Treatment of autoimmune disorders with NK cells

Abstract

Provided herein are, among other things, methods for treating a patient suffering from an autoimmune disease.

Description

Technical Field

[0001] This application claims the benefits of U.S. Provisional Application Serial No. 63 / 466,589, filed May 15, 2023; U.S. Provisional Application Serial No. 63 / 513,154, filed July 12, 2023; U.S. Provisional Application Serial No. 63 / 603,850, filed November 29, 2023; U.S. Provisional Application Serial No. 63 / 556,636, filed February 22, 2024; and U.S. Provisional Application Serial No. 63 / 642,218, filed May 3, 2024. The entire foregoing is incorporated herein by reference. Background Technology

[0002] High levels of autoreactive immune cells are associated with autoimmune indications. In autoimmune patients (e.g., lupus), incomplete or reduced depletion of pathogenic B cells when using antibodies (e.g., rituximab) alone can lead to limited efficacy or disease relapse.

[0003] This invention addresses these and other deficiencies in the art. Summary of the Invention

[0004] NK cells are immune cells that engage with tumor cells via a complex array of receptors on their cell surface and through antibody-dependent cell cytotoxicity (ADCC). To initiate ADCC, NK cells bind to antibodies via the CD16 receptor on their surface. NK cells may have advantages over other immune cells, such as T cells used in CAR-T cell therapy and other cell therapies. Among the exemplary advantages, NK cells can be used as allogeneic therapy, meaning that NK cells from a single donor can be safely used in one or more patients without requiring 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 provide an important treatment option for patients with autoimmune diseases, producing better efficacy and results than antibodies alone, which generate pathogenic B cells through deeper and longer-term depletion.

[0006] Furthermore, umbilical cords with preferred characteristics that enhance clinical activity (e.g., high affinity for CD16 and killer cell immunoglobulin-like receptor (KIR) B-haplotype) can be selected by utilizing diverse cord blood banks as sources of NK cells.

[0007] As described in this article, administration of allogeneic NK cells can enhance a patient's ADCC response, for example, when receiving monoclonal antibody therapy.

[0008] Therefore, this article provides methods for depleting B cells in patients, such as methods for treating patients with autoimmune diseases such as systemic lupus erythematosus (SLE).

[0009] This document describes a method for treating a patient suffering from an autoimmune disease, the method comprising administering a population of natural killer cells (NK cells) and an antibody targeting immune cells, wherein the NK cells are allogeneic to the patient. In some embodiments, the immune cells participate in an autoimmune response. In some embodiments, the immune cells are B cells. In some embodiments, the antibody is a B cell depletion antibody, such as 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 KIR-B haplotyped and homozygous for the CD16 158V polymorphism.

