Treatment of autoimmune disorders using NK cells

Abstract

In particular, methods for treating patients suffering from autoimmune diseases are provided herein.

Description

[Technical Field]

[0001] Claim of priority This application claims the interests of U.S. Provisional Patent Application No. 63 / 466,589 filed on 15 May 2023, U.S. Provisional Patent Application No. 63 / 513,154 filed on 12 July 2023, U.S. Provisional Patent Application No. 63 / 603,850 filed on 29 November 2023, U.S. Provisional Patent Application No. 63 / 556,636 filed on 22 February 2024, and U.S. Provisional Patent Application No. 63 / 642,218 filed on 3 May 2024. The entire contents of these applications are incorporated herein by reference. [Background technology]

[0002] High levels of autoreactive immune cells are associated with signs of autoimmunity. In patients with autoimmune diseases (e.g., lupus), incomplete depletion or reduction of pathogenic B cells using antibodies alone (e.g., rituximab) may result in limited efficacy or disease relapse.

[0003] This invention addresses these and other shortcomings in the art. [Overview of the Initiative]

[0004] NK cells are immune cells that can bind to tumor cells via a complex array of receptors on their cell surface and via antibody-dependent cell-mediated cytotoxicity (ADCC). To induce ADCC, NK cells bind to antibodies via the CD16 receptor on their surface. NK cells may have advantages compared to other immune cells, such as T cells used in CAR-T cell therapy and other cell therapies. An exemplary advantage is that NK cells can be used as allogeneic therapy, meaning that NK cells from a single donor can be safely used in one or many patients without the need for HLA matching, gene editing, or other genetic manipulation. Allogeneic NK cells with antitumor 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).

[0005] Allogeneic NK cells can offer an important treatment option for patients with autoimmune disorders, for example, by leading to significantly and longer-lasting depletion of pathogenic B cells compared to antibodies alone, resulting in better efficacy and outcomes.

[0006] Furthermore, by utilizing diverse umbilical cord blood banks as a source of NK cells, it is possible to select umbilical cords that possess desirable characteristics for enhancing clinical activity (e.g., high-affinity CD16 and killer cell immunoglobulin-like receptor (KIR) B-haplotype).

[0007] The administration of allogeneic NK cells as described herein can enhance the ADCC response in patients, for example, when receiving monoclonal antibody therapy.

[0008] Therefore, in particular, a method for depleting B cells in a patient is provided herein, for example, to treat a patient suffering from an autoimmune disorder, such as systemic lupus erythematosus (SLE).

[0009] Methods for treating patients suffering from autoimmune disorders, comprising the step of administering a population of natural killer cells (NK cells) and antibodies targeting immune cells, wherein the NK cells are allogeneic to the patient, are described herein. In some embodiments, the immune cells are involved in the autoimmune response. In some embodiments, the immune cells are B cells. In some embodiments, the antibody is a B cell depletion antibody, e.g., a B cell depletion monoclonal antibody (mAb). In some embodiments, the antibody is an antibody targeting human CD19 and / or human CD20. In some embodiments, the NK cells are of the KIR-B haplotype and homozygous for the CD16 158V polymorphism.

[0010] In some embodiments, autoimmune diseases include acromegaly, acquired aplastic anemia, acquired hemophilia, primary agammaglobulinemia, alopecia areata, ankylosing spondylitis (AS), anti-NMDA receptor encephalitis, antiphospholipid syndrome (APS) / severe antiphospholipid syndrome (CAPS) / Asherson syndrome, arteriosclerosis, autoimmune Addison's disease (AAD), autoimmune autonomic ganglion disorder (AAG) / autoimmune autonomic neuropathy / autoimmune gastrointestinal motility disorder (AGID), autoimmune encephalitis / acute disseminated encephalomyelitis (ADEM), autoimmune gastritis, autoimmune hemolytic anemia. AIHA, autoimmune hepatitis (AIH), autoimmune hyperlipidemia, autoimmune hypophysitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune myelofibrosis, autoimmune myocarditis, autoimmune oophoritis, autoimmune pancreatitis (AIP), polyglandular autoimmune syndrome, type I, type II & III (APS1, APS2, APS3, APECED), autoimmune progesterone dermatitis, autoimmune retinopathy (AIR), autoimmune sudden hearing loss (SNHL), Barlow's disease, Behçet's disease, birdshot chorioretinopathy / birdshot Uveitis, bullous pemphigoid, Castleman disease, celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic urticaria (CU), Churg-Strauss syndrome / granulomatosis with eosinophilia / polyangiitis (EGPA), Cogan syndrome, cold agglutinin disease, CREST syndrome / limited cutaneous systemic sclerosis, Crohn's disease (CD), Cronkhite-Canada syndrome (CSS), idiopathic organizing pneumonia (COP), herpetic dermatitis, dermatomyositis, type 1 diabetes, discoid lupus, Dressler syndrome / post-myocardial infarction / post-pericardiotomy syndrome, eczema / atopic dermatitis Inflammation, endometriosis, eosinophilic esophagitis, eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, fibrotic alveolar septitis / idiopathic pulmonary fibrosis (IPF), giant cell arteritis / temporal arteritis / Houghton's disease, giant cell myocarditis, glomerulonephritis, Goodpasture syndrome / anti-GBM / anti-TBM disease, granulomatosis with polyangiitis (GPA) / Wegener's granulomatosis, Graves' disease / thyroid eye disease, Guillain-Barré syndrome (GBS), Hashimoto's thyroiditis / chronic lymphocytic thyroiditis / autoimmune thyroiditis, Henoch-Schönlein purpura / IgA vasculitis, hidradenitis suppurativa,Hearst's disease / acute hemorrhagic leukoencephalitis (AHLE), hypogammaglobulinemia, IgA nephropathy / Berge's disease, immune-mediated necrotizing myopathy (IMNM), immune thrombocytopenia (ITP) / autoimmune thrombocytopenic purpura / autoimmune thrombocytopenia, inclusion body myositis, IgG4-related sclerosing disease (ISD), interstitial cystitis, juvenile idiopathic arthritis / adult-onset Still's disease, juvenile polymyositis / juvenile dermatomyositis / juvenile myositis, Kawasaki disease, Lambert-Eaton myasthenia dysthenia syndrome (LEMS), leukocytosis-destructive vasculitis, lichen planus, lichen sclerosing, woody conjunctivitis, linear IgA disease (LAD) Linear IgA bullous dermatosis (LABD), lupus nephritis, Lyme disease / chronic Lyme disease / post-treatment Lyme disease syndrome (PTLDS), lymphocytic colitis / microscopic colitis, lymphocytic hypophysitis / autoimmune hypophysitis, Meniere's disease, microscopic polyangiitis (MPA) / ANCA-associated vasculitis, mixed connective tissue disease (MCTD), Mohlen's ulcer, Mucher-Habermann disease, multifocal motor neuropathy, multiple sclerosis (MS), myalgic encephalomyelitis (ME) / chronic fatigue syndrome (CFS), myasthenia gravis (MG), narcolepsy, neuromyelitis optica / Devic's disease, ocular scarring pemphigoid Opsoclonus myoclonus syndrome (OMS), relapsing rheumatoid arthritis, paraneoplastic cerebellar degeneration, paraneoplastic pemphigus, Parley-Romberg syndrome (PRS) / hemifacial atrophy (HFA) / progressive hemifacial atrophy, paroxysmal nocturnal hemoglobinuria (PNH), peripheral uveitis / ciliary tonsillar atrophy, PANS / PANDAS, personality-turner syndrome, pemphigus of pregnancy / herpes of pregnancy, pemphigus foliaceus, pemphigus vulgaris, pernicious anemia, POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, postural orthostatic tachycardia syndrome (POTS), primary biliary liver disease Cirrhosis (PBC) / Primary biliary cholangitis, Primary sclerosing cholangitis (PSC), Psoriasis, Palmoplantar pustulosis, Psoriatic arthritis, Idiopathic pulmonary fibrosis (IPF), Pure red cell aplasia (PRCA), Pyoderma gangrene, Rasmussen's encephalitis, Raynaud's syndrome / phenomenon, Reactive arthritis / Reiter's syndrome, Reflex sympathetic dystrophy syndrome (RSD) / Complex regional pain syndrome (CRPS), Relapsing polychondritis, Restless legs syndrome (RLS) / Willis-Ekbom disease, Rheumatic fever, Rheumatoid arthritis, Sarcoidosis, Schmidt's syndrome / Autoimmune polyendocrine syndrome type II, Scleritis, Scleroderma,The following conditions are selected: sclerosing mesentericitis / mesentile panniculitis, creeping choroidopathy, Sjögren's syndrome, generalized rigidity syndrome (SPS), small fiber sensory neuropathy, systemic lupus erythematosus (SLE), subacute bacterial endocarditis (SBE), subacute cutaneous lupus, Suzac syndrome, Sydenham's chorea, sympathetic ophthalmitis, Takayasu's arteritis (vasculitis), testicular autoimmune disease (vasculitis, orchitis), Tolosa-Hunt syndrome, transverse myelitis (TM), tubulointerstitial nephritis-uveitis syndrome (TINU), ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), uveitis (anterior / intermediate / posterior), vasculitis, VEXAS syndrome, vitiligo, Vogt-Koyanagi-Harada syndrome (VKH), and combinations thereof.

[0011] In some embodiments, the autoimmune disorder is systemic lupus erythematosus (SLE). In some embodiments, the patient has lupus nephritis. In some embodiments, the patient has relapsed after treatment with anti-CD19 and / or anti-CD20 antibodies. In some embodiments, the patient has experienced disease progression after treatment with autologous stem cell transplantation or chimeric antigen receptor T-cell therapy (CAR-T).

[0012] In some embodiments, 1 × 10^8 to 1 × 10^10 NK cells are administered to the patient. In some embodiments, 1 × 10^9 to 8 × 10^9 NK cells are administered to the patient. The method according to any one of the claims, wherein 4 × 10^8, 1 × 10^9, 4 × 10^9, or 8 × 10^9 NK cells are administered to the patient. In some embodiments, 5 × 10^8, 1 × 10^9, or 4 × 10^9 NK cells are administered to the patient.

[0013] In some embodiments, the antibody is selected from Table 1, Table 2, or Table 3. In some embodiments, the antibody is rituximab, obinutuzumab, or tafacitamab. In some embodiments, the antibody is rituximab. In some embodiments, the antibody is obinutuzumab. In some embodiments, the antibody is tafacitamab.

[0014] In some embodiments, a population of 1 × 10^9 to 5 × 10^9 NK cells is administered to the patient. In some embodiments, a population of 2 × 10^9 NK cells or so, a population of 4 × 10^9 NK cells or so, a population of 5 × 10^8 NK cells or so, or a population of 1 × 10^9 NK cells or so is administered to the patient.

[0015] In some embodiments, 500 to 1500 mg of antibody or around that amount is administered to the patient. In some embodiments, 100 mg of antibody or around that amount is administered to the patient.

[0016] In some embodiments, the patient is subjected to lymphocyte depletion chemotherapy before treatment. In some embodiments, lymphocyte depletion chemotherapy is non-myeloablative chemotherapy. In some embodiments, lymphocyte depletion chemotherapy includes treatment with at least one of cyclophosphamide and fludarabine. In some embodiments, lymphocyte depletion chemotherapy includes treatment with cyclophosphamide and fludarabine. In some embodiments, cyclophosphamide is administered at a dose of 100-500 mg / m². 2 It is administered at a dose of / day. In some embodiments, cyclophosphamide is 250 or 300 mg / m². 2 It is administered at a dose of 500 mg / m². In some embodiments, cyclophosphamide is administered at a dose of 500 mg / m². 2 It is administered at a dose of 10-50 mg / m². In some embodiments, fludarabine is 10-50 mg / m². 2 It is administered at a dose of / day. In some embodiments, fludarabine is 30 mg / m². 2 It is administered daily.

[0017] In some embodiments, the method further includes the step of administering IL-2. In some embodiments, 1 × 10^6 IU / m³ 2IL-2 is administered to the patient. In some embodiments, 6 million IU of IL-2 is administered to the patient. In some embodiments, IL-2 administration is performed 1 to 4 hours before NK cell administration. In some embodiments, NK cell and antibody administration is performed weekly. In some embodiments, NK cells and antibodies are administered weekly for 3 to 8 or 4 to 8 weeks. In some embodiments, lymphocyte depletion is performed on days 1, 2, and 3 of the treatment cycle. In some embodiments, NK cells are administered on days 6, 13, and 20 or 6, 9, 13, and 16 of the treatment cycle. In some embodiments, NK cells are administered in units of 2 billion or 4 billion cells or approximately 2 billion or 4 billion cells per dose. In some embodiments, if administered, NK cells are administered on days 6 and 13 in units of 4 billion or approximately 4 billion cells. In some embodiments, if administered, NK cells are administered on days 9, 16, and 20 in units of 2 billion or approximately 2 billion cells. In some embodiments, NK cells are administered on days 6, 13, and 20 in the form of 5 × 10^8, 1 × 10^9, or 4 × 10^9 NK cells, or approximately 5 × 10^8, 1 × 10^9, or 4 × 10^9 NK cells. In some embodiments, antibodies are administered on days 2 and 13 of the treatment cycle. In some embodiments, NK cell administration is performed weekly, and antibody administration is performed every other week.

[0018] In some embodiments, the NK cells are not genetically modified.

[0019] 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, less than 1% of the NK cells are CD3+, less than 1% of the NK cells are CD19+, and less than 1% of the NK cells are CD14+. In some embodiments, each dose of NK cells is between 1 × 10^9 and 5 × 10^9 NK cells. In some embodiments, each dose of NK cells is between 1 × 10^9 and 5 × 10^9 NK cells.

[0020] In some embodiments, the patient receives a dose of a CD20-targeted antibody before the first administration of NK cells.

[0021] In some embodiments, the enlarged natural killer cells are enlarged umbilical cord blood natural killer cells.

[0022] In some embodiments, the expanded natural killer cell population includes at least 60%, for example, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% CD16+ cells. In some embodiments, the expanded natural killer cell population includes at least 60%, for example, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% NKG2D+ cells. In some embodiments, the expanded natural killer cell population includes at least 60%, for example, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% NKp46+ cells. In some embodiments, the expanded natural killer cell population includes at least 60%, for example, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% NKp30+ cells. In some embodiments, the expanded natural killer cell population includes 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 natural killer cell population includes 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 natural killer cell population includes 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 natural killer cell population includes less than 20% or less, 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 includes less than 20% or less CD19+ cells, for example, less than 10%, less than 5%, less than 1%, less than 0.5%, or 0%. In some embodiments, the expanded natural killer cell population includes less than 20% or less CD38+ cells, for example, less than 10%, less than 5%, less than 1%, less than 0.5%, or 0%.

[0023] In some embodiments, natural killer cells do not contain the CD16 transgene. In some embodiments, natural killer cells do not express the exogenous CD16 protein. In some embodiments, enlarged natural killer cells are not genetically modified. In some embodiments, enlarged natural killer cells are derived from the same umbilical cord blood donor.

[0024] In some embodiments, the population of NK cells includes at least 100 million enlarged natural killer cells, for example, 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 enlarged natural killer cells.

[0025] In some embodiments, a population of NK cells is produced by a method that includes the steps of (a) obtaining seed cells containing 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 group of Hut78 cells engineered to express membrane-bound IL-21, mutated TNFα, and 4-1BBL genes, thereby producing an expanded population of natural killer cells.

[0026] In some embodiments, a population of NK cells is produced by a method for producing a population of expanded natural killer cells, comprising the steps of (a) obtaining seed cells containing natural killer cells from umbilical cord blood; (b) depleting seed cells of CD3+ cells; (c) expanding the natural killer cells by culturing the depleted seed cells with a first group of Hut78 cells engineered to express membrane-bound IL-21, mutant TNFα, and 4-1BBL genes to produce an expanded master cell bank population of natural killer cells; and (d) expanding the expanded master cell bank population of natural killer cells by culturing a second group of Hut78 cells engineered to express membrane-bound IL-21, mutant TNFα, and 4-1BBL genes to produce expanded natural killer cells.

[0027] In some embodiments, the population of NK cells is produced by a method that, after step (c), further comprises the steps of (i) freezing a master cell bank population of expanded natural killer cells in a plurality of containers; and (ii) thawing a container containing aliquots of the master cell bank population of expanded natural killer cells, wherein the step of expanding the master cell bank population of expanded natural killer cells in step (d) includes the step of expanding aliquots of the master cell bank population of expanded natural killer cells.

[0028] In some embodiments, the umbilical cord blood is derived from a donor who has the KIR-B haplotype and is homozygous for the CD16 158V polymorphism.

[0029] In some embodiments, a population of NK cells is generated by a method that includes the step of expanding natural killer cells from umbilical cord blood by at least 10,000 times, for example, 15,000 times, 20,000 times, 25,000 times, 30,000 times, 35,000 times, 40,000 times, 45,000 times, 50,000 times, 55,000 times, 60,000 times, 65,000 times, or 70,000 times.

[0030] In some embodiments, the enlarged population of natural killer cells is neither enriched nor sorted after enlargement.

[0031] In some embodiments, the percentage of NK cells expressing CD16 in the expanded natural killer cell population is the same as or higher than the percentage of natural killer cells in umbilical cord blood-derived seed cells. In some embodiments, the percentage of NK cells expressing NKG2D in the expanded natural killer cell population is the same as or higher than the percentage of natural killer cells in umbilical cord blood-derived seed cells. In some embodiments, the percentage of NK cells expressing NKp30 in the expanded natural killer cell population is the same as or higher than the percentage of natural killer cells in umbilical cord blood-derived seed cells. In some embodiments, the percentage of NK cells expressing NKp44 in the expanded natural killer cell population is the same as or higher than the percentage of natural killer cells in umbilical cord blood-derived seed cells. In some embodiments, the percentage of NK cells expressing NKp46 in the expanded natural killer cell population is the same as or higher than the percentage of natural killer cells in umbilical cord blood-derived seed cells. 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 umbilical cord blood-derived seed cells.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this invention pertains. 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 and not intended to limit. All publications, patent applications, patents, sequences, database entries and other references referenced herein are incorporated herein by reference in their entirety. In case of any conflict, including definitions, this specification shall prevail.

[0033] Other features and advantages of the present invention will become apparent from the following detailed description and figures, as well as from the claims.

[0034] Built-in by reference All publications, patents, and patent applications referenced herein are incorporated herein by reference to the same extent as each individual publication, patent, or patent application is incorporated by reference specifically and individually.

[0035] Novel features of the present invention are described in detail in the appended claims. This patent or application file includes at least one drawing produced in color. A copy of this patent or publication of the patent application, including the color drawing, will be provided by the authorities upon request and payment of the necessary fees. The features and merits of the present invention will be better understood by referring to the following detailed description and appended drawings that describe exemplary embodiments in which the principles of the present invention are utilized. [Brief explanation of the drawing]

[0036] [Figure 1] This series of plots shows that AB-101, combined with an anti-CD20 antibody, mediates the death of B cells in healthy donors in vitro. [Figure 2]This graph shows that AB-101, combined with an anti-CD19 or anti-CD20 antibody, mediates the death of B cells in healthy donors in vitro. [Figure 3] This figure shows a representative set of FACS plots for apoptosis in SLE B cells. [Figure 4] This figure shows the specific AB-101-mediated lysis of SLE B cells in combination with anti-CD19 or anti-CD20 mAb. The four bars in four sets for each antibody represent the percentage of caspase-positive B cells for each AB101:PBMC ratio (left to right: 0, 0.2, 1, and 2) and each antibody level (left to right: no antibody, 0.01 μg / mL, 0.1 μg / mL, and 1 μg / mL antibody). [Figure 5] This is a series of plots showing that AB-101, combined with an anti-CD20 antibody, results in minimal T cell death. [Figure 6] This series of plots shows that AB-101, combined with an anti-CD20 antibody, mediates the death of SLE donor B cells in a 4-hour cytotoxicity assay. [Figure 7] This figure shows the enhanced SLE B cell death observed with the combination of rituximab and AB-101. PBMCs from SLE patients were isolated from peripheral blood and mixed with thawed AB-101 for 4 hours, either with anti-CD20 antibody and rituximab or without. The percentage of caspase-positive B cells was determined by flow cytometry. Data are expressed as mean + / - SD of two wells. Representative data from one SLE patient sample is shown. Four sets of four bars represent the percentage of caspase-positive B cells for each AB101:PBMC ratio (left to right: 0, 0.2, 1, and 2) and each antibody level (left to right: no antibody, 0.01 μg / mL, 0.1 μg / mL, and 1 μg / mL antibody). [Figure 8] This figure shows an example of a treatment regimen. DLT = Dose-limiting toxicity; FU = Follow-up; EOT = End of treatment; Flu = Fludarabine; Ritux = Rituximab; Obi = Obinutuzumab; CRR = Complete renal response; Cyclo = Cyclophosphamide. [Figure 9]This figure shows the enhanced SLE B cell death observed with the combination of obinutuzumab and AB-101. PBMCs from SLE patients were isolated from peripheral blood and mixed with thawed AB-101, either with or without anti-CD20 antibody and obinutuzumab, for 4 hours. The percentage of caspase-positive B cells was determined by flow cytometry. Data are expressed as mean + / - SD of two wells. Representative data from one SLE patient sample is shown. The four bars in four sets for each antibody represent the percentage of caspase-positive B cells for each AB101:PBMC ratio (left to right: 0, 0.2, 1, and 2) and each antibody level (left to right: no antibody, 0.01 μg / mL, 0.1 μg / mL, and 1 μg / mL antibody). [Figure 10] This is a schematic diagram of administration to a humanized NSG model. Obin = obinutuzumab. [Figure 11] This figure shows the in vivo activity of AB-101 and obinutuzumab in a humanized NSG model. HuNSG mice were treated as follows: vehicle (PBS IP, frozen medium IV), obinutuzumab (150 μg / kg), AB-101 (1 × 10⁷ cells), or AB-101 + obinutuzumab on day 0 or days 0 and 7 of the study. Blood was collected from mice at baseline (i.e., before the first treatment) and at specified time points (A). Kinetics of peripheral blood B cell count by flow cytometry analysis are shown as the percentage change in human CD19+ B cells from baseline ± SEM (cells / μl) (B). B cells were gated for CD19 rather than CD20 due to potential interference with the CD20 detection reagent in flow cytometry. For the specified groups, data for day 7 are after a single dose of Obin, AB-101, or obin+AB-101, and data for day 14 are after two doses of Obin, AB-101, or obin+AB-101. Obin = obinutuzumab. The six sets of six bars show the percentage (cells / μL) relative to baseline for each day after treatment (left to right: 7, 14, 21, and 28) and for each treatment (left to right: vehicle, Obin (D0), Obin (D0, D7), AB-101 (D0, D7), Obin+AB-101 (D0), Obin+AB-101 (D0, D7)). [Figure 12] This figure shows weight change in the humanized NSG model. Mean (±SD) weight change is shown for all groups, and there are no significant differences in percentage weight change between the vehicle, Obin, AB-101, or Obin+AB-101 combination treatment groups. SD, standard deviation. [Figure 13] This figure shows the pharmacokinetic profile of AB-101. The distribution of AB-101 in several tissues of NSG mice was determined by calculating the amount of AB-101 DNA per μg of mouse blood / tissue DNA. The data are shown as the mean concentration (±sem) of AB-101 DNA in each organ and represent six mice (3 males, 3 females) per time point. [Figure 14] This figure shows the characterization of B cells from SLE and healthy donors. Using the gating shown in the top row, B cell subsets from SLE and healthy donors were evaluated by flow cytometry. Data are shown mean + SEM. *p=<0.05. [Figure 15] This figure shows the characterization of NK cells from SLE and healthy donors. Flow cytometry characterized NK cell subsets and surface markers from SLE and healthy donor-derived NK cells. Data are shown mean + SEM. *p<0.05, **p<0.005. [Modes for carrying out the invention]

[0037] In particular, natural killer (NK) cells, such as enlarged and stimulated NK cells, methods for generating NK cells, pharmaceutical compositions comprising NK cells, and methods for treating patients suffering from autoimmune disorders with NK cells are provided herein.

