cancer, head and neck cancer, gastric cancer, liver cancer, pancreatic cancer, renal cancer, urothelial cancer, prostate cancer, testicular cancer, breast cancer, cervical cancer, ovarian cancer, melanoma, lymphoma, and combinations thereof.

[0365] In some embodiments, the CD38+ cancer is prostate cancer.

[0366] In some embodiments, the CD38+ cancer is a lymphoma.

[0367] In some embodiments, the CD38+ cancer is multiple myeloma.

[0368] B.Patient In the present invention, suitable patients for the compositions and methods include those suffering from, diagnosed with, or suspected of suffering from a cell proliferation and / or differentiation disorder, such as cancer. Patients undergoing the procedures referred to in the present invention generally respond better to the methods and compositions of the present invention, in part because the pharmaceutical compositions are allogeneic and generally identify target cells with antibodies rather than targeting proliferating cells. Therefore, there are fewer off-target effects, and patients are more likely to complete a therapeutic regimen without substantial adverse off-target effects.

[0369] In some embodiments, the therapeutic methods provided herein can be used to treat an individual (e.g., a human, monkey, dog, cat, mouse) diagnosed with or suspected of suffering from a cell proliferation and / or differentiation disorder, such as cancer. In some embodiments, the individual is a mammal. In some embodiments, the individual is a human.

[0370] As used herein, an individual refers to a mammal, including, for example, a human.

[0371] In some embodiments, the mammal is selected from the group consisting of armadillo, donkey, bat, bear, beaver, cat, chimpanzee, cow, coyote, deer, dog, dolphin, elephant, fox, panda, gibbon, giraffe, goat, gopher, hedgehog, hippopotamus, horse, humpback whale, jaguar, kangaroo, koala, leopard, deer, llama, lynx, mole, monkey, mouse, narwhal, orangutan, stag beetle, otter, bull, pig, polar bear, porcupine, puma, rabbit, raccoon, rat, rhino, sheep, squirrel, tiger, walrus, weasel, wolf, zebra, goat, horse, and combinations thereof.

[0372] In some embodiments, the mammal is a human.

[0373] The individual, e.g., a human individual, may be a child, e.g., 0 or about 0 to 14 or about 14 years of age. The individual may be a youth, e.g., 15 or about 15 to 24 or about 24 years of age. The individual may be an adult, e.g., 25 or about 25 to 64 or about 64 years of age. The individual may be an elderly individual, e.g., 65 years or older.

[0374] In some embodiments, the individual may be a human who has developed one or more symptoms associated with a cell proliferation and / or differentiation disorder, e.g., cancer, e.g., a tumor. Various stages of cancer can be treated using any of the therapeutic methods provided herein. For example, cancer stages include, but are not limited to, early stage, advanced, locally advanced, in remission, refractory, recurrent after remission, and progressive. In some embodiments, the individual is in an early stage of cancer. In other embodiments, the individual is in an advanced stage of cancer. In various embodiments, the individual is suffering from stage 1, stage 2, stage 3, or stage 4 cancer. The therapeutic methods described herein can promote tumor reduction or shrinkage, reduce or inhibit tumor growth or cancer cell proliferation, and / or induce, increase, or promote tumor cell killing. In some embodiments, the individual is in cancer remission. The therapeutic methods described herein can prevent or delay cancer metastasis or recurrence.

[0375] In some embodiments, the individual is at risk for developing, or has genetic or other (e.g., risk factors for), a diagnosed or undiagnosed cell proliferative and / or differentiative disorder, e.g., cancer.

[0376] As used herein, an "at risk" individual is one who is at risk for developing a condition to be treated, e.g., a cell proliferation and / or differentiation disorder, e.g., cancer. In general, an "at risk" individual may or may not have detectable disease, and may or may not have developed detectable disease prior to the treatment methods described in the present invention. "At risk" means that the individual has one or more so-called risk factors, which are measurable parameters associated with the development of a disease or condition and are known in the art. For example, an at risk individual may have one or more risk factors that are measurable parameters associated with the development of cancer. An individual who has such one or more risk factors has a higher probability of developing cancer than an individual who does not have such risk factors. In general, risk factors can include, for example, age, sex, race, diet, history of previous disease, presence of precursor disease, genetic (e.g., hereditary) considerations, and environmental exposures. In some embodiments, individuals at risk of developing cancer encompass, for example, individuals who have had the disease among their relatives, as well as individuals who have a risk determined by genetic or biochemical marker analysis.

[0377] In conjunction with this, the individual may be undergoing one or more standard therapies, such as chemotherapy, radiation therapy, immunotherapy, surgery, or a combination thereof, whereby the one or more kinase inhibitors may be administered before, during, or after the chemotherapy, radiation therapy, immunotherapy, surgery, or a combination thereof.

[0378] In certain embodiments, the individual may be a human who is (i) substantially refractory to one or more chemotherapeutic agents, or (ii) has relapsed after being treated with chemotherapy, or both (i) and (ii). In some embodiments, the individual is refractory to two or more, three or more, or four or more chemotherapeutic agents (including standard or experimental chemotherapy).

[0379] In some embodiments, the patient has been diagnosed with or has been diagnosed with a CD38+ cancer.

[0380] In some embodiments, the patient has been diagnosed with or has been diagnosed with CD38+ cancer by immunohistochemical staining of cancer biopsy or surgical sample. In some embodiments, the patient has been diagnosed with or has been diagnosed with CD38+ cancer by chromogenic in situ hybridization. In some embodiments, the patient has been diagnosed with or has been diagnosed with CD38+ cancer by fluorescent in situ hybridization of cancer biopsy or surgical sample. In some embodiments, the patient has been diagnosed with or has been diagnosed with CD38+ cancer by genetic analysis, for example, by identifying CD38 mutant cancer, for example, somatic mutation of CD38 gene.

[0381] In some embodiments, the patient is a multiple myeloma patient who is not a candidate for autologous hematopoietic stem cell transplantation (ASCT). In some embodiments, the patient is relapsed or refractory after undergoing ASCT treatment.

[0382] In some cases, the patient is a multiple myeloma patient whose cancer is relapsed or refractory after administration of a therapeutic agent selected from the group consisting of one or more steroids, alkylating agents, and / or anthracyclines, proteasome inhibitors, or immunomodulatory agents.

[0383] In some embodiments, the patient has a cancer comprising one or more of the mutation sets set forth in Table 7, an insertion or deletion polymorphism in the CD38 gene, a copy number alteration in the CD38 gene, a methylation mutation in the CD38 gene, or a combination thereof.

[0384] In some embodiments, the patient has a chromosomal translocation associated with the cancer, e.g., a CD38+ cancer. In some embodiments, the patient has a fusion gene associated with the cancer, e.g., a CD38+ cancer.

[0385] In some embodiments, the patient has a chromosomal translocation associated with the cancer, e.g., a CD38+ cancer. In some embodiments, the patient has a fusion gene associated with the cancer, e.g., a CD38+ cancer.

[0386] [Table 7-1]

[0387] [Table 7-2]

[0388] C. Lymphocyte depletion In some embodiments, the patient is lymphodepleted prior to treatment.

[0389] Exemplary lymphodepleting chemotherapy regimens, along with associated useful biomarkers, are described in WO2016 / 191756 and WO2019 / 079564, which are incorporated herein by reference in their entireties. In certain embodiments, the lymphodepleting chemotherapy regimen comprises administering cyclophosphamide (200 mg / m 2 / day~2000mg / m 2 / day) and fludarabine (20 mg / m 2 / day~900mg / m 2 / day).

[0390] In some embodiments, lymphodepletion is achieved with cyclophosphamide at or about 250-500 or about 500 mg / m 2 Administration, for example, of cyclophosphamide 250 or about 250 to 500 or about 500, 250, 400, 500, about 250, about 400 or about 500 mg / m 2 This includes administration of

[0391] In some embodiments, lymphodepletion is with fludarabine 20 or about 20 mg / m 2 / day~40 or about 40mg / m 2 / day, for example, 30 or about 30 mg / m 2 / day administration.

[0392] In some embodiments, lymphodepletion comprises administration of both cyclophosphamide and fludarabine.

[0393] In some embodiments, the patient is receiving cyclophosphamide (250 mg / m 2 / day) and fludarabine (30 mg / m 2 / day) intravenously to deplete lymphocytes.

[0394] In some embodiments, the patient is receiving cyclophosphamide (500 mg / m 2 / day) and fludarabine (30 mg / m 2 / day) intravenously to deplete lymphocytes.

[0395] In some embodiments, lymphodepletion is performed up to 5 days prior to the first administration of NK cells. In some embodiments, lymphodepletion is performed up to 7 days prior to the first administration of NK cells.

[0396] In some embodiments, lymphodepletion is performed daily for three consecutive days (ie, days -5 to -3) beginning five days prior to the primary administration of NK cells.

[0397] In some embodiments, lymphodepletion is performed on days −5, −4 and −3.

[0398] D. Administration 1.NK cells In some embodiments, the NK cells are administered as part of a pharmaceutical composition, e.g., a pharmaceutical composition described herein. The cells are administered after thawing, optionally without any additional manipulation if the cryoprotectant is suitable for extemporaneous administration. For a given individual, a therapeutic regimen often involves administering multiple aliquots or doses of NK cells over time, often from a common batch or donor.

[0399] In some embodiments, the NK cells, e.g., the NK cells described herein, are administered at a concentration of 1×10 NK cells per dose. 8 or about 1 x 10 8 ~8×10 9 or about 8 x 10 9 In some embodiments, the NK cells are administered at a dose of 1×10 cells per dose. 8 or about 1 x 10 8 , 1×10 9 or about 1 x 10 9 , 4×10 9 or about 4 x 10 9 , or 8 × 10 9 or about 8 x 10 9 Each dose is administered.

[0400] In some embodiments, the NK cells are administered weekly. In some embodiments, the NK cells are administered weekly for at or about 4 weeks. In some embodiments, the NK cells are administered weekly for at or about 8 weeks.

[0401] In some embodiments, the NK cells are administered every 4 weeks or about 4 weeks every other week. In some embodiments, the NK cells are administered every 8 weeks or about 8 weeks every other week. In some embodiments, the NK cells are administered every 9, 10, 11, 12, 13, 14, 15, or 16, or about 9, about 10, about 11, about 12, about 13, about 14, about 15, or about 16 weeks every other week.

[0402] In some embodiments, the NK cells are administered monthly for at or about 4 months. In some embodiments, the NK cells are administered monthly for at or about 8 months. In some embodiments, the NK cells are administered monthly for at or about 12 months. In some embodiments, the NK cells are administered monthly for at or about 16 months.

[0403] In some embodiments, the first administration course comprises weekly, biweekly or monthly administration of NK cells as described above, and the second administration course comprises weekly, biweekly or monthly administration of NK cells, hi some embodiments, the second administration course continues until the patient experiences cancer progression, toxicity or intolerance.

[0404] In some embodiments, the NK cells are cryopreserved in an infusion-ready media, for example, a cryopreservation composition suitable for intravenous administration, as described herein.

[0405] In some embodiments, the NK cells are at 1×10 cells per vial. 8 or about 1 x 10 8 ~8×10 9 or about 8 x 10 9 In some embodiments, the NK cells are frozen and stored in single dose vials.

[0406] In some embodiments, the cells are thawed, for example in a 37° C. water bath, prior to administration.

[0407] In some embodiments, a thawed vial of NK cells is aseptically transferred to a single dose container, e.g., a dose bag, e.g., using a vial adapter and a sterile syringe. The NK cells can be administered from the container to the patient as an IV infusion via a Y-type blood / solution set filter by gravity.

[0408] In some embodiments, the NK cells are administered as soon as possible after thawing, preferably within 90 minutes, e.g., within 80 minutes, 70 minutes, 60 minutes, 50 minutes, 40 minutes, 30 minutes, 20 minutes, or 10 minutes. In some embodiments, the NK cells are administered within 30 minutes after thawing.