[0010] In some implementation schemes, autoimmune diseases are selected from: acromegaly, acquired aplastic anemia, acquired hemophilia, primary agammaglobulinemia, alopecia areata, ankylosing spondylitis (AS), anti-NMDA receptor encephalitis, antiphospholipid syndrome (APS) | catastrophic antiphospholipid syndrome (CAPS) / Asherson syndrome, arteriosclerosis, autoimmune Addison's disease (AAD), autoimmune autonomic ganglionosis (AAG) / autoimmune autonomic dysfunction | autoimmune gastrointestinal motility disorder (AGID), autoimmune encephalitis | acute disseminated encephalomyelitis (ADEM), autoimmune gastritis, autoimmune hemolytic anemia (AIHA), autoimmune hepatitis (AI) H), autoimmune hyperlipidemia, autoimmune hypophysitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune myelofibrosis, autoimmune myocarditis, autoimmune oophoritis, autoimmune pancreatitis (AIP), type I, II, and III autoimmune polyglandular syndrome (Type 1 APS, Type 2 APS, Type 3 APS, APECED), autoimmune progesterone dermatitis, autoimmune retinopathy (AIR), autoimmune sudden sensorineural hearing loss (SNHL), Barlow's disease, Behcet's disease, shotgun-like chorioretinopathy / shotgun-like uveitis, bullous pemphigoid, Kassman's disease, celiac disease, Chagas disease, chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), chronic urticaria (CU), Chag-Strauss syndrome / eosinophilic granulomatous polyangiitis (EGPA), Cogan syndrome, cold agglutinin disease, CREST syndrome | cutaneous systemic scleroderma, Crohn's disease (CD), Cronkhite-Canada syndrome (CSS), cryptogenic organizing pneumonia (COP), herpetiform dermatitis, dermatomyositis, type 1 diabetes mellitus, discoid lupus, Dressler syndrome / post-myocardial infarction / post-pericardiotomy syndrome, eczema / atopic dermatitis, endometriosis, eosinophilic esophagitis, eosinophilic fasciitis, erythema nodosum, primary mixed Cryoglobulinemia, Evans syndrome, fibrotic alveolitis / idiopathic pulmonary fibrosis (IPF), giant cell arteritis / temporal arteritis / Houghton's disease, giant cell myocarditis, glomerulonephritis, Goodbast syndrome / anti-GBM / anti-TBM disease, granulomatous polyangiitis (GPA) / Wegener's granulomatosis, Graves' disease / thyroid ophthalmopathy, Guillain-Barré syndrome (GBS), Hashimoto's thyroiditis / chronic lymphocytic thyroiditis / autoimmune thyroiditis, Henno-Schlan purpura / IgA vasculitis, hidradenitis suppurativa, Hurst's disease / acute hemorrhagic leukoencephalitis (AHLE), hypogammaglobulinemia, IgA nephropathy / Beger's disease, immune-mediated necrotizing myopathy (IMNM)Immune thrombocytopenic purpura (ITP), inclusion body myositis, IgG4-associated sclerotic disease (ISD), interstitial cystitis, juvenile idiopathic arthritis, adult-onset Still's disease, juvenile polymyositis, juvenile dermatomyositis, juvenile myositis, Kawasaki disease, Lambert-Eton myasthenic syndrome (LEMS), leukocytic clotting 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, lymphocytes Hypophysitis / Autoimmune Hypophysitis, Meniere's Disease, Microscopic Polyangiitis (MPA) / ANCA-associated vasculitis, Mixed Connective Tissue Disease (MCTD), Mollen's Ulcer, Mueller-Hacker Disease, Multifocal Motor Neuropathy, Multiple Sclerosis (MS), Myalgic Encephalomyelitis (ME) / Chronic Fatigue Syndrome (CFS), Myasthenia Gravis (MG), Narcolepsy, Neuromyelitis Optic / Devik's Disease, Ocular Cicatricial Pemphigoid, Ocular Clonic-Myoclonic Syndrome (OMS), Recurrent Rheumatism, Paraneoplastic Cerebellar Degeneration, Paraneoplastic Pemphigus, Parry-Romberg Syndrome (PRS) / Hypoplastic Facial Atrophy (HFA) / Progressive Hemifacial Atrophy, Paroxysmal Nocturnal Hemoglobinuria (PNH) Peripheral uveitis / spasm, PANS / PANDAS, Parson-Turner syndrome, pemphigoid of pregnancy / herpes gestationis, 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 gangrenosa, Rasmussen encephalitis, Raynaud's syndrome / phenomenon, reactive arthritis / Reiter's syndrome, reflex sympathetic dystrophy. Complex regional pain syndrome (RSD) / complex regional pain syndrome (CRPS), recurrent polychondritis, restless legs syndrome (RLS) / Willis-Eckbond disease, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome / type II autoimmune polyendocrine syndrome, scleritis, scleroderma, sclerosing mesenteric / mesenteric panniculitis, creeping choroidal lesions, Sjögren's syndrome, stiff-person syndrome (SPS), small fiber sensory neuropathy, systemic lupus erythematosus (SLE), subacute bacterial endocarditis (SBE), subacute cutaneous lupus, Sussac syndrome, Sidnum chorea, sympathetic ophthalmia, Goran's arteritis (vasculitis), testicular autoimmune disease (vasculitis, orchitis), Tolosa-Hunter syndrome,Transverse myelitis (TM), tubulointerstitial nephritis-uveitis syndrome (TINU), ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), pre- / middle / post-uveitis, vasculitis, VEXAS syndrome, vitiligo, Vogt-Koyanagi-Harada syndrome (VKH), and combinations thereof.

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

[0012] In some implementation schemes, the patient is administered 1×10 8 Up to 1×10 10 One NK cell. In some implementations, the patient is administered 1×10⁶ NK cells. 9 Up to 8×10 9 NK cells. The method according to any one of the preceding claims, wherein the patient is administered 4 × 10⁴ NK cells. 8 1×10 9 4×10 9 Or 8×10 9 NK cells. In some implementations, the patient is administered 5 × 10⁶ NK cells. 8 1×10 9 Or 4×10 9 NK cells.

[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 tafasitamab. In some embodiments, the antibody is rituximab. In some embodiments, the antibody is obinutuzumab. In some embodiments, the antibody is tafasitamab.

[0014] In some implementation schemes, the patient is administered 1×10 9 Up to 5×10 9 A population of NK cells. In some implementations, the patient is administered 2 × 10⁶ cells. 9 A population of NK cells or approximately 2 × 10 9 A population of NK cells, 4 × 10 9 A population of NK cells or approximately 4 × 10 9 A population of NK cells, 5 × 10 8 A population of NK cells or approximately 5 × 10 8A population of NK cells, or 1×10 9 A population of NK cells or approximately 1 × 102 9 A population of NK cells.

[0015] In some implementations, the patient is administered 500 to 1500 mg of antibody, or about 500 to 1500 mg of antibody. In some implementations, the patient is administered 100 mg of antibody, or about 100 mg of antibody.