[0038] I. Enlargement and stimulation of natural killer cells In some cases, NK cells are enlarged and stimulated, for example, as described in International Publication No. 2022216813, the entire contents of which are incorporated herein by reference.

[0039] In some cases, for example, after being expanded and stimulated ex vivo as described herein, the expanded and stimulated NK cell population not only has a number / density that cannot naturally exist in the human body (e.g., as described above), but also differs in phenotypic characteristics (e.g., gene expression and / or surface protein expression) from the starting material or other naturally occurring NK cell populations.

[0040] In some cases, the starting NK cell source is a sample derived from a single individual, e.g., a single umbilical cord blood unit that has not been expanded ex vivo. Thus, in some cases, expanded and stimulated NK cells share a common lineage, i.e., they all arise from the expansion of the starting NK cell source and therefore share a genotype mediated by the clonal proliferation of a population of cells derived from a single organism. Furthermore, they may not exist naturally at the densities achieved by the expansion ex vivo and may also differ from the starting NK cell source in phenotypic characteristics.

[0041] In some cases, a population of enlarged and stimulated NK cells includes at least 100 million enlarged 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 enlarged natural killer cells.

[0042] In some embodiments, the enlarged and stimulated NK cells include at least 80%, for example, at least 90%, at least 95%, at least 99%, or 100% CD56+CD3- cells.

[0043] In some embodiments, the enlarged and stimulated NK cells do not contain the CD16 transgene.

[0044] In some embodiments, enlarged and stimulated NK cells do not express exogenous CD16 protein.

[0045] Enlarged and stimulated NK cells may be characterized by surface expression of one or more of the following, for example: CD16, CD56, CD3, CD38, CD14, CD19, NKG2D, NKp46, NKp30, DNAM-1, and NKp44.

[0046] The expression levels of surface proteins described herein are, in some cases, achieved without positive selection for specific reference surface proteins. For example, in some cases, an NK cell source, e.g., a single umbilical cord unit, contains both the KIR B allele of the KIR receptor family and the 158V / V variant of CD16, and gating for CD56+CD3- expression enriches + and depletes CD3(+), but no selection of other surface protein expression occurs during expansion and stimulation.

[0047] In some embodiments, for example as described above, the enlarged and stimulated NK cells derived from the enlargement and stimulation of a single umbilical cord blood unit include at least 60%, for example, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% NKG2D+ cells.

[0048] In some embodiments, for example as described above, the enlarged and stimulated NK cells derived from the enlargement and stimulation of a single umbilical cord blood unit include at least 60%, for example, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% NKp46+ cells.

[0049] In some embodiments, for example as described above, the enlarged and stimulated NK cells derived from the enlargement and stimulation of a single umbilical cord blood unit include at least 60%, for example, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% NKp30+ cells.

[0050] In some embodiments, for example as described above, the enlarged and stimulated NK cells derived from, for example, a single umbilical cord blood unit, contain at least 60%, for example, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% DNAM-1+ cells.

[0051] In some embodiments, for example as described above, the enlarged and stimulated NK cells derived from the enlargement and stimulation of a single umbilical cord blood unit include at least 60%, for example, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% NKp44+ cells.

[0052] In some embodiments, for example as described above, the enlarged and stimulated NK cells derived from, for example, a single umbilical cord blood unit, contain at least 60%, for example, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% CD94+(KLRD1) cells.

[0053] In some embodiments, for example as described above, the enlarged and stimulated NK cells derived from the enlargement and stimulation of a single umbilical cord blood unit contain 20% or less, for example, 10% or less, 5% or less, 1% or less, or 0% CD3+ cells.

[0054] In some embodiments, for example as described above, the enlarged and stimulated NK cells derived from the enlargement and stimulation of a single umbilical cord blood unit contain 20% or less, for example, 10% or less, 5% or less, 1% or less, or 0% CD14+ cells.

[0055] In some embodiments, for example as described above, the enlarged and stimulated NK cells derived from the enlargement and stimulation of a single umbilical cord blood unit contain 20% or less, for example, 10% or less, 5% or less, 1% or less, or 0% CD19+ cells.

[0056] In some embodiments, for example as described above, the enlarged and stimulated NK cells derived from the enlargement and stimulation of a single umbilical cord blood unit contain 20% or less, for example, 10% or less, 5% or less, 1% or less, or 0% CXCR+ cells.

[0057] In some embodiments, for example as described above, the enlarged and stimulated NK cells derived from the enlargement and stimulation of a single umbilical cord blood unit contain 20% or less, for example, 10% or less, 5% or less, 1% or less, or 0% CD122+(IL2RB) cells.

[0058] In some embodiments, for example as described above, the enlarged and stimulated NK cells derived from the enlargement and stimulation of a single umbilical cord blood unit contain 90% or more, for example, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% CD3-CD14-CD19-CD16+CD56- cells.

[0059] As described herein, the inventors have surprisingly demonstrated that NK cells enlarged and stimulated by the method herein express CD16 at high levels throughout the entire enlargement and stimulation process, resulting in a cell population that is highly CD16-expressing. The high expression of CD16 eliminates the need to manipulate the enlarged cells to express CD16, which is important for inducing ADCC and is therefore a surprising and unexpected benefit of the enlargement and stimulation method described herein. Accordingly, in some embodiments, for example as described above, the enlarged and stimulated NK cells derived from, for example, a single umbilical cord blood unit contain more than 50%, for example, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% CD16+ NK cells.

[0060] In some embodiments, for example as described above, the enlarged and stimulated NK cells derived from the enlargement and stimulation of a single umbilical cord blood unit contain both the KIR B allele of the KIR receptor family and the 158V / V variant of CD16, and contain more than 50%, for example, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% CD16+ NK cells.

[0061] In some embodiments, for example as described above, the percentage of CD16-expressing expanded and stimulated NK cells derived from a single umbilical cord blood unit is equal to or higher than the percentage of natural killer cells in umbilical cord blood-derived seed cells.

[0062] In some embodiments, as described above, for example, the percentage of expanded and stimulated NK cells expressing NKG2D, derived from expanded and stimulated single umbilical cord blood units, is equal to or higher than the percentage of natural killer cells in umbilical cord blood-derived seed cells.

[0063] In some embodiments, for example as described above, the percentage of expanded and stimulated NK cells expressing NKp30, derived from a single umbilical cord blood unit, is equal to or higher than the percentage of natural killer cells in umbilical cord blood-derived seed cells.

[0064] In some embodiments, for example as described above, the percentage of expanded and stimulated NK cells expressing DNAM-1, derived from expanded and stimulated single umbilical cord blood units, is equal to or higher than the percentage of natural killer cells in umbilical cord blood-derived seed cells.

[0065] In some embodiments, for example as described above, the percentage of expanded and stimulated NK cells expressing NKp44, derived from expanded and stimulated single umbilical cord blood units, is equal to or higher than the percentage of natural killer cells in umbilical cord blood-derived seed cells.

[0066] In some embodiments, for example as described above, the percentage of expanded and stimulated NK cells expressing NKp46, derived from a single umbilical cord blood unit, is equal to or higher than the percentage of natural killer cells in umbilical cord blood-derived seed cells.

[0067] As described herein, the inventors have also demonstrated, to their surprise, that NK cells enlarged and stimulated by the method described herein also express CD38 at low levels. CD38 is an effective target for 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 Cancers,” Expert Opinion on Investigational Drugs 29(11):1295-1308 (2020).

[0068] Therefore, in some embodiments, for example as described above, the enlarged and stimulated NK cells derived from the enlargement and stimulation of a single umbilical cord blood unit contain 80% or less CD38+ cells, for example, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or 20% or less CD38+ cells.

[0069] In some embodiments, for example as described above, the enlarged and stimulated NK cells derived from the enlargement and stimulation of a single umbilical cord blood unit contain both the KIR B allele of the KIR receptor family and the 158V / V variant of CD16, and contain less than 80% CD38+ cells, e.g., 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or less CD38+ cells.

[0070] In some embodiments, for example as described above, the enlarged and stimulated NK cells derived from the enlargement and stimulation of a single umbilical cord blood unit include both the KIR B allele of the KIR receptor family and the 158V / V variant of CD16, and include less than 80% CD38+ cells, e.g., 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or 20% CD38+ cells and more than 50% CD16+ NK cells, e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% CD16+ NK cells.

[0071] In some embodiments, for example as described above, the enlarged and stimulated NK cells derived from the enlargement and stimulation of a single umbilical cord blood unit include both the KIR B allele of the KIR receptor family and the 158V / V variant of CD16, and i) more than 50%, e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of CD16+ NK cells; and / or ii) less than 80% of CD38+ cells, e.g., 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or 20% or less of CD38+ cells; and / or iii) 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 6 0%, 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 viiii) 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) less than 20%, e.g., less than 10%, less than 5%, less than 1%, or 0% CD3+ cells; and / or x) less than 20%, e.g., less than 10%, less than 5%, less than 1%, or 0% CD14+ cells; and / or xi) less than 20%, e.g., less than 10%, less than 5%, less than 1%, or 0% CD19+ cells; and / or xii) less than 20%, e.g., less than 10%, less than 5%, less than 1%, or 0% CXCR+ cells;and / or xiii) containing 20% ​​or less, e.g., 10% or less, 5% or less, 1% or less, or 0% CD122+(IL2RB) cells;

[0072] In some embodiments, NK cells are manipulated to alter, for example, reduce, the expression of one or more inhibitory receptor genes.

[0073] In some embodiments, the inhibitory receptor gene is an HLA-specific inhibitory receptor. In some embodiments, the inhibitory receptor gene is a non-HLA-specific inhibitory receptor.

[0074] 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.

[0075] Pharmaceutical compositions containing natural killer cells as described herein, and dosing units of the pharmaceutical compositions as described herein, are also provided herein.

[0076] In some cases, a drug dose unit contains between 100 million and 1.5 billion cells, for example, 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.

[0077] Pharmaceutical compositions typically include a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes salines, solvents, dispersions, coatings, antimicrobial and antifungal agents, isotonic and absorption retardants, etc., that are suitable for pharmaceutical administration.

[0078] In some embodiments, the pharmaceutical composition comprises a) natural killer cells as described herein; and b) a cryopreservation composition. Suitable cryopreservation compositions are described herein.

[0079] In some embodiments, the composition is frozen. In some embodiments, the composition is frozen for at least 3 months, for example, 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.

[0080] In some embodiments, after thawing, at least 60% of the natural killer cells, for example, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% are viable.

[0081] In some embodiments, the pharmaceutical composition comprises: a) the cryopreserved composition described herein; and b) therapeutic cells, for example, the engineered NK cells described herein.

[0082] In some embodiments, the pharmaceutical composition further comprises c) a buffer solution. Suitable buffer solutions are described herein, for example, similar to the cryopreserved composition.

[0083] In some embodiments, the pharmaceutical composition comprises 1×10 7 or about 1×10 7 ~1×10 9 or about 1×10 9 cells / mL. In some embodiments, the pharmaceutical composition comprises 1×10 8 cells / mL. In some embodiments, the pharmaceutical composition comprises about 1×10 8 cells / mL.

[0084] In some embodiments, the pharmaceutical composition comprises 1×10 8 or about 1×10 8 ~1×10 10 or about 1×10 10 cells / mL.

[0085] In some embodiments, the pharmaceutical composition further comprises an antibody or an antigen-binding fragment thereof, for example, the antibodies described herein.

[0086] Pharmaceutical compositions are typically formulated to suit the intended route of administration. Examples of routes of administration include parenteral administration, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.

[0087] Methods for formulating appropriate 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, a solution or suspension used for parenteral, intradermal, or subcutaneous application may contain the following components: sterile diluents, e.g., water for injection, saline, non-volatile oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antimicrobial agents, e.g., benzyl alcohol or methylparaben; antioxidants, e.g., ascorbic acid or sodium bisulfite; chelating agents, e.g., ethylenediaminetetraacetic acid; buffers, e.g., acetic acid, citric acid, or phosphoric acid; and agents for adjusting osmotic pressure, e.g., sodium chloride or dextrose. The pH can be adjusted with an acid or base, e.g., hydrochloric acid or sodium hydroxide. Parenteral preparations can be sealed in glass or plastic ampoules, disposable syringes, or multi-dose vials.

[0088] Pharmaceutical compositions suitable for injection may include sterile aqueous solutions (if water-soluble) or dispersions and sterile powders for the immediate preparation of sterile, injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL® (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and fluid enough to be easily injected. It must be stable under manufacturing and storage conditions and protected from microbial contamination, such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol) and suitable mixtures thereof. Adequate fluidity can be maintained, for example, by the use of coating agents such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial activity can be achieved by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferable to include isotonic agents in the composition, such as sugars, polyalcohols, such as mannitol, sorbitol, and sodium chloride. Sustained absorption of the injectable composition can be achieved by including absorption-delaying agents, such as aluminum monostearate and gelatin, in the composition.

[0089] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound, along with one or a combination of the components listed above, into a suitable solvent, and subsequently sterilizing by filtration, if necessary. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other required components from those listed above. For sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying, which yield a powder of the active component + any further desired components from a previously sterile filtered solution.

[0090] Examples of suitable pharmaceutical compositions are described, for example, in International Publication No. 2017 / 135631 and International Publication No. 2022 / 0133061, each of which is incorporated herein by reference in its entirety.

[0091] II. Antibodies Methods described herein include the step of administering an antibody, for example, an antibody that targets immune cells, for example, immune cells involved in autoimmune reactions, for example, B cells. In some cases, the antibody is a B cell depletion antibody, for example, a B cell depletion monoclonal antibody (mAb). In some cases, the antibody is an antibody that targets CD20 and / or CD19. In some cases, methods described herein include the step of administering multiple antibodies, for example, multiple antibodies that target immune cells, for example, immune cells involved in autoimmune reactions, for example, B cells. In some cases, one or more of the antibodies are B cell depletion antibodies, for example, B cell depletion monoclonal antibodies (mAb). In some cases, the antibodies are selected from antibodies that target CD20 and / or CD19, for example, as described herein.

[0092] The term "antibody" refers to an immunoglobulin molecule or its immunologically active portion, i.e., the antigen-binding portion.

[0093] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, such that, for example, the individual antibodies constituting the population are identical except for any naturally occurring mutations that may be present in small amounts. The antibody may be monoclonal. The antibody may be a human antibody or a humanized antibody. The term “monoclonal antibody” encompasses intact monoclonal antibodies and full-length monoclonal antibodies, as well as antibody fragments (e.g., Fab, Fab', F(ab')2, Fv), single-chain antibodies (e.g., scFv), fusion proteins containing antibody fragments, and any other modified immunoglobulin molecules containing at least one antigen-binding site. Furthermore, “monoclonal antibody” refers to antibodies produced by several techniques, including, but not limited to, hybridoma generation, phage library display, recombinant expression, and transgenic animals.

[0094] The term "chimeric antibody" refers to an antibody in which part of the heavy chain and / or light chain originates from a first source or species, but the remainder of the heavy chain and / or light chain originates from a different source or species.

[0095] As used herein, the term “humanized antibody” refers to an antibody comprising human heavy chain variable regions and light chain variable regions, in which native CDR residues are replaced with corresponding CDR residues derived from a non-human antibody (e.g., mouse, rat, rabbit, or non-human primate), and the non-human antibody possesses desired specificity, affinity, and / or activity. In some embodiments, one or more framework region residues of the human heavy chain or light chain variable region are replaced with corresponding residues of the non-human antibody. Furthermore, the humanized antibody may contain residues not found in either the human or non-human antibody. In some embodiments, these modifications are made to further refine and / or optimize the antibody's properties. In some embodiments, the humanized antibody includes at least a portion of the immunoglobulin constant region (e.g., CH1, CH2, CH3, Fc), typically those of human immunoglobulin.

[0096] As used herein, the term “human antibody” means an antibody produced by a human being, and / or an antibody having an amino acid sequence corresponding to an antibody produced using any technique known to those skilled in the art for the production of a human antibody. These techniques include, but are not limited to, phage display libraries, yeast display libraries, transgenic animals, recombinant protein synthesis, and B-cell hybridoma techniques.

[0097] An "antibody fragment" may include a portion of an intact antibody, preferably the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and polyspecific antibodies formed from antibody fragments.

[0098] The terms “epitope” and “antigen determinant” are used interchangeably herein and refer to a portion of an antigen or target that a particular antibody can recognize and bind to. When the antigen or target is a polypeptide, epitopes can be formed from both continuous amino acids and discontinuous amino acids juxtaposed by the tertiary folding of the protein. Epitopes formed from continuous amino acids (also called linear epitopes) are typically retained during protein denaturation, while epitopes formed by tertiary folding (also called conformational epitopes) are typically lost during protein denaturation. Epitopes typically contain at least three, more commonly, at least five, six, seven, or eight to ten amino acids in a unique spatial conformation. Epitopes can be predicted using one of the numerous software bioinformatics tools available on the internet. Epitopes on target proteins can be characterized by analyzing the interactions of amino acid residues in the antigen / antibody complex using X-ray crystallography.

[0099] "Fv" contains a minimal antibody fragment that includes a complete antigen recognition and binding site. This region consists of a dimer of one heavy-chain variable domain and one light-chain variable domain, bound by a robust non-covalent bond. In this configuration, three CDRs of each variable domain interact to define the antigen-binding site on the surface of the VH-VL dimer. Collectively, six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of Fv containing only three antigen-specific CDRs) has the ability to recognize and bind to the antigen, albeit with lower affinity than the entire binding site. The Fab fragment also includes a constant domain of the light chain and a first constant domain (CH1) of the heavy chain. The Fab fragment differs from the Fab' fragment by the addition of several residues at the carboxyl terminus of the heavy-chain CH1 domain, including one or more cysteines derived from the antibody hinge region. Fab'-SH is the herein designation for Fab', where the cysteine ​​residue of the constant domain has a free thiol group. The F(ab')2 antibody fragment was originally generated as a pair with the Fab' fragment, which has a hinged cysteine ​​in between. Other chemical couplings of antibody fragments are also known.

[0100] Depending on the amino acid sequence of the constant domain of the heavy chain, immunoglobulins can be assigned to different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgB1, IgG2, IgG3, IgG4, IgA, and IgA2. A "single-chain Fv" or "sFv" antibody fragment contains the VH and VL domains of the antibody, and these domains are present in a single polypeptide chain. In some cases, the Fv polypeptide further contains a polypeptide linker between the VH and VL domains, which allows the sFv to form a structure desirable for antigen binding.

[0101] In various embodiments, the antibody or its antigen-binding fragment includes a human antibody or a humanized antibody. The humanized form of a non-human (e.g., mouse) antibody is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (e.g., Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of the antibody) containing a minimal sequence derived from a non-human immunoglobulin. The humanized antibody contains a human immunoglobulin (recipient antibody), in which residues in the recipient's complementarity-determining region (CDR) are replaced with CDR residues from a non-human species (donor antibody), such as mouse, rat, or rabbit, having the desired specificity, affinity, and ability. In some cases, Fv framework residues of the human immunoglobulin are replaced with corresponding non-human residues. The humanized antibody may also contain residues not found in the recipient antibody or in the transferred CDR or framework sequence. Generally, humanized antibodies contain substantially all of at least one, typically two, variable domains, with all or substantially all of the CDR region corresponding to that of a non-human immunoglobulin and all or substantially all of the FR region being that of a human immunoglobulin consensus sequence. Methods for humanizing non-human antibodies are well known in the art.

[0102] An antibody that "bounds to," "specifically binds to," or "is specific to" a particular polypeptide or epitope on a particular polypeptide is one that binds to that particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope. As used herein, the term "specifically binds" refers to a conjugate (e.g., an antibody) that interacts with a particular antigen, epitope, protein, or target molecule more frequently, more rapidly, for a longer duration, with greater affinity, or in any combination of the above, than alternatives. A conjugate (e.g., an antibody) that specifically binds to an antigen can be identified, for example, by immunoassays, ELISAs, surface plasmon resonance (SPR) assays (e.g., Biacore) or other techniques known to those skilled in the art. A conjugate that specifically binds to an antigen binds to the target antigen with a higher affinity than its affinity to different antigens. The different antigens may be related antigens. In some embodiments, a binder that specifically binds to an antigen binds to the target antigen with an affinity at least 20 times greater than its affinity to a different antigen, for example, at least 30 times, at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, or at least 100 times greater than its affinity to a different antigen. In some embodiments, a binder that specifically binds to a particular antigen binds to a different antigen with such a low affinity that binding cannot be detected using the assay described herein or an assay known in the art. In some embodiments, affinity is measured using SPR technique, for example, in a Biacore system or another system known to those skilled in the art.

[0103] In some embodiments, the antibody or its antigen-binding fragment is an antibody, for example, a full-length antibody containing an Fc domain with at least one heavy chain. In some embodiments, the antibody is a recombinant antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is an IgA, IgD, IgE, IgG, or IgM antibody. In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is an IgG2 antibody. In some embodiments, the antibody is an IgG3 antibody. In some embodiments, the antibody is an IgG4 antibody.

[0104] In some embodiments, the antibody is an antibody fragment containing an antigen-binding site. In some embodiments, the antibody is an scFv. In some embodiments, the antibody is a disulfide-bonded scFv. In some embodiments, the antibody is a bispecific or multispecific antibody. In some embodiments, the antibody is a monovalent antibody. In some embodiments, the antibody is a single-specific antibody. In some embodiments, the antibody is a bivalent antibody. In some embodiments, the antibody is isolated. In some embodiments, the antibody is substantially pure. In some embodiments, the binder is a polyclonal antibody. Polyclonal antibodies can be prepared by any method known to those skilled in the art.

[0105] In some embodiments, the binder is a monoclonal antibody. Monoclonal antibodies can be prepared by any method known to those skilled in the art. In some embodiments, the binder is a humanized antibody. Various methods for generating humanized antibodies are known in the art. In some embodiments, the binder is a human antibody. Human antibodies can be prepared using various techniques known in the art.

[0106] In some embodiments, the binder is an scFv antibody, Fv, Fab, F(ab')2, F(ab'), or a bispecific antibody.