[0409] In some embodiments, the pharmaceutical composition is administered intravenously via a syringe.

[0410] In some embodiments, the patient is administered 1 ml, 4 ml, or 10 ml of the medication intravenously via a syringe.

[0411] 2. Antibodies

[0412] In some embodiments, the NK cells described herein, e.g., a pharmaceutical composition comprising the NK cells referred to herein, are administered in combination with an antibody, e.g., an antibody described herein, e.g., a CD38 antibody. In some embodiments, the antibody is administered together with the NK cells as part of a pharmaceutical composition. In some embodiments, the antibody is administered separately from the NK cells, e.g., as part of a separate pharmaceutical composition. The antibody may be administered before, after, or simultaneously with the administration of the NK cells.

[0413] In some embodiments, the dose or administration of the antibody comprises administering 1,800 mg of the antibody to the patient.

[0414] In some embodiments, the antibody is administered prior to administering the NK cells. In some embodiments, the antibody is administered after administering the NK cells.

[0415] In some embodiments, the NK cells are administered at least 30 minutes, 60 minutes, 90 minutes, 120 minutes, 150 minutes, 180 minutes, 210 minutes, or 240 minutes after completing antibody administration.

[0416] In some embodiments, the NK cells are administered the day after administering the antibody.

[0417] In some embodiments, NK cells are administered at each dose and the antibody is administered only during a portion of the doses, for example, in some embodiments, NK cells are administered once per week and the antibody is administered once every other week or month.

[0418] In some embodiments, the antibody is administered weekly. In some embodiments, the antibody is administered weekly for at or about 4 weeks. In some embodiments, the NK cells are administered weekly for at or about 8 weeks.

[0419] In some embodiments, the antibody is administered every 4 weeks or about 4 weeks every other week. In some embodiments, the antibody is administered every 8 weeks or about 8 weeks every other week. In some embodiments, the antibody is administered every 9, 10, 11, 12, 13, 14, 15, or 16, or about 9, about 10, about 11, about 12, about 13, about 14, about 15, or about 16 weeks every other week.

[0420] In some embodiments, the antibody is administered monthly for at or about 4 months. In some embodiments, the NK cells are administered monthly for at or about 8 months. In some embodiments, the NK cells are administered monthly for at or about 12 months. In some embodiments, the NK cells are administered monthly for at or about 16 months.

[0421] In some embodiments, the antibody is delivered weekly for the first 8 doses, then biweekly for the next 8 doses, and then at 4 week intervals thereafter, and administration may continue until disease progression.

[0422] In some embodiments, the first administration course comprises weekly, biweekly or monthly administration of the antibody, as described above, and the second administration course comprises weekly, biweekly or monthly administration of the antibody. In some embodiments, the first administration course comprises weekly, biweekly or monthly administration of the antibody and the NK cells, as described above, and the second administration course comprises weekly, biweekly or monthly administration of the antibody and the NK cells. In some embodiments, the second administration course continues until the patient experiences cancer progression, toxicity or intolerance.

[0423] In some embodiments, the antibody is administered every week for eight weeks. In some embodiments, the antibody is administered every two weeks for eight weeks.

[0424] In some embodiments, a dose of the antibody is provided prior to the first administration of the cells. In some embodiments, a reduced dose of the antibody is provided prior to the first administration of the cells.

[0425] 3. Cytokines In some embodiments, a cytokine is administered to the patient.

[0426] In some embodiments, the cytokines are administered together with the NK cells as part of a pharmaceutical composition. In some embodiments, the cytokines are administered separately from the NK cells, e.g., as part of a separate pharmaceutical composition.

[0427] In some embodiments, the cytokine is IL-2.

[0428] In some embodiments, IL-2 is administered subcutaneously.

[0429] In some embodiments, IL-2 is administered 1-4 hours or about 1 to about 4 hours after administration of NK cells is completed. In some embodiments, IL-2 is administered at least 1 hour after administration of NK cells is completed. In some embodiments, IL-2 is administered within a maximum of 4 hours after administration of NK cells is completed. In some embodiments, IL-2 is administered within a minimum of 1 hour and a maximum of 4 hours after administration of NK cells is completed.

[0430] In some embodiments, IL-2 is up to 10,000,000 IU / m 2 , for example up to 1,000,000, 2,000,000, 3,000,000, 4,000,000, 5,000,000, 6,000,000, 7,000,000, 8,000,000, 9,000,000 or 10,000,000 IU / m 2 It is administered at .

[0431] In some embodiments, IL-2 is at or about 1,000,000, 2,000,000 or about 2,000,000, or 3,000,000 or about 3,000,000, or 4,000,000 or about 4,000,000, or 5,000,000 or about 5,000,000, or 6,000,000 or about 6,000,000, or 7,000,000 or about 7,000,000, or 8,000,000 or about 8,000,000, or 9,000,000 or about 9,000,000, or 10,000,000 IU / m 2 It is administered at .

[0432] In some embodiments, IL-2 is administered at 1×10 6 IU / m 2 or about 1 x 10 6 IU / m 2 In some embodiments, IL-2 is administered at 2×10 6 IU / m 2 or about 2 x 10 6 IU / m 2 It is administered at .

[0433] In some embodiments, IL-2 is administered to a patient at a dose of 1×10 6 IU / m 2 is administered in less than

[0434] In some embodiments, IL-2 is administered to the patient in a flat dose. In some embodiments, a flat dose of 6,000,000 IU or about 6,000,000 IU is administered to the patient.

[0435] In some embodiments, no IL-2 is administered to the patient.

[0436] E. Administration An "effective amount" is an amount sufficient to achieve a beneficial or desired result. For example, a therapeutic content is an amount that achieves a desired therapeutic effect. Such an amount may be the same as or different from the amount necessary to prevent the onset of a disease or disease symptoms, i.e., a prophylactically effective amount. An effective amount is administered in one or more administrations, applications, or dosages. The therapeutically effective amount (i.e., an effective dosage) of a therapeutic compound is determined by the therapeutic compound selected. The composition can be administered one or more times per day or one or more times per week, including once every two days. Those skilled in the art will recognize that certain factors may affect the dosage and time required to effectively treat an individual, including, but not limited to, the severity of the disease or disorder, previous treatments, the general health and / or age of the individual, and other diseases present. In conjunction with this, treating an individual with a therapeutically effective amount of a therapeutic compound according to the present invention may include a single treatment or a series of treatments.

[0437] Dosage, toxicity and therapeutic efficacy of therapeutic compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine the LD50 (the dose lethal to 50% of the population) and ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxicity and therapeutic effect is the therapeutic index, which can be expressed as the ratio LD50 / ED50. Compounds with high therapeutic indices are preferred. Compounds with toxic side effects may be used, but care must be taken to design systems that target delivery of such compounds to diseased tissue sites in order to minimize potential damage to non-infected cells and reduce side effects.

[0438] Data obtained from cell culture assays and animal studies can be used to formulate a dosage range for use in humans. The dosage of such compounds may be within a circulating concentration range that includes the ED50 with little or no toxicity. Dosages may vary within such ranges depending on the dosage form employed and the route of administration utilized. For any compound used in the methods of the invention, the therapeutically effective amount can be estimated initially by cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound that achieves half the maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Plasma levels can be measured, for example, by high performance liquid chromatography.

[0439] F. Combination Therapy In some embodiments, the methods include administering the NK cell and CD38 targeting antibodies described herein in combination with other therapies, such as antibodies, NK cell engagers, antibody drug conjugates (ADCs), chemotherapeutic drugs, such as small molecule drugs, immune checkpoint inhibitors, and combinations thereof.

[0440] 1. Small molecule / chemotherapeutic drugs In some embodiments, the additional therapy is a small molecule drug. In some embodiments, the additional therapy is a chemotherapy drug. In some embodiments, the additional therapy is a small molecule chemotherapy drug. Such small molecule drugs can include current standard of care regimens to which adaptive NK cell therapy is added. In some cases, the use of NK cells as described herein can enhance the effect of small molecule drugs, such as by enhancing the efficacy of the small molecule drug, reducing the amount of the small molecule drug required to achieve a desired effect, or reducing the toxicity of the small molecule drug.

[0441] In some embodiments, the drug is a steroid. In some cases, the steroid is selected from the group consisting of dexamethasone, methylprednisolone, triamcinolone, prednisolone, prednisone, betamethasone, and combinations thereof. In some embodiments, the drug is a proteasome inhibitor. In some cases, the proteasome inhibitor is selected from the group consisting of bortezomib, carfilzomib, and combinations thereof. In some embodiments, the drug is a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is selected from the group consisting of melphalan, lenalidomide, thalidomide, pomalidomide, and combinations thereof. In some embodiments, the additional therapy includes the use of multiple small molecule drugs, such as any steroid, proteasome inhibitor, and combination of the aforementioned chemotherapeutic agents. For example, the additional therapy can include administering bortezomib, melphalan, and prednisone. As another example, the additional therapy can include administering lenalidomide and dexamethasone. As yet another example, the additional therapy can include administering bortezomib, thalidomide, and dexamethasone. As yet another example, the additional therapy can include administering bortezomib and dexamethasone. As yet another example, the additional therapy can include administering pomalidomide and dexamethasone. As yet another example, the additional therapy can include administering carfilzomib and dexamethasone. Some of these embodiments can additionally include administering an immunomodulatory agent, such as, for example, a checkpoint inhibitor as described herein.

[0442] In some embodiments, the drug is [(1S,2S,3R,4S,7R,9S,10S,12R,15S)-4-acetyloxy-1,9,12-trihydroxy-15-[(2R,3S)-2-hydroxy-3-[(2-methylpropan-2-yl)oxycarbonylamino]-3-phenylpropanoyl]oxy-10,14,17,17-tetramethyl-11-oxo-6-oxatetracyclo[11.3.1.0 3,10 .0 4,7 ]heptadeth-13-en-2-yl]benzoate (docetaxel) or a pharma- ceutically acceptable salt thereof.

[0443] In some embodiments, the drug is [(1S,2S,3R,4S,7R,9S,10S,12R,15S)-4,12-diacetyloxy-15-[(2R,3S)-3-benzamido-2-hydroxy-3-phenylpropanoyl]oxy-1,9-dihydroxy-10,14,17,17-tetramethyl-11-oxo-6-oxatetracyclo[11.3.1.0 3,10 .0 4,7 ]heptadeth-13-en-2-yl]benzoate (paclitaxel) or a pharma- ceutically acceptable salt thereof.

[0444] In some embodiments, the drug is 6-N-(4,4-dimethyl-5H-1,3-oxazol-2-yl)-4-N-[3-methyl-4-([1,2,4]triazolo[1,5-a]pyridin-7-yloxy)phenyl]quinazoline-4,6-diamine (tucatinib) or a pharma- ceutical acceptable salt thereof.

[0445] In some embodiments, the drug is pentyl N-[1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-methyloxolan-2-yl]-5-fluoro-2-oxopyrimidin-4-yl]carbamate (capecitabine) or a pharma- ceutically acceptable salt thereof.

[0446] In some embodiments, the drug is an azanide; cyclobutane-1,1-dicarboxylic acid; platinum(2+) (carboplatin) or a pharma- ceutically acceptable salt thereof.

[0447] In some embodiments, the drug is methyl(1R,9R,10S,11R,12R,19R)-11-acetyloxy-12-ethyl-4-[(12S,14R)-16-ethyl-12-methoxycarbonyl-1,10-diazatetracyclo[12.3.1.0 3,11 .0 4,9 ]Octadeca-3(11),4,6,8,15-pentaen-12-yl]-10-hydroxy-5-methoxy-8-methyl-8,16-diazapentacyclo[10.6.1.0 1,9 .0 2,7 .0 16,19 ]nonadeca-2,4,6,13-tetraene-10-carboxylate (vinorelbine) or a pharma- ceutically acceptable salt thereof.