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

[0017] In some implementations, the method further includes administering IL-2. In some implementations, the patient is administered 1×10⁻⁶ IL-2. 6 IU / m 2 IL-2. In some embodiments, the patient is administered 6 million IU of IL-2. In some embodiments, IL-2 administration occurs within 1 to 4 hours of 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 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 of the treatment cycle, or on days 6, 9, 13, and 16. In some embodiments, NK cells are administered in doses of 2 billion or 4 billion cells, or approximately 2 billion or 4 billion cells. In some embodiments, if administered, NK cells are administered on days 6 and 13 in doses of 4 billion cells, or approximately 4 billion cells. In some embodiments, if administered, NK cells are administered on days 9, 16, and 20 in doses of 2 billion cells, or approximately 2 billion cells. In some implementations, NK cells are administered at 5 × 10⁻⁶ on days 6, 13, and 20. 8 1×109 Or 4×10 9 One NK cell, or approximately 5 × 10 8 1×10 9 Or 4×10 9 NK cell administration. In some embodiments, the antibody is 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 implementations, 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 administration of NK cells is 1 × 10⁻⁶. 9 Up to 5×10 9 One NK cell. In some implementations, each administration of NK cells is 1 × 10⁶ cells. 9 Up to 5×10 9 NK cells.

[0020] In some implementations, patients receive a dose of CD20-targeting antibody before the first dose of NK cells.

[0021] In some implementations, the expanded natural killer cells are expanded umbilical cord blood natural killer cells.

[0022] In some embodiments, the expanded natural killer cell 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. In some embodiments, the expanded natural killer cell 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. In some embodiments, the expanded natural killer cell 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. In some embodiments, the expanded natural killer cell 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. In some embodiments, the expanded natural killer cell population comprises at least 60%, such as 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 comprises at least 60%, such as 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 comprises less than 20%, such as less than 10%, less than 5%, less than 1%, less than 0.5%, or 0%, CD3+ cells. In some embodiments, the expanded natural killer cell population comprises less than 20%, such as less than 10%, less than 5%, less than 1%, less than 0.5%, or 0%, CD19+ cells. In some implementations, the expanded natural killer cell population contains less than 20%, such as less than 10%, less than 5%, less than 1%, less than 0.5%, or 0% CD38+ cells.

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

[0024] In some implementations, the NK cell population contains at least 100 million expanded natural killer cells, such as 200 million, 250 million, 300 million, 400 million, 500 million, 600 million, 700 million, 750 million, 800 million, 900 million, 1 billion, 2 billion, 3 billion, 4 billion, 5 billion, 6 billion, 7 billion, 8 billion, 9 billion, 10 billion, 15 billion, 20 billion, 25 billion, 50 billion, 75 billion, 80 billion, 90 billion, 100 billion, 200 billion, 250 billion, 300 billion, 400 billion, 500 billion, 600 billion, 700 billion, 800 billion, 900 billion, 1 trillion, 2 trillion, 3 trillion, 4 trillion, 5 trillion, 6 trillion, 7 trillion, 8 trillion, 9 trillion, or 10 trillion expanded natural killer cells.

[0025] In some implementations, the NK cell population is generated by methods including: (a) obtaining seed cells containing natural killer cells from umbilical cord blood; (b) depleting CD3+ cells from the seed cells; and (c) generating expanded natural killer cells by culturing the depleted seed cells with a first-plurality of Hut78 cells engineered to express membrane-bound IL-21, mutant TNFα, and 4-1BBL genes, thereby generating an expanded natural killer cell population.

[0026] In some implementations, the NK cell population is generated by methods including: (a) obtaining seed cells containing natural killer cells from umbilical cord blood; (b) depleting CD3+ cells in the seed 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, mutated TNFα, and 4-1BBL genes, thereby generating an expanded natural killer cell population; and (d) expanding the expanded natural killer cell population by culturing the expanded natural killer cell master cell population with a second plurality of Hut78 cells engineered to express membrane-bound IL-21, mutated TNFα, and 4-1BBL genes, thereby generating an expanded natural killer cell population.

[0027] In some embodiments, the NK cell population is generated by further comprising the following methods after step (c): (i) freezing the expanded natural killer cell master cell bank population in multiple containers; and (ii) thawing containers containing aliquots of the expanded natural killer cell master cell bank population, wherein step (d) amplifying the expanded natural killer cell master cell bank population includes amplifying aliquots of the expanded natural killer cell master cell bank population.

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

[0029] In some implementations, the NK cell population is generated by a method comprising expanding natural killer cells derived from umbilical cord blood by at least 10,000-fold, such as 15,000-fold, 20,000-fold, 25,000-fold, 30,000-fold, 35,000-fold, 40,000-fold, 45,000-fold, 50,000-fold, 55,000-fold, 60,000-fold, 65,000-fold, or 70,000-fold.

[0030] In some implementations, the expanded natural killer cell population is not enriched or sorted after expansion.

[0031] In some embodiments, the percentage of CD16-expressing NK cells in the expanded natural killer cell population is the same as or higher than the percentage of natural killer cells from the cord blood 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 from the cord blood 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 from the cord blood 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 from the cord blood 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 from the cord blood seed cells. In some embodiments, the percentage of DNAM-1-expressing NK cells in the expanded natural killer cell population is the same as or higher than the percentage of natural killer cells from the cord blood seed cells.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. This document describes the methods and materials used in this invention; other suitable methods and materials known in the art may also be used. Materials, methods, and examples are illustrative and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification, including the definitions, shall prevail.