[0107] In some embodiments, bispecific antibodies exhibit reduced toxicity and / or side effects. In some embodiments, bispecific antibodies exhibit reduced toxicity and / or side effects compared to a mixture of two individual antibodies or an antibody as a single agent. In some embodiments, bispecific antibodies exhibit an increased therapeutic index. In some embodiments, bispecific antibodies exhibit an increased therapeutic index compared to a mixture of two individual antibodies or an antibody as a single agent. Several techniques for producing bispecific antibodies are known to those skilled in the art. In some embodiments, bispecific antibodies include a heavy chain constant region with modifications to amino acids that are part of the interface between two heavy chains. These modifications are made to enhance heterodimerization and generally to reduce or eliminate homodimerization. In some embodiments, bispecific antibodies are produced using a knob-in-to-hole (KIH) strategy. In some embodiments, bispecific antibodies include a variant hinge region that prevents the formation of disulfide bonds between identical heavy chains (e.g., to reduce homodimerization). In some embodiments, bispecific antibodies include heavy chains with amino acid changes that alter electrostatic interactions. In some embodiments, the bispecific antibody contains a heavy chain having amino acid changes that alter the hydrophobic / hydrophilic interaction. The bispecific antibody may be an intact antibody or an antibody fragment containing an antigen-binding site.

[0108] Binding agents with a binding titer greater than 2 are also being considered. In some embodiments, triplicate or quadruplicate antibodies are generated.

[0109] In some cases, the antibody or its antigen-binding fragment is an IgG, IgA, or IgE antibody or its antigen-binding fragment. In some cases, the antibody or its antigen-binding fragment is an IgG antibody or its antigen-binding fragment. In some cases, the antibody or its antigen-binding fragment is an IgG1, IgG3, or IgG4 antibody or its antigen-binding fragment. In some cases, the antibody or its antigen-binding fragment is an IgG1 antibody or its antigen-binding fragment.

[0110] In some cases, the antibody or its antigen-binding fragment is an antibody or combination of antibodies selected from Table 1, Table 2, or Table 3. In some cases, the antibody or its antigen-binding fragment is an IgG antibody selected from Table 1, Table 2, or Table 3. In some cases, the antibody or its antigen-binding fragment is an IgG1 antibody selected from Table 1, Table 2, or Table 3. In some cases, the patient suffers from a disorder for which the antibody or its antigen-binding fragment is approved as a therapeutic agent (e.g., by a regulatory authority such as the U.S. Food and Drug Administration or the European Medicines Agency), as reflected in Table 1, Table 2, or Table 3.

[0111] In some cases, an antibody or its antigen-binding fragment is modified to enhance the binding of its Fc domain to an activating receptor (e.g., an Fcγ receptor) compared to an unmodified antibody.

[0112] [Table 1]

[0113] [Table 2]

[0114] [Table 3-1]

[0115] [Table 3-2]

[0116] [Table 3-3]

[0117] In some cases, the antibody or its antigen-binding fragment is an NK cell conjugate, such as a bispecific or tripspecific antibody, that cross-links an NK cell activating receptor (e.g., CD16A, NKG2D, NKp30, or NKp46) with a molecule specific to disease cells (e.g., tumor cells). See, for example, Demaria et al., “Natural Killer Cell Engagers in Cancer Immunotherapy: Next Generation of Immuno-Oncology Treatments,” European Journal of Immunology 51(8):doi.org / 10.1002 / eji.202048953 (2021).

[0118] Mai. Pharmaceutical composition A pharmaceutical composition containing natural killer cells as described herein and a dosing unit of the pharmaceutical composition as described herein are provided herein.

[0119] In some cases, a drug dose unit may contain between 100 million and 1.5 billion cells, for example, 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 or around that number.

[0120] In some cases, a drug dose unit contains between 100 million and 10 billion cells, for example, 100 million, 200 million, 300 million, 400 million, 500 million, 600 million, 700 million, 800 million, 900 million, 1 billion, 1.5 billion, 2 billion, 2.5 billion, 3 billion, 3.5 billion, 4 billion, 4.5 billion, 5 billion, 5.5 billion, 6 billion, 6.5 billion, 7 billion, 7.5 billion, 8 billion, 8.5 billion, 9 billion, 9.5 billion, 10 billion cells or around that number.

[0121] Pharmaceutical compositions typically include a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes salines, solvents, dispersions, coatings, antimicrobial and antifungal agents, isotonic and absorption retardants, etc., that are suitable for pharmaceutical administration.

[0122] In some embodiments, the pharmaceutical composition comprises a) natural killer cells as described herein; and b) a cryopreservation composition.

[0123] Suitable cryopreservation compositions are described herein.

[0124] In some embodiments, the composition is frozen. In some embodiments, the composition is frozen for at least three months, for example, at least six months, at least nine months, at least twelve months, at least fifteen months, at least eighteen months, at least twenty-four months, or at least thirty-six months.

[0125] In some embodiments, after thawing, at least 60%, for example, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% of natural killer cells remain viable.

[0126] In some embodiments, the pharmaceutical composition comprises a) the cryopreservation composition described herein; and b) therapeutic cells.

[0127] In some embodiments, the therapeutic cells are animal cells. In some embodiments, the therapeutic cells are human cells.

[0128] In some embodiments, the therapeutic cells are immune cells. In some embodiments, the immune cells are selected from basophils, eosinophils, neutrophils, mast cells, monocytes, macrophages, neutrophils, dendritic cells, natural killer cells, B cells, T cells, and combinations thereof.

[0129] In some embodiments, the immune cells are natural killer (NK) cells. In some embodiments, the natural killer cells are enlarged and stimulated by the methods described herein.

[0130] In some embodiments, the pharmaceutical composition further comprises c) a buffer solution. Suitable buffer solutions are described herein, for example, as are those for cryopreservation compositions.

[0131] In some embodiments, the pharmaceutical composition is 1 × 10 cells 7 Or approximately 1 x 10 7 ~1 × 10 9 Or approximately 1 x 10 9 Contains cells / mL. In some embodiments, the pharmaceutical composition contains 1 × 10 cells. 8 Contains cells / mL. In some embodiments, the pharmaceutical composition contains approximately 1 × 10 cells. 8 Contains 1 / mL.

[0132] In some embodiments, the pharmaceutical composition is 1 × 10 cells 8 Or approximately 1 x 10 8 ~1 × 10 10 Or approximately 1 x 10 10 Contains 1 / mL.

[0133] In some embodiments, the pharmaceutical composition further comprises an antibody or an antigen-binding fragment thereof, for example, the antibody described herein.

[0134] Pharmaceutical compositions are typically formulated to suit the intended route of administration. Examples of routes of administration include parenteral administration, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.

[0135] Methods for formulating appropriate 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, a solution or suspension used for parenteral, intradermal, or subcutaneous application may contain the following components: sterile diluents, e.g., water for injection, saline, non-volatile oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antimicrobial agents, e.g., benzyl alcohol or methylparaben; antioxidants, e.g., ascorbic acid or sodium bisulfite; chelating agents, e.g., ethylenediaminetetraacetic acid; buffers, e.g., acetic acid, citric acid, or phosphoric acid; and agents for adjusting osmotic pressure, e.g., sodium chloride or dextrose. The pH can be adjusted with an acid or base, e.g., hydrochloric acid or sodium hydroxide. Parenteral preparations can be sealed in glass or plastic ampoules, disposable syringes, or multi-dose vials.

[0136] Pharmaceutical compositions suitable for injection may include sterile aqueous solutions (if water-soluble) or dispersions and sterile powders for the immediate preparation of sterile, injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL® (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and fluid enough to be easily injected. It must be stable under manufacturing and storage conditions and protected from microbial contamination, such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol) and suitable mixtures thereof. Adequate fluidity can be maintained, for example, by the use of coating agents such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial activity can be achieved by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferable to include isotonic agents in the composition, such as sugars, polyalcohols, such as mannitol, sorbitol, and sodium chloride. Sustained absorption of the injectable composition can be achieved by including absorption-delaying agents, such as aluminum monostearate and gelatin, in the composition.

[0137] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound, along with one or a combination of the components listed above, into a suitable solvent, and subsequently sterilizing by filtration, if necessary. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other required components from those listed above. For sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying, which yield a powder of the active component + any further desired components from a previously sterile filtered solution.

[0138] IV. Treatment Methods The NK cells described herein are used to treat autoimmune disorders.

[0139] Accordingly, methods for treating patients suffering from disorders, such as autoimmune disorders, such as autoreactive immune cells, such as autoreactive B cells, are also provided herein, comprising the step of administering NK cells, such as NK cells as described herein, and CD19 and / or CD20 targeted antibodies, such as antibodies as described herein.

[0140] As used herein, the terms “treatment,” “to treat,” and “to treat” mean to reverse, alleviate, delay the onset, or inhibit the progression of an autoimmune disorder (e.g., the disorders described herein, e.g., systemic lupus erythematosus ("SLE")). In some embodiments, treatment may be administered after the onset of one or more symptoms. In other embodiments, treatment may be administered in the absence of symptoms. For example, treatment may be administered to highly susceptible individuals before the onset of symptoms (e.g., taking into account a medical history of the symptoms and / or genetic or other susceptibility factors). Treatment may also be continued after the symptoms have subsided, for example, to prevent or delay their relapse.

[0141] As used herein, “delaying” the onset of a disease or disorder or one or more of its symptoms means delaying, preventing, slowing, blocking, stabilizing, and / or postponing the onset of the disease, disorder or its symptoms. This delay can be of varying lengths depending on the disease and / or the medical history of the subject receiving treatment. As will be apparent to those skilled in the art, a sufficient or significant delay can effectively encompass prevention in that the subject does not develop the disease, disorder or its symptoms. For example, a method of “delaying” the onset of an autoimmune disorder is a method that reduces the probability of the disease occurring and / or the severity of the disease within a given time frame compared to not using the method. Such a comparison can be based on clinical studies using a statistically significant number of subjects.

[0142] As used herein, “prevention” or “preventing” refers to a regimen that prevents the onset of a disease or disorder so that the clinical symptoms of the disease do not occur. Therefore, “prevention” relates to the application of therapy (e.g., administration of a therapeutic substance) to a subject before signs of the disease can be detected in the subject, and / or before a particular stage of the disease. A subject may be an individual at risk of developing a disease or disorder, or at risk of disease progression, such as the development of proliferative lupus nephritis. For example, an individual with one or more risk factors known to be associated with the development or onset of a disease or disorder. For example, an individual may have a mutation associated with the development or progression of an autoimmune disease (e.g., SLE). Furthermore, it will be understood that prevention may not completely prevent the onset of a disease or disorder. In some cases, prevention includes reducing the risk of developing a disease or disorder. Reducing the risk may not completely eliminate the risk of developing a disease or disorder.

[0143] In some cases, the “increased” or “enhanced” amount refers to an increase of 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40 or 50 times or more (e.g., 100, 500, 1000 times) (including all integers and decimals in between and greater than 1, e.g., 2.1, 2.2, 2.3, 2.4, etc.). This may also include 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% of the amount or level described herein.

[0144] In some cases, the amount of “reduced,” “reduced,” or “less” means a reduction of about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40 or 50 times or more (e.g., 100, 500, 1000 times) (including all integers and decimals in between and greater than 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.). This may also include 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%, or at least 90%, at least 100%, at least 150%, at least 200%, at least 500%, or at least 1000% of the amount or level described herein.

[0145] A method for depleting immune cells, such as autoreactive immune cells, such as autoreactive B cells, is also provided herein, comprising the step of administering NK cells, such as NK cells as described herein, and CD19 and / or CD20-targeted antibodies, such as antibodies as described herein. In some cases, the immune cells are CD19+ immune cells, such as CD19+ autoreactive immune cells, such as CD19+ autoreactive B cells. In other cases, the immune cells are CD20+ immune cells, such as CD20+ autoreactive immune cells, such as CD20+ autoreactive B cells. In some cases, the immune cells are CD19+ / CD20-, CD19+ / CD20+, or CD19- / CD20+ immune cells, for example, CD19+ / CD20-, CD19+ / CD20+, or CD19- / CD20+ autoreactive immune cells, for example, CD19+ / CD20-, CD19+ / CD20+, or CD19- / CD20+ autoreactive B cells.

[0146] In some cases, immune cells (e.g., autoreactive immune cells) include or consist of B cells (e.g., autoreactive B cells). In other cases, B cells are CD19+ / CD20- B cells, CD19+ / CD20+ B cells, CD19- / CD20+ B cells, or a combination thereof.

[0147] In some cases, B cells are selected from anorectal B cells, appendiceal B cells, lymph node sinus B cells, lymph node mantle zone B cells, monocyte-like B cells, CD19-positive B cells, and combinations thereof.

[0148] In some cases, CD19-positive B cells are selected from immature B cells, mature B cells, progenitor B cells, transitional B cells, and combinations thereof.

[0149] In some cases, immature B cells are selected from CD38-negative immature B cells, fraction E immature B cells, and combinations thereof.

[0150] In some cases, mature B cells are selected from B-1 B cells, B-2 B cells, Be cells, Peyer's patch B cells, follicular B cells, fraction F mature B cells, germinal center B cells, marginal zone B cells of lymph nodes, marginal zone B cells of the spleen, memory B cells, naive B cells, plasmablasts, regulatory B cells, and combinations thereof.

[0151] In some cases, B-1B cells are selected from B-1aB cells, B-1bB cells, and combinations thereof. In some cases, B-2B cells are selected from Peyer's patch B cells, follicular B cells, fraction F mature B cells, and combinations thereof. In some cases, follicular B cells are selected from Bm1B cells, Bm2B cells, and combinations thereof. In some cases, fraction F mature B cells are Bm1B cells. In some cases, Be cells are selected from Be1 cells, Be2 cells, and combinations thereof. In some cases, germinal center B cells are selected from Bm2'B cells, Bm3B cells, Bm3-delta B cells, Bm4B cells, centroblasts, centrocells, tonsil germinal center B cells, and combinations thereof.

[0152] In some cases, memory B cells are selected from Bm5 B cells, IgD-negative memory B cells, IgM memory B cells, class-converted memory B cells, double-negative memory B cells, non-converted memory B cells, and combinations thereof.

[0153] In some cases, IgD-negative memory B cells are selected from Bm5 B cells, CD38-negative IgG memory B cells, IgD-negative CD38-positive IgG memory B cells, IgM memory B cells, double-negative memory B cells, and combinations thereof. In other cases, double-negative memory B cells are selected from IgG-negative double-negative memory B cells, IgG-positive double-negative memory B cells, and combinations thereof.

[0154] In some cases, class-conversion memory B cells are selected from IgA memory B cells, IgE memory B cells, IgG memory B cells, IgG-negative class-conversion memory B cells, and combinations thereof. In some cases, IgG memory B cells are selected from CD38-negative IgG memory B cells, CD38-positive IgG memory B cells, and combinations thereof. In some cases, CD38-positive IgG memory B cells are selected from IgD-negative CD38-positive IgG memory B cells, IgD-positive CD38-positive IgG memory B cells, and combinations thereof. In some cases, IgG-negative class-conversion memory B cells are selected from CD38-positive IgG-negative class-conversion memory B cells, CD38-positive IgG-negative class-conversion memory B cells, and combinations thereof. In some cases, CD38-negative IgG-negative class-conversion memory B cells are selected from CD24-negative CD38-negative IgG-negative class-conversion memory B cells, CD24-positive CD38-negative IgG-negative class-conversion memory B cells, and combinations thereof. In some cases, CD38-positive IgG-negative class-converted memory B cells are selected from B220 low CD38-positive IgG-negative class-converted memory B cells, B220-positive CD38-positive IgG-negative class-converted memory B cells, and combinations thereof. In some cases, B220-positive CD38-positive IgG-negative class-converted memory B cells are B220 low CD38-positive IgG-negative class-converted memory B cells. In some cases, double-negative memory B cells are selected from IgG-negative double-negative memory B cells, IgG-positive double-negative memory B cells, and combinations thereof. In some cases, non-converted memory B cells are selected from CD38-negative non-converted memory B cells, CD38-positive non-converted memory B cells, and combinations thereof. In some cases, CD38-negative non-converted memory B cells are selected from B220 low CD38-negative non-converted memory B cells, B220-positive CD38-negative non-converted memory B cells, and combinations thereof. In some cases, B220-positive CD38-negative non-converted memory B cells are B220 low CD38-negative non-converted memory B cells. In some cases, CD38-positive non-converting memory B cells are selected from B220 low CD38-positive non-converting memory B cells, B220-positive CD38-positive non-converting memory B cells, and combinations thereof.In some cases, B220-positive CD38-positive non-converted memory B cells are B220-low CD38-positive non-converted memory B cells. In some cases, naive B cells are selected from CD38-negative naive B cells, CD38-positive naive B cells, and combinations thereof. In some cases, CD38-positive naive B cells are selected from B220-low CD38-positive naive B cells, B220-positive CD38-positive naive B cells, and combinations thereof. In some cases, B220-positive CD38-positive naive B cells are B220-low CD38-positive naive B cells. In some cases, plasmablasts are selected from CD86-positive plasmablasts, IgA plasmablasts, IgD plasmablasts, IgD plasmablasts, IgE plasmablasts, IgG plasmablasts, IgM plasmablasts, and combinations thereof.

[0155] In some cases, progenitor B cells are selected from fraction B / C progenitor B cells, fraction C' progenitor B cells, fraction D progenitor B cells, late progenitor B cells, pre-BI cells, pre-B-II cells, and combinations thereof. In some cases, pre-B-II cells are selected from large pre-B-II cells, small pre-B-II cells, and combinations thereof. In some cases, large pre-B-II cells are selected from pre-BCR-negative large pre-B-II cells, pre-BCR-positive large pre-B-II cells, and combinations thereof. In some cases, pre-BCR-positive large pre-B-II cells are CD38-positive pre-BCR-positive cells. In some cases, small pre-B-II cells are CD22-positive, CD38-low small pre-B cells.

[0156] In some cases, transitional B cells are selected from T1B cells, T2B cells, T3B cells, and combinations thereof.

[0157] A. Disability The methods and compositions disclosed herein are used for targeting several disorders, such as autoimmune disorders. The advantage of the approach herein is that allogeneic cells are used in combination with the administration of exogenous antibodies to specifically target immune cells, such as B cells.

[0158] In some cases, autoimmune disorders are caused by autoantibodies and / or autoreactive B cells.

[0159] In some cases, autoimmune disorders include acromegaly, acquired aplastic anemia, acquired hemophilia, primary agammaglobulinemia, alopecia areata, ankylosing spondylitis (AS), anti-NMDA receptor encephalitis, antiphospholipid syndrome (APS) / severe antiphospholipid syndrome (CAPS) / Asherson syndrome, arteriosclerosis, autoimmune Addison's disease (AAD), autoimmune autonomic ganglion disorder (AAG) / autoimmune autonomic neuropathy / autoimmune gastrointestinal motility disorder (AGID), autoimmune encephalitis / acute disseminated encephalomyelitis (ADEM), autoimmune gastritis, and autoimmune hemolytic anemia (AIHA). ), autoimmune hepatitis (AIH), autoimmune hyperlipidemia, autoimmune hypophysitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune myelofibrosis, autoimmune myocarditis, autoimmune oophoritis, autoimmune pancreatitis (AIP), polyglandular autoimmune syndrome, type I, type II & III (APS1, APS2, APS3, APECED), autoimmune progesterone dermatitis, autoimmune retinopathy (AIR), autoimmune sudden hearing loss (SNHL), Barlow's disease, Behçet's disease, birdshot chorioretinopathy / birdshot grape Femoritis, bullous pemphigoid, Castleman disease, celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic urticaria (CU), Churg-Strauss syndrome / granulomatosis with eosinophilia / polyangiitis (EGPA), Cogan syndrome, cold agglutinin disease, CREST syndrome / limited cutaneous systemic sclerosis, Crohn's disease (CD), Cronchite-Canada syndrome (CSS), idiopathic organizing pneumonia (COP), herpetic dermatitis, dermatomyositis, type 1 diabetes, discoid lupus, Dressler syndrome / post-myocardial infarction / post-pericardiotomy syndrome, eczema / atopic dermatitis, Endometriosis, eosinophilic esophagitis, eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, fibrotic alveolar septitis / idiopathic pulmonary fibrosis (IPF), giant cell arteritis / temporal arteritis / Houghton's disease, giant cell myocarditis, glomerulonephritis, Goodpasture syndrome / anti-GBM / anti-TBM disease, granulomatosis with polyangiitis (GPA) / Wegener's granulomatosis, Graves' disease / thyroid eye disease, Guillain-Barré syndrome (GBS), Hashimoto's thyroiditis / chronic lymphocytic thyroiditis / autoimmune thyroiditis, Henoch-Schönlein purpura / IgA vasculitis, hidradenitis suppurativa,Hearst's disease / acute hemorrhagic leukoencephalitis (AHLE), hypogammaglobulinemia, IgA nephropathy / Berge's disease, immune-mediated necrotizing myopathy (IMNM), immune thrombocytopenia (ITP) / autoimmune thrombocytopenic purpura / autoimmune thrombocytopenia, inclusion body myositis, IgG4-related sclerosing disease (ISD), interstitial cystitis, juvenile idiopathic arthritis / adult-onset Still's disease, juvenile polymyositis / juvenile dermatomyositis / juvenile myositis, Kawasaki disease, Lambert-Eaton myasthenia dysthenia syndrome (LEMS), leukocytosis-destructive vasculitis, lichen planus, lichen sclerosing, woody conjunctivitis, linear IgA disease (LAD) Linear IgA bullous dermatosis (LABD), lupus nephritis, Lyme disease / chronic Lyme disease / post-treatment Lyme disease syndrome (PTLDS), lymphocytic colitis / microscopic colitis, lymphocytic hypophysitis / autoimmune hypophysitis, Meniere's disease, microscopic polyangiitis (MPA) / ANCA-associated vasculitis, mixed connective tissue disease (MCTD), Mohlen's ulcer, Mucher-Habermann disease, multifocal motor neuropathy, multiple sclerosis (MS), myalgic encephalomyelitis (ME) / chronic fatigue syndrome (CFS), myasthenia gravis (MG), narcolepsy, neuromyelitis optica / Devic's disease, ocular scarring pemphigoid Opsoclonus myoclonus syndrome (OMS), relapsing rheumatoid arthritis, paraneoplastic cerebellar degeneration, paraneoplastic pemphigus, Parley-Romberg syndrome (PRS) / hemifacial atrophy (HFA) / progressive hemifacial atrophy, paroxysmal nocturnal hemoglobinuria (PNH), peripheral uveitis / ciliary tonsillar atrophy, PANS / PANDAS, personality-turner syndrome, pemphigus of pregnancy / herpes of pregnancy, pemphigus foliaceus, pemphigus vulgaris, pernicious anemia, POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, postural orthostatic tachycardia syndrome (POTS), primary biliary liver disease Cirrhosis (PBC) / Primary biliary cholangitis, Primary sclerosing cholangitis (PSC), Psoriasis, Palmoplantar pustulosis, Psoriatic arthritis, Idiopathic pulmonary fibrosis (IPF), Pure red cell aplasia (PRCA), Pyoderma gangrene, Rasmussen's encephalitis, Raynaud's syndrome / phenomenon, Reactive arthritis / Reiter's syndrome, Reflex sympathetic dystrophy syndrome (RSD) / Complex regional pain syndrome (CRPS), Relapsing polychondritis, Restless legs syndrome (RLS) / Willis-Ekbom disease, Rheumatic fever, Rheumatoid arthritis, Sarcoidosis, Schmidt's syndrome / Autoimmune polyendocrine syndrome type II, Scleritis, Scleroderma,Selected from sclerosing mesentericitis / mesentile panniculitis, creeping choroidopathy, Sjögren's syndrome, generalized rigidity syndrome (SPS), small-diameter fibrosensory neuropathy, systemic lupus erythematosus (SLE), subacute bacterial endocarditis (SBE), subacute cutaneous lupus, Suzac syndrome, Sydenham's chorea, sympathetic ophthalmitis, Takayasu's arteritis (vasculitis), testicular autoimmune (vasculitis, orchitis), Tolosa-Hunt syndrome, transverse myelitis (TM), tubulointerstitial nephritis-uveitis syndrome (TINU), ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), uveitis (anterior / intermediate / posterior), vasculitis, VEXAS syndrome, vitiligo, Vogt-Koyanagi-Harada syndrome (VKH), and combinations thereof.