[0448] In some embodiments, the drug is N-[3-chloro-4-[(3-fluorophenyl)methoxy]phenyl]-6-[5-[(2-methylsulfonylethylamino)methyl]furan-2-yl]quinazolin-4-amine (lapatinib) or a pharma- ceutically acceptable salt thereof.

[0449] In some embodiments, the drug is (E)-N-[4-[3-chloro-4-(pyridin-2-ylmethoxy)anilino]-3-cyano-7-ethoxyquinolin-6-yl]-4-(dimethylamino)but-2-enamide (neratinib) or a pharma- ceutically acceptable salt thereof.

[0450] In some embodiments, the drug is 6-acetyl-8-cyclopentyl-5-methyl-2-[(5-piperazin-1-ylpyridin-2-yl)amino]pyrido[2,3-d]pyrimidin-7-one (palbociclib) or a pharma- ceutically acceptable salt thereof.

[0451] In some embodiments, the drug is 7-cyclopentyl-N,N-dimethyl-2-[(5-piperazin-1-ylpyridin-2-yl)amino]pyrrolo[2,3-d]pyrimidine-6-carboxamide (ribociclib) or a pharma- ceutically acceptable salt thereof.

[0452] In some embodiments, the drug is N-[5-[(4-ethylpiperazin-1-yl)methyl]pyridin-2-yl]-5-fluoro-4-(7-fluoro-2-methyl-3-propan-2-ylbenzimidazol-5-yl)pyrimidin-2-amine (abemaciclib) or a pharma- ceutically acceptable salt thereof.

[0453] In some embodiments, the drug is (1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28E,30S,32S,35R)-1,18-dihydroxy-12-[(2R)-1-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]propan-2-yl]-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-11,36-dioxa-4-azatricyclo[30.3.1.0 4,9 ]Hexatriaconta-16,24,26,28-tetraene-2,3,10,14,20-pentone (everolimus) or a pharma- ceutically acceptable salt thereof.

[0454] In some embodiments, the drug is (2S)-1-N-[4-methyl-5-[2-(1,1,1-trifluoro-2-methylpropan-2-yl)pyridin-4-yl]-1,3-thiazol-2-yl]pyrrolidine-1,2-dicarboxamide (alpelisib) or a pharma- ceutical acceptable salt thereof.

[0455] In some embodiments, the drug is 4-[[3-[4-(cyclopropanecarbonyl)piperazine-1-carbonyl]-4-fluorophenyl]methyl]-2H-phthalazin-1-one (olaparib) or a pharma- ceutically acceptable salt thereof.

[0456] In some embodiments, the drug is (11S,12R)-7-fluoro-11-(4-fluorophenyl)-12-(2-methyl-1,2,4-triazol-3-yl)-2,3,10-triazatricyclo[7.3.1.0 5,13 ]trideca-1,5(13),6,8-tetraen-4-one (talazoparib) or a pharma- ceutically acceptable salt thereof.

[0457] In some embodiments, the drug is N-[2-[2-(dimethylamino)ethyl-methylamino]-4-methoxy-5-[[4-(1-methylindol-3-yl)pyrimidin-2-yl]amino]phenyl]prop-2-enamide (osimertinib) or a pharma- ceutical acceptable salt thereof.

[0458] In some embodiments, the drug is N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholin-4-ylpropoxy)quinazolin-4-amine (gefitinib) or a pharma- ceutically acceptable salt thereof.

[0459] In some embodiments, the drug is N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine (erlotinib) or a pharma- ceutically acceptable salt thereof.

[0460] In some embodiments, the drug is (E)-N-[4-(3-chloro-4-fluoroanilino)-7-[(3S)-oxolan-3-yl]oxyquinazolin-6-yl]-4-(dimethylamino)but-2-enamide (afatinib) or a pharma- ceutical acceptable salt thereof.

[0461] In some embodiments, the drug is azan; dichloroplatinum (cisplatin, platinol) or a pharma- ceutically acceptable salt thereof.

[0462] In some embodiments, the drug is an azanide; cyclobutane-1,1-dicarboxylic acid; platinum(2+) (carboplatin) or a pharma- ceutically acceptable salt thereof.

[0463] In some embodiments, the drug is 4-amino-1-[(2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]pyrimidin-2-one (gemcitabine) or a pharma- ceutically acceptable salt thereof.

[0464] In some embodiments, the drug is (2S)-2-[[4-[2-(2-amino-4-oxo-3,7-dihydropyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]amino]pentanedioic acid (pemetrexed) or a pharma- ceutically acceptable salt thereof.

[0465] In some embodiments, the drug is N,N-bis(2-chloroethyl)-2-oxo-1,3,2λ 5 -oxazaphosphinan-2-amine (cyclophosphamide) or a pharma- ceutically acceptable salt thereof.

[0466] In some embodiments, the drug is (2R,3S,4S,5R)-2-(6-amino-2-fluoropurin-9-yl)-5-(hydroxymethyl)oxolane-3,4-diol (fludarabine) or a pharma- ceutically acceptable salt thereof.

[0467] In some embodiments, the drug is (7S,9S)-7-[(2R,4S,5S,6S)-4-amino-5-hydroxy-6-methyloxan-2-yl]oxy-6,9,11-trihydroxy-9-(2-hydroxyacetyl)-4-methoxy-8,10-dihydro-7H-tetracene-5,12-dione (doxorubicin) or a pharma- ceutically acceptable salt thereof.

[0468] In some embodiments, the drug is methyl(1R,9R,10S,11R,12R,19R)-11-acetyloxy-12-ethyl-4-[(13S,15S,17S)-17-ethyl-17-hydroxy-13-methoxycarbonyl-1,11-diazatetracyclo[13.3.1.0 4,12 .0 5,10 ]nonadeca-4(12),5,7,9-tetraen-13-yl]-8-formyl-10-hydroxy-5-methoxy-8,16-diazapentacyclo[10.6.1.0 1,9 .0 2,7 .0 16,19 ]nonadeca-2,4,6,13-tetraene-10-carboxylate (vincristine) or a pharma- ceutically acceptable salt thereof.

[0469] In some embodiments, the drug is (8S,9S,10R,13S,14S,17R)-17-hydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-6,7,8,9,12,14,15,16-octahydrocyclopenta[a]phenanthrene-3,11-dione (prednisone) or a pharma- ceutically acceptable salt thereof.

[0470] In some embodiments, the drug is N,3-bis(2-chloroethyl)-2-oxo-1,3,2λ 5 -oxazaphosphinan-2-amine (ifosfamide) or a pharma- ceutically acceptable salt thereof.

[0471] In some embodiments, the drug is (5S,5aR,8aR,9R)-5-[[(2R,4aR,6R,7R,8R,8aS)-7,8-dihydroxy-2-methyl-4,4a,6,7,8,8a-hexahydropyrano[3,2-d][1,3]dioxin-6-yl]oxy]-9-(4-hydroxy-3,5-dimethoxyphenyl)-5a,6,8a,9-tetrahydro-5H-[2]benzofuro[6,5-f][1,3]benzodioxol-8-one (etopside) or a pharma- ceutical acceptable salt thereof.

[0472] In some embodiments, the drug is (8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-17-(2-hydroxyacetyl)-10,13,16-trimethyl-6,7,8,11,12,14,15,16-octahydrocyclopenta[a]phenanthren-3-one (dexamethasone) or a pharma- ceutically acceptable salt thereof.

[0473] In some embodiments, the drug is (8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-17-(2-hydroxyacetyl)-10,13,16-trimethyl-6,7,8,11,12,14,15,16-octahydrocyclopenta[a]phenanthren-3-one (cytarabine) or a pharma- ceutically acceptable salt thereof.

[0474] 2. NK cell engagers In some embodiments, the additional therapy is a NK cell engager, for example, a bispecific or trispecific antibody.

[0475] In some embodiments, the NK cell engager is a bispecific antibody against CD16 and a disease-associated antigen, e.g., a cancer-associated antigen, e.g., a cancer antigen described herein, e.g., CD38. In some embodiments, the NK cell engager is a trispecific antibody against CD16 and two disease-associated antigens, e.g., a cancer-associated antigen, e.g., an antigen described herein.

[0476] 3. Checkpoint inhibitors In some embodiments, the additional therapy is an immune checkpoint inhibitor.

[0477] In some embodiments, the immune checkpoint inhibitor is selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, and combinations thereof.

[0478] In some embodiments, the immune checkpoint inhibitor is selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a VISTA inhibitor, a BTLA inhibitor, a TIM-3 inhibitor, a KIR inhibitor, a LAG-3 inhibitor, a TIGIT inhibitor, a CD-96 inhibitor, a SIRPα inhibitor, and combinations thereof.

[0479] In some embodiments, the immune checkpoint inhibitor is selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a LAG-3 (CD223) inhibitor, a TIM-3 inhibitor, a B7-H3 inhibitor, a B7-H4 inhibitor, an A2aR inhibitor, a CD73 inhibitor, an NKG2A inhibitor, a PVRIG / PVRL2 inhibitor, a CEACAM1 inhibitor, a CEACAM5 inhibitor, a CEACAM6 inhibitor, a FAK inhibitor, a CCL2 inhibitor, a CCR2 inhibitor, a LIF inhibitor, a CD47 inhibitor, a SIRPα inhibitor, a CSF-1 inhibitor, an M-CSF inhibitor, a CSF-1R inhibitor, an IL-1 inhibitor, an IL-1R3 inhibitor, an IL-RAP inhibitor, an IL-8 inhibitor, a SEMA4D inhibitor, an Ang-2 inhibitor, a CELVER-1 inhibitor, an Axl inhibitor, a phosphatidylserine inhibitor, and combinations thereof.

[0480] In some embodiments, the immune checkpoint inhibitor is selected from the agents listed in Table 8, or combinations thereof.

[0481] [Table 8]

[0482] In some embodiments, the immune checkpoint inhibitor is an antibody.

[0483] In some embodiments, the PD-1 is selected from the group consisting of pembrolizumab, nivolumab, toripalimab, cemiplimab-rwlc, sintilimab, and combinations thereof.

[0484] In some embodiments, the PD-L1 inhibitor is selected from the group consisting of atezolizumab, durvalumab, avelumab, and combinations thereof.

[0485] In some embodiments, the CTLA-4 inhibitor is ipilimumab.

[0486] In some embodiments, the PD-1 inhibitor is selected from the group of inhibitors shown in Table 9.

[0487] [Table 9-1]

[0488] [Table 9-2]

[0489] [Table 9-3]

[0490] In some embodiments, the PD-L1 inhibitor is selected from the group of inhibitors shown in Table 10.

[0491] [Table 10-1]

[0492] [Table 10-2]

[0493] In some embodiments, the CTLA-4 inhibitor is selected from the group of inhibitors shown in Table 11.

[0494] [Table 11]

[0495] In some embodiments, the immune checkpoint inhibitor is a small molecule drug. Small molecule checkpoint inhibitors are described, for example, in WO2015 / 034820A1, WO2015 / 160641A2, WO2018 / 009505A1, WO2017 / 066227A1, WO2018 / 044963A1, WO2018 / 026971A1, WO2018 / 045142A1, WO2018 / 005374A1, WO2017 / 202275A1, WO2017 / 202273A1, WO2017 / 202276A1, W O2018 / 006795A1, WO2016 / 142852A1, WO2016 / 142894A1, WO2015 / 033301A1, WO2015 / 033299A1, WO2016 / 142886A2, WO2 016 / 142833A1, WO2018 / 051255A1, WO2018 / 051254A1, WO2017 / 205464A1, US2017 / 0107216A1, WO2017 / 070089A1, WO201 7 / 106634A1, US2017 / 0174679A1, US2018 / 0057486A1, WO2018 / 013789A1, US2017 / 0362253A1, WO2017 / 192961A1, WO20 17 / 118762A1, US2014 / 199334A1, WO2015 / 036927A1, US2014 / 0294898A1, US2016 / 0340391A1, WO2016 / 039749A1, WO201 US2017 / 0252432A1, WO2016 / 126646A1, WO2015 / 044900A1, US2015 / 0125491A1, WO2015 / 033303A1, WO2016 / 142835A1, WO2019 / 008154A1, WO2019 / 008152A1 and WO2019023575A1.