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

[0034] By incorporating references

[0035] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication, patent or patent application is specifically and individually indicated to be incorporated herein by reference. Attached Figure Description

[0036] The novel features of the invention are specifically set forth in the appended claims. The patent or application documents include at least one color-drawn drawing. Upon request and payment of the necessary fees, the Patent Office will provide a copy of the patent or patent application publication with color drawings. A better understanding of the features and advantages of the invention will be obtained by referring to the following detailed description and the accompanying drawings, in which illustrative embodiments utilizing the principles of the invention are described, and in which:

[0037] Figure 1 This is a set of images showing the in vitro mediated killing of healthy donor B cells by AB-101 combined with anti-CD20 antibody.

[0038] Figure 2 This is a diagram showing how AB-101, in combination with anti-CD19 or anti-CD20 antibodies, mediates the killing of healthy donor B cells in vitro.

[0039] Figure 3 This is a representative FACS image set of SLE B cell apoptosis.

[0040] Figure 4 This diagram shows AB-101-mediated SLE B-cell-specific lysis in combination with anti-CD19 or anti-CD20 monoclonal antibodies. Four bar charts are presented for each antibody, with four bars per chart showing the percentage of caspase-positive B cells. Each bar chart corresponds to the AB101:PBMC ratio (from left to right: 0, 0.2, 1, and 2), and each chart corresponds to the level of each antibody (from left to right: no antibody, 0.01 µg / mL, 0.1 µg / mL, and 1 µg / mL antibody).

[0041] Figure 5 This is a set of images showing that AB-101 combined with anti-CD20 antibody results in minimal killing of T cells.

[0042] Figure 6 This is a set of graphs showing the killing of SLE donor B cells mediated by AB-101 combined with anti-CD20 antibody in a 4-hour cytotoxicity assay.

[0043] Figure 7Enhanced SLE B-cell killing observed with the combined use of rituximab and AB-101 is shown. PBMCs from SLE patients were isolated from peripheral blood and mixed with thawed AB-101, with or without anti-CD20 antibody rituximab, for 4 hours. The percentage of caspase-positive B cells was determined by flow cytometry. Data are presented as mean + / - SD of replicate wells. Representative data from a single SLE patient sample are shown. Four bar charts are presented for each antibody, with four bars per chart showing the percentage of caspase-positive B cells. Each bar chart corresponds to the AB101:PBMC ratio (from left to right: 0, 0.2, 1, and 2), and each bar chart corresponds to the antibody level (from left to right: no antibody, 0.01 µg / mL, 0.1 µg / mL, and 1 µg / mL antibody).

[0044] Figure 8 Examples of treatment regimens are shown. DLT = Dose-limiting toxicity; FU = Follow-up; EOT = End of treatment; Flu = Fludarabine; Ritux = Rituximab; Obi = Oxtuzumab; CRR = Complete renal response; Cyclo = Cyclophosphamide.

[0045] Figure 9 Enhanced SLE B-cell killing observed with the combined use of oxutuzumab and AB-101 is shown. PBMCs from SLE patients were isolated from peripheral blood and mixed with thawed AB-101, with or without anti-CD20 antibody oxutuzumab, for 4 hours. The percentage of caspase-positive B cells was determined by flow cytometry. Data are presented as mean + / - SD of replicate wells. Representative data from a single SLE patient sample are shown. Four bar charts are presented for each antibody, with four bars per chart showing the percentage of caspase-positive B cells. Each bar chart corresponds to the AB101:PBMC ratio (from left to right: 0, 0.2, 1, and 2), and each bar chart corresponds to the antibody level (from left to right: no antibody, 0.01 µg / mL, 0.1 µg / mL, and 1 µg / mL antibody).

[0046] Figure 10 This diagram illustrates the dosing regimen for the humanized NSG model. Obin = Otutuzumab.

[0047] Figure 11 This study demonstrated the in vivo activity of AB-101 and oxotuzumab in a humanized NSG model. HuNSG mice were treated on day 0, or on days 0 and 7, with the following: solvent (intraperitoneal PBS, intravenous frozen medium), oxotuzumab (150 μg / kg), and AB-101 (1×10⁻⁶). 7(1 cell) or AB-101 plus obituzumab. Blood samples were collected from mice at baseline (i.e., before the first treatment) and at specified time points (A). The kinetics of peripheral blood B cell counts analyzed by flow cytometry are expressed as percentage change from baseline in human CD19+ B cells (cells / μl) ± SEM (B). B cells were gated for CD19 rather than CD20 due to the potential blocking effect of the CD20 assay reagent on flow cytometry. For the specified groups, data on day 7 were obtained after a single dose of obin, AB-101, or obin + AB-101, and data on day 14 were obtained after two doses of obin, AB-101, or obin + AB-101. Obin = obituzumab. Six groups of six columns show the percentage (cells / µL) of baseline for each treatment (from left to right: solvent, Obin (D0), Obin (D0, D7), AB-101 (D0, D7), Obin + AB-101 (D0), Obin + AB-101 (D0, D7) for each treatment at each treatment post-treatment number of days (from left to right: 7, 14, 21, and 28).