[0160] In some embodiments, the treatment involves improvement of the symptoms of a patient or patient population (e.g., as described herein). In some cases, the improvement is compared to a baseline or threshold amount. In some cases, the baseline or threshold amount is a value that is normally considered to be within a normal (e.g., healthy) range. In some cases, the baseline or threshold amount is based on pre-treatment and post-treatment values ​​(e.g., the patient's own baseline score before treatment or the baseline score of the patient population before treatment).

[0161] In some cases, improvement is measured after administration, for example, within a treatment cycle. In some cases, the response is measured within one week after administration, for example, within 1, 2, 3, 4, 5, 6, or 7 days. In some cases, the response is measured after the last dose of the first treatment cycle (for example, as described herein). In some cases, the response is measured after the last dose of the second treatment cycle (for example, as described herein). In some cases, the response is measured within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 12 months after administration, for example, the last dose of the treatment cycle.

[0162] 1. Lupus In some cases, the autoimmune disorder is systemic lupus erythematosus (SLE). In other cases, the autoimmune disorder is lupus nephritis.

[0163] Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by the production of autoantibodies, abnormal B lymphocyte function, and loss of immune tolerance to endogenous nuclear material, which can lead to systemic autoimmunity that can damage various tissues and organs (Pisetsky 2001, Anders 2020).

[0164] SLE can lead to arthritis, renal failure, inflammation of the heart and lungs, changes in the central nervous system (CNS), vasculitis, severe skin rashes, and blood disorders such as anemia, leukopenia, and thrombocytopenia. The manifestation of SLE symptoms varies from patient to patient, and it can take years to make an accurate diagnosis.

[0165] Initial therapy for SLE typically consists of glucocorticoids combined with either mycophenolate mofetil (MMF) or cyclophosphamide. Rational alternatives to initial therapy include MMF combined with either a calcineurin inhibitor (voclosporine or tacrolimus) or belimumab, or cyclophosphamide combined with belimumab. Patients who fail initial therapy should switch to alternative therapies, including the use of intravenous cyclophosphamide and / or anti-CD20 monoclonal antibodies (mAbs).

[0166] Lupus nephritis (LN) is a form of glomerulonephritis and constitutes one of the most severe organ manifestations of SLE, and in many cases, LN is the primary manifestation leading to a diagnosis of SLE (Singh S 2009, Pons-Estel GL 2011). The goals of LN treatment are to achieve rapid remission of active disease, prevent renal flare, prevent progression of chronic kidney disease (CKD), reduce morbidity and mortality, minimize treatment-related toxicity, and preserve fertility. Standard care includes the use of immunosuppressants, such as glucocorticoids and cyclophosphamide (CYC) or mycophenolate mofetil (MMF), as well as adjuvant therapy, based on the histological disease classification. Immunosuppressive therapy is primarily used to treat class III and class IV LN and is divided into two phases: an induction phase with intensive immunosuppression, usually lasting 3–6 months; and a maintenance phase with long-term, less intensive treatment to prevent renal flare.

[0167] However, conventional immunosuppressive treatments are not uniformly effective. The importance of achieving a complete or partial response has been demonstrated by the Lupus Nephritis Collaborative. Patients who achieved complete remission had better 10-year patient survival and renal survival rates (95% and 94%, respectively) compared to those who achieved partial remission (76% and 45%, respectively) (Korbert SM 2000). In patients who did not respond to therapy, the 10-year patient survival and renal survival rates were 46% and 13%, respectively (Chen YE 2008) (Houssiau FA 2004, Houssiau F and Ginzler, 2008).

[0168] The management of patients with refractory disease varies depending on the first-line medication used for induction therapy, clinical factors, and local practices. Switching to a different first-line induction medication is a recommended initial approach for patients with refractory LN and is recommended in both the EULAR / ERA-EDTA and American College of Rheumatology guidelines (Yo JH 2019). Generally, patients resistant to CYC are treated with MMF, and patients resistant to MMF are treated with CYC. In addition to switching immunosuppressants, some patients are treated with a 3-day intravenous pulse of glucocorticoids (Hahn BH 2012).

[0169] LN is histologically classified into six distinct classes, each representing a different symptomatic manifestation and severity of renal complications in SLE (Bajema IM 2018). The rationale for classifying LN into different classes is based on differences in prognosis. While Class VI LN essentially presents with renal atrophy in end-stage renal disease (ESKD), patients with Class III, IV, or V LN, rather than Class I or II LN, are at imminent risk of progression to chronic kidney disease (CKD), which reduces kidney lifespan (Romagnani P 2017, Mackay M 2019). Our understanding of the genetic and pathogenic basis of LN has advanced considerably over the past few decades. However, despite this increase in knowledge and improved treatment options, LN remains a substantial cause of morbidity and mortality among patients with SLE. The 5-year mortality rate for lN decreased between 1975 and 1995, but has remained stable since then, and the rate of progression to end-stage renal disease (ESKD) has remained unchanged (Croca SC 2011, Anders 2020).

[0170] B cells play a major role in the pathogenesis of lnephrosis (LN), making them an attractive therapeutic target. Rituximab (RTX) is a chimeric mouse-human monoclonal antibody against the B cell surface molecule CD20. Several non-controlled studies have reported the efficacy of RTX in patients with refractory LN (Lan 2012). However, RTX did not show benefit in the EXPLORER trial, which excluded patients with severely active LN (Merril JY 2010). Furthermore, a randomized, double-blind, placebo-controlled trial (LUNAR) comparing RTX to placebo in addition to standard care with prednisolone and MMF in patients with incident LN also failed to show a significant therapeutic effect (Rovin BH 2012). However, patients who received RTX showed a higher complete response rate for proteinuria (32% vs. 9%). This reduction in proteinuria persisted up to 78 weeks, increasing the likelihood that longer follow-up periods could yield significant differences. While the study also found higher partial response rates in the RTX group and in a pre-specified subgroup of African American patients compared to the overall patient population, the trial was not powerful enough to detect differences in partial response rates (Yo JH 2019). Recent post-hoc analyses of the LUNAR trial have shown substantial variability in peripheral blood B cell depletion in patients with LN treated with RTX. Achieving complete peripheral depletion (0 peripheral B cells / μL), as well as the speed and duration of complete depletion, was associated with the complete response at 78 weeks (Gomez Mendez LM 2018). This was hypothesized to involve autoreactive pathogenic B cells persisting within the lymphoid structure and renal tubulointerstitium (Ahuja A 2007).

[0171] Despite the LUNAR trial failing to meet its primary endpoint, the advocacy for RTX use in refractory LN persists, primarily based on observational evidence, especially in patients with inadequate responses to first-line induction regimens. A systematic review of published case reports and case series on the efficacy of RTX in patients with refractory LN showed that 300 patients followed up for 60 weeks maintained complete and partial response rates in 87%, 76%, 67%, and 76% of patients in classes III, IV, V, and mixed classes, respectively (Weidenbusch M 2013).

[0172] Obinutuzumab is an anti-CD20 monoclonal antibody glycoengineered to enhance antibody-dependent cell-mediated cytotoxicity (ADCC). It possesses a type II binding conformation that leads to a greater direct cell-destroying effect and more restricted internal distribution than monoclonal antibodies. These properties result in a much more pronounced and sustained B-cell depletion compared to rituximab [Reddy V 2017]. Obinutuzumab was tested in a small phase II trial called NOBILITY, as better B-cell depletion, particularly in the kidney itself, can increase CRR rates [Furie 2020]. Obinutuzumab was compared to PBO in a rapidly tapered glucocorticoid with moderate doses in an MMF background. The percentage of patients achieving a complete renal response (CRR) was higher in patients given obinutuzumab compared to those receiving placebo, reaching 35% at week 76 (vs. 23%) and 41% at week 104 (vs. 23%). Almost all patients still had very low peripheral B cell counts (CD19+ count ≤ 5 cells / uL) at week 52, a finding significantly different from that of the LUNAR trial [Rovin BH 2012], where only half of RTX-treated patients had undetectable peripheral B cells one year after treatment. Further confirmation may be obtained from a large phase III trial (NCT04221477).

[0173] While the contributions of pathogenic B and T cells in SLE have been well studied, the role of innate immune cells has not been adequately demonstrated. Natural killer (NK) cells are a crucial component of the innate immune system, providing immune surveillance by recognizing healthy cells and eliminating stressed cells, including virus-infected and cancer cells.

[0174] NK cell dysfunction has been reported in SLE. NK cells are reduced in number, exhibit decreased cytotoxicity, impaired differentiation, and altered cytokine production in the peripheral blood of SLE patients (Humbel, 2021, Liu, 2021, Lu, 2022). Interestingly, the decrease in NK cell count correlated with disease activity, with greater reductions observed in patients with higher disease activity (Humbel, 2021). NK cell dysfunction in SLE may contribute to disease pathogenesis by impairing the clearance of apoptotic cells and immune complexes, leading to the release of autoantigens and activation of autoreactive B and T cells. Furthermore, altered cytokine production by NK cells may contribute to the chronic inflammation observed in SLE.

[0175] In addition to decreased NK cell count and dysfunction, certain subsets of peripheral SLE NK cells have been reported to exhibit increased IFNγ production and an activated phenotype (Liu, 2021). However, the data are inconsistent across studies and may be related to disease stage and treatment drugs. Furthermore, most of the knowledge regarding the characterization of NK cells in SLE comes from peripheral blood; little is known about the function of tissue-resident NK cells. Renal immune cell profiles in SLE patients were recently evaluated by single-cell RNA sequencing, identifying two distinct clusters of NK cells (Azari, 2019). Further research is needed to determine how specific NK cell subsets contribute to the pathogenesis of SLE.

[0176] In some cases, treatment for SLE includes improvement in the overall renal response (ORR) (e.g., as described above). In some cases, the improvement is compared to the patient's ORR before treatment. In some cases, the improvement is compared to a baseline or threshold level. In some cases, the baseline or threshold level is a value that is normally considered to be within a normal (e.g., healthy) range. In some cases, the baseline or threshold level is based on the patient's own level before treatment, for example. In some cases, the improvement is full renal response (CRR). In some cases, CRR is a urinary protein-to-creatinine ratio (UPCR) of <0.5 and / or normal renal function (serum creatinine <ULN) without a worsening of baseline serum creatinine by more than 15%. In some cases, the improvement is partial renal response (PRR). In some cases, PRR is a reduction of 50% or more in UPCR from baseline to a value of <1 (<3 if baseline UPCR was ≥3) and / or no increase of >15% in serum creatinine from baseline. In some cases, the response is measured after administration, within the treatment cycle. In some cases, the response is measured within one week after administration, e.g., within 1, 2, 3, 4, 5, 6, or 7 days. In some cases, the response is measured after the last dose of the first treatment cycle (e.g., as described herein). In some cases, the response is measured after the last dose of the second treatment cycle (e.g., as described herein). In some cases, the response is measured within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 12 months after administration, e.g., the last dose of the treatment cycle. In some cases, the response (e.g., CRR or PERR) is measured at 12, 22, 52, 76, and / or 104 weeks.

[0177] In some cases, treatment for SLE includes improvement in one or more of the following (e.g., as described above): SLE Disease Activity Index (SLEDAI), Hybrid SELENA-SLEDAI (Systemic Lupus Erythematosus Disease Activity Index), Systemic Lupus Activity Measure (SLAM), C3 complement levels, and C4 complement levels. In some cases, improvement occurs in weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 of the treatment cycle.

[0178] 2. Rheumatoid arthritis (RA) In some cases, the autoimmune disorder is rheumatoid arthritis (RA).

[0179] RA is a systemic autoimmune disease with an annual incidence of 41 / 100,000 in the United States and Nordic countries (Myasoedova, 2021). It is characterized by symmetrical inflammatory polyarthritis, but often with extraarticular features and is frequently associated with a poor prognosis. Refractory RA has emerged as an area of ​​unmet needs. Most patients are adequately managed with methotrexate and other first-line disease-modifying antirheumatic drugs (DMARDs, e.g., sulfasalazine, hydroxychloroquine, leflunomide), a certain percentage of patients require biological disease-modifying antirheumatic drugs (bDMARDs, e.g., infliximab, rituximab, etanercept, tocilizumab) and / or targeted synthetic disease-modifying antirheumatic drugs (tsDMARDs, e.g., baricitinib, tofacitinib), and further subsections are resistant to multiple drugs. Recent observational studies have adopted a provisional definition of refractory RA based on the number of unsuccessful DMARDs, estimating the prevalence of refractory RA to be between 6 and 21% of all patients treated with conventional DMARDs (Melville, 2020).

[0180] In some cases, treatment for RA includes one or more improvements (e.g., as described above) in the participant's response as assessed by the Rheumatoid Arthritis Disease Activity Score DAS28, the Clinical Disease Activity Index (CDAI), and the EULAR decision criteria. In some cases, the improvement occurs in weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 of the treatment cycle.

[0181] 3. Pemphigus vulgaris (PV) In some cases, the autoimmune disorder is pemphigus vulgaris (PV).

[0182] Pemphigus is a group of autoimmune bullous disorders affecting mucous membranes and / or skin. The most common type of pemphigus is pemphigus vulgaris (PV), which accounts for approximately 80% of all pemphigus cases. Pemphigus vulgaris is an autoimmune skin disease caused by desmoglein (Dsg)-specific autoantibodies of cadherin that bind to epithelial desmosomes, resulting in herpes and erosions of the mucous membranes or skin. Although various modalities have been attempted, the disease is difficult to manage. Targeting pathogenic immune pathways with anti-CD20 monoclonal antibodies, BAFF inhibitors, IL-17 blockade, mTOR pathway inhibitors, p38 MAPK, BTK inhibitors, and TNF-α inhibitors have all been attempted in the management of PV (Abulikemu, 2023). However, the disease remains difficult to manage, patients experience relapses, and develop resistance or intolerance to existing treatments.

[0183] In some cases, treatment for PV includes improvements in the Pemphigus Disease Area Index (PDAI), time to disease relapse, number of disease relapses, time to first complete remission as assessed by PDAI, changes in health-related QoL as measured by the Dermatology Life Quality Index (DLQI), and one or more improvements (e.g., as described above) in the blood levels of DSG1 and DSG3. In some cases, the improvement occurs in weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 of the treatment cycle.

[0184] 4. Granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA) In some cases, the autoimmune disorder is granulomatosis with polyangiitis (GPA) and / or microscopic polyangiitis (MPA).

[0185] Granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA) are forms of small-to-medium vasculitis. GPA and MPA are rare diseases with prevalence rates of 24–160 patients and 39–94 patients per million, respectively. While no validated diagnostic criteria exist, the 1990 American College of Rheumatology (ACR) classification criteria (for GPA) and the 2012 Chapel Hill Consensus Conference classification are helpful in defining these diseases for clinical trial purposes because their clinical manifestations make them suitable for innovative therapeutic approaches. Both GPA and MPA generally cause pulmonary and / or renal symptom manifestations, with GPA often affecting the upper respiratory tract. Due to these clinical similarities, GPA and MPA are often studied together in clinical trials. A multidisciplinary approach is required for the diagnosis and management of vasculitis due to its systemic nature (Koike, 2022).

[0186] In some cases, GPA and / or MPA treatment includes one or more improvements (e.g., as described above) in the Birmingham vasculitis activity score, time to disease relapse, number of disease relapses, time to first complete remission, and health-related quality of life (QoL) changes as measured by the Dermatology Quality of Life Index (DLQI). In some cases, the improvement occurs in week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 of the treatment cycle.

[0187] B.Patient Suitable patients for whom the compositions and methods of this specification are appropriate include, for example, patients suffering from, having been diagnosed with, or suspected of having, an autoimmune disorder as described herein.

[0188] In some embodiments, the treatment methods provided herein can be used to treat subjects (e.g., humans, monkeys, dogs, cats, mice) that have been diagnosed with or suspected of having an autoimmune disorder as described herein. In some embodiments, the subjects are mammals. In some embodiments, the subjects are humans.

[0189] In some embodiments, the patient has been diagnosed with or has been diagnosed with an autoimmune disorder, such as an autoimmune disorder as described herein. In some cases, the patient is resistant, relapsing, or refractory after initial treatment for the disorder. In some cases, the patient has failed at least two lines of prior treatment in line with standard care (SOC).

[0190] In some cases, the patient has systemic lupus erythematosus (SLE).

[0191] In some cases, a patient's SLE is diagnosed according to the 2010 American College of Rheumatology (ACR) / European League Against Rheumatism (EULAR) classification criteria for RA (Kay, 2012).

[0192] In some cases, the patient has a total Systemic Lupus Erythematosus Disease Activity Index (SLEDAI-2K) score of 6 or higher prior to treatment. In other cases, the patient has a total SLEDAI-2K score of 8 or higher prior to treatment, excluding alopecia, mucosal ulcers, and fever.

[0193] In some cases, patients have attempted and failed with conventional SLE therapies (e.g., two conventional therapies for at least 12 weeks), including, for example, antimalarial drugs, corticosteroids, immunosuppressants (e.g., mycophenolate mofetil, methotrexate, azathioprine), and / or biological agents (e.g., belimumab, aniflorumab, and rituximab). In other cases, patients have received antimalarial drugs (e.g., hydroxychloroquine, chloroquine, quinacrine) at a stable dose for at least 12 weeks prior to the first treatment, and possibly at least 6 weeks prior to the first administration of NK cells. In some cases, patients receive immunomodulatory drugs (e.g., mycophenolate mofetil (MMF) / mycophenolate, azathioprine / 6-mercaptopurine, leflunomide, methotrexate, or concomitant folic acid, calcineurin inhibitors and / or cyclosporine A, or alone) at a stable dose for at least 12 weeks prior to the first administration of NK cells.

[0194] In some cases, the patient has lupus nephritis. In some cases, the patient has class I, class II, class III, class IV, class V, and / or class VI lupus nephritis. In some cases, the patient has micromesangial lupus nephritis, mesangial proliferative lupus nephritis, focal lupus nephritis, diffuse lupus nephritis, membranous lupus nephritis, and / or advanced sclerosing lupus nephritis. In some cases, the lupus nephritis is refractory, for example, the patient did not improve 3-4 months after the previous treatment, did not achieve a partial response after 6-12 months, and / or did not achieve a full response 2 years after the previous treatment.

[0195] In some cases, the patient has evidence of active disease on renal biopsy based on the modified NIH lupus nephritis activity and chronicity index (Bajema IM 2018). In other cases, the patient has a detectable anti-double-stranded DNA antibody titer.

[0196] In some cases, the patient is a subject (e.g., an adult subject) with class III or IV lupus nephritis, with or without the presence of class V, according to the 2018 ISN / RPS criteria (Bajema IM 2018). In some cases, the patient is relapsed or refractory after initial therapy for SLE. In some cases, initial therapy includes one or more of the following: glucocorticoids in combination with either mycophenolate mofetil (MMF) or cyclophosphamide; MMF in combination with either a calcineurin inhibitor (e.g., voclosporine or tacrolimus) or belimumab; cyclophosphamide in combination with belimumab; intravenous cyclophosphamide; anti-CD20 monoclonal antibody (mAb); or intravenous cyclophosphamide in combination with an anti-CD20 monoclonal antibody (mAb). In some cases, the patient did not improve within 3-4 months of prior treatment, did not achieve a partial response 6-12 months after prior treatment, or did not achieve a complete response 2 years after prior treatment. In some cases, the patient has failed at least two lines of prior treatment in line with standard care (SOC) for subjects with lupus nephritis.

[0197] In some cases, the patient has rheumatoid arthritis (RA).

[0198] In some cases, a patient's RA is diagnosed according to the American College of Rheumatology (ACR) / European League Against Rheumatism (EULAR) 2010 classification criteria for RA (Kay, 2012). In some cases, a patient has received prior treatment with biological disease-modifying antirheumatic drugs ("bDMARDs," e.g., infliximab, rituximab, etanercept, tocilizumab) and / or targeted synthetic disease-modifying antirheumatic drugs ("tsDMARDs," e.g., baricitinib, tofacitinib) and is considered refractory by one or more of the following: lack of benefit of bDMARD or tsDMARD; lack of benefit of at least two bDMARDs; lack of benefit of at least one bDMARD and one tsDMARD; or intolerance to one or more lines of prior therapy (e.g., one, two, or three lines of prior therapy), including bDMARDs and / or tsDMARDs. In some cases, the lack of benefit includes one or more inadequate improvements in joint count, physical function, and disease activity. In some cases, the patient has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 swollen joints (SJCs) and / or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 tender joints (TJCs). In some cases, the patient has at least 6 swollen joints (SJCs) and 6 tender joints (TJCs).

[0199] In some cases, the patient has pemphigus vulgaris (PV).

[0200] In some cases, patients have active lesions; are positive for anti-desmoglein Dsg1 or Dsg3; and have one or more pemphigus disease area index scores >80% (Shimizu, 2014).

[0201] In some cases, the patient has granulomatosis with polyangiitis (GPA) and / or microscopic polyangiitis (MPA). In some cases, the patient has one or more of the following on the Birmingham Vasculitis Activity Score Version 3 (BVASv3): ≥1 for the “primary” item, ≥3 for the “other” item, and ≥2 for the “renal” item.

[0202] C. Lymphocyte depletion In some embodiments, the patient is subjected to lymphocyte depletion before treatment.

[0203] Exemplary lymphocyte depletion chemotherapy regimens, along with relevant beneficial biomarkers, are described in International Publication Nos. 2016 / 191756 and International Publication Nos. 2019 / 079564, which are incorporated herein by reference in their entirety. In certain embodiments, the lymphocyte depletion chemotherapy regimen includes cyclophosphamide (200 mg / m²). 2 2000 mg / m² per day 2 The dose (per day) and fludarabine (20 mg / m²) 2 900 mg / m² per day 2 The procedure includes the step of administering a dose (over a period of / day) to the patient.

[0204] In some embodiments, lymphocyte depletion is 100 or about 100-1500 or about 1500 mg / m² 2 Cyclophosphamide, for example, 250 or approximately 250 to approximately 500 or approximately 500 mg / m² 2 Cyclophosphamide, for example, 250 or about 250-500, 250, 400, 500 or about 500, 250, 400, 500, about 250, about 400, or about 500 mg / m² 2 This includes the administration of cyclophosphamide. In some embodiments, lymphocyte depletion is 500 or about 500 mg / m². 2 This includes the administration of cyclophosphamide. In some embodiments, lymphocyte depletion is 100 or about 100 mg / m². 2 This includes the administration of cyclophosphamide.