[0496] In some embodiments, the PD-1 inhibitor is 2-[[4-amino-1-[5-(1-amino-2-hydroxypropyl)-1,3,4-oxadiazol-2-yl]-4-oxobutyl]carbamoylamino]-3-hydroxypropanoic acid (CA-170).

[0497] In some embodiments, the immune checkpoint inhibitor is (S)-1-(3-bromo-4-((2-bromo-[1,1'-biphenyl]-3-yl)methoxy)benzyl)piperidine-2-carboxylic acid.

[0498] In some embodiments, the immune checkpoint inhibitor is a peptide. See, e.g., Sasikumar et al., "Peptide and Peptide-Inspired Checkpoint Inhibitors: Protein Fragments to Cancer Immunotherapy," Medicine in Drug Discovery 8:100073 (2020).

[0499] VII. Mutants In some embodiments, the fusion proteins or components thereof described herein, or the NK cell genotypes described herein are at least 80%, e.g., at least 85%, 90%, 95%, 98% or 100% identical to the amino acid sequence of the exemplary sequences (e.g., provided herein), e.g., include mutations described herein, or in addition have up to 1%, 2%, 5%, 10%, 15% or 20% differences as residues of the exemplary sequences replaced with, e.g., conservative mutations. In preferred embodiments, the variants maintain the desired activity of the parent sequence.

[0500] To determine the sequence identity of two nucleic acid sequences, the sequences are aligned for optimal comparison (e.g., gaps are introduced in one or both of the first amino acid or nucleic acid sequence and the second amino acid or nucleic acid sequence for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). The length of the reference sequence aligned for comparison purposes is at least 80% of the length of the reference sequence, and in some embodiments, at least 90% or 100%. Nucleotides are then compared at corresponding amino acid or nucleotide positions. If a position in the first sequence contains a nucleotide identical to a corresponding position in the second sequence, then the molecules are identical at that position (as used herein, nucleic acid "identity" is the same as nucleic acid "homology"). The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the length of each gap and the number of gaps that should be introduced to optimally align the two sequences.

[0501] The percent identity between a subject polypeptide or nucleic acid sequence (i.e., the query) and a second polypeptide or nucleic acid sequence (i.e., the target) can be determined in a variety of ways within the skill of the art, for example, by Smith Waterman alignment (Smith, T F and MS Waterman (1981) J Mol Biol 147:195-7); GeneMatcher Plus TM, "BestFit" as incorporated in Schwarz and Dayhof (1979) Atlas of Protein Sequence and Structure, Dayhof, MO, Ed, pp 353-358 (Smith and Waterman, Advances in Applied Mathematics, 482-489 (1981)); the BLAST program (Basic Local Alignment Search Tool; (Altschul, S. F., W. Gish, et al. (1990) J Mol Biol215:403-10), BLAST-2, BLAST-P, BLAST-N, BLAST-X, WU-BLAST-2, ALIGN, ALIGN-2, CLUSTAL or Megalign (DNASTAR) software is used to determine the alignment. In addition, a person skilled in the art can determine the appropriate parameters for determining the alignment, such as any algorithm required to achieve maximum alignment over the entire length of the sequences being compared. In general, for target proteins or nucleic acids, the comparison length can be any length of the target, up to and including the full length of the target (e.g., 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%). For the purposes of the present invention, the identity percentage is based on the full length of the query sequence.

[0502] For purposes of the present invention, alignment and determination of percent identity of two sequences may be accomplished using the Blossum62 scoring matrix, applying a gap penalty of 12, a gap extension penalty of 4 and a frameshift gap penalty of 5.

[0503] Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

[0504] VIII.Definitions

[0505] Unless otherwise specified, all technical terms, notations, and other technical and scientific terms or terminology used in this application are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. In some cases, terms having commonly understood meanings are defined in this application for clarity and / or ease of reference, but the inclusion of such definitions in the present invention should not necessarily be interpreted as meaning a substantial difference from what is commonly understood in the art.

[0506] Throughout this application, various embodiments are presented in a range format. Descriptions in range format are primarily for convenience and brevity and should not be construed as inflexible limitations on the scope of the disclosure. Thus, a description of a range should be considered to specifically refer to each individual value within the range, as well as all subranges, to the extent possible. For example, a description of a range such as 1-6 should be considered to specifically refer to each individual value within the range, e.g., 1, 2, 3, 4, 5, and 6, as well as subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc. This applies regardless of the breadth of the range.

[0507] As used in the specification and claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "sample" includes a plurality of samples, including a mixture of samples.

[0508] The terms "ascertaining," "measuring," "assessing," "determining," "assaying," and "analyzing" are used interchangeably herein and often refer to types of measurements. These terms include determining (e.g., detecting) the presence or absence of an element. These terms can encompass quantitative, qualitative, or quantitative and qualitative determinations. Assessment can be relative or absolute. "Detecting the presence of" can encompass determining the amount of something present, other than ascertaining the presence or absence, depending on the context.

[0509] The terms "individual", "person" or "patient" are often used interchangeably herein.

[0510] The term "in vivo" is used to refer to phenomena that take place in the body of an individual.

[0511] The term "in vitro" is used to refer to a phenomenon that takes place outside the body of an individual. An in vitro analysis is not performed on an individual. Rather, it is performed on a sample that is isolated from an individual. An example of an in vitro analysis that is performed on a sample is an "in vitro" analysis.

[0512] The term "in vitro" is used to refer to phenomena that take place in a container for holding experimental reagents such that the material is separated from the biological source from which it is obtained. In vitro analysis can encompass cell-based analysis using living or dead cells. In vitro analysis can also encompass acellular analysis, which does not use intact cells.

[0513] As used herein, the term "about" in reference to a number means that 10% of the number in question can be added or subtracted from that number. The term "about" in reference to a range means from the lowest value minus 10% of the range to the highest value plus 10% of the range.

[0514] As used herein, the term "buffer solution" refers to an aqueous solution that consists of a mixture of a weak acid and its counterbase, or vice versa.

[0515] As used herein, the term "cell culture medium" refers to a mixture for in vitro cell growth and proliferation that contains factors essential for cell growth and proliferation, such as sugars, amino acids, various nutrients, inorganic substances, etc.

[0516] A buffer solution as used herein is not a cell culture medium.

[0517] As used herein, the term "bioreactor" refers to a culture device in which a range of conditions that affect cell culture, such as dissolved oxygen concentration, dissolved carbon dioxide concentration, pH, and temperature, can be continuously controlled.

[0518] As used herein, the term "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked. This term encompasses vectors as self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which it is introduced. Some vectors are suitable for transferring the nucleic acid molecules or polynucleotides of the present invention. Some vectors are capable of directing the expression of a nucleic acid to which they are operably linked. Such vectors are referred to herein as expression vectors.

[0519] The term "operably linked" refers to two or more nucleic acid sequences or polypeptide elements that are typically physically linked and in a functional relationship to each other. For example, a promoter is operably linked to a coding sequence if it is capable of initiating or regulating the transcription or expression of the coding sequence, in which case the coding sequence should be understood to be "under the control of" the promoter.

[0520] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably to refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells encompass "engineered cells," "transformants," and "transformed cells," which encompass the primary engineered (e.g., transformed) cell and its derived progeny, regardless of the number of subcultures. The progeny may not be completely identical in nucleic acid makeup to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as that screened or selected from the originally transformed cell are also included in the invention.

[0521] Where appropriate, host cells can be stably or transiently transfected with a polynucleotide encoding a fusion protein as described herein.

[0522] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter of the described invention. EXAMPLES

[0523] IX. Working Examples The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

[0524] Example 1: Off-the-shelf NK cell therapy platform An example of a method for expanding and stimulating NK cells is shown in FIG.

[0525] High affinity variants of the receptor CD16 (158V / V variant, see e.g., Koene et al., "FcγRIIIa-158V / F Polymorphism Influences the Binding of IgG by Natural Killer Cell FcgammaRIIIa, Independently of the FcgammaRIIIa-48L / R / H Phenotype", Blood 90:1109-14 (1997)) and KIR-B genotypes (KIR B alleles of the KIR receptor system, see e.g., Hsu et al., "The Killer Cell Immunoglobulin-Like Receptor (KIR) Genomic Region: Gene-Order, Haplotypes and Allelic Polymorphism", Immunological Review 190:40-52 (2002); and Pyo et al., "Different Patterns of Evolution in the Centromeric and Telomeric Regions of Group A and B Haplotypes of the Human Killer Cell FDA-approved frozen umbilical cord blood single units bearing the Ig-like Receptor Locus (see PLoS One 5:e15115 (2010)) were selected as the NK cell source.

[0526] Cord blood units were thawed and centrifuged to remove the freezing medium. The cell preparation was then T cell depleted using the QuadroMACS cell sorting system (Miltenyi) and CD3 (T cell) MicroBeads. Total nucleated cells (TNC) 6 × 10 8 Populations were labeled with MicroBeads and separated using a QuadroMACS instrument and buffer. After T cell depletion, the remaining cells, mainly monocytes and NK cells, were washed and collected in antibiotic-free medium (CellgroSCGM). Cell preparations were subsequently analyzed for total nucleated cell count, viability and % CD3+ cells. Cord blood NK cells were CD3-depleted as shown in Figure 1.

[0527] CD3- cell preparations were seeded into gas-permeable cell expansion bags with growth medium. Cells were co-cultured with replicatively engineered HuT-78 (eHUT-78) feeder cells to enhance expansion against a master cell bank (MCB) population. CellgroSCGM culture medium was initially supplemented with anti-CD3 antibody (OKT3), human plasma, glutamine, and IL-2.

[0528] As shown in FIG. 1, during any of the co-culture steps, the NK cells were selectively engineered to introduce a CAR into the NK cells, for example, using a lentiviral vector.

[0529] The cells were grown as static cultures at 37°C in a 5% CO2 balanced air atmosphere for 12-16 days, with additional medium changes at 2-4 day intervals. When the cultures were amplified 100-fold or more, the cultured cells were harvested and then suspended in freezing medium and packed into cryobags. In this case, 10 cells per bag or vial were grown during such co-culture. 8 Eighty bags or vials were made. The cryobags were frozen in a vapor-phase liquid nitrogen (LN2) tank at below -150°C using a controlled rate freezer. Such cryopreserved NK cells derived from FDA-cleared umbilical cord units were used as a master cell bank (MCB).

[0530] To produce the drug product, the frozen cell bags obtained from the MCB were thawed and the freezing medium was then removed. The thawed cells were inoculated into disposable culture bags and co-cultured with feeder cells, e.g., eHUT78 feeder cells, to produce the drug product. In this case, the cells were cultured in a 50 L bioreactor (e.g., 4,000-8,000 cryovials, each 100 mL) to produce several thousand drug products per cord blood unit. 9The cells / vial are mixed with a cryopreservation composition and then frozen in multiple storage containers such as cryovials. The drug product is an off-the-shelf infusion ready product available for direct injection. Each drug product lot can be injected into hundreds to thousands of patients (e.g., 100-1,000 patients, e.g., a target dose of 4 x 10 9 cell).