[0048] Figure 12 Shows weight changes in the humanized NSG model. The mean (±SD) weight change is shown for all groups; there were no significant differences in the percentage of weight change among the solvent, Obin, AB-101, or Obin + AB-101 combined treatment groups. SD, standard deviation.

[0049] Figure 13 The pharmacokinetic profile of AB-101 is shown. The distribution of AB-101 in multiple tissues of NSG mice was determined by calculating the amount of AB-101 DNA per μg of mouse blood / tissue DNA. The data show the mean concentration (± sem) of AB-101 DNA in each organ, representing 6 mice (3 males and 3 females) at each time point.

[0050] Figure 14 Characterization of B cells from SLE and healthy donors is shown. B cell subsets from SLE and healthy donors were assessed by flow cytometry using the gating shown in the top row. Data are presented as mean + SEM. p = <0.05.

[0051] Figure 15 Characterization of NK cells from SLE and healthy donors. NK cell subsets and surface markers from SLE and healthy donors were characterized by flow cytometry. Data are shown as mean ± SEM. p<0.05, p<0.005. Detailed Implementation

[0052] This article provides, for example, natural killer (NK) cells such as expanded and stimulated NK cells, methods for generating NK cells, pharmaceutical compositions containing NK cells, and methods for treating patients with diseases such as autoimmune disorders using NK cells.

[0053] I. Proliferation and stimulation of natural killer cells

[0054] In some cases, such as the amplification and stimulation of NK cells as described in WO2022216813, the aforementioned literature is incorporated into the text in its entirety by reference.

[0055] In some cases, for example, after in vitro expansion and stimulation as described herein, the expanded and stimulated NK cell populations not only have a number / density that cannot naturally exist in the human body (e.g., as described above), but they also differ from the original source material or other naturally occurring NK cell populations in terms of their phenotypic characteristics (e.g., gene expression and / or surface protein expression).

[0056] In some cases, the originating NK cells are derived from samples from a single individual, such as a single umbilical cord blood unit that has not been expanded in vitro. Therefore, in some cases, the expanded and stimulated NK cells share a common lineage; that is, they are all generated from expansions of originating NK cells and thus share a common genotype through clonal expansion of a cell population originating from a single organism. However, they cannot naturally exist at the density achieved through in vitro expansion and also differ in phenotypic characteristics from those of originating NK cells.

[0057] In some cases, the population of expanded and stimulated NK cells contains at least 100 million expanded natural killer cells, such as 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, 1 trillion, 2 trillion, 3 trillion, 4 trillion, 5 trillion, 6 trillion, 7 trillion, 8 trillion, 9 trillion, or 10 trillion expanded natural killer cells.

[0058] In some implementations, the expanded and stimulated NK cells contain at least 80%, for example at least 90%, at least 95%, at least 99%, or 100% CD56+CD3- cells.

[0059] In some implementations, the expanded and stimulated NK cells do not contain the CD16 transgene.

[0060] In some implementations, the amplified and stimulated NK cells do not express exogenous CD16 protein.

[0061] For example, amplified and stimulated NK cells can be characterized by surface expression of more than one of, such as CD16, CD56, CD3, CD38, CD14, CD19, NKG2D, NKp46, NKp30, DNAM-1, and NKp44.

[0062] In some cases, the surface protein expression levels described herein are achieved without positive selection for the specific surface protein mentioned. For example, in some cases, NK cell sources such as a single umbilical cord unit contain both the KIR B allele of the KIR receptor family and the 158V / V variant of CD16, and are positively enriched and CD3(+) depleted, for example by gating CD56+CD3- expression, but without selection for other surface protein expression during amplification and stimulation.

[0063] In some implementations, for example, the expanded and stimulated NK cells from a single umbilical cord blood unit, such as as described above, contain at least 60%, for example, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% NKG2D+ cells.

[0064] In some implementations, for example, expanded and stimulated NK cells from a single umbilical cord blood unit, such as those described above, contain at least 60%, for example, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% NKp46+ cells.

[0065] In some implementations, for example, expanded and stimulated NK cells from a single umbilical cord blood unit, such as those described above, contain at least 60%, for example, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% NKp30+ cells.

[0066] In some implementations, for example, expanded and stimulated NK cells from a single umbilical cord blood unit, such as those described above, 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.

[0067] In some implementations, for example, the expanded and stimulated NK cells from a single umbilical cord blood unit, such as those described above, contain at least 60%, for example, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% NKp44+ cells.

[0068] In some implementations, for example, the expanded and stimulated NK cells from a single umbilical cord blood unit, such as those described above, 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.

[0069] In some implementations, for example, expanded and stimulated NK cells from a single umbilical cord blood unit, as described above, contain less than or equal to 20%, for example less than or equal to 10%, less than or equal to 5%, less than or equal to 1%, or 0% CD3+ cells.