[0205] In some embodiments, lymphocyte depletion is 20 mg or about 20 mg / m³. 2 / day ~40mg or approximately 40mg / m² 2 Fludarabine per day, e.g., 30 or approximately 30 mg / m² 2 Includes administration per day.

[0206] In some embodiments, lymphocyte depletion involves the administration of both cyclophosphamide and fludarabine.

[0207] In some embodiments, the patient receives cyclophosphamide (250 mg / m²). 2 ( / day) and fludarabine (30 mg / m²) 2 Lymphocyte depletion is induced by intravenous administration ( / day).

[0208] In some embodiments, the patient receives cyclophosphamide (500 mg / m³). 2 ( / day) and fludarabine (30 mg / m²) 2 Lymphocyte depletion is induced by intravenous administration ( / day).

[0209] In some embodiments, lymphocyte depletion is performed within 5 days prior to the first dose of NK cells. In some embodiments, lymphocyte depletion is performed within 7 days prior to the first dose of NK cells.

[0210] In some embodiments, lymphocyte depletion is initiated 5 days before the first dose of NK cells and performed daily for 3 consecutive days.

[0211] In some embodiments, the first dose of NK cells is administered on day 6, and lymphocyte depletion is performed on days 1, 2, and 3.

[0212] D. Administration Methods comprising the administration of NK cells (for example, as described herein) are described herein. In some cases, NK cells are administered as part of a therapy further comprising the administration of one or more further agents, including, for example, antibodies (for example, as described herein), cytokines (for example, as described herein), lymphocyte depletion agents (for example, as described herein), corticosteroids (for example, prednisone, prednisolone, dexamethasone, methylprednisolone), analgesics, antipyretics and / or antihistamines. For example, a patient may be pre-treated / pre-medicated prior to the infusion of NK cells, as described herein, for example, as shown in Table 13 or Table 14.

[0213] 1.NK cells In some embodiments, NK cells are administered as part of a pharmaceutical composition, for example, the pharmaceutical composition described herein. The cells are administered after thawing, and in some cases without any further handling if the cryoprotective substance is suitable for immediate administration. For a given individual, the therapeutic regimen often involves the administration of multiple aliquots or doses of NK cells over time, which can be obtained from a common batch or donor. In some embodiments, the NK cells, for example, the NK cells described herein, are 1 × 10⁶ NK cells per dose. 8 Or approximately 1 x 10 8 ~8×10 9 Or approximately 8 x 10 9 It is administered individually. In some embodiments, NK cells are administered as 1 × 10⁶ NK cells per dose. 8 Or approximately 1 x 10 8 , 1 x 10 9 Or approximately 1 x 10 9 , 4×10 9 Or approximately 4 x 10 9 , or 8×10 9 Or approximately 8 x 10 9 They are administered individually. In some cases, NK cells are administered in doses of 4 billion or approximately 4 billion cells. In other cases, NK cells are administered in doses of 2 billion or approximately 2 billion cells.

[0214] In some embodiments, NK cells are administered weekly. In some embodiments, NK cells are administered for 3 to 8 weeks or approximately 3 to 8 weeks. In some embodiments, NK cells are administered for 4 to 8 weeks or approximately 4 to 8 weeks. In some embodiments, NK cells are administered weekly for 4 weeks or approximately 4 weeks. In some embodiments, NK cells are administered weekly for 8 weeks or approximately 8 weeks.

[0215] In some embodiments, NK cells are administered on days 6, 9, 13, and 16 of the treatment cycle. In some embodiments, NK cells are administered on days 6, 13, and 20 of the treatment cycle.

[0216] In some embodiments, the administration is repeated over one or more treatment cycles following, for example, the administration planned above. In some embodiments, the further treatment cycle is 2 to 12 months or about 2 to 12 months after the previous treatment cycle, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months or around that time.

[0217] In some embodiments, NK cells are cryopreserved in an injection-compatible medium, such as a cryopreservation composition suitable for intravenous administration, as described herein.

[0218] In some embodiments, NK cells are present in 1 × 10⁶ vials. 8 Or approximately 1 x 10 8 ~8×10 9 Or approximately 8 x 10 9 The cells are cryopreserved in vials containing individual cells. In some embodiments, NK cells are cryopreserved in vials containing single doses.

[0219] In some embodiments, the cells are thawed before administration, for example, in a water bath at 37°C.

[0220] In some embodiments, thawed vials of NK cells are aseptically transferred to single-dose containers, such as dosing bags, using, for example, vial adapters and sterile syringes. The NK cells can then be administered to the patient from the container via a Y-type blood / solution set filter by gravity as an IV infusion.

[0221] In some embodiments, NK cells are administered as quickly as practically possible after thawing, preferably less than 90 minutes, for example, 80, 70, 60, 50, 40, 30, 20, or less than 10 minutes. In some embodiments, NK cells are administered within 30 minutes of thawing.

[0222] In some embodiments, the pharmaceutical composition is administered intravenously via syringe.

[0223] In some embodiments, 1 mL, 4 mL, or 10 mL of the drug product is administered intravenously to the patient via syringe.

[0224] 2. Antibodies In some embodiments, the NK cells described herein, for example, a pharmaceutical composition comprising the NK cells described herein, are administered in combination with one or more antibodies, for example, one or more antibodies described herein, for example, B-cell depletion antibodies, for example, CD19 and / or CD20 antibodies, for example, rituximab and / or obinutuzumab. In some embodiments, the antibodies are administered together with the NK cells as part of the pharmaceutical composition. In some embodiments, the antibodies are administered separately from the NK cells, for example, as part of a separate pharmaceutical composition. The antibodies may be administered before the administration of the NK cells, following the administration of the NK cells, or concurrently with the administration of the NK cells.

[0225] In some embodiments, the antibody is administered before the NK cells. In some embodiments, the antibody is administered after the NK cells.

[0226] In some embodiments, NK cells are administered at least 30, 60, 90, 120, 150, 180, 210, or 240 minutes after the completion of antibody administration.

[0227] In some embodiments, NK cells are administered the day after the antibody is administered.

[0228] In some embodiments, NK cells are administered with each dose, while antibodies are administered with some doses. For example, in some embodiments, NK cells are administered once a week, and antibodies are administered once a month.

[0229] In some embodiments, the antibody is administered weekly for 8 weeks. In some embodiments, the antibody is administered every two weeks for 8 weeks.

[0230] In some embodiments, a certain dose of antibody is administered before a first dose of cells. In some embodiments, a reduced dose of antibody is administered before a first dose of cells.

[0231] In some embodiments, the antibody is administered on days 2 and 13 of the treatment cycle. In some embodiments, the antibody is administered on days 1 and 15 of the treatment cycle.

[0232] 3. Cytokines In some embodiments, cytokines are administered to the patient.

[0233] In some embodiments, cytokines are administered together with NK cells as part of a pharmaceutical composition. In some embodiments, cytokines are administered separately from NK cells, for example, as part of a separate pharmaceutical composition. In some embodiments, the cytokine is IL-2.

[0234] In some embodiments, cytokines are not administered to the patient.

[0235] E. Dosage "Effective amount" is an amount sufficient to produce a beneficial or desired result. For example, a therapeutic amount is an amount that achieves the desired therapeutic effect. This amount may be the same as or different from a prophylactically effective amount, which is the amount necessary to prevent the onset of a disease or disease symptoms. An effective amount can be administered in one or more administrations, applications or dosages. The therapeutically effective amount (i.e., the effective dosage) of a therapeutic compound depends on the therapeutic compound selected. The composition can be administered once or more times per day to once or more times per week; including once every other day. It will be appreciated by those skilled in the art that certain factors, including but not limited to the severity of the disease or disorder, previous treatment, the overall health status and / or age of the subject, and other diseases present, can affect the dosage and timing required to effectively treat the subject. Further, treatment of a subject using a therapeutically effective amount of a therapeutic compound described herein can include a single treatment or a series of treatments.

[0236] The dosage, toxicity and therapeutic effect of a therapeutic compound can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine LD50 (the dose that causes 50% of the population to die) and ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between the toxic effect and the therapeutic effect is the therapeutic index, which can be expressed as the LD50 / ED50 ratio. Compounds showing a high therapeutic index are preferred. Compounds showing toxic side effects can be used, but care should be taken to design a delivery system that targets such compounds to the diseased tissue site to minimize potential damage to non-infected cells and thereby reduce side effects.

[0237] Data obtained from cell culture assays and animal studies can be used to establish a range of dosage amounts for use in humans. The dosage amount of such a compound can be within a range of circulating concentrations that includes an ED50 with little or no toxicity. The dosage amount can vary within this range depending on the dosage form used and the route of administration utilized. For any compound used in the methods of the invention, a therapeutically effective amount can first be estimated from cell culture assays. Dosages can be formulated in animal models to achieve a range of circulating plasma concentrations that includes the IC50 (i.e., the test compound concentration that achieves half-maximal inhibition of symptoms) determined in cell culture. Such information can be used to more accurately determine useful dosages in humans. Plasma levels can be measured, for example, by high performance liquid chromatography, flow cytometry or molecular assays.

[0238] F. Treatment Cycles In some cases, treatment includes administration of NK cells and an antibody (such as those described herein) over the course of a treatment cycle. For example, in some cases, a treatment cycle includes lymphodepletion, followed by administration of NK cells and a B cell-depleting antibody. In some cases, the B cell-depleting antibody is administered prior to the first administration of NK cells. In some cases, the B cell-depleting antibody is administered both prior to and after the first administration of NK cells (e.g., spaced about two weeks apart, such as on days 2 and 13). In some cases, NK cells are administered one or more times (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times) during a treatment cycle, for example, weekly. In some cases, NK cells are administered about weekly after lymphodepletion (e.g., on days 6, 13, 20, etc.). In some cases, NK cells are administered on days 6, 13 and 20. In some cases, the treatment cycle is repeated one or more times, for example, 1, 2, 3, 4, 5 or 6 times. In some cases, the treatment cycles are spaced about one month or more months apart, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months apart.

[0239] For example, an exemplary treatment cycle (for example, for SLE) includes lymphocyte depletion at the start of the treatment cycle (e.g., before NK cell administration). In some cases, lymphocyte depletion includes a 3-day consecutive (days 1-3) lymphocyte depletion regimen to induce lymphocyte depletion and create an optimal environment for NK cell expansion in vivo. In this example, a B-cell depletion antibody (e.g., rituximab or obinutuzumab) is administered twice (with an interval of approximately 2 weeks) as an intravenous infusion (e.g., 1000 mg) during the treatment cycle, optionally after methylprednisolone premedication, to reduce the risk of infusion-related reactions. In some cases, NK cells are administered by IV infusion after the application of the lymphocyte depletion regimen and after the first dose of the antibody. In some cases, the treatment cycle includes administration of NK cells according to one of the dose levels in Table 4. Lymphocyte depletion regimens may include, for example, fludarabine and cyclophosphamide regimens, such as fludarabine at 30 mg / m² on days 1, 2, and 3 at the start of each treatment cycle. 2 It is administered as follows: cyclophosphamide is 1000 mg / m² on day 3 of each treatment cycle. 2 It is administered as follows. For example, the antibody is administered at a dose of 1000 mg on days 2 and 13 of each treatment cycle. In some cases, the first dose of AB-101 is administered at least 48 hours after the last infusion of the lymphocyte depletion regimen for each treatment cycle, using the administration scheme described in Table 4. In other cases, the second treatment cycle is administered, for example, approximately 24 weeks after the first dose of NK cells in the first treatment cycle.

[0240] [Table 4]

[0241] In another example, a treatment cycle (for example, for SLE) includes lymphocyte depletion at the start of the treatment cycle (e.g., before NK cell administration). In this example, lymphocyte depletion includes a 3-day consecutive lymphocyte depletion regimen (days 1-3), and a B-cell depletion antibody (e.g., rituximab or obinutuzumab) is administered twice during the treatment cycle, optionally after premedication, as an intravenous infusion (e.g., 1000 mg) (with an interval of approximately 2 weeks between doses, e.g., on days 2 and 13) to reduce the risk of infusion-related reactions (e.g., as described herein). In some cases, NK cells are administered by IV infusion after the application of the lymphocyte depletion regimen and after the first dose of the antibody. In some cases, the treatment cycle includes administration of NK cells according to one of the dose levels in Table 5. Lymphocyte depletion regimens may include, for example, fludarabine and cyclophosphamide regimens, such as fludarabine at 30 mg / m² on days 1, 2, and 3 at the start of each treatment cycle. 2 It is administered as follows: cyclophosphamide is 1000 mg / m² on day 3 of each treatment cycle. 2 It is administered as follows. For example, the antibody is administered at a dose of 1000 mg on days 2 and 13 of each treatment cycle. In some cases, the first dose of AB-101 is administered at least 48 hours after the last infusion of the lymphocyte depletion regimen for each treatment cycle, using the administration scheme described in Table 5. In some cases, AB-101 is administered on days 6, 13 and 20 of the treatment cycle. In some cases, the second treatment cycle is administered, for example, approximately 24 weeks after the first dose of NK cells in the first treatment cycle.

[0242] [Table 5]

[0243] V. mutant In some embodiments, the fusion protein or its components or the NK cell genotype described herein is at least 80%, e.g., at least 85%, 90%, 95%, 98%, or 100%, identical to the amino acid sequence of the exemplary sequence (e.g., provided herein), and has differences of up to 1%, 2%, 5%, 10%, 15%, or 20% of residues of the exemplary sequence, including, for example, the mutations described herein, or, in addition to the mutations described herein, replaced by, for example, conservative mutations. In preferred embodiments, the mutant retains the desired activity of the parent.

[0244] To determine the percentage of identity between two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (for example, gaps can be introduced into one or both of the first and second amino acid or nucleic acid sequences 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 at corresponding amino acid or nucleotide positions are then compared. If a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then these molecules are identical at that position (as used herein, nucleic acid "identity" is equivalent to nucleic acid "homology"). The percentage of identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap.

[0245] The percentage of identity between a target polypeptide or nucleic acid sequence (i.e., the query) and a second polypeptide or nucleic acid sequence (i.e., the target) can be determined using publicly available computer software, such as Smith-Waterman alignment (Smith, TF and MS Waterman (1981) J Mol Biol 147:195-7); GeneMatcher Plus™, Schwarz and Dayhof (1979) Atlas of Protein Sequence and Structure, Dayhof, MO, Ed, pp 353-358, and "BestFit" (Smith and Waterman, Advances in Applied Mathematics, 482-489 (1981)); and the BLAST program (Basic Local Alignment Search Tool; (Altschul, SF, W. Gish, et al. (1990) J Mol Biol 215: The alignment is determined in various ways within the scope of the art using software such as 403-10), BLAST-2, BLAST-P, BLAST-N, BLAST-X, WU-BLAST-2, ALIGN, ALIGN-2, CLUSTAL, or Megalign (DNASTAR). Furthermore, those skilled in the art can determine appropriate parameters for measuring the alignment, including any algorithm required to achieve the greatest alignment over the length of the sequences being compared. Generally, for target proteins or nucleic acids, the length of comparison can be any length up to (or 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 this disclosure, the identity percentage is compared to the full length of the query sequence.

[0246] In this disclosure, the comparison of sequences between two sequences and the determination of the identity percentage can be achieved using a Blossum 62 score matrix with 12 gap penalties, 4 gap stretching penalties, and 5 frameshift gap penalties.

[0247] 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.

[0248] VI.Definitions Unless otherwise defined, all technical terms, notations, and other technical and scientific terms or terms used herein are intended to have the same meaning as those commonly understood by those skilled in the art in which the claimed subject matter relates. In some cases, terms that have a commonly understood meaning are defined herein for clarity and / or for immediate reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference from those commonly understood in the art.

[0249] Throughout this application, various embodiments may be presented in range form. It should be understood that range form descriptions are merely for convenience and brevity and should not be interpreted as a firm limitation on the scope of this disclosure. Therefore, range descriptions should be considered to specifically disclose not only the individual numbers within the range, but also all possible subranges. For example, a range description such as 1–6 should be considered to specifically disclose not only the individual numbers within the range, e.g., 1, 2, 3, 4, 5, and 6, but also subranges such as 1–3, 1–4, 1–5, 2–4, 2–6, 3–6, etc. This applies regardless of the width of the range.

[0250] As used herein and in the claims, the singular forms “a,” “an,” and “the” include plural references unless otherwise specified in the context. For example, the term “one sample” includes multiple samples, including mixtures of multiple samples.

[0251] The terms “determining,” “measuring,” “evaluating,” “assessing,” and “analyzing” are often used interchangeably herein to refer to forms of measurement. These terms include determining whether an element is present or not (e.g., detection). These terms may include quantitative determination, qualitative determination, or both quantitative and qualitative determination. Assessment may be relative or absolute. “Detecting the presence of something” may, depending on the context, include determining the amount of something that is present, in addition to determining whether something is present or not.

[0252] The terms “subject,” “individual,” and “patient” are often used interchangeably in this specification.

[0253] The term "in vivo" is used to describe events that occur within the body of a subject.

[0254] The term "ex vivo" is used to describe events that occur outside the body of a subject. Ex vivo assays are not performed on the subject itself; rather, they are performed on samples isolated from the subject. An example of an ex vivo assay performed on a sample is an "in vitro" assay.

[0255] The term "in vitro" is used to describe what happens when research reagents are contained in a container that holds them, so that the material is separated from the biological source from which the research reagents were obtained. In vitro assays can include cell-based assays in which live or dead cells are used. In vitro assays can also include cell-free assays in which intact cells are not used.

[0256] As used herein, a number preceded by the term "about" refers to that number plus or minus 10% of that number. A range preceded by the term "about" refers to that range minus 10% of its minimum value and plus 10% of its maximum value.

[0257] As used herein, the term "buffer solution" refers to an aqueous solution consisting of a mixture of a weak acid and its conjugate base or vice versa.

[0258] As used herein, the term "cell culture medium" refers to a mixture for in vitro cell growth and proliferation that contains elements essential for cell growth and proliferation, such as sugars, amino acids, various nutrients, and inorganic substances.

[0259] As used herein, a buffer solution is not a cell culture medium.

[0260] As used herein, the term "bioreactor" refers to a culture device that can continuously control a series of conditions affecting cell culture, such as dissolved oxygen concentration, dissolved carbon dioxide concentration, pH, and temperature.

[0261] As used herein, the term "vector" refers to a nucleic acid molecule that can propagate another nucleic acid that is ligated thereto. This term includes vectors as self-replicating nucleic acid constructs and vectors that are integrated into the genome of the introduced host cell. Some vectors are suitable for delivering the nucleic acid molecules or polynucleotides of the present application. A particular vector can direct the expression of a nucleic acid to which it is operably linked. Such vectors are referred to herein as expression vectors.

[0262] The term "operatably linked" typically refers to two or more nucleic acid sequences or polypeptide elements that are physically linked and functionally related to one another. For example, if a promoter can induce or regulate the transcription or expression of a coding sequence, then that promoter is operatably linked to the coding sequence, and in that case, the coding sequence should be understood as being "under the control" of the promoter.

[0263] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include “engineered cells,” “transformed cells,” and “transformed cells,” including engineered (e.g., transformed) primary cells and their progeny, regardless of passage number. Progeny do not have to be completely identical in nucleic acid content to the parent cells and may contain mutations. Mutant progeny having the same function or biological activity as those screened or selected in the initially transformed cells are included herein.

[0264] Host cells can be stably or transiently transfected with polynucleotides encoding fusion proteins as described herein, as appropriate.

[0265] Section headings used herein are for structural purposes only and should not be construed as limiting the subject matter described herein.

[0266] VII. References

[0267] [Table 6-1]

[0268] [Table 6-2]

[0269] [Table 6-3]

[0270] [Table 6-4]

[0271] [Table 6-5]

[0272] [Table 6-6]

[0273] [Table 6-7]

[0274] [Table 6-8]

[0275] VIII. Examples The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention.

[0276] [Example 1] AB-101 AB-101 is a general-purpose, ready-made, cryopreserved allogeneic umbilical cord blood-derived NK cell therapy product containing ex vivo-enlarged and activated effector cells, designed to enhance the ADCC antitumor response in patients, e.g., those treated with monoclonal antibodies or NK cell conjugates. AB-101 was prepared as described, for example, in International Publication No. 2022 / 133056.

[0277] [Example 2] AB-101, combined with an anti-CD20 antibody, mediates the death of B cells in healthy donors in vitro. In a 4-hour cell death assay, AB-101 was combined with healthy donor PBMCs (in a ratio of 0.2:1 or 1:1), with or without antibody. Donor B cells (CD19+CD20+) were detected by flow cytometry, and the percentage of apoptotic cells was detected with caspase 3 / 7 dye. The numbers in the plots represent the percentage of caspase-positive B cells. The results show that AB-101 combined with anti-CD20 antibody (lower panel) resulted in ADCC and increased B cell death compared to obinutuzumab alone or AB-101+PBMC (without antibody) in the upper panel. As shown in Figure 1, this combination mediated B cell death in healthy donors.

[0278] [Example 3] AB-101, when combined with an anti-CD19 or anti-CD20 antibody, mediates the death of B cells in healthy donors in vitro. In a 4-hour cell death assay, AB-101 was combined with PBMCs from healthy donors (in a ratio of 0.2:1 or 1:1), combined with 0.01 or 0.1 ug / mL of antibody, or not combined. Donor B cells (CD19+CD20+) were detected by flow cytometry, and the percentage of apoptotic cells was detected with caspase 3 / 7 dye. The results show that AB-101 combined with antibody induced ADCC and increased B cell death compared to PBMC+ antibody in the absence of AB-101 (symbol on the y axis). Obinutuzumab combined with AB-101 was the most potent combination for demonstrating B cell death. As shown in Figure 2, AB-101 combined with anti-CD19 (tafacitamab) or anti-CD20 antibody (rituximab or obinutuzumab) mediates B cell death in vitro from healthy donors.

[0279] ADCCs of AB-101 were evaluated in combination with anti-CD20 (rituximab and obinutuzumab) or anti-CD19 (tafacitamab) monoclonal antibodies against healthy human peripheral blood mononuclear cells (PBMCs) and PBMCs isolated from SLE patients. PBMCs were incubated with or without antibodies at different effector (NK cell) vs. target (PBMC) ratios. Apoptotic (caspase-positive) cells were quantified by flow cytometry.

[0280] Whole blood was collected from healthy donors or SLE donors in ACD-A anticoagulant and transported overnight at ambient temperature. PBMCs were isolated and the number of viable cells was determined. The PBMCs were resuspended in assay medium, and the cells were counted at 2 × 10⁶. 5 Cells were plated in cells / well. AB-101 was thawed in a 37°C water bath. After thawing, AB-101 was washed in culture medium, centrifuged, and cell aliquots were counted. The number of cells in AB-101 was 5 × 10⁶ cells. 6 The concentration was adjusted to cells / mL. AB-101 was further diluted in culture medium, and 4 × 10 cells were added. 4 cells / well or 2 × 10⁶ cells 5 Cells were added to PBMCs at a rate of one cell / well, and E:T ratios of 1:1 and 0.2:1 were obtained. Antibodies (0.01, 0.1, or 1 μg / mL of rituximab, obinutuzumab, or tafacitamab) were added to the coculture, and the plates were incubated for 3.5 hours in a 37°C incubator with 5% CO2. Caspase 3 / 7 Green was added to each well, and the cultures were incubated for a further 30 minutes at 37°C. After the incubation period, the cocultures were prepared for analysis by flow cytometry. Due to antibody interference with the detection antibody in flow cytometry, B cells were identified as CD19+ cells for rituximab and obinutuzumab-treated cells, and as CD20+ cells for tafacitamab-treated cells. Specific lysis (%) of target cells was calculated as follows: Specific lysis (%) = (Sample well - Spontaneity) / (100 - Spontaneity) * 100.