[0531] Example 2: Amplification of feeder cells As an example, suitable feeder cells, e.g., eHut-78 cells, were thawed from frozen stocks and expanded and cultured in 125 ml flasks in culture medium containing RPMI1640 (Life Technologies) 89% v / v, inactivated fetal bovine serum (FBS) (Life Technologies) (10% v / v) and glutamine (hyclone) (2 mM) at or about 37°C under 3-7% CO2 or about 3-7% CO2 for 18-24 days or about 18-24 days. Cells were split into 125 ml-2 liter flasks every 2-3 days. Cells were harvested by centrifugation and gamma irradiated. The harvested and irradiated cells were mixed with cryopreservation medium (Cryostor CS10) in 2 ml cryovials, then frozen in a controlled rate freezer at a cooling rate of about 15°C per 5 min to a final temperature of −90°C or about −90°C, and then transferred to a liquid nitrogen tank or freezer for cooling to a final temperature of −150°C or about −150°C.

[0532] After freezing, cell viability should be greater than 70% of the original cell number (wherein at least 1.0 × 10 8 viable cells / ml), >85% of the cells expressed tmTNF-α, >85% of the cells expressed mbIL-21+, and >85% of the cells expressed 4-1BBL.

[0533] Example 3: Expansion and stimulation of NK cells As an example, suitable NK cells can be produced using HuT-78 cells transduced to express 4-1BBL, membrane-bound IL-21 and mutant TNFα ("eHut-78P cells") as feeder cells. The feeder cells were suspended in 1% (v / v) CellGro medium and irradiated with 20,000 cGy in a gamma ray radiation device. Seed cells (e.g., CD3-depleted PBMCs or CD3-depleted umbilical cord blood cells) were cultured on the feeder cells in CellGro medium containing human plasma, glutamine, IL-2 and OKT-3 at 37°C as static culture. The cells were split at 2-4 day intervals. The total culture time is 19 days. The NK cells were collected by centrifugation and cryopreserved. The thawed NK cells are administered to the patient in an infusion medium composed of: phosphate buffered saline (PBS 1x, FujiFilm Irvine) (50% v / v), albumin (human) (20% v / v OctaPharma albumin solution: human albumin ≥ 96% protein 200 g / l, sodium 130-160 mmol; ≤ 2 mmol potassium, 0.064-0.096 mmol / g protein N-acetyl-DL-tryptophan, 0.064-0.096 mmol / g protein, caprylic acid, ad. 1000 ml water), dextran 40 / dextrose (25% v / v Hospira dextran 40 / dextrose injection, USP containing: 10 g / 100 ml dextran 40 and 5 g / 100 ml aqueous dextrose (dextrose hydrous) aqueous solution) and dimethyl sulfoxide (DMSO) (5% v / v Avantor DMSL solution, density 1.101 g / cm 3 , 20℃).

[0534] In some cases, the seed cells are CD3-depleted cord blood cells. The cell fraction can be depleted of CD3 cells by immunomagnetic sorting using the CliniMACS T cell depletion set (LS Depletion set (162-01) Miltenyi Biotec).

[0535] Preferably, the cord blood cells are selected to express CD16 with V / V polymorphism at F158 (Fc gamma RIIIa-158 V / V genotype) (Musolino et al. 2008 J Clin Oncol 26:1789). Preferably, the cord blood cells are of the KIR-B haplotype.

[0536] Example 4: Cord blood NK cells sorted for KIR-B and CD16 158V / V express low levels of CD38 after expansion NK cells were expanded as described in Example 1 to generate AB-101 cells using two cord blood samples from different donors selected for KIR-B and CD16 158v / v, and one unselected donor cord blood sample (control group). The cell purity (CD56+CD3-%) obtained by flow cytometry is shown in Figure 9. As shown in Figures 10 and 11, CD38 expression was lower in KIR-B / 158V / V NK cells as a group (% positive, Figure 10) and individually (mean fluorescence intensity of positive cells, Figure 11) compared to unselected NK cells.

[0537] Example 5: Phenotype of expanded and stimulated NK-cells As an example, NK cells derived from umbilical cord blood units were expanded and stimulated with eHut-78 cells using the expansion and stimulation steps described in Example 1. As shown in Figure 4, the resulting population of expanded and stimulated NK cells consistently exhibited high CD16(158V) and activating NK cell receptor expression.

[0538] Example 6: AB-101 AB-101 is a universal, standardized, cryopreserved, allogeneic cord blood-derived NK cell therapy product that contains ex vivo expanded and activated effector cells designed to enhance ADCC anti-tumor responses in patients, e.g., patients treated with monoclonal antibodies or NK cell engagers. AB-101 is composed of cord blood-derived mononuclear cells (CBMCs) enriched for NK cells by T lymphocyte depletion and co-cultured with engineered replicating defective T cell feeder cells supplemented with IL-2 and anti-CD3 antibody (OKT3).

[0539] AB-101 is an FDA approved cord blood derived allogeneic NK-cell product specifically designed for use in combination with therapeutic monoclonal antibodies (mAbs) to treat hematologic and solid tumors. The AB-101 manufacturing process produces an NK cell product with the following attributes:

[0540] A consistent NK cell profile. High expression of surface receptors such as CD16 and tumor antigen-binding / activating receptors, e.g. NKG2D, NKp46, Nkp30 and NKp44, to which the antibodies bind.

[0541] · KIR-B-haplotype. KIR-B haplotypes are associated with improved clinical outcomes in the haploidentical transplant setting and higher therapeutic potential in the allogeneic setting.

[0542] CD16 F158V polymorphism. The high affinity CD16 F158V variant that binds mAb Fc appears to promote improved antibody-dependent cellular cytotoxicity (ADCC).

[0543] Non-transformed NK cells. Genetic enhancement or gene editing is not required for or part of the AB-101 drug product.

[0544] The components and composition of AB-101 are listed in Table 12. AB-101 inhibits NK cells (CD16 + , CD56 + ) AB-101 is composed of T cells, B cells and macrophages (≤0.2% CD3 + , ≤1.0% CD19 + , ≤1.0% CD14 + The residual rate of eHuT-78P feeder cells used in culturing AB-101 is ≦0.2% of the drug.

[0545] [Table 12]

[0546] Initial stability studies indicate that AB-101 is stable for up to six months in vapor phase liquid nitrogen. Long-term stability studies to evaluate product stability beyond six months are underway and updated stability information will be included in the Certificate of Analysis.

[0547] The creation of the AB-101 drug consists of the following core steps (Figure 5): · Thawing of FDA cleared umbilical cord blood units (Hemacord, BLA125937). Removal of freezing-storage medium from cord blood units (CBUs) CD3 depletion using the FDA-cleared Vario MACS cell sorting system (Miltenyi) · Amplification and co-culture in bags with engineered feeder cells (eHuT-78 cells) Testing and cryopreservation of AB-101 master cell bank (approximately 200 bags) Co-culture with thawed (1 bag), amplified and engineered HuT-78 cells Further amplification in bioreactors ·Collect and recharge (1×10 9 NK cells / vial) ·AB-101 Frozen storage of pharmaceuticals (approximately 150 vials) Extensive characterization to ensure concentration, purity, potency and stability.

[0548] Such a production process reproducibly produces large quantities of highly pure and active AB-101 therapeutic NK cells, as shown in Table 13. Data points represent results from three independent cord blood units.

[0549] [Table 13]

[0550] Purity: eHuT-78P residue (eHuT-78P cell residue) The eHuT-78P cell residues in AB-101 pharmaceuticals are measured by flow cytometry (FACS). The eHuT-78 residues in AB-101 DP are detected by quantifying live CD3+4-1BBLhigh+ eHuT-78P using FACS. Since eHuT-78 is derived from the HuT-78 cell line expressing CD3 as cutaneous T lymphocytes, a FACS gating strategy is utilized to sequentially gate singlets, 7-AAD and CD3+4-1BBL+. The HuT-78 cell line was transduced with 4-1BB ligand (4-1BBL), membrane tumor necrosis factor-α (mTNF-α) and membrane-bound IL-21 (mbIL-21). The eHuT-78 single cells strongly expressing the three genes were selected, and research, master and working cell banks were sequentially established. Of these three genes, 4-1BBL was identified as the most highly expressed in the AB-101 cell bank and the final drug product and was therefore used in the FACS gating strategy.

[0551] Potency (cytotoxicity in AB-101 DP cells:K562 cells 10:1) The efficacy of AB-101 drugs is determined by evaluating their cellular cytotoxicity ability against K562 tumor cells. Cytotoxicity of drugs is evaluated by fluorimetric analysis. K562 tumor cells are stained with 30 μM calcein-AM (Molecular probes) for 1 hour at 37°C. Drug samples and labeled tumor cells are co-cultured in triplicate in 96-well plates at 37°C and 5% CO2 for 4 hours in the dark. To provide spontaneous maximum release, targets are supplemented with RPMI1640 medium supplemented with 10% FBS or 2% Triton-X100. To determine background fluorescence, each well is supplemented with RPMI1640 medium supplemented with 10% FBS or 2% Triton-X100. Fluorescence is measured at excitation 485 nm and emission 535 nm using a fluorescence reader. Specific cytotoxicity % is calculated by the following formula:

[0552]

number

[0553] Potency (cytotoxicity in AB-101 DP cells:Ramos cells 10:1) The same methods and calculations described above will be used to determine the efficacy of the AB-101 drug by assessing its cellular cytotoxicity potential against Ramos tumor cells. Details for this test are currently being finalized.

[0554] Example 7: Phenotypic Characterization of AB-101 The purity as well as the expression of antibody-engaging CD16 and activating, inhibitory and chemokine receptors in various AB-101 batches were determined by flow cytometric measurements.

[0555] AB-101 purity was determined using cell surface markers: AB-101 batches were observed to contain >99% CD3-CD56+ NK cells and <0.1% CD3+, CD14+ and CD19+ cells. CD16 expression was measured in AB-101. 95.11±2.51% of AB-101 cells were CD16+, with mean and median MFI for CD16 of 15311±6186 and 13097±5592, respectively. NK cells are known to express a variety of NK-specific activating and inhibitory receptors. In the various AB-101 batches tested, >80% of cells expressed CD16, NKG2A, NKG2D, CD94, NKp30, 2B4, Tim-3, and CD44, 40-70% of cells expressed NKp44, NKp46, and DNAM-1, approximately 30% of cells expressed CD161 and CD96, 15% of cells expressed CXCR3, and less than 5% of cells expressed other activation inhibitory receptors.

[0556] Two GMP batches of AB-101 were included in this study to evaluate the phenotypic characteristics of the cells at three different stages of the production process: cord blood cells after CD3+ cell depletion; an intermediate product, the Master Cell Bank (MCB), and the AB-101 final drug product (DP). Purity and NK cell receptors were measured in the CD3-depleted cells, MCB, and DP, respectively. The results first confirmed that NK cells derived from the CB exhibited an immature NK phenotype. The NK phenotype matured during the production process. At the MCB stage, more than 90% of the cells expressed phenotypic characteristics already present in mature NK cells, with other cell type markers at <0.1%. The expression levels of most of the NK cell-specific receptors increased throughout the production process, from the CD3-depleted cells, to the MCB, and finally to the DP.

[0557] List of abbreviations: NK: natural killer cell; mAb: monoclonal antibody; TNF-α: tumor necrosis factor alpha; CXCR: CXC chemokine receptor; DNAM-1: DNAX accessory molecule-1; CRACC: CD2-like receptor-activated cytotoxicity cells; ILT2: Ig-like transcript 2; Tim-3: T-cell immunoglobulin mucin-3; 7AAD: 7-amino-actinomycin D; ULBP: UL16-binding protein; MICA / B: MHC class I chain-associated proteins A and B; RAE1: ribonucleic acid efflux 1; H60: NKG2D interacts with two cell surface ligands associated with class I MHC molecules; MULT1: murine UL16-binding protein-like transcript 1; MHC: major histocompatibility complex; HLA: human leukocyte antigen.