[0070] In some implementations, for example, expanded and stimulated NK cells from a single umbilical cord blood unit, as described above, contain less than or equal to 20%, for example less than or equal to 10%, less than or equal to 5%, less than or equal to 1%, or 0% CD14+ cells.

[0071] In some implementations, for example, expanded and stimulated NK cells from a single umbilical cord blood unit, as described above, contain less than or equal to 20%, for example less than or equal to 10%, less than or equal to 5%, less than or equal to 1%, or 0% CD19+ cells.

[0072] In some implementations, for example, expanded and stimulated NK cells from a single umbilical cord blood unit, as described above, contain less than or equal to 20%, for example less than or equal to 10%, less than or equal to 5%, less than or equal to 1% or 0% CXCR+ cells.

[0073] In some implementations, for example, expanded and stimulated NK cells from a single umbilical cord blood unit, such as those described above, contain less than or equal to 20%, for example less than or equal to 10%, less than or equal to 5%, less than or equal to 1%, or 0% of CD122+ (IL2RB) cells.

[0074] In some implementations, for example, expanded and stimulated NK cells from a single umbilical cord blood unit, such as those described above, contain more than 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% CD3-CD14-CD19-CD16+CD56- cells.

[0075] As described herein, the inventors have demonstrated, surprisingly, that NK cells amplified and stimulated by the methods described herein express CD16 at high levels throughout the amplification and stimulation process, resulting in a cell population with high CD16 expression. This high CD16 expression eliminates the need to engineer the amplified cells to express CD16 (which is important for initiating ADCC), and thus leads to the surprising and unexpected benefits of the amplification and stimulation methods described herein. Therefore, in some embodiments, amplified and stimulated NK cells, for example from a single umbilical cord blood unit as described above, contain 50% or more, for example, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% CD16+ NK cells.

[0076] In some implementations, for example, expanded and stimulated NK cells from a single umbilical cord blood unit, such as those described above, 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.

[0077] In some implementations, the percentage of CD16+-expressing, for example, amplified and stimulated NK cells from a single cord blood unit as described above is the same as or higher than the percentage of natural killer cells from cord blood seed cells.

[0078] In some implementations, the percentage of expanded and stimulated NK cells expressing NKG2D, for example, from a single cord blood unit as described above, is the same as or higher than the percentage of natural killer cells from cord blood seed cells.

[0079] In some implementations, the percentage of expanded and stimulated NK cells expressing NKp30, for example, from a single cord blood unit as described above, is the same as or higher than the percentage of natural killer cells from cord blood seed cells.

[0080] In some implementations, the percentage of amplified and stimulated NK cells expressing DNAM-1, for example, from a single umbilical cord blood unit as described above, is the same as or higher than the percentage of natural killer cells from seed cells derived from umbilical cord blood.

[0081] In some implementations, the percentage of expanded and stimulated NK cells expressing NKp44, for example, from a single cord blood unit as described above, is the same as or higher than the percentage of natural killer cells from cord blood seed cells.

[0082] In some implementations, the percentage of expanded and stimulated NK cells expressing NKp46, for example, from a single cord blood unit as described above, is the same as or higher than the percentage of natural killer cells from cord blood seed cells.

[0083] As described herein, the inventors have also demonstrated, surprisingly, that NK cells amplified and stimulated by the methods described herein express CD38 at low levels. CD38 is an effective target for certain cancer therapies, such as 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).

[0084] Therefore, in some embodiments, amplified and stimulated NK cells, for example from a single umbilical cord blood unit as described above, contain less than or equal to 80% CD38+ cells, such as less than or equal to 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or 20% CD38+ cells.

[0085] In some embodiments, for example, expanded and stimulated NK cells from a single umbilical cord blood unit, such as those described above, contain both the KIR B allele of the KIR receptor family and the 158V / V variant of CD16, and contain less than or equal to 80% CD38+ cells, such as less than or equal to 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or 20% CD38+ cells.

[0086] In some embodiments, for example, expanded and stimulated NK cells from a single umbilical cord blood unit, such as those described above, contain both the KIR B allele of the KIR receptor family and the 158V / V variant of CD16, and contain less than or equal to 80% CD38+ cells, such as less than or equal to 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or 20% CD38+ cells, and more than 50%, such as 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% CD16+ NK cells.