[0281] Cytotoxicity experiments using healthy human PBMCs co-cultured with AB-101 showed enhanced AB-101-mediated B cell death at different E:T ratios in the presence of anti-CD20 or anti-CD19 antibodies (Table 6). Obinutuzumab combined with AB-101 was more effective than rituximab or tafacitamab in inducing B cell apoptosis at lower E:T ratios, likely due to enhanced antibody glycoengineering. PBMCs and AB-101 alone showed little to no increase in B cell apoptosis (Table 6). PBMC samples (n=3) isolated from SLE patients were tested in the same co-culture system with AB-101 as normal human PBMCs. B cell apoptosis was measured after adding anti-CD20 or anti-CD19 antibodies to the co-culture. Enhancement of ADCC against SLE B cells was observed in an antibody concentration-dependent and E:T ratio-dependent manner when AB-101 was combined with anti-CD20 or anti-CD19 antibodies (Figures 3 and 4). At a 1 μg / mL antibody concentration and a 1:1 E:T ratio, enhanced cytotoxicity of SLE B cells was observed with obinutuzumab (range 78.6–95.4%), tafacitamab (range 24.2–76.9%), and rituximab (19.2–62.2%) compared to the minimum B cell death with human IgG1 control antibody (range 2.3–8.3%) (Table 7, representative donor). The specificity of cell death in SLE PBMC samples was evaluated by examining the combined effects on SLE T cells. In the presence of AB-101 and any of the three antibodies tested, little to no off-target apoptosis was observed in this cell population (Table 7, representative donor).

[0282] In conclusion, AB-101 combined with anti-CD19 and anti-CD20 mAb demonstrated that it kills SLE B cells via the ADCC mechanism. This killing was specific to B cells, as no significant effect was observed on SLE T cells under the same co-culture conditions.

[0283] [Table 7-1]

[0284] [Table 7-2]

[0285] Figure 3 shows representative FACS plots illustrating the gating of dead / dying caspase 3 / 7+CD19+SLE B cells in PBMCs alone (top left) or PBMCs with AB-101 (bottom left), with anti-CD20 antibody (center), or with AB-101+anti-CD20 antibody in a 1:1 E:T ratio (right). Lymphocytes were first gated by forward scattering (FSC) and side scattering (SSC), followed by single cells. B cells were gated by CD45 + CD14 - CD3 - CD56 - CD16 - and CD19 + It was gated as such.

[0286] PBMCs from SLE patients were isolated from peripheral blood and combined with thawed AB-101 for 4 hours, either with anti-CD20, rituximab, or obinutuzumab (upper panel) or anti-CD19, tafacitamab, or human IgG1 isotype control (lower panel), or without. The percentage of caspase-positive B cells was determined by flow cytometry. Figure 4. Data are expressed as mean ± SD of two wells.

[0287] [Table 8]

[0288] [Example 4] AB-101, when combined with an anti-CD20 antibody, results in minimal T cell death. AB-101 was cultured with or without the antibody, along with PBMCs from healthy donors. After 4 hours, the percentage of caspase-positive T cells (CD3+) was determined by flow cytometry. The addition of AB-101 and obinutuzumab resulted in the smallest increase in T cell death compared to PBMCs alone. As shown in Figure 5, AB-101 (obinutuzumab) combined with an anti-CD20 antibody resulted in the smallest T cell (CD3+) death.

[0289] [Example 5] In a 4-hour cytotoxicity assay, AB-101 combined with an anti-CD20 antibody mediated the death of B cells in SLE donors. AB-101 was cultured with PBMCs from SLE donors, either with or without the antibody. After 4 hours, the percentage of caspase-positive B cells (CD19+) was determined by flow cytometry. The combination of AB-101 + obinutuzumab (0.1 or 1 μg / mL) resulted in increased B cell death from SLE donors. In the presence of the antibody, a higher ratio of AB-101 led to greater B cell death. As shown in Figure 6, in a 4-hour cytotoxicity assay, AB-101 combined with anti-CD20 (obinutuzumab) antibody mediated B cell death from SLE donors.

[0290] [Example 6] Humanized NSG mouse model To evaluate ADCC against human B cells in vivo using a combination of AB-101 and the anti-CD20 mAb, obinutuzumab, we used a CD34+ humanized NSG (huNSG) mouse model. The CD34+ huNSG mouse model is specialized for studying human hematopoietic and immune systems. Briefly, this model consists of NSG mice conditioned with whole-body irradiation and subsequently transplanted with human umbilical cord blood-derived CD34+ hematopoietic stem cells (Ishikawa 2005). These huNSG mice, approximately 12-16 weeks post-transplant, have high levels of human B cells and moderate levels of T cells, as well as a small population of myeloid cells in their peripheral blood, but very few NK cells. Given the high levels of human B cells, this model was considered suitable for evaluating the effects of treatment on B cell levels and general animal health.

[0291] As experimental recipients, we used 19-23 week old female CD34+ humanized NSG (NOD.Cg-Prkdc scid Il2rg tm1Wjl We used mice (SzJ). Upon receipt, the animals were acclimatized for 7 days before being put into the study. Based on the human CD45+CD19+B cell transplantation level, the animals were assigned to treatment groups on day 0 to ensure that the group mean was similar between groups.

[0292] The selected doses and schedules for AB-101 and obinutuzumab administration were based on previously published preclinical studies using obinutuzumab in similar humanized models (Bacac M, 2018), as well as internal studies using B-cell lymphoma xenografts and pilot studies using huNSG mice. The selected 150 μg / kg obinutuzumab dose was considered to be a dose level that would partially deplete human B cells while leaving an opportunity to observe ADCC with this combination.

[0293] The administration scheme is shown in Figure 10. In the monotherapy group, AB-101 (cells 1 × 10) 7AB-101 (individual dose) was administered to huNSG mice as a single slow bolus intravenous injection (IV) on days 0 and 7, while obinutuzumab was administered by intraperitoneal (IP) injection on day 0 or on days 0 and 7. For the combination group, obinutuzumab was administered first, followed by AB-101 on day 0 or on days 0 and 7. Vehicle animals were treated similarly to the combination group and received IP and IV injections on days 0 and 7. Peripheral blood was collected on days 2 (baseline), 7, 14, 21, and 28. Blood collection on day 7 was performed prior to the second dose of either test substance. Human B cell depletion was evaluated in peripheral blood and tissue after treatment with AB-101, obinutuzumab, or a combination of AB-101 and obinutuzumab. Toxicity was assessed by mortality / cage-side observation, clinical observation results, body weight measurements, clinical chemistry (alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, serum urea nitrogen, creatinine, calcium, total bilirubin, phosphorus, total protein, lipemia index, hemolysis index), and macroscopic and microscopic assessments of organs and selected tissues.

[0294] The efficacy of AB-101, obinutuzumab, and AB-101 in combination with obinutuzumab was evaluated by weekly monitoring of changes in CD19+ B cell levels in peripheral blood from baseline (-2 days) after treatment (Figure 11). On day 7, the single-dose obinutuzumab + AB-101 combination resulted in comparable CD19+ B cell depletion (mean 28.15% ± 4, groups 5 and 6, n=8) compared to obinutuzumab alone (mean 29.65% ± 3, groups 2 and 3, n=8). However, when comparing the percentage change in CD19+ B cells at day 21 for either two doses of obinutuzumab (mean 49.3% ± 11, n=5) or the combination of AB-101 and obinutuzumab (mean 33.6% ± 3, n=5), there was a tendency for enhanced B cell depletion with this combination (Table 8). For comparison, the mean percentage change in CD19+ B cells from baseline in the vehicle group and the AB-101 monotherapy group was 59.67 ± 17.6 and 72.3% ± 11.1, respectively. In the presence of AB-101 monotherapy or AB-101 in combination with obinutuzumab, little to no off-target apoptosis (T cells, myeloid cells) was observed.

[0295] [Table 9] For the specified groups, data for day 7 are after a single dose of obinutuzumab, AB-101, or obinutuzumab + AB-101, and data for day 14 are after two doses of obinutuzumab, AB-101, or obinutuzumab + AB-101. In this section, the day 7 data for groups 2 and 3 and groups 5 and 6 were averaged because the test substance was administered once at this point. Comparable CD19+ B cell depletion was observed with obinutuzumab alone (mean 29.65% ± 3, n=8) versus obinutuzumab + AB-101 (mean 28.15% ± 4, n=8). Obin = obinutuzumab, AVG = mean, SEM = standard error of mean.

[0296] The effects of the treatments on toxicity endpoints were evaluated. No deaths or clinical observations were observed in any of the treatment groups. There were no significant differences in percentage change in body weight among the vehicle, obinutuzumab, AB-101, or the combination of AB-101 and obinutuzumab (Figure 12). There were no effects associated with AB-101 or obinutuzumab, either as monotherapy or in combination, among the clinical chemistry parameters across all groups. Inter-individual variability and mean values ​​were sporadic, consistent with biological variability, and / or negligible, and were considered unrelated to test agent administration.

[0297] Autopsy revealed no macroscopic findings related to the test substance in any organ from the treatment group. No microscopic findings related to AB-101 or obinutuzumab administration were reported.

[0298] In summary, the efficacy and tolerability of AB-101 in combination with the anti-CD20 mAb, obinutuzumab, were evaluated in a CD34+ humanized NSG mouse model. Given the high levels of human B cells, this was considered appropriate for evaluating the effects of the treatment on B cell levels and general animal health. AB-101 in combination with obinutuzumab showed a tendency toward enhanced B cell depletion compared to AB-101 or obinutuzumab alone. Off-target apoptosis (T cells, myeloid cells) was little to no in vivo, and no toxicity associated with this combination was observed.

[0299] The in vivo distribution and pharmacokinetics (PK) of AB-101 were determined using an NSG mouse model. Vehicle (PBS, dextran, human albumin, DMSO) and AB-101 cells (0.5 × 10⁶ cells) were used. 7 Individual / mouse, 2 × 10 cells 7 The drug (0.25 mL / mouse) was administered intravenously in a total of eight doses. Animals in the vehicle group and the AB-101 group were sacrificed at 4 hours after the final dose infusion, and at 1, 3, 7, 14, and 78 days (n=3 male mice and n=3 female mice per time point).

[0300] The pharmacokinetics of AB-101 were determined using the qPCR method. AB-101 DNA was detected against the background of NSG mouse matrix DNA using a set of primers / probes specific to the human β-globin gene.

[0301] AB-101 cells were detected primarily in highly perfused tissues (lungs, spleen, heart, and liver) and at the injection site, starting 4 hours post-administration and continuing until 3 days post-administration of the final dose of AB-101 (day 53). At 7 days post-administration of the final dose (day 57), AB-101 cells were detected in the lungs (3 out of 6 samples), spleen (5 out of 6 samples), and injection site (5 out of 6 samples). At 14 and 28 days post-administration of the final dose (64 and 78 days, respectively), AB-101 cells were detected in 2 and 1 injection site samples, respectively. The sporadic occurrences and low concentrations observed in injection site samples at 64 and 78 days do not appear to indicate systemic persistence of AB-101.

[0302] The results of the in vivo distribution study indicate that the distribution of AB-101 cells in vivo is consistent with the intravenous administration route of the cell product, and that these cells lack the potential for long-term persistence and are removed after 7 days post-administration, with no evidence of permanent transplantation. The pharmacokinetic profile of AB-101 is shown in Figure 13.

[0303] Toxicological studies were conducted as dose-range exploration studies (DRFs) and GLP toxicological studies. The objective of the DRF studies was to identify the safe dose range of AB-101 cells in NSG mice after multiple intravenous administrations. The objective of the GLP toxicity studies was to evaluate the toxicity of AB-101 in NSG mice after repeated intravenous administrations of the doses identified in the DRF studies.

[0304] Mice possess two mutations in the NOD / ShiLtJ genetic background: severe combined immunodeficiency (SCID) and a complete null allele of the common gamma chain of the IL2 receptor (IL2rgnull). The SCID mutation is located in the DNA repair complex protein Prkdc and leads to a deficiency of mouse B cells and T cells. The IL2rgnull mutation interferes with cytokine signaling via multiple receptors, leading to a deficiency of functional NK cells.

[0305] The objective of the DRF study was to identify a safe dose range for AB-101 cells in NSG mice after multiple intravenous administrations. The regimen tested was eight weekly intravenous administrations via the tail vein. The proposed test dose range in the preclinical toxicological evaluation was 4 × 10 cells per dose. 9 It was calculated to result in greater exposure to the AB-101 product than an equivalent human dose. Cells tested in the DRF study: 0.1 × 10⁶ 7 ~2.5×10 7 The individual / dose / animal dose range provides a sufficient safety margin for expected clinical administration. The mouse-to-human dose was scaled non-proportionally (Nair 2016): 0.5 × 10⁶ cells for a 70 kg patient. 7 The individual / animal medium dose level is equivalent to a human dose of 14 × 10 cells. 9 Each cell corresponds to 2.5 × 10⁶ cells. 7 The high dose level per individual / animal is 70 × 10 cells. 9 Individualized responses were used. Outcomes from the DRF study were used to inform the dosing of the GLP toxicological study. The experimental design for the DRF study is shown in Table 9.

[0306] [Table 10]

[0307] The administered formulation was freshly prepared each day from the frozen test material stock vial. The cell test material and vehicle material were thawed by immersion in a water bath set at 37°C. Once thawed, the test material was diluted in the vehicle to the target cell count (within ±15%) for administration to animals. A pilot preliminary test was conducted to assess the stability of the cell test material in the formulation during benchtop storage and its suitability for syringes.

[0308] Vehicle controls or cell studies were administered to groups 1-3 on days 1 and 3, then weekly starting from day 12 (days 1, 3, 12, 19, 26, 33, 40, 47, and 54). Cell studies were administered to group 4 only on days 1 and 3, and to group 5 weekly (days 1, 8, 15, 22, 29, 36, 43, and 50). The animals were administered by slow IV bolus injection via the tail vein (over 30-40 seconds). The dose level for groups 1-3 was 0, equivalent to 0.1 × 10⁶ cells. 7 Individual and cell 0.5 × 10 7 The number of cells is 2.5 × 10 for groups 4 and 5. 7 The sample was administered in a dose volume of 0.25 mL.

[0309] Following the first dose, animals in group 4 exhibited decreased activity and / or cold skin upon contact. These observations resolved before the next dose. After the second dose (day 3), animals in group 4 exhibited clinical and / or veterinary observations including significant decreased activity, hunched posture, ruffled hair, partially closed eyes, pallor, loss of skin elasticity, cold skin upon contact, and irregular breathing. Due to the severity of these findings, early euthanasia was planned for these animals on day 3.

[0310] Mild swelling (feet / limbs) and / or impaired limb function and / or limb splay were observed in all groups, including vehicle control animals. These findings were present in most animals and, due to their mild nature and the absence of painful responses on palpation, were considered harmless and vehicle-related.

[0311] In both Groups 3 and 5, findings related to the test item of tremors and a disheveled appearance were noted. Additional clinical and / or veterinary findings related to the test item in Group 5 consisted of a moderate decrease in activity, ataxia, slow respiration, partially / fully closed eyes, cold skin on contact, and loss of skin elasticity. These findings in Group 5 were mainly noted after the 6th dose, and the animals usually recovered by the next day. After the 8th dose, the animals in Group 5 showed a marked decrease in activity, dehydration symptoms, weakness, ataxia, partially closed eyes, irregular respiration, a disheveled appearance, a low posture, splaying of the limbs, and cold skin on contact. Despite the severity of the findings noted after the 8th dose, all animals recovered by the next day. The findings in Group 5 were considered harmful because the severity of the observations increased with the increasing number of doses administered. The findings in Group 3 were not considered harmful due to the low incidence and mild nature of the observations. In Group 2, no findings related to the test item were noted.

[0312] Conclusion. AB-101 was administered as a weekly injection for 8 weeks at dose levels in the range of 0.1×10 7 to 2.5×10 7 cells / dose / animal. Among the different dose levels tested in the DRF study, at the dose level of 2.5×10 7 cells after the 8th dose, adverse clinical observations were noted, including a marked decrease in activity, dehydration symptoms, weakness, ataxia, partially closed eyes, irregular respiration, a disheveled appearance, a low posture, splaying of the limbs, and cold skin on contact. These findings were considered harmful despite recovery because of the increase in severity and number of the findings noted in these animals after the 8th dose. Non-adverse test item-related changes were noted at the dose levels of 0.5×10 7 and 2.5×10 7 cells. Non-adverse clinical observations, including tremors, a disheveled appearance, and piloerection, were noted at the dose level of 0.5×10 7 cells. At the dose level of 2.5×10 7At each dose level, changes in clinical pathology endpoints were observed, including non-adverse test article-related changes such as decreased weight gain and decreases in red blood cell volume and reticulocytes, as well as increases in white blood cells and neutrophils. Anatomical pathology findings were not observed in the animals of the DRF study. As a conclusion, the dose of 2.5×10 cells administered intravenously to NSG mice once a week for 8 weeks was determined as the maximum tolerated dose (MTD). 7

[0313] The purpose of the GLP toxicity study was to evaluate the acute and delayed toxicity of AB-101 in NSG mice after repeated intravenous administration. The doses for the GLP toxicity study were selected according to the results obtained from the DRF study. In the DRF study, adverse clinical observations were noted at a dose of 2.5×10 cells after the 8th dose. Therefore, a cell number of 2×10 was used as the highest dose for the GLP toxicity study. AB-101 was administered to mice once a week for 8 weeks. The doses of 0.5×10 cells / animal and 2×10 cells / animal used in the GLP toxicity study provided a sufficient safety margin for the expected clinical administration (for a 70 kg patient, 0.5×10 cells / animal corresponded to an equivalent human cell dose of 14×10 cells, and 2×10 cells / animal corresponded to an equivalent human cell dose of 56×10 cells). The dose from mouse to human was scaled non-proportionally. The experimental plan for this study is shown in Table 10. 7 7 6 7 7 9 7 9

[0314]

Table 11

[0315] Similar to the DRF study, the dosing formulation was freshly prepared on each dosing day from the frozen test article stock vial.

[0316] ​​​​​​​​​Cellular samples and vehicle controls were administered by intravenous injection via the tail vein. The dose was delivered by a slow bolus injection (over a period of 30-40 seconds). Administration was performed once a week for 8 weeks.

[0317] 0.5 × 10 in mice 7 and 2 × 10 7 Once-weekly intravenous administration of AB-101 at live cell dose levels resulted in no study-related deaths, changes in body weight, or changes in ophthalmic, clinicopathological, or anatomical pathological endpoints. Based on the absence of adverse findings, the no-observed-adverse-effect level (NOEL) is 2 × 10⁶. 7 This was the dose level for living cells.

[0318] [Example 7] Treatment of lupus nephritis with AB-101 and B-cell depletion mAbs B cells are recognized as a crucial mediator of SLE pathogenesis, and anti-CD20 mAbs promote direct B cell death via antibody-dependent cell-mediated cytotoxicity (ADCC) and apoptosis induction. However, studies have shown that B cell depletion is incomplete in the tissues of certain patients after rituximab treatment, leading to the escape of pathogenic B cells and preventing an effective reset of the immune system. NK cells in SLE patients are shown to be deficient in peripheral blood, accompanied by reduced cytotoxicity and defective ADCC, which may contribute to rituximab-induced incomplete B cell depletion. Various nonclinical studies have shown that AB-101 can enhance anti-CD20-mediated ADCC against malignant B lymphocytes expressing CD20 in vitro and in vivo, and is not adversely affected by glucocorticoids. Furthermore, AB-101 has been shown to induce B cell apoptosis in combination with rituximab in PBMCs isolated from SLE patients. Early clinical data from rituximab and AB-101 in patients with advanced B-cell malignancies have shown that this treatment approach is generally well-tolerated and can induce a significant response in malignant B-cell damage.

[0319] AB-101 is an unmanipulated, allogeneic, ready-made, cryopreserved, umbilical cord blood-derived natural killer (NK) cell therapy. The umbilical cord blood units (CBUs) are pre-selected for the KIR-B haplotype and a naturally occurring high-affinity variant (158V / V) of CD16 associated with ADCC enhancement, resulting in a highly active NK cell product without the need for further manipulation.

[0320] AB-101 is a commercially scaled, cryopreserved allogeneic NK cell product in injection-ready medium. The starting material is FDA-approved umbilical cord blood units with the following attributes: selected killer immunoglobulin-like receptor B (KIR B) haplotype: exhibits a more active phenotype (Cooley, 2009); selected V / V CD16 polymorphism at F158: exhibits higher affinity binding to the mAb Fc domain for enhanced antibody-dependent cell-mediated cytotoxicity [ADCC] (Musolino, 2008).

[0321] In preclinical studies, AB-101 demonstrated direct, specific, and potent killing of multiple tumor cell lines in vitro and in vivo when combined with tumor-targeted monoclonal antibodies. AB-101 also demonstrated the ability to secrete cytokines such as tumor necrosis factor alpha (TNFα) and interferon-gamma (IFNγ) upon activation. AB-101 is currently being studied in Phase 1 / 2 clinical trials as monotherapy in subjects with B-cell origin r / r NHL, in combination with rituximab (ClinicalTrials.gov:NCT04673617), and in combination with AFM13 (a bispecific CD30 / CD16 antibody designed to redirect and enhance natural killer (NK) cell-mediated antibody-dependent cell-mediated cytotoxicity) in patients with r / r HL and CD30+ PTCL (NCT05883449).

[0322] NK cells are unique in their ability to induce a rapid cytolytic response without the need for antigen presentation or prior sensitization (Miller 2013; Malmberg 2017). This is partially achieved through the integration of signals delivered from both inhibitory and activating NK cell receptors, and this combined modulation of activity allows for the recognition of a wide range of malignant or virus-infected cell types while avoiding inappropriate targeting of healthy cells and tissues (Vivier 2008; Guillerey, Huntington, and Smyth 2016). In addition to direct cytotoxicity, NK cells can exert their effects via antibody-dependent cell-mediated cytotoxicity (ADCC), where they bind to the crystallizable fragment (Fc) portion of tumor-targeting antibodies covering the surface of target cells, lysing the target cells via the release of cytotoxic factors (e.g., perforin, granzymes).