[0558] The purity of AB-101 is expressed as CD3-CD56+ cells for NK cells, CD3+ cells for T cells, CD14+ cells for monocytes, and CD19+ cells for B cells. A total of nine batches of AB-101 were assayed for purity. The results were 99.27 ± 0.59% (mean ± SD) CD3-CD56+ cells, 0.02 ± 0.03% CD3+ cells, 0.10 ± 0.12% CD14+ cells, and 0.02 ± 0.04% CD19+ cells (Figure 6). Thus, it was verified that AB-101 is composed of highly pure NK cells with almost no other types of cells present as impurities.

[0559] Comparison of purity of CD3-depleted cells, MCB and DP produced under GMP conditions Two GMP batches of AB-101 were used to analyze the purity of the AB-101 starting material (CD3-depleted cells), intermediate product (Master Cell Bank, MCB) and final drug product (DP). 50-60% of the cells in the CD3-depleted cell fraction were NK cells, and the percentage increased to over 90% in the MCB and DP. 20-30% of the CD3-depleted cell fraction were CD14+ and CD19+ cells, and the percentage of such cells decreased to less than 0.1% in the MCB and DP, indicating the purity of the AB-101 MCB and the final drug product of AB-101 (Figure 7, Table 14).

[0560] [Table 14]

[0561] Comparison of NK cell receptors in CD3-depleted cells, MCB and DP produced under GMP conditions GMP batches of AB-101 were further used to analyze the expression of various NK cell receptors in the AB-101 starting material (CD3-depleted cells), intermediate product (Master Cell Bank, MCB) and final drug product (DP). It was observed that several NK cell and activating receptors such as CD16, NKG2D, NKG2C, NKp30, NKp44, NKp46 and DNAM-1 were expressed at higher levels in the MCB and final drug product when compared to the AB-101 starting material (CD3-depleted cells). CD57 expression was lower in the MCB and final drug product compared to the AB-101 starting material (CD3-depleted cells) (Figure 8, Table 15). Overall, the data showed increased expression of NK cell activating receptors in the MCB and final drug product, implying that AB-101 is effective against tumors.

[0562] [Table 15]

[0563] Conclusion The use of surface marker analysis demonstrated the identity and purity of the AB-101 product and batch-to-batch consistency. Furthermore, extensive evaluation of NK-specific activating and inhibitory cell surface markers established a consistent profile of the AB-101 product after the production amplification step. CB-derived NK cells have an immature phenotype, such as high expression of NKG2A and low expression of NKG2C, CD62L, CD57, IL-2R, CD16, and DNAM-1, compared to peripheral blood (PB)-derived NK cells, and CB-derived NK cells with an immature phenotype are known to have low cytotoxicity against tumor cells. The data from this experiment demonstrate that the allogeneic umbilical cord blood (CB)-derived NK cell product, i.e., AB-101, expresses high levels of key activating receptors, which implies potentially better cytotoxicity against tumor cells.

[0564] Example 8: AB-101 Toxicology The non-clinical toxicity of AB-101 was evaluated in a GLP study in NSG mice. The study was designed to evaluate the acute and delayed toxicity profile of AB-101. AB-101 was administered at two dose levels, i.e., 0.5×10 7 and 2 × 10 7 The cells / animals tested in this study. The proposed test dose range is 4×10 9 The product was designed to provide a level of exposure greater than the highest human equivalent dose planned to be delivered per cell / dose. Based on the relative growth factor (Nair 2016), the 0.5×10 7 Cells / mouse: 14 × 10 9 Cells / human equivalent: 2 x 10 7 Cells / mouse: 56 × 10 9The corresponding doses per cell / human are extrapolated to a patient weighing 70 kg. AB-101 was administered intravenously via the tail vein once weekly for 8 weeks. Acute toxicity of AB-101 was assessed 3 days after the 8th (i.e., last) dose. Delayed toxicity was assessed at the end of a 28-day recovery period after the 8th dose. Survival, body weight, clinical observations, and palpation were recorded for each animal for the life of the study. All animals underwent gross necropsy at the time of euthanasia, with sample collection for blood analysis, clinical chemistry, and histopathology analysis.

[0565] Each group consisted of 20 animals, 10 of each sex, to evaluate findings in both sexes and for powered statistical analysis. A vehicle-treated control group was included for comparison with the AB-101-treated groups. To minimize treatment bias, animals were assigned to dose groups by a computer-generated (weight order) random procedure, with separate random assignment of females and males. The study adhered to GLP guidelines, including data recording.

[0566] No mortality or adverse clinical observations were recorded related to administration of AB-101 at any of the dose levels evaluated. All minor observations that emerged were typical of mice and were not considered related to administration of AB-101. Body and organ weight changes were similar between dose groups and at the dates evaluated after treatment (53 days for the acute toxicity group and 78 days for the delayed toxicity group). There were no AB-101-related changes in hematology and clinical chemistry parameters or gross necropsy findings identified in animals at the time of euthanasia in either the acute or delayed toxicity groups. All variations between individuals and mean clinical chemistry values, regardless of statistical significance, were considered sporadic, consistent with biological and procedure-related deviations and / or minor in size, and therefore not related to administration of AB-101. There were no AB-101-related microscopic findings. In conclusion, the results of the GLP toxicity studies showed no significant differences in toxicity up to 2 × 10 7These results suggest that AB-101 is well tolerated in NSG mice following repeated cell / dose / animal dosing.

[0567] Example 9: Cryopreservation of NK cells AB-101 cells were prepared according to the process shown in Figure 5. At the end of the culture period, Sartorius kSep (R) The cells were harvested using a 400 disposable automated centrifuge system at a relative centrifugal force (RCF): 800-1200g and a flow rate of 60-120 ml / min, and washed twice with phosphate buffered saline (PBS). After washing, the cells were diluted with (1) albumin (human); (2) dextran 40; (3) DMSO; and (4) PBS to a target concentration of 1 × 10 8 AB-101 cells were formulated at 1000 cells / ml (Cryopreservation Composition Example #1, Table 4). The suspension formulation was placed in a 10 ml AT-Closed vial. (R) After filling to a target volume of 11 ml, the tubes were labeled and placed in a controlled rate freezer for frozen storage at ≦-135°C.

[0568] Stability studies were performed at time = 0 for initial release testing data. The stability storage freezer was a validated vapor phase LN2 storage freezer set to maintain a temperature ≦-135°C. At the sterility time point, 10% of the batch size or 4 vials, whichever was greater, was tested. Tests were thawed at 37°C to mimic clinical thawing conditions.

[0569] As shown in Table 16, it was confirmed that the viability and activity of cryopreserved AB-101 was preserved for at least 9 months.

[0570] [Table 16]

[0571] A "bedside" short-term stability study was performed to characterize the stability of AB-101 during handling immediately prior to administration. Samples were thawed, transferred to 10 ml syringes, filtered, and the contents were stored in Falcon tubes at the indicated temperatures for the indicated times. The harvested products were analyzed. Short-term stability data for the two AB-101 lots are shown in Table 17.

[0572] [Table 17]

[0573] Example 10: Cord blood NK cells sorted for KIR-B and CD16 158v / v express low CD38 after expansion NK cells were expanded as described in Example 6 to generate AB-101 cells using two cord blood samples from different donors selected for KIR-B and CD16 158 v / v, and one non-selected donor cord blood sample (control group). The cell purity (CD56+CD3-%) obtained by flow cytometry is shown in Figure 9. As shown in Figures 10 and 11, CD38 expression was lower in KIR-B / 158 v / v NK cells as a group (% positive, Figure 10) and individually (mean fluorescence intensity of positive cells, Figure 11) compared to non-selected NK cells.

[0574] Example 11: Surface Protein Expression of AB-101 NK cells were expanded as described in Example 1. Surface protein expression of the starting NK cell source (umbilical cord blood gated for CD56+ / CD3- expression, n=3) was compared to the expanded resulting NK cells (n=16). As shown in Figure 12, CD16 expression was high in the resulting cells and increased compared to the starting cells. Expression of NKG2D, CD94, NKp30, NKp44 and NKp46 was also increased, whereas expression of CXCR4 and CD122 was decreased.

[0575] Example 12: In vitro AB-101 + Daratumumab experiment Different lots of AB-101 and different lots of PB-NK cells (peripheral blood-derived CD56+ NK cells) were evaluated for viability, purity, and CD38 expression upon thawing.

[0576] Upon thawing, AB-101 cells had a viability of 94.85% ± 0.92% (Figure 13) and were (CD56 + CD3 - The NK cell purity was 98.60% ± 0.76%, as defined by CD56 + CD16 + The population was 87.43 ± 8.61% (mean ± SD).

[0577] CD38 expression was examined on AB-101 cells at thawing and after 24 hours of culture in RPMI 1640 medium supplemented with 10% FBS and 1000 IU / ml IL-2. At thawing and 24 hours after thawing, % CD38 expression (mean ± SD) was low and unchanged in AB-101 donor lots 21AB101PG004 (22.3% ± 0.85% and 23.4 ± 0.57%, respectively) and 20AB101PG005 (21.5% ± 2.55% and 21.2 ± 0.71, respectively). The geometric mean fluorescence intensity of CD38 expression was low and stable for AB-101 lots, 21AB101PG004 (9806±86.27 and 10340±1476.44, respectively) and 20AB101PG005 (8979±90.51 and 9056±1443.91, respectively). For AB-101 lot 19AB101PN004, CD38 expression intensity was high and stable at both time points, with MFIs of 31735±747.51 and 28396.00±3162.18, respectively. Data were obtained using cells from two vials for each donor (two vials for each donor examined).

[0578] In comparison, PB-NK cells from three healthy donors had better viability (93.27% ± 4.82%), purity (93.8% ± 2.43%) and CD16 expression (95.13% ± 0.74%), but higher CD38 expression compared to AB-101 cells (Figure 14). On average, CD56+ PB-NK cells expressed 88.47% ± 7.98% CD38 at thawing and 87.67% ± 8.6% CD38 after 24 h of culture. The MFI of CD38 expression in whole donors was 25,467.33 ± 6318.3 and 19,429.67 ± 3870.87 at thawing and after culture, respectively.

[0579] The effect of daratumumab on NK viability was examined in the presence of various concentrations of daratumumab using fratricide and complement fixation assays.

[0580] To confirm the effect of daratumumab on NK cell fratricide, AB-101 cells were cultured for 4 hours in the presence of various concentrations of daratumumab. Cell viability was maintained unaffected in the presence of IgG1 control antibody or daratumumab at concentrations ranging from 300 μg / ml to 0.41 μg / ml. The data show that AB-101 cells did not kill each other by ADCC in the presence of daratumumab (Figure 15). Interestingly, AB-101 donor lot 19AB101PN004 did not exhibit fratricide despite high CD38 surface expression.

[0581] To examine the effect of complement-mediated cell lysis, AB-101 cells were incubated with IgG control antibody or daratumumab in the presence of human complement serum. The Burkitt's lymphoma cell line Daudi was included as a control in the analysis. AB-101 cells did not lyse in the presence of daratumumab and complement despite CD38 expression, whereas the tumor cell line Daudi reached saturation levels of cell lysis at the lowest daratumumab concentration tested in this experiment (0.41 μg / ml) (Figure 16).