[0087] In some embodiments, for example, expanded and stimulated NK cells from a single umbilical cord blood unit, such as those described above, comprise both the KIR B allele of the KIR receptor family and the 158V / V variant of CD16, and comprise: i) more than 50%, for example 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% CD16+ NK cells; and / or ii) less than or equal to 80% CD38+ cells, for example less than or equal to 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or 20% CD38+ cells; and / or iii) at least 60%, for example 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%, for example at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% NKG2D+ cells. 00% of NKp46+ cells; and / or v) at least 60%, for example at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% of NKp30+ cells; and / or vi) at least 60%, for example at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% of DNAM-1+ cells; and / or vii) at least 60%, for example at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% of NKp44+ cells; and / or viiii) at least 60%, for example at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% of CD94+ cells. (KLRD1) cells; and / or ix) less than or equal to 20%, for example less than or equal to 10%, less than or equal to 5%, less than or equal to 1% or 0% of CD3+ cells; and / or x) less than or equal to 20%, for example less than or equal to 10%, less than or equal to 5%, less than or equal to 1% or 0% of CD14+ cells; and / or xi) less than or equal to 20%, for example less than or equal to 10%, less than or equal to 5%, less than or equal to 1% or 0% of CD19+ cells; and / or xii) less than or equal to 20%, for example less than or equal to 10%, less than or equal to 5%, less than or equal to 1% or 0% of CXCR+ cells; and / or xiii) less than or equal to 20%, for example less than or equal to 10%, less than or equal to 5%, less than or equal to 1% or 0% of CD122+ (IL2RB) cells.

[0088] In some implementations, NK cells are engineered to alter (e.g., reduce) the expression of more than one inhibitory receptor gene.

[0089] 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.

[0090] In some implementations, 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.

[0091] This document also provides pharmaceutical compositions comprising the natural killer cells described herein and dosage units of the pharmaceutical compositions described herein.

[0092] In some cases, the dosage unit contains between 100 million and 1.5 billion cells, such as 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.

[0093] Pharmaceutical compositions typically include pharmaceutically acceptable carriers. As used herein, the term "pharmaceutically acceptable carrier" includes saline, solvent, dispersion medium, coating, antibacterial and antifungal agents, isotonic agents, and absorption delay agents that are compatible with drug administration.

[0094] 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.

[0095] In some embodiments, the composition is frozen. In some embodiments, the composition has been frozen for at least three months, such as at least six months, at least nine months, at least 12 months, at least 15 months, at least 18 months, at least 24 months, or at least 36 months.

[0096] In some implementations, at least 60%, such as at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% of the natural killer cells are viable after thawing.

[0097] In some embodiments, the pharmaceutical composition comprises: a) the cryopreserved composition described herein; and b) therapeutic cells, such as engineered NK cells described herein.

[0098] In some embodiments, the pharmaceutical composition further comprises: c) a buffer solution. Suitable buffer solutions are described herein, such as those used for cryopreservation of the composition.

[0099] In some embodiments, the pharmaceutical composition contains 1×10 7 Or approximately 1×10 7Up to 1×10 9 Or approximately 1×10 9 Cells / mL. In some embodiments, the pharmaceutical composition contains 1 × 10⁻⁶ cells / mL. 8 Cells / mL. In some embodiments, the pharmaceutical composition contains about 1 × 10⁻⁶ cells / mL. 8 Cells / mL.

[0100] In some embodiments, the pharmaceutical composition contains 1×10 8 Or approximately 1×10 8 Up to 1×10 10 Or approximately 1×10 10 Cells / mL.

[0101] In some embodiments, the pharmaceutical composition further comprises an antibody or an antigen-binding fragment thereof, such as the antibody described herein.

[0102] Pharmaceutical compositions are typically formulated to be compatible with their 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.

[0103] Methods for preparing suitable pharmaceutical compositions are known in the art, see, for example, Remington: The Science and Practice of Pharmacy, 21st ed., 2005; and Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY). For example, solutions or suspensions for parenteral, intradermal, or subcutaneous application may include the following components: sterile diluents, such as water for injection, saline solution, fixed oils, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates, or phosphates; and agents for adjusting tension, such as sodium chloride or glucose. pH may be adjusted with acids or bases (such as hydrochloric acid or sodium hydroxide). Parenteral preparations may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0104] Suitable pharmaceutical compositions for injectable use may include sterile aqueous solutions (in the case of water solubility) or dispersions, as well as sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, antibacterial water, Cremophor EL... TM(BASF, Parsippany, NJ) or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and should be a fluid sufficient for easy injection. It should be stable under manufacturing and storage conditions and must be preserved to prevent contamination by microorganisms such as bacteria and fungi. The carrier can 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. For example, appropriate flowability can be maintained by using coatings such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, it will be preferred to include isotonic agents in the composition, such as sugars, polyols such as mannitol, sorbitol, and sodium chloride. Prolonged absorption of the injectable composition can be achieved by including agents that delay absorption (e.g., aluminum monostearate and gelatin) in the composition.

[0105] Sterile injectable solutions can be prepared by mixing the desired amount of the active compound with one or a combination of the ingredients listed above (as needed) in a suitable solvent, followed by filtration and sterilization. Typically, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and any other desired ingredients from those listed above. In the case of sterile powders used to prepare sterile injectable solutions, preferred methods of preparation include vacuum drying and freeze-drying, which produce a powder of the active ingredient plus any additional desired ingredients from a previously sterile filtered solution.

[0106] Examples of suitable pharmaceutical compositions are described, for example, in WO2017 / 135631 and WO2022 / 0133061, which are incorporated herein by reference in their entirety.