[0323] In SLE, NK cell dysfunction includes defective ADCCs (Kozlowski, 1982; Green, 2005). Anti-CD20 antibodies utilize ADCCs as part of their mechanism of action to kill malignant B cells in cancer (Manches, 2003; Boross, 2012) and to deplete pathogenic B cells in SLE (Albert, 2008). As mentioned above, treatment of SLE / LN patients with rituximab did not show a significant therapeutic effect and was associated with varying degrees of B cell depletion (Rovin, 2012). Obinutuzumab treatment of SLE / LN patients showed improved efficacy and more rapid, significant, and persistent B cell depletion compared to that observed with rituximab (Furie, 2020); however, not all patients responded, thus indicating that an area of ​​unmet needs still exists. Substitution of endogenous SLE NK cells with allogeneic NK cell products is thought to provide competent effector cells to enhance the activity of anti-CD20 antibodies. AB-101 is combined with these antibodies to enhance ADCC-mediated B cell depletion. ADCC is mediated through the expression of the Fcγ receptor (FcγR) on the surface of NK cells, i.e., CD16 (or FcγRIII). CD16 is expressed in ≥80% of AB-101, making CD16 an ideal cell therapy candidate for combination with anti-CD20 antibodies in SLE / LN.

[0324] To evaluate the cytotoxicity, in vivo distribution, efficacy, and safety of AB-101 against tumor cell lines, the nonclinical pharmacology of AB-101 was studied in both in vitro and in vivo studies.

[0325] In relation to the mechanism of action of ADCC, the cytotoxic activity of AB-101 was evaluated against B-cell lymphoma or leukemia cell lines, normal human peripheral blood mononuclear cells (PBMCs), and PBMCs isolated from SLE patients, in combination with anti-CD20 (rituximab and obinutuzumab) or anti-CD19 (tafacitamab) monoclonal antibodies. Tumor cells or PBMCs were incubated with or without the antibody at different effector (NK cells) versus target (tumor cells or PBMCs) ratios (E:T ratio). Apoptotic (caspase-positive) cells were quantified by flow cytometry.

[0326] All three antibodies enhanced the ADCC activity of AB-101 against B-cell lymphoma or leukemia cell lines. Cytotoxic experiments using normal human PBMCs co-cultured with AB-101 showed enhanced AB-101-mediated B-cell death at different E:T ratios in the presence of anti-CD20 or anti-CD19 antibodies. Enhancement of ADCC against SLE B cells was observed in an antibody concentration-dependent and E:T ratio-dependent manner when AB-101 was combined with rituximab (Figure 7) or obinutuzumab (Figure 9). The specificity of cell death in SLE PBMC samples was evaluated by examining the effect of the combinations on SLE T cells. In the presence of AB-101 and any of the three antibodies tested, little to no off-target apoptosis was observed in this cell population.

[0327] PBMCs from SLE patients were isolated from peripheral blood and combined with thawed AB-101 for 4 hours, either with anti-CD20 antibody and rituximab, or without. The percentage of caspase-positive B cells was determined by flow cytometry. Data are expressed as mean + / - SD of two wells. Representative data from one SLE patient sample are shown.

[0328] The combination of AB-101 and a B-cell depletion mAb (e.g., rituximab or obinutuzumab) may be an effective means of inducing significant B-cell depletion and further inhibiting the replenishment of short-lived plasmablasts, thereby resetting the immune system in these patients. This may provide a meaningful, long-lasting clinical response in patients with advanced proliferative lupus nephritis.

[0329] The patient is eligible to receive combination antihypertensive and antiproteinuria therapy using renin-angiotensin system blockade (e.g., angiotensin-converting enzyme [ACE] inhibitors or angiotensin II receptor blockers).

[0330] [Example A] In this example, adult subjects with class III or IV lupus nephritis, with or without the presence of class V, who have relapsed or have not responded to previous standard care approaches, are treated with AB-101 + B-cell depleting mAb (e.g., rituximab) after a lymphocyte depletion regimen. AB-101 and rituximab are administered to patients over a total of up to two treatment cycles, spaced 24 weeks apart.

[0331] The subjects receive a 3-day (days 1-3) lymphocyte depletion regimen at the start of each treatment cycle to induce lymphocyte depletion and create an optimal environment for the expansion of AB-101 in vivo. Rituximab is administered twice (approximately 2 weeks apart) as a 1000 mg intravenous infusion at the start of each treatment cycle, following methylprednisolone premedication, to reduce the risk of infusion-related reactions. AB-101 is administered at one of three levels (Table 11) after the application of the lymphocyte depletion regimen and after the first dose of rituximab.

[0332] [Table 12]

[0333] The lymphocyte depletion regimen is a fludarabine and cyclophosphamide regimen, with fludarabine administered at 30 mg / m² on days 1, 2, and 3 at the start of each treatment cycle. 2 The drug is administered as follows: cyclophosphamide is given at 1000 mg / m² on the third day of each treatment cycle. 2 It is administered as follows.

[0334] Rituximab is administered at a dose of 1000 mg on days 2 and 13 of each treatment cycle.

[0335] The first dose of AB-101 is administered at least 48 hours after the last infusion of the lymphocyte depletion regimen for each treatment cycle, using the administration scheme described in Table 11.

[0336] [Example B] In this example, using the Common Terminology Criteria for Adverse Events (CTCAE) v.5.0 criteria, adult subjects with relapsed / refractory lupus nephritis class III or IV, with or without the presence of class V, were treated with AB-101 + B-cell depletion mAb (e.g., rituximab, obinutuzumab) after a lymphocyte depletion regimen. As shown in Figure 8, AB-101 and the B-cell depletion mAb were administered to patients over a total of up to two treatment cycles, spaced 24 weeks apart. Subjects with a complete renal response (CRR) at week 22 received only the assigned mAb, if appropriate. Subjects who had not achieved a complete renal response received a second cycle of the initial treatment regimen.

[0337] All subjects receive a 3-day consecutive (days 1-3) lymphocyte depletion regimen consisting of fludarabine (days 1-3) and cyclophosphamide (day 3) during the first treatment cycle to induce lymphocyte depletion and create an optimal environment for the expansion of AB-101 in vivo. Rituximab or obinutuzumab is administered twice (approximately 2 weeks apart) as an intravenous infusion of 1000 mg in each treatment cycle, following methylprednisolone premedication, to reduce the risk of infusion-related reactions. AB-101 is administered on two different schedules (see below) after the application of the lymphocyte depletion regimen and after the first dose of rituximab.

[0338] Treat seven cohorts of patients with AB-101, either with or without a B-cell depletion antibody, as follows:

[0339] [Table 13]

[0340] The monotherapy DL1 cohort will receive AB-101 as monotherapy on days 6, 13, and 20 of cycle 1.

[0341] Fludarabine (30 mg / m²) 2 ) is administered at the start of each treatment cycle, on days 1, 2, and 3; dose adjustments may be applied due to renal failure or other medical conditions. Cyclophosphamide (1000 mg / m²) 2) is administered on day 3 of each treatment cycle. AB-101 is administered as an IV infusion via gravity at a rate of 5–10 mL / min on days 6, 13, and 20 of each treatment cycle. Patients may be pre-treated with one or more of the following: acetaminophen (500–1,000 mg administered orally), diphenhydramine 12.5–25 mg IV or 25 mg administered orally (e.g., if required to treat hypersensitivity reactions to AB-101), and / or second-generation oral antihistamines (e.g., cetirizine, fexofenadine, or loratadine).

[0342] The first dose of AB-101 in each cycle should be administered at least 48 hours after the last infusion of the lymphocyte depletion (LD) regimen.

[0343] Rituximab or obinutuzumab (1000 mg, if administered) is administered via infusion (50–400 mg / hour) on days 1 and 15 of each treatment cycle. To reduce the incidence and severity of infusion-related reactions (IRRs), for example, patients may be pre-treated with one or more corticosteroids (prednisone, prednisolone, dexamethasone, or methylprednisolone), analgesics / antipyretics, and / or antihistamines, as outlined in Table 13. Patients may also be pre-treated as outlined in Table 14. Patient doses may also be adjusted as outlined in Table 14.

[0344] [Table 14]

[0345] [Table 15]

[0346] [Example 8] Treatment of autoimmune disorders using NK cell and B cell depletion mAbs This study evaluates the safety and activity of allogeneic NK cells combined with a B-cell depletion antibody (rituximab) in adult subjects with one of the following disease conditions who have failed previous treatment approaches and are considered "resistant / refractory" by researchers using clinical sign-specific disease criteria (see below): rheumatoid arthritis (RA); pemphigus vulgaris (PV); granulomatosis with polyangiitis (GPA) / microscopic polyangiitis (MPA); and systemic lupus erythematosus (SLE).

[0347] Disease-specific inclusion criteria Rheumatoid arthritis: i. A verified diagnosis of RA that meets the American College of Rheumatology (ACR) / European League Against Rheumatism (EULAR) classification criteria for RA (Kay, 2012). ii. The patient has previously received treatment with a biological disease-modifying antirheumatic drug (bDMARD, e.g., infliximab, rituximab, etanercept, tocilizumab) and / or a targeted synthetic disease-modifying antirheumatic drug (tsDMARD, e.g., baricitinib, tofacitinib) and is considered refractory for any of the following reasons: a. In the researchers' opinion, there is a lack of benefit for at least two bDMARDs or one bDMARD and one tsDMARD. b. Lack of benefit may include insufficient improvement in joint count, physical function, or disease activity. b. Intolerance to at least two lines of prior therapy, including DMARDs and / or tsDMARDs. iii. A minimum of 6 swollen joints (SJCs) and 6 tender joints (TJCs).

[0348] Pemphigus vulgaris: i. A confirmed diagnosis of pemphigus vulgaris with active lesions. ii. Positive for anti-desmoglein Dsg1 or Dsg3. iii. Pemphigus disease area index score >80% (Shimizu, 2014).

[0349] Granulomatosis with polyangiitis (GPA) / Microscopic polyangiitis (MPA): i. Clinical diagnosis of granulomatosis with polyangiitis (GPA) or microscopic polyangiitis (MPA). ii. Having a "primary" item of ≥1, a "other" item of ≥3, or a "renal" item of ≥2 on the Birmingham Vasculitis Activity Score Version 3 (BVASv3).

[0350] Systemic lupus erythematosus: i. Diagnosis of SLE according to the 2019 European League Against Rheumatism / American College of Rheumatology (EULAR / ACR) classification criteria. ii. The total score of the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI-2K) with a score of ≥6 at the time of screening. iii. Positive for anti-double-stranded deoxyribonucleic acid (dsDNA) antibody.

[0351] The patient is administered 1) NK cells and 2) a B cell depletion antibody (rituximab). The administered NK cells (AB-101) are cryopreserved, infusion-ready suspension cells, composed of non-modified, ex vivo-enlarged allogeneic umbilical cord blood-derived NK cells, and are produced from FDA-approved umbilical cord blood units that have been pre-selected for a) V / V CD16 polymorphism at F158 for enhancement of antibody-dependent cell-mediated cytotoxicity (ADCC) and b) killer immunoglobulin-like receptor B (KIR-B) haplotype.

[0352] All subjects will receive one treatment cycle. The treatment cycle will consist of fludarabine (25 mg / m² on days 1, 2, and 3). 2 (For patients with impaired renal function, the dosage should be reduced or eliminated.) and cyclophosphamide (1000 mg / m² on day 3) 2The regimen is initiated with a 3-day (days 1-3) lymphocyte depletion regimen consisting of ) ). Rituximab is administered intravenously twice (1000 mg on days 2 and 13) after methylprednisolone premedication to reduce the risk of infusion-related reactions. NK cells are administered as 1B (1 × 10^9) cells on days 6, 13, and 20. The first dose of NK cells should be administered at least 48 hours (up to 7 days) after the last infusion of the lymphocyte depletion regimen. The infusion schedule, cycle duration, and schedule of study drug administration may be modified based on newly obtained safety, pharmacokinetic, and biomarker data, as determined by the researchers.

[0353] Research evaluation / endpoints Safety: Safety will be assessed throughout the study by monitoring adverse events (AEs), concomitant medications, physical examination, vital signs, and laboratory findings. Except for the adverse events listed below, the severity of AEs will be graded according to CTCAE version 5.0: Cytokine release syndrome (CRS); the severity of CRS events will be assessed according to the American Society for Transplantation and Cell Therapy (ASTCT) grading (Lee, 2019); Immunoeffector cell-associated neurotoxicity syndrome (ICANS); the severity of ICANS events will be assessed according to the American Society for Transplantation and Cell Therapy (ASTCT) grading (Lee, 2019); Graft-versus-host disease (GvHD); the diagnosis and grading of GvHD should follow the criteria of the Mount Sinai Acute GvHD International Consortium (MAGIC) (Harris, 2016).

[0354] Primary activity endpoints: RA: Change from baseline in DAS28 at weeks 12 and 24; PV: Change from baseline in Pemphigus Disease Area Index (PDAI) at weeks 12 and 24; GPA / MPA: Change from baseline in Birmingham Vasculitis Activity Score at weeks 12 and 24; SLE: Change from baseline in SLE Disease Activity Index (SLEDAI) at weeks 12 and 24.

[0355] Exploratory endpoints (changes from baseline at weeks 12 and 24). Rheumatoid arthritis: changes in Clinical Disease Activity Index (CDAI); changes in participant response as assessed by EULAR criteria. Pemphigus vulgaris: time to disease relapse and number of disease relapses; time to first complete remission as assessed by PDAI; changes in health-related QoL (by Dermatological Quality of Life Index (DLQI)); blood levels of DSG1 and 3. Granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA): time to disease relapse and number of disease relapses; time to first complete remission; changes in health-related QoL (by Dermatological Quality of Life Index (DLQI)). Systemic lupus erythematosus (SLE): changes in Systemic Lupus Activity Scale (SLAM); C3 and C4 complement levels.

[0356] [Example 9] Characterization of SLE donor cells We characterized the B cells and NK cells of SLE and healthy donors. As shown in Figure 14, SLE donors had altered B cell subsets, with an increase in transitional B cells and a decrease in activated memory B cells. As shown in Figure 15, SLE donors had reduced total NK, CD16+, and NKG2D, but reduced CD56 bright CD16 neg NK cells were increased.

[0357] Other Embodiments While the present invention has been described with further detail, it should be understood that the foregoing description is intended to illustrate the scope of the invention, not to limit it, and that the scope of the invention is defined by the appended claims. Other aspects, advantages, and modifications are within the following claims.

Claims

1. A method for treating a patient suffering from an autoimmune disorder, comprising the step of administering a population of natural killer cells (NK cells) and antibodies that target immune cells, wherein the NK cells are allogeneic to the patient.

2. The method according to claim 1, wherein the immune cells are involved in an autoimmune reaction.

3. The method according to claim 1 or claim 2, wherein the immune cells are B cells.

4. The method according to any one of claims 1 to 3, wherein the antibody is a B-cell depletion antibody.

5. The method according to any one of claims 1 to 4, wherein the antibody is an antibody that targets human CD19 and / or human CD20.

6. The method according to any one of claims 1 to 5, wherein the NK cells are of the KIR-B haplotype and are homozygous for the CD16 158V polymorphism.

7. The aforementioned autoimmune diseases include acromegaly, acquired aplastic anemia, acquired hemophilia, primary agammaglobulinemia, alopecia areata, ankylosing spondylitis (AS), anti-NMDA receptor encephalitis, antiphospholipid syndrome (APS) | severe antiphospholipid syndrome (CAPS) / Asherson syndrome, arteriosclerosis, autoimmune Addison's disease (AAD), autoimmune autonomic ganglion disorder (AAG) / autoimmune autonomic neuropathy | autoimmune gastrointestinal motility disorder (AGID), autoimmune encephalitis | acute disseminated encephalomyelitis (ADEM), autoimmune gastritis, autoimmune hemolytic anemia (AIHA), and autoimmune hepatitis. (AIH), autoimmune hyperlipidemia, autoimmune hypophysitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune myelofibrosis, autoimmune myocarditis, autoimmune oophoritis, autoimmune pancreatitis (AIP), polyglandular autoimmune syndrome, type I, type II & III (APS1, APS2, APS3, APECED), autoimmune progesterone dermatitis, autoimmune retinopathy (AIR), autoimmune sudden hearing loss (SNHL), Barlow's disease, Behçet's disease, birdshot chorioretinopathy / birdshot uveitis, bullous pemphigoid, Sslmann disease, celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic urticaria (CU), Churg-Strauss syndrome / granulomatosis with eosinophilic polyangiitis (EGPA), Cogan syndrome, cold agglutinin disease, CREST syndrome / limited cutaneous systemic sclerosis, Crohn's disease (CD), Cronchite-Canada syndrome (CSS), idiopathic organizing pneumonia (COP), herpetic dermatitis, dermatomyositis, type 1 diabetes, discoid lupus, Dressler syndrome / post-myocardial infarction / post-pericardiotomy syndrome, eczema / atopic dermatitis, endometriosis, eosinophilic esophagitis, eosinophils Myofasciitis, erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, fibrotic alveolar septitis / idiopathic pulmonary fibrosis (IPF), giant cell arteritis / temporal arteritis / Houghton's disease, giant cell myocarditis, glomerulonephritis, Goodpasture syndrome / anti-GBM / anti-TBM disease, granulomatosis with polyangiitis (GPA) / Wegener's granulomatosis, Graves' disease / thyroid eye disease, Guillain-Barré syndrome (GBS), Hashimoto's thyroiditis / chronic lymphocytic thyroiditis / autoimmune thyroiditis, Henoch-Schönlein purpura / IgA vasculitis, hidradenitis suppurativa, Hearst's disease / acute hemorrhagic leukoencephalitis (AHLE),Hypogammaglobulinemia, IgA nephropathy / Berge's disease, immune-mediated necrotizing myopathy (IMNM), immune thrombocytopenia (ITP) / autoimmune thrombocytopenic purpura / autoimmune thrombocytopenia, inclusion body myositis, IgG4-related sclerosing disease (ISD), interstitial cystitis, juvenile idiopathic arthritis / adult-onset Still's disease, juvenile polymyositis | juvenile dermatomyositis | juvenile myositis, Kawasaki disease, Lambert-Eaton myasthenia dysthenia syndrome (LEMS), leukocytosis-destroying vasculitis, lichen planus, lichen sclerosing, woody conjunctivitis, linear IgA disease (LAD) | linear IgA bullous dermatosis (LABD), lupus Nephritis, Lyme disease / chronic Lyme disease / post-treatment Lyme disease syndrome (PTLDS), lymphocytic colitis / microscopic colitis, lymphocytic hypophysitis / autoimmune hypophysitis, Meniere's disease, microscopic polyangiitis (MPA) / ANCA-associated vasculitis, mixed connective tissue disease (MCTD), Mohlen's ulcer, Mucher-Habermann disease, multifocal motor neuropathy, multiple sclerosis (MS), myalgic encephalomyelitis (ME) / chronic fatigue syndrome (CFS), myasthenia gravis (MG), narcolepsy, neuromyelitis optica / Devic's disease, ocular scarring pemphigoid, opsoclonus myoclonus syndrome (OMS) ), relapsing rheumatoid arthritis, paraneoplastic cerebellar degeneration, paraneoplastic pemphigus, Parley-Romberg syndrome (PRS) / hemifacial atrophy (HFA) / progressive hemifacial atrophy, paroxysmal nocturnal hemoglobinuria (PNH), peripheral uveitis / ciliary tonsillar atrophy, PANS / PANDAS, personality-Turner syndrome, pemphigus of pregnancy / herpes of pregnancy, pemphigus foliaceus, pemphigus vulgaris, pernicious anemia, POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, postural orthostatic tachycardia syndrome (POTS), primary biliary cirrhosis (PBC) / primary biliary cholangitis, primary sclerosing Cholangitis (PSC), psoriasis, palmoplantar pustulosis, psoriatic arthritis, idiopathic pulmonary fibrosis (IPF), pure red cell aplasia (PRCA), pyoderma gangrenosum, Rasmussen's encephalitis, Raynaud's syndrome, reactive arthritis / Reiter's syndrome, reflex sympathetic dystrophy syndrome (RSD) / complex regional pain syndrome (CRPS), relapsing polychondritis, restless legs syndrome (RLS) / Willis-Ekbom disease, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt's syndrome / autoimmune polyendocrine syndrome type II, scleritis, scleroderma, sclerosing mesentericitis / mesenteral panniculitis, creeping choroidopathy,The method according to any one of claims 1 to 6, selected from Sjögren's syndrome, generalized rigidity syndrome (SPS), small-diameter fiber sensory neuropathy, systemic lupus erythematosus (SLE), subacute bacterial endocarditis (SBE), subacute cutaneous lupus, Suzac syndrome, Sydenham's chorea, sympathetic ophthalmitis, Takayasu's arteritis (vasculitis), testicular autoimmunity (vasculitis, orchitis), Tolosa-Hunt syndrome, transverse myelitis (TM), tubulointerstitial nephritis uveitis syndrome (TINU), ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), uveitis | anterior / intermediate / posterior, vasculitis, VEXAS syndrome, vitiligo, Vogt-Koyanagi-Harada syndrome (VKH), and combinations thereof.

8. The method according to claim 7, wherein the autoimmune disorder is systemic lupus erythematosus (SLE).

9. The method according to any one of the claims, wherein the patient has lupus nephritis.

10. The method according to claim 7, wherein the autoimmune disorder is rheumatoid arthritis (RA).

11. The method according to claim 7, wherein the autoimmune disorder is pemphigus vulgaris (PV).

12. The method according to claim 7, wherein the autoimmune disorder is granulomatosis with polyangiitis (GPA).

13. The method according to claim 7, wherein the autoimmune disorder is microscopic polyangiitis (MPA).

14. The method according to any one of the claims, wherein the patient has relapsed after treatment with an anti-CD19 and / or anti-CD20 antibody.

15. The method according to any one of the claims, wherein the patient has experienced disease progression after treatment with autologous stem cell transplantation or chimeric antigen receptor T-cell therapy (CAR-T).

16. 1 x 10 ８ ~1 x 10 １０ The method according to any one of the claims, wherein NK cells are administered to the patient.

17. 1 x 10 ９ ~8 x 10 ９ The method according to any one of the claims, wherein NK cells are administered to the patient.

18. 4 x 10 ８ , 1 x 10 ９ , 4 x 10 ９ or 8 x 10 ９ The method according to any one of the claims, wherein NK cells are administered to the patient.

19. The method according to any one of the claims, wherein 5 × 10⁸, 1 × 10⁹, or 4 × 10⁹ NK cells are administered to the patient.

20. The method according to any one of the claims, wherein the antibody is selected from Table 1, Table 2, or Table 3.

21. The method according to claim 20, wherein the antibody is rituximab, obinutuzumab, or tafacitamab.

22. The method according to claim 20, wherein the antibody is rituximab.

23. The method according to claim 20, wherein the antibody is obinutuzumab.

24. The method according to claim 20, wherein the antibody is tafacitamab.

25. The method according to any one of the claims, wherein the patient is subjected to lymphocyte depletion chemotherapy before the procedure.

26. The method according to claim 25, wherein the lymphocyte depletion chemotherapy is a non-myeloablative chemotherapy.

27. The method according to claim 25 or claim 26, wherein the lymphocyte depletion chemotherapy comprises treatment with at least one of cyclophosphamide and fludarabine.

28. The method according to claim 27, wherein the lymphocyte depletion chemotherapy comprises treatment with cyclophosphamide and fludarabine.

29. The cyclophosphamide is administered at 100 to 500 mg / m ２ / day, and the method according to any one of claims 27 to 28.

30. The cyclophosphamide is 250 or 300 mg / m² ２ The method according to claim 29, administered on a daily basis.

31. The aforementioned cyclophosphamide is 500 mg / m². ２ The method according to claim 29, administered on a daily basis.