[0582] Conclusion The mechanism of action of daratumumab is to induce ADCC and CDC responses against opsonized tumor cells (de Weers M, Tai YT, van der Veer MS, et al. Daratumumab, a novel therapeutic human CD38 monoclonal antibody, induces killing of multiple myeloma and other hematological tumors. J Immunol 2011(Feb1);186(3):1840-8) and macrophage-mediated ADCP (Overdijk MB, Verploegen S, Bogels M, et al. Antibody-mediated phagocytosis contributes to the anti-tumor activity of the therapeutic antibody daratumumab in lymphoma and multiple myeloma. MAbs 2015;7(2):311-21). Natural killer cells express CD38 and are therefore depleted in patients treated with daratumumab.Therefore, it is preferable to supplement with allogeneic NK cell therapy that can tolerate daratumumab treatment without being depleted (Naeimi Kararoudi M, Nagai Y, Elmas E et al. CD38 deletion of human primary NK cells eliminates daratumumab-induced fratricide and boosts their effector activity. Blood 2020 (Nov 19); 136 (21): 2416-27; Gurney M, Stikvoort A, Nolan E, et al. CD38 knockout natural killer cells expressing an affinity optimized CD38 chimeric antigen receptor successfully target acute myeloid leukemia with reduced effector cell fratricide. Haematologica 2020 (Dec 30); Online ahead of print. doi: 10.3324 / haematol. 2020.271908; Woan KV, Kim H, Bjordahl R, et al. Harnessing features of adaptive NK cells to generate iPSC derived NK cells for enhanced immunotherapy.Cell Stem Cell2021(Dec2);28(12):2062 2075.e5). To evaluate the suitability of AB-101 for clinical combination with daratumumab, we examined the expression of CD38 on AB-101 cells and investigated the effect of daratumumab on the viability of AB-101.

[0583] The data reported in this application demonstrate that <23% of AB-101 GMP lot cells express CD38 at low intensity. CD38 expression on AB-101 cells did not induce fratricide or CDC in the presence of daratumumab. These results are consistent with clinical findings such as:

[0584] · The intensity of CD38 expression on MM cells has been shown to positively correlate with clinical response to daratumumab in the GEN501 and SIRUS studies (Nijhof IS,Casneuf T,van Velzen J,et al.CD38 expression and complement inhibitors affect response and resistance to daratumumab therapy in myeloma.Blood2016(Aug18);128(7):95 70).

[0585] Peripheral blood mononuclear cells collected from the majority of MM patients continue to induce ADCC in the presence of daratumumab, indicating the presence of activated NK cells (Casneuf T, Adams III HC, van de Donk NWCJ, et al. Deep immune profiling of patients treated with lenalidomide and dexamethasone with or without daratumumab. Leukemia 2021; 35: 573 584; Wang Y, Zhang Y, Hughes T, et al. Fratricide of NK cells in daratumumab therapy for multiple myeloma overcome by ex vivo-expanded autologous NK cells. Clin Cancer Res 2018(Aug15); 24(16): 4006-17).

[0586] After treatment with daratumumab, the NK cell repertoire present in MM patients was CD38 low / (Wang Y, Zhang Y, Hughes T, et al.Fratricide of NK cells in daratumumab therapy for multiple myeloma overcome by ex vivo-expanded autologous NK cells.Clin Cancer Res2018(Aug15);24(16):4006-17).

[0587] The majority of peripheral blood NK cells express highly CD38 on their surface, and a subset expresses CD38 low / - As a result, daratumumab has a depleting effect on the majority of circulating NK cells. In contrast, in the present invention, the majority of AB-101 GMP lot cells are negative for CD38 expression, and a subset of cells (<23%) are negative for CD38 low It was found that the low CD38 positivity rate of AB-101 cells was similar to that of peripheral blood-derived NK cells produced by CD38 knockout for combination with daratumumab (Naeimi Kararoudi M, Nagai Y, Elmas E et al.CD38 deletion of human primary NK cells eliminates daratumumab-induced fratricide and boosts their effector activity.Blood2020(Nov19);136(21):2416-27;Gurney M,Stikvoort A,Nolan E,et al.CD38 knockout natural killer cells expressing an affinity optimized CD38 chimeric antigen receptor successfully target acute myeloid leukemia with reduced effector cell fratricide.Haematologica2020(Dec30);Online ahead of print.doi:10.3324 / haematol.2020.271908).

[0588] Interestingly, one of the AB-101 donors (Engineering Lot) strongly expressed CD38, but the cells did not undergo cytolysis by fratricide or CDC mechanisms in the presence of daratumumab. This donor, unlike the GMP lot, was not selected for KIR-B haplotype or CD16 158V / V allele. In conclusion, AB-101 GMP lot cells express low CD38, are resistant to daratumumab-induced fratricide and CDC, and are compatible with daratumumab combination without gene editing to knock out CD38 expression.

[0589] Example 13: Effect of steroids on AB-101 + daratumumab ADCC Three different lots of AB-101 cells were treated with dexamethasone and methylprednisolone for 48 hours in the presence of IL-2 50 IU / ml or 500 IU / ml at the following concentrations: dexamethasone 0.1 μM, 1 μM, 10 μM; methylprednisolone 0.08 μg / ml, 0.4 μg / ml, 2 μg / ml. The steroid-treated AB-101 cells were washed and then co-cultured with the following cell lines (as well as + and IgG controls) at a 5:1 E:T ratio: K562, Daudi, RPMI-8226, and NCI-H929.

[0590] In the case of K562, dexamethasone treatment reduced the basal cytotoxicity of AB-101 cells at all concentrations tested, except for one cell lot. Cytotoxicity was restored upon treatment with AB-101 and 500 IU / ml IL-2. Dexamethasone treatment of AB-101 cells enhanced basal cytotoxicity against Daudi and NCI-H929 during steroid treatment, especially when combined with 500 IU / ml IL-2. Dexamethasone treatment did not inhibit the ADCC potential of AB-101 cells. See Figures 17, 18, 19 and 20. Methylprednisolone showed a similar pattern, indicating that the combination of steroid treatment with AB-101 + daratumumab is advantageous.

[0591] Example 14: Daratumumab induces cytokine secretion cell line The cell lines used in this study are listed in Table 18.

[0592] [Table 18]

[0593] Effect of daratumumab concentration on antibody-dependent cellular cytotoxicity responses by AB-101 cells To investigate the effect of daratumumab concentration on ADCC, AB-101 cells were co-cultured with the CD38+ multiple myeloma cell line MM.1S and the CD38- cell line K562 at an E:T ratio of 5:1. Daratumumab or control IgG was added to the co-cultures at concentrations ranging from 300 μg / ml to 0.4 μg / ml. After 4 hours of co-culture, samples were analyzed by flow cytometry. Daratumumab induced ADCC against the MM.1S tumor cell line compared to the IgG1 control antibody. Peak ADCC was observed at daratumumab 1-11 μg / ml. K562 was used as a control for ADCC analysis, and as expected, no difference in AB-101 cell cytotoxicity was observed between the daratumumab or IgG control groups.

[0594] Based on the observations in this study, a daratumumab concentration of 2 μg / ml was selected for subsequent ADCC analysis in this study.

[0595] Short-term antibody-dependent cell-mediated cytotoxicity The ADCC potential of AB-101 cells in combination with daratumumab was tested against multiple myeloma cell lines-RPMI8226, NCI-H929, MM.1S and MM.1R. Burkitt's lymphoma cell line Daudi, which highly expresses CD38, and CML cell line K562, which lacks CD38 expression, were used as positive and negative controls, respectively. AB-101 and tumor targets were co-cultured in the presence of 2μg / ml daratumumab or control IgG1 antibody at E:T ratios of 10:1, 5:1, 2.5:1, 1.25:1, 0.6:1 and 0.3:1. After 4 hours of co-culture, samples were analyzed by flow cytometry.

[0596] ADCC of the three AB-101 donors against each tumor cell line was examined. For Daudi, the mean daratumumab-mediated ADCC ranged from 79.46% ± 4.72% (mean ± SD) at an E:T ratio of 10:1 to 36.99% ± 11.68% at an E:T ratio of 0.31:1, whereas for the IgG1 control group, it was 48.96% ± 9.98% at an E:T ratio of 10:1 and 10.2% ± 3.58% at an E:T ratio of 0.31:1 (Figure 21, Figure 22, Figure 23, Figure 24, Figure 25, and Figure 26). ADCC against the multiple myeloma cell line NCI-H929 ranged from 46.44% ± 11.19% at an E:T ratio of 10:1 to 3.38% ± 0.77% at an E:T ratio of 0.31:1; for RPMI8226, it ranged from 58.22% ± 8.71% at an E:T ratio of 10:1 to 8.75% ± 2.31% at an E:T ratio of 0.31:1; for MM.1R, it ranged from 42.36% ± 11.02% at an E:T ratio of 10:1 to 4.88% ± 2.77% at an E:T ratio of 0.31:1; and for MM.1S, it ranged from 19.84% ± 7.32% at an E:T ratio of 10:1 to 2.34% ± 2.33% at an E:T ratio of 0.31:1. The K562 cell line showed no difference in AB-101-mediated cytolysis in the presence of daratumumab compared to the IgG1 control antibody, as expected: % specific cytolysis against K562 was 36.88% ± 5.98% in the presence of daratumumab at an E:T ratio of 10:1, compared to 37.52% ± 8.55% in the presence of the IgG1 control antibody.

[0597] Daratumumab induced a stronger ADCC response from AB-101 cells compared to the IgG1 control group. The ADCC level was dependent on the E:T ratio. Improved tumor cell cytolysis in the presence of daratumumab was significantly higher than the IgG1 control group at all E:T ratios for Daudi, NCI-H929, and RPMI8226, and at the top three E:T ratios for MM.1R. MM.1S showed no significant difference in cytolysis except at an E:T ratio of 10:1. As expected, K562 cell lysis was similar between the IgG1 and daratumumab groups.

[0598] Statistical analysis was performed using GraphPad Prism software with a non-parametric Wilcoxon Matched-Pairs Signed Rank Test between the IgG control group and daratumumab, and significance is shown as adjusted P value.

[0599] Daratumumab-induced cytokine secretion in vitro Activation-induced cytokine secretion of AB-101 cells in response to daratumumab was examined against multiple myeloma cell lines-RPMI8226, NCI-H929, MM.1S, MM.1R, and with Daudi and K562 as positive and negative controls, respectively. Co-cultures of AB-101 and tumor cells were set up at a 1:1 E:T ratio (100,000 effector cells / well) in the presence of 2 μg / ml daratumumab or IgG1 control antibody. Array analytes were analyzed for IFNγ and TNFα by ELISA.

[0600] The analyzed groups included (1) tumor cells alone, (2) AB-101 alone, (3) AB-101 + tumor cells, (4) AB-101 + tumor cells + IgG1 control group, and (5) AB-101 + tumor cells + daratumumab (Figures 27, 28, and 29). IFNγ secretion by donor 21AB101PG004 was significantly higher than that of Daudi (920.33 ± 24.42 pg / ml / 10 6cells) and RPMI8226 (595.33 ± 25.97 pg / ml / 10 6 cells), was superior in the presence of daratumumab compared to the IgG1 control group (Daudi: 298.00 ± 11.53 pg / ml / 10 6 Cells and RPMI8226: 284.33 ± 48.99 pg / ml / 10 6 cells); donor 19AB101PN004 was Daudi (525.33 ± 54.59 pg / ml / 10 6 cells) versus the IgG1 control group (130.67±14.57pg / ml / 10 6 In contrast, the daratumumab-dependent IFN-γ secretion from these donors was not elicited by other cell lines.

[0601] Similarly, donor 21AB101PG004 had a higher antibody titer than the IgG1 control (281.33±48.75pg / ml / 10 6 cells), compared with daratumumab (726.00 ± 374.01 pg / ml / 10 6 cells), and 19AB101PN004 secreted more TNF-α in the presence of MM.1R (697.67 ± 223.55 pg / ml / 10 6 IgG control group (308.00±80.54pg / ml / 10 6 Cells secreted more TNF-α than controls.

[0602] Donor 20AB101PG005 secreted substantially higher amounts of IFNγ to all tumor cell lines tested in the presence of daratumumab compared to the IgG1 control group. 20AB101PG005 also secreted significantly higher amounts of IFNγ to Daudi (2002.67 ± 645.34 pg / ml / 10 in the presence of daratumumab compared to the IgG1 control group. 6 cells), RPMI8226 (1791.67±586.29pg / ml / 10 6 cells) and NCI-H929 (1752.67 ± 639.66 pg / ml / 10 6 Cells secreted more TNF-α than controls.