32. The aforementioned fludarabine is 10 to 50 mg / m². ２ The method according to any one of claims 27 to 31, administered on a daily basis.

33. The aforementioned fludarabine is 30 mg / m² ２ The method according to claim 31, administered on a daily basis.

34. The method according to any one of the claims, further comprising the step of administering IL-2.

35. 1 x 10 ６ IU / m ２ The method according to claim 34, wherein IL-2 is administered to the patient.

36. The method according to claim 34, wherein 6 million IU of IL-2 is administered to the patient.

37. The method according to any one of claims 34 to 36, wherein IL-2 is administered within 1 to 4 hours of the administration of NK cells.

38. The method according to any one of the claims, wherein the administration of the NK cells and the antibody is performed weekly.

39. The method according to any one of the claims, wherein the NK cells and the antibody are administered weekly for 4 to 8 weeks.

40. The method according to any one of the claims, wherein lymphocyte depletion is performed on the first, second, and third days of the treatment cycle.

41. The method according to any one of the claims, wherein the NK cells are administered on days 6, 13, and 20 of the treatment cycle or on days 6, 9, 13, and 16.

42. The method according to any one of the claims, wherein the NK cells are administered in the form of 2 billion or 4 billion cells or approximately 2 billion or 4 billion cells per administration.

43. The method according to any one of the claims, wherein, when administered, the NK cells are administered on days 6 and 13 in the form of 4 billion cells or approximately 4 billion cells.

44. The method according to any one of the claims, wherein, when administered, the NK cells are administered in the form of 2 billion cells or approximately 2 billion cells on days 9, 16, and 20.

45. The method according to any one of the claims, wherein the NK cells are administered on days 6, 13, and 20 in the form of 5 × 10⁸, 1 × 10⁹, or 4 × 10⁹ NK cells, or approximately 5 × 10⁸, 1 × 10⁹, or 4 × 10⁹ NK cells.

46. The method according to any one of the claims, wherein the antibody is administered on the 2nd and 13th days of the treatment cycle.

47. The method according to any one of the claims, wherein the administration of NK cells is performed weekly, and the administration of the antibody is performed every other week.

48. The method according to any one of the claims, wherein the NK cells are not genetically modified.

49. The method according to any one of the claims, wherein at least 70% of the NK cells are CD56+ and CD16+.

50. The method according to any one of the claims, wherein at least 85% of the NK cells are CD56+ and CD3-.

51. The method according to any one of the claims, wherein 1% or less of the NK cells are CD3+, 1% or less of the NK cells are CD19+, and 1% or less of the NK cells are CD14+.

52. Each dose of NK cells is 1 x 10 ９ ~5 x 10 ９ The method according to any one of the claims, wherein the administration of individual NK cells.

53. Each dose of NK cells is 1 x 10 ９ ~5 x 10 ９ The method according to claim 34, wherein the administration of individual NK cells.

54. The method according to any one of the claims, wherein the patient receives a dose of the CD20-targeted antibody before a first dose of NK cells.

55. The method according to any one of the claims, wherein the enlarged natural killer cells are enlarged umbilical cord blood natural killer cells.

56. The method according to any one of the claims, wherein the expanded population of natural killer cells comprises at least 60%, for example, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% CD16+ cells.

57. The method according to any one of the claims, wherein the expanded population of natural killer cells comprises at least 60%, for example, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% NKG2D+ cells.

58. The method according to any one of the claims, wherein the expanded population of natural killer cells comprises at least 60%, for example, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% NKp46+ cells.

59. The method according to any one of the claims, wherein the expanded population of natural killer cells comprises at least 60%, for example, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% NKp30+ cells.

60. The method according to any one of the claims, wherein the expanded population of natural killer cells comprises at least 60%, for example, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% DNAM-1+ cells.

61. The method according to any one of the claims, wherein the expanded population of natural killer cells comprises at least 60%, for example, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% NKp44+ cells.

62. The method according to any one of the claims, wherein the expanded population of natural killer cells comprises less than 20%, for example, 10% or less, 5% or less, 1% or less, 0.5% or less, or 0% CD3+ cells.

63. The method according to any one of the claims, wherein the expanded population of natural killer cells comprises less than or equal to 20% CD14+ cells, for example, 10% or less, 5% or less, 1% or less, 0.5% or less, or 0%.

64. The method according to any one of the claims, wherein the expanded population of natural killer cells comprises less than or equal to 20% CD19+ cells, for example, 10% or less, 5% or less, 1% or less, 0.5% or less, or 0%.

65. The method according to any one of the claims, wherein the expanded population of natural killer cells comprises less than 20% or less, for example, 10% or less, 5% or less, 1% or less, 0.5% or less, or 0% CD38+ cells.

66. The method according to any one of the claims, wherein the natural killer cells do not contain a CD16 transgene.

67. The method according to any one of the claims, wherein the natural killer cells do not express exogenous CD16 protein.

68. The method according to any one of the claims, wherein the enlarged natural killer cells are not genetically modified.

69. The method according to any one of the claims, wherein the enlarged natural killer cells are derived from the same umbilical cord blood donor.

70. The method according to any one of the claims, wherein the population of NK cells comprises at least 100 million enlarged natural killer cells, for example, 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 enlarged natural killer cells.

71. The aforementioned population of NK cells, (a) A step of obtaining seed cells containing natural killer cells from umbilical cord blood; (b) A step of depleting the seed cells of the CD3+ cells; (c) A step of expanding the natural killer cells by culturing the depleted seed cells together with a first group of Hut78 cells manipulated to express membrane-bound IL-21, mutant TNFα, and 4-1BBL genes, thereby generating expanded natural killer cells. The method according to any one of the claims, comprising, and thereby produced by a method for generating the expanded population of natural killer cells.

72. The aforementioned population of NK cells, (a) A step of obtaining seed cells containing natural killer cells from umbilical cord blood; (b) A step of depleting the seed cells of the CD3+ cells; (c) A step of expanding the natural killer cells by culturing the depleted seed cells together with a first group of Hut78 cells engineered to express membrane-bound IL-21, mutant TNFα, and 4-1BBL genes, thereby generating an expanded master cell bank population of natural killer cells; and (d) A step of expanding the master cell bank population of expanded natural killer cells by culturing a second group of Hut78 cells engineered to express membrane-bound IL-21, mutant TNFα, and 4-1BBL genes to generate expanded natural killer cells. The method according to any one of the claims, comprising, and thereby produced by a method for generating the expanded population of natural killer cells.

73. The aforementioned population of NK cells, after step (c), (i) the step of freezing the expanded natural killer cell master cell bank population in multiple containers; and (ii) Thawing a container containing aliquots of the master cell bank population of enlarged natural killer cells. Produced by a method that further includes, The method according to claim 71 or 72, wherein the step of expanding the master cell bank population of expanded natural killer cells in step (d) includes the step of expanding the aliquots of the master cell bank population of expanded natural killer cells.

74. The method according to any one of claims 71 to 73, wherein the umbilical cord blood is derived from a donor having the KIR-B haplotype and being homozygous for the CD16 158V polymorphism.

75. The method according to any one of claims 71 to 74, wherein the population of NK cells is generated by a method comprising the step of expanding the natural killer cells from umbilical cord blood by at least 10,000 times, for example, 15,000 times, 20,000 times, 25,000 times, 30,000 times, 35,000 times, 40,000 times, 45,000 times, 50,000 times, 55,000 times, 60,000 times, 65,000 times or 70,000 times.

76. The method according to any one of claims 71 to 75, wherein the enlarged population of natural killer cells is neither enriched nor sorted after enlargement.

77. The method according to any one of claims 71 to 76, wherein 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 the seed cells derived from umbilical cord blood.

78. The method according to any one of claims 71 to 77, wherein 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 the seed cells derived from umbilical cord blood.

79. The method according to any one of claims 71 to 78, wherein 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 the seed cells derived from umbilical cord blood.

80. The method according to any one of claims 71 to 79, wherein 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 the umbilical cord blood-derived seed cells.

81. The method according to any one of claims 71 to 80, wherein 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 the seed cells derived from umbilical cord blood.

82. The method according to any one of claims 71 to 81, wherein 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 the seed cells derived from umbilical cord blood.

83. A method for treating systemic lupus erythematosus (SLE), A method comprising the step of administering antibodies targeting a population of natural killer cells (NK cells) and B cells to a patient diagnosed with SLE, wherein the NK cells are allogeneic to the patient, the NK cells are of the KIR-B haplotype, and the NK cells are homozygous for the CD16 158V polymorphism.

84. The method according to claim 83, wherein the patient has lupus nephritis (LN).

85. The method according to claim 83 or 84, wherein the antibody is an anti-CD19 antibody and / or an anti-CD20 antibody.

86. The method according to claim 85, wherein the antibody is rituximab, obinutuzumab, or tafacitamab.

87. The method according to claim 85, wherein the antibody is rituximab.

88. The method according to any one of claims 83 to 87, wherein a population of 1 × 10⁹ to 5 × 10⁹ NK cells is administered to the patient.

89. The method according to claim 88, wherein a population of 2 × 10^9 NK cells or a fraction thereof is administered to the patient.

90. The method according to claim 88, wherein a population of 4 × 10^9 NK cells or a fraction thereof is administered to the patient.

91. The method according to claim 88, wherein a population of 5 × 10^8 NK cells or a fraction thereof is administered to the patient.

92. The method according to claim 88, wherein a population of 1 × 10^9 NK cells or a fraction thereof is administered to the patient.

93. The method according to any one of claims 83 to 90, wherein 500 to 1500 mg of the antibody or approximately that amount is administered to the patient.

94. The method according to claim 93, wherein 100 mg of the antibody or a fraction thereof is administered to the patient.

95. The method according to any one of claims 83 to 94, wherein lymphocyte depletion chemotherapy is administered to the patient prior to the administration of the NK cells.

96. The method according to claim 95, wherein the lymphocyte depletion chemotherapy is a non-myeloablative chemotherapy.

97. The method according to claim 95 or claim 96, wherein the lymphocyte depletion chemotherapy comprises treatment with at least one of cyclophosphamide and fludarabine.

98. The method according to claim 97, wherein the lymphocyte depletion chemotherapy comprises treatment with cyclophosphamide and fludarabine.

99. The cyclophosphamide is present in a concentration of 100 to 1500 mg / m². ２ The method according to any one of claims 97 to 98, administered on a daily basis.

100. The cyclophosphamide is 500 or 1000 mg / m² ２ The method according to claim 99, administered on a daily basis.

101. The aforementioned cyclophosphamide is 1000 mg / m². ２ The method according to claim 99, administered on a daily basis.

102. The aforementioned fludarabine is 10 to 50 mg / m². ２ The method according to any one of claims 97 to 101, administered on a daily basis.

103. The aforementioned fludarabine is 30 mg / m² ２ The method according to claim 102, administered on a daily basis.

104. The method according to any one of claims 83 to 103, comprising a first treatment cycle.

105. The method according to claim 104, wherein the first treatment cycle includes the administration of lymphocyte depletion chemotherapy prior to the administration of the NK cells.

106. The method according to claim 105, wherein the administration of lymphocyte depletion chemotherapy includes the administration of cyclophosphamide and fludarabine.

107. The method according to claim 106, wherein fludarabine is administered on the first, second, and third days of the treatment cycle.

108. The aforementioned fludarabine is 30 mg / m² ２ The method according to claim 106 or 107, administered at a rate of one day.

109. The method according to any one of claims 106 to 108, wherein the cyclophosphamide is administered on the third day of the treatment cycle.

110. The method according to any one of claims 106 to 109, wherein the NK cells are administered at least 48 hours after the last dose of the lymphocyte-depleting chemotherapy.

111. The aforementioned cyclophosphamide is 100 mg / m² ２ The method according to any one of claims 106 to 110, administered on a daily basis.

112. The method according to any one of claims 104 to 111, wherein the antibody is administered on the 2nd and 13th days of the treatment cycle.

113. The method according to any one of claims 104 to 112, wherein the antibody is rituximab.

114. The method according to claim 113, wherein the antibody is administered in an amount of 1000 mg.

115. The method according to any one of claims 104 to 114, wherein the NK cells are administered on one or more days among days 6, 9, 13, 16, and 20.

116. The method according to claim 115, wherein the NK cells are administered on days 6, 13, and 20.

117. The method according to claim 115, wherein the NK cells are administered on days 6, 9, 13, and 16.

118. The method according to any one of claims 104 to 114, wherein the NK cells are administered in a quantity of 2 × 10^9 cells on days 6 and 13, and in a quantity of 1 × 10^9 cells on day 20.

119. The method according to any one of claims 104 to 114, wherein the NK cells are administered in a quantity of 4 × 10^9 cells on days 6 and 13, and in a quantity of 2 × 10^9 cells on day 20.

120. The method according to any one of claims 104 to 114, wherein the NK cells are administered in a quantity of 4 × 10^9 cells on days 6 and 13, and in a quantity of 2 × 10^9 cells on days 9 and 16.

121. The method according to any one of claims 104 to 120, further comprising a second treatment cycle.

122. The method according to claim 121, wherein the second treatment cycle includes the administration of lymphocyte depletion chemotherapy prior to the administration of the NK cells.

123. The method according to claim 122, wherein the administration of lymphocyte depletion chemotherapy constitutes the second treatment cycle, and comprises the administration of cyclophosphamide and fludarabine.

124. The method according to claim 123, wherein fludarabine is administered on the first, second, and third days of the second treatment cycle.

125. The fludarabine is administered at a dose of 30 mg / m² during the second treatment cycle. ２ The method according to claim 123 or 124, administered on a daily basis.

126. The method according to any one of claims 123 to 125, wherein the cyclophosphamide is administered on the third day of the second treatment cycle.

127. The method according to any one of claims 122 to 126, wherein the NK cells are administered during the second treatment cycle at least 48 hours after the last dose of the lymphocyte-depleting chemotherapy.

128. The cyclophosphamide is administered at a dose of 100 mg / m² during the second treatment cycle. ２ The method according to any one of claims 122 to 126, administered on a daily basis.

129. The method according to any one of claims 121 to 128, wherein the antibody is administered on the 2nd and 13th days of the second treatment cycle.

130. The method according to any one of claims 121 to 129, wherein the antibody administered during the second treatment cycle is rituximab.

131. The method according to claim 130, wherein the antibody is administered at a dose of 1,000 mg during the second treatment cycle.

132. The method according to any one of claims 121 to 131, wherein the NK cells are administered on one or more days among days 6, 9, 13, 16, and 20 of the second treatment cycle.

133. The method according to claim 132, wherein the NK cells are administered on days 6, 13, and 20 of the second treatment cycle.

134. The method according to claim 132, wherein the NK cells are administered on days 6, 9, 13, and 16 of the second treatment cycle.

135. The method according to any one of claims 121 to 132, wherein the NK cells are administered in a quantity of 2 × 10^9 cells on days 6 and 13 of the second treatment cycle, and in a quantity of 1 × 10^9 cells on day 20.

136. The method according to any one of claims 121 to 132, wherein the NK cells are administered in a quantity of 4 × 10^9 cells on days 6 and 13 of the second treatment cycle, and in a quantity of 2 × 10^9 cells on day 20.

137. The method according to any one of claims 121 to 132, wherein the NK cells are administered in a quantity of 4 × 10^9 cells on days 6 and 13 of the second treatment cycle, and in a quantity of 2 × 10^9 cells on days 9 and 16.

138. The method according to any one of claims 121 to 137, wherein the second treatment cycle is administered 18 to 30 weeks after the first dose of NK cells in the first treatment cycle.

139. The method according to claim 138, wherein the second treatment cycle is administered approximately 24 weeks after the first dose of NK cells in the first treatment cycle.

140. The method according to any one of claims 83 to 139, wherein the patient has lupus nephritis class III or IV.

141. The method according to claim 140, wherein the patient has lupus nephritis class III.

142. The method according to claim 140, wherein the patient has lupus nephritis class IV.

143. The method according to any one of claims 84 to 142, wherein the patient has lupus nephritis class V.

144. The method according to any one of claims 83 to 143, wherein the patient is recurrent or refractory after receiving prior treatment for SLE.

145. The method according to claim 144, wherein the preceding treatment is selected from the group consisting of glucocorticoids in combination with either mycophenolate mofetil (MMF) or cyclophosphamide, MMF in combination with either a calcineurin inhibitor (e.g., voclosporine or tacrolimus) or belimumab, cyclophosphamide in combination with belimumab, intravenous cyclophosphamide, an anti-CD20 monoclonal antibody (mAb), or intravenous cyclophosphamide in combination with an anti-CD20 monoclonal antibody (mAb), or a combination thereof.

146. The method according to any one of claims 83 to 145, wherein the overall renal response (ORR) of the patient is improved after the treatment.

147. The method according to claim 146, wherein the improvement is a complete renal response (CRR).

148. The method according to claim 147, wherein CRR is measured as a urinary protein-to-creatinine ratio (UPCR) <0.5 and / or normal renal function without a baseline serum creatinine worsening of more than 15%.

149. The method according to claim 148, wherein normal renal function is measured as serum creatinine below ULN.

150. The method according to claim 146, wherein the improvement is a partial renal response (PRR).

151. The method according to claim 150, wherein PRR is measured as a reduction of 50% or more in UPCR from a baseline value to a value of <3.

152. The method according to claim 151, wherein PRR is measured as a reduction of 50% or more in UPCR from baseline to a value of <1.

153. The method according to any one of claims 146 to 152, wherein the ORR is measured within 1, 2, 3, 4, 5, 6, or 7 days after administration.

154. The method according to any one of claims 146 to 152, wherein the ORR is measured within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 12 months after administration.

155. The method according to any one of the claims, wherein the NK cells are not genetically modified.

156. The method according to any one of the claims, wherein at least 70% of the NK cells are CD56+ and CD16+.

157. The method according to any one of the claims, wherein at least 85% of the NK cells are CD56+ and CD3-.

158. The method according to any one of the claims, wherein 1% or less of the NK cells are CD3+, 1% or less of the NK cells are CD19+, and 1% or less of the NK cells are CD14+.

159. The method according to any one of the claims, wherein the NK cells are enlarged umbilical cord blood natural killer cells.

160. The method according to any one of the claims, wherein the population comprises at least 60%, for example, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% CD16+ cells.

161. The method according to any one of the claims, wherein the population comprises at least 60%, for example, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% NKG2D+ cells.

162. The method according to any one of the claims, wherein the population comprises at least 60%, for example, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% NKp46+ cells.

163. The method according to any one of the claims, wherein the population comprises at least 60%, for example, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% NKp30+ cells.

164. The method according to any one of the claims, wherein the population comprises at least 60%, for example, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% DNAM-1+ cells.

165. The method according to any one of the claims, wherein the population comprises at least 60%, for example, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% NKp44+ cells.

166. The method according to any one of the claims, wherein the population comprises less than 20%, for example, 10% or less, 5% or less, 1% or less, 0.5% or less, or 0% CD3+ cells.

167. The method according to any one of the claims, wherein the population comprises less than 20% or less, for example, 10% or less, 5% or less, 1% or less, 0.5% or less, or 0% CD14+ cells.

168. The method according to any one of the claims, wherein the population comprises less than 20% or less, for example, 10% or less, 5% or less, 1% or less, 0.5% or less, or 0% CD19+ cells.

169. The method according to any one of the claims, wherein the population comprises less than 20% or less, for example, 10% or less, 5% or less, 1% or less, 0.5% or less, or 0% CD38+ cells.

170. The method according to any one of the claims, wherein the natural killer cells do not contain a CD16 transgene.

171. The method according to any one of the claims, wherein the natural killer cells do not express exogenous CD16 protein.

172. The method according to any one of the claims, wherein the natural killer cells are not genetically modified.

173. The method according to any one of the claims, wherein the natural killer cells are derived from the same umbilical cord blood donor.

174. The method according to any one of the claims, wherein the population of NK cells comprises at least 100 million enlarged natural killer cells, for example, 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 enlarged natural killer cells.

175. The aforementioned population of NK cells, (a) A step of obtaining seed cells containing natural killer cells from umbilical cord blood; (b) A step to deplete the seed cells of CD3+ cells; (c) A step of expanding the natural killer cells by culturing the depleted seed cells together with a first group of Hut78 cells manipulated to express membrane-bound IL-21, mutant TNFα, and 4-1BBL genes, thereby generating expanded natural killer cells. The method according to any one of the claims, comprising, and thereby produced by a method for generating the expanded population of natural killer cells.

176. The aforementioned population of NK cells, (a) A step of obtaining seed cells containing natural killer cells from umbilical cord blood; (b) A step of depleting the seed cells of the CD3+ cells; (c) A step of expanding the natural killer cells by culturing the depleted seed cells together with a first group of Hut78 cells engineered to express membrane-bound IL-21, mutant TNFα, and 4-1BBL genes, thereby generating an expanded master cell bank population of natural killer cells; and (d) A step of expanding the master cell bank population of expanded natural killer cells by culturing a second group of Hut78 cells engineered to express membrane-bound IL-21, mutant TNFα, and 4-1BBL genes to generate expanded natural killer cells. The method according to any one of the claims, comprising, and thereby produced by a method for generating the expanded population of natural killer cells.

177. The aforementioned population of NK cells, after step (c), (i) the step of freezing the expanded natural killer cell master cell bank population in multiple containers; and (ii) Thawing a container containing aliquots of the master cell bank population of enlarged natural killer cells. Produced by a method that further includes, The method according to claim 175 or claim 176, wherein the step of expanding the master cell bank population of expanded natural killer cells in step (d) includes the step of expanding the aliquots of the master cell bank population of expanded natural killer cells.

178. The method according to any one of claims 175 to 177, wherein the umbilical cord blood is derived from a donor having the KIR-B haplotype and being homozygous for the CD16 158V polymorphism.

179. The method according to any one of the claims, wherein the population of NK cells is generated by a method comprising the step of expanding natural killer cells from umbilical cord blood by at least 10,000 times, for example, 15,000 times, 20,000 times, 25,000 times, 30,000 times, 35,000 times, 40,000 times, 45,000 times, 50,000 times, 55,000 times, 60,000 times, 65,000 times or 70,000 times.

180. The method according to any one of claims 175 to 179, wherein the enlarged population of natural killer cells is neither enriched nor sorted after enlargement.

181. The method according to any one of the claims, wherein the percentage of NK cells expressing CD16 in the population is equal to or higher than the percentage of natural killer cells in the seed cells derived from umbilical cord blood.

182. The method according to any one of the claims, wherein the percentage of NK cells expressing NKG2D in the population is equal to or higher than the percentage of natural killer cells in the seed cells derived from umbilical cord blood.

183. The method according to any one of the claims, wherein the percentage of NK cells expressing NKp30 in the population is equal to or higher than the percentage of natural killer cells in the seed cells derived from umbilical cord blood.

184. The method according to any one of the claims, wherein the percentage of NK cells expressing NKp44 in the population is equal to or higher than the percentage of natural killer cells in the seed cells derived from umbilical cord blood.

185. The method according to any one of the claims, wherein the percentage of NK cells expressing NKp46 in the population is equal to or higher than the percentage of natural killer cells in the seed cells derived from umbilical cord blood.

186. The method according to any one of the claims, wherein the percentage of NK cells expressing DNAM-1 in the population is equal to or higher than the percentage of natural killer cells in the seed cells derived from umbilical cord blood.

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