[0603] K562 induced higher levels of secretion of IFNγ and TNFα in all three donors, independent of daratumumab.

[0604] Discussion and Conclusion The data presented in this invention demonstrate that AB-101 cells, when combined with daratumumab, can lyse multiple myeloma cell lines via ADCC. The anti-tumor function was significantly superior to the combination of AB-101 with the IgG1 control. AB-101 cells also responded to daratumumab-mediated activation by secreting higher levels of the pro-inflammatory / immunomodulatory cytokines IFNγ and TNFα.

[0605] AB-101 GMP lot cells have a high percentage of CD16 receptor expression (21AB101PG004=86.3% and 20AB101PG005=93.1%) and are naturally negative for CD38 expression, with a fraction of cells (<23%) expressing CD38. low The three AB-101 donors tested in this study were insensitive to daratumumab-induced fratricide. Collectively, these data support the hypothesis that AB-101 cells can be effectively combined with daratumumab in a clinical setting for multiple myeloma indications.

[0606] [Table 19-1]

[0607] [Table 19-2]

[0608] [Table 19-3]

[0609] Other embodiments Although the present invention has been described in conjunction with a detailed description, it should be understood that the above description is for purposes of illustration only and is not intended to limit the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

Claims

1. A combination pharmaceutical comprising as active ingredients a population of natural killer cells (NK cells) and an antibody targeting human CD38 in the same or separate compositions, said NK cells being allogeneic to the patient, of the KIR-B haplotype, and homozygous for the CD16 158V polymorphism to the patient, for treating a CD38+ cancer patient.

2. 2. The pharmaceutical combination of claim 1, wherein the cancer is selected from the group consisting of glioma, thyroid cancer, lung cancer, colorectal cancer, head and neck cancer, gastric cancer, liver cancer, pancreatic cancer, renal cancer, urothelial cancer, prostate cancer, testicular cancer, breast cancer, cervical cancer, ovarian cancer, melanoma, lymphoma, and combinations thereof.

3. The pharmaceutical combination according to claim 1 , wherein the cancer is myeloma.

4. The combination pharmaceutical of claim 3, wherein the cancer is multiple myeloma.

5. The pharmaceutical combination of claim 1, wherein the patient relapsed after treatment with an anti-CD38 antibody.

6. The pharmaceutical combination of claim 1, wherein the patient has transitioned to disease progression following treatment with autologous stem cell transplant or chimeric antigen receptor T-cell therapy (CAR-T).

7. NK cells 1×10 ８ ~1×10 １０ The pharmaceutical combination according to claim 1 , comprising

8. The combination pharmaceutical of claim 1, wherein the antibody is daratumumab, isatuximab, or a biosimilar thereof.

9. The pharmaceutical combination of claim 1, wherein the patient is administered lymphodepleting chemotherapy prior to treatment.

10. The pharmaceutical combination of claim 9, wherein the lymphodepleting chemotherapy is a non-myeloablative chemotherapy.

11. The pharmaceutical combination according to claim 9 or 10, wherein the lymphodepleting chemotherapy comprises treatment with one or more of cyclophosphamide and fludarabine.

12. The cyclophosphamide is administered at a dose of 100-500 mg / m ２ The pharmaceutical combination according to claim 11 , wherein the administration is on a daily basis.

13. The fludarabine is 10-50 mg / m ２ The pharmaceutical combination according to claim 11 , wherein the administration is on a daily basis.

14. The pharmaceutical combination according to claim 1, which additionally comprises IL-2.

15. The IL-2 is 1 x 10 ６ IU / m ２ The pharmaceutical combination according to claim 14, wherein the pharmaceutical combination is administered 16. The combination pharmaceutical of claim 14, wherein the IL-2 is administered at 6,000,000 IU.

17. The pharmaceutical combination according to any one of claims 14 to 16, wherein the administration of IL-2 is performed within 1-4 hours of the administration of the NK cells.

18. The pharmaceutical combination of claim 1 , wherein the NK cells are not genetically modified.

19. The pharmaceutical combination of claim 1 , wherein the NK cells are at least 70% CD56+ and CD16+.

20. The pharmaceutical combination of claim 1, wherein the NK cells are at least 85% CD56+ and CD3-.

21. The pharmaceutical combination of claim 1, wherein no more than 1% of the NK cells are CD3+, no more than 1% of the NK cells are CD19+, and no more than 1% of the NK cells are CD14+.

22. Each administration of NK cells contains 1×10 NK cells. ９ ~5×10 ９ The pharmaceutical combination according to claim 1 , wherein each of the two is administered individually.

23. The pharmaceutical combination of claim 1 , wherein the patient is administered a dose of a CD38-targeting antibody prior to the first administration of NK cells.

24. The pharmaceutical combination of claim 1 , wherein the natural killer cells comprise expanded cord blood natural killer cells.

25. The pharmaceutical combination of claim 1 , wherein the population of natural killer cells comprises at least 60% CD16+ cells.

26. The pharmaceutical combination of claim 1, wherein the population of natural killer cells comprises at least 60% NKG2D+ cells.

27. The pharmaceutical combination of claim 1 , wherein the population of natural killer cells comprises at least 60% NKp46+ cells.

28. The pharmaceutical combination of claim 1 , wherein the population of natural killer cells comprises at least 60% NKp30+ cells.

29. The pharmaceutical combination of claim 1, wherein the population of natural killer cells comprises at least 60% DNAM-1+ cells.

30. The pharmaceutical combination of claim 1 , wherein the population of natural killer cells comprises at least 60% NKp44+ cells.

31. The pharmaceutical combination of claim 1, wherein the population of natural killer cells contains less than 20% or no CD3+ cells.

32. The pharmaceutical combination of claim 1, wherein the population of natural killer cells contains less than 20% or no CD14+ cells.

33. The pharmaceutical combination of claim 1, wherein the population of natural killer cells contains less than 20% or no CD19+ cells.

34. The pharmaceutical combination of claim 1, wherein the population of natural killer cells contains less than 20% or no CD38+ cells.

35. The pharmaceutical combination of claim 1 , wherein the natural killer cells do not contain the CD16 transgene.

36. The pharmaceutical combination according to claim 1 , wherein the natural killer cells do not express exogenous CD16 protein.

37. The pharmaceutical combination of claim 1 , wherein the natural killer cells are not genetically engineered.

38. The pharmaceutical combination of claim 1 , wherein the natural killer cells are derived from the same umbilical cord blood donor.

39. 2. The pharmaceutical combination of claim 1, wherein the population of NK cells is produced by a method comprising: (a) obtaining seed cells comprising natural killer cells from umbilical cord blood; (b) depleting the seed cells of CD3+ cells; (c) expanding the natural killer cells by culturing the depleted seed cells with a first plurality of Hut78 cells engineered to express membrane-bound IL-21, mutant TNFα, and a 4-1BBL gene to produce expanded natural killer cells; This produces an expanded population of natural killer cells.

40. 2. The pharmaceutical combination of claim 1, wherein the population of NK cells is produced by a method comprising: (a) obtaining seed cells comprising natural killer cells from umbilical cord blood; (b) depleting the seed cells of CD3+ cells; (c) expanding the natural killer cells by culturing the depleted seed cells with a first plurality of Hut78 cells engineered to express membrane-bound IL-21, mutant TNFα, and the 4-1BBL gene to produce a master cell bank population of expanded natural killer cells; and (d) expanding the master cell bank population of expanded natural killer cells by culturing with a second plurality of Hut78 cells engineered to express membrane-bound IL-21, mutant TNFα, and 4-1BBL genes to produce expanded natural killer cells; This produces an expanded population of natural killer cells.

41. The population of NK cells, after step (c), (i) freezing the master cell bank population of the expanded natural killer cells in multiple containers; and (ii) a method further comprising the step of thawing a container containing an aliquot of the master cell bank population of amplified natural killer cells; The pharmaceutical combination of claim 39 or 40, wherein in step (d), expanding the master cell bank population of expanded natural killer cells comprises expanding an aliquot of the master cell bank population of expanded natural killer cells.

42. The pharmaceutical combination of claim 39 or 40, wherein the cord blood is from a donor who is KIR-B haplotype and homozygous for the CD16 158V polymorphism.

43. The pharmaceutical combination of claim 1, additionally comprising a steroid.

44. 44. The pharmaceutical combination of claim 43, wherein the steroid is selected from the group consisting of dexamethasone, methylprednisolone, triamcinolone, prednisolone, prednisone, betamethasone, and combinations thereof.

45. The pharmaceutical combination of claim 43, wherein the steroid is administered within 1-4 hours from the time of administration of the NK cells.

46. 44. The pharmaceutical combination of claim 43, wherein the steroid does not reduce or eliminate the ADCC activity of NK cells.

47. The combination drug is administered at a dose of 1×10 ６ IU / m ２ or 6 x 10 ６ The pharmaceutical combination according to claim 43, further comprising IL-2 in an amount of IU.

48. The combination pharmaceutical of claim 47, wherein the IL-2 is administered within 1-4 hours from the time of administration of the NK cells.

49. Amplified CD16 ＋ / CD38 ｌｏｗ A composition comprising a population of NK cells.

50. 50. The composition of claim 49, wherein the NK cells express CD38 at a lower level than a naturally occurring heterogeneous population of NK cells.

51. The combination pharmaceutical of claim 1, wherein the NK cells exhibit ADCC activity against CD38+ tumor cells in the presence of a CD38-targeting antibody.

52. The method includes the steps of: ＋ / CD38 ｌｏｗ Methods for generating a population of NK cells: Obtaining a cord blood sample containing NK cells from a donor homozygous for the KIR-B haplotype or CD16 158V / V genotype; and NK cells to CD4 ＋ Expanding in vitro in the presence of a T cell line.

53. The method includes the steps of: ＋ / CD38 ｌｏｗ Methods for enriching a population of NK cells: Obtaining a cord blood sample containing NK cells from a donor homozygous for the KIR-B haplotype or CD16 158V / V genotype; and NK cells to CD4 ＋ Expanding in vitro in the presence of a T cell line.

54. Cord blood samples containing NK cells were analyzed using CD38 ｈｉｇｈ 54. The method of claim 52 or 53, comprising a population of NK cells.

55. Cord blood samples containing NK cells were analyzed using CD38 ｌｏｗ 54. The method of claim 52 or 53, comprising a population of NK cells.

56. CD16 ＋ / CD38 ｌｏｗ 54. The method of claim 52 or 53, wherein the population of NK cells is not genetically engineered.

57. CD16 ＋ / CD38 ｌｏｗ 54. The method of claim 52 or 53, wherein the population of NK cells has not been genetically engineered to alter CD38 expression.

58. Medicinal combinations for treating CD38+ cancer patients, comprising: 1) A population of NK cells, The NK cells are CD38 ＋ and The cells were then subjected to CD38 immunoreaction in the presence of an anti-CD38 antibody. ＋ Mediates ADCC against cancer cells; The cells were then subjected to the addition of CD38 to the NK cell population in the presence of anti-CD38 antibodies. ＋ a population that does not substantially mediate ADCC against NK cells; and 2) Anti-CD38 antibody.

59. The pharmaceutical combination of claim 58, wherein the NK cells are allogeneic to the patient and are of the KIR-B haplotype and homozygous for the CD16 158V polymorphism to the patient.

60. A composition for treating a patient suffering from a CD38+ cancer comprising as an active ingredient a population of natural killer cells (NK cells), said population of NK cells being administered in combination with an antibody targeting human CD38, said NK cells being allogeneic to the patient, being of the KIR-B haplotype and being homozygous for the CD16 158V polymorphism.

61. A composition for treating a patient suffering from a CD38+ cancer comprising as an active ingredient an antibody targeting human CD38, said antibody targeting human CD38 being administered in combination with a population of natural killer cells (NK cells), said NK cells being allogeneic to the patient, being of the KIR-B haplotype, and being homozygous for the CD16 158V polymorphism.

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