[0107] II. Antibodies

[0108] The methods described herein include administering antibodies, such as antibodies targeting immune cells, such as immune cells involved in autoimmune responses, such as B cells. In some cases, the antibody is a B cell-depleting antibody, such as a B cell-depleting monoclonal antibody (mAb). In some cases, the antibody is a CD20 and / or CD19-targeting antibody. In some cases, the methods described herein include administering multiple antibodies, such as multiple antibodies targeting more than one type of immune cell, such as more than one type of immune cell involved in autoimmune responses, such as B cells. In some cases, more than one antibody is a B cell-depleting antibody, such as a B cell-depleting monoclonal antibody (mAb). In some cases, the antibody is selected from, for example, CD20 and / or CD19-targeting antibodies as described herein.

[0109] The term "antibody" refers to an immunoglobulin molecule or its immunologically active portion, i.e., the antigen-binding portion.

[0110] As used herein, the term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous population of antibodies (e.g., the individual antibodies constituting the population are identical, except for possible naturally occurring mutations present in small quantities). Antibodies can be monoclonal. Antibodies can be human antibodies or humanized antibodies. The term "monoclonal antibody" encompasses complete 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 such antibodies prepared using a variety of techniques, including but not limited to hybridoma production, phage library display, recombinant expression, and transgenic animals.

[0111] The term "chimeric antibody" refers to an antibody in which a portion of the heavy chain and / or light chain is derived from a first source or species, while the remainder of the heavy chain and / or light chain is derived from a different source or species.

[0112] As used herein, the term "humanized antibody" refers to an antibody comprising a human heavy chain variable region and a light chain variable region, wherein native CDR residues are replaced by residues of the corresponding CDR from a non-human antibody (e.g., a mouse, rat, rabbit, or non-human primate), wherein the non-human antibody has the desired specificity, affinity, and / or activity. In some embodiments, more than one framework region residue of the human heavy chain or light chain variable region is replaced by the corresponding residue from the non-human antibody. Furthermore, the humanized antibody may contain residues not found in either the human antibody or the non-human antibody. In some embodiments, these modifications are made to further improve and / or optimize antibody properties. In some embodiments, the humanized antibody comprises at least a portion of the immunoglobulin constant region (e.g., CH1, CH2, CH3, Fc), typically the constant region of human immunoglobulins.

[0113] As used herein, the term "human antibody" refers to an antibody having an amino acid sequence corresponding to that of antibodies produced by humans, and / or an antibody prepared using any technique known to those skilled in the art for the preparation of human antibodies. These techniques include, but are not limited to, phage display libraries, yeast display libraries, transgenic animals, recombinant protein production, and B-cell hybridoma techniques.

[0114] An "antibody fragment" may include a portion of a complete antibody, preferably the antigen-binding region or variable region of the complete antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0115] The terms “epitope” and “antigenic determinant” are used interchangeably herein, referring to the portion of an antigen or target that can be recognized and bound by a specific antibody. When the antigen or target is a polypeptide, an epitope can be formed from consecutive amino acids and spatially adjacent (juxtaposed) non-consecutive amino acids through the ternary folding of the protein. Epitopes formed from consecutive amino acids (also known as linear epitopes) are generally retained after protein denaturation, while epitopes formed from ternary folding (also known as conformational epitopes) are generally lost after protein denaturation. Epitopes typically contain at least 3, more often at least 5, 6, 7, or 8 to 10 amino acids with a unique spatial conformation. Epitopes can be predicted using any of the many software bioinformatics tools available on the internet. X-ray crystallography can be used to characterize epitopes on target proteins by analyzing the amino acid residue interactions of antigen / antibody complexes.

[0116] The “Fv” comprises the smallest antibody fragment containing a complete antigen recognition and binding site. This region consists of a dimer composed of a heavy chain variable domain and a light chain variable domain in close, non-covalent bonding. In this configuration, the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Overall, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of the Fv containing only the three CDRs specific to the antigen) has the ability to recognize and bind to the antigen, although its affinity is lower than that of the complete binding site. The Fab fragment also contains 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 in that several residues are added to the carboxyl terminus of the heavy chain CH1 domain, including more than one cysteine ​​residue from the antibody hinge region. Fab'-SH is the name given to Fab' in this paper, where the cysteine ​​residues of the constant domain have a free thiol group. The F(ab')2 antibody fragment was originally generated as a Fab' fragment pair with a hinge cysteine ​​residue between them. Other chemical conjugation methods for antibody fragments are also known.

[0117] Immunoglobulins can be classified into different classes based on the amino acid sequence of their constant domains in their heavy chain. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further subdivided into subclasses (isotypes), such as IgB1, IgG2, IgG3, IgG4, IgA1, and IgA2. A "single-chain Fv" or "sFv" antibody fragment contains the antibody's VH and VL domains, which are present within a single polypeptide chain. In some cases, the Fv polypeptide further includes a polypeptide linker between the VH and VL domains, enabling the sFv to form the structure required for antigen binding.

[0118] In various embodiments, the antibody or its antigen-binding fragment comprises a human antibody or a humanized antibody. Humanized forms of non-human (e.g., mouse) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of the antibody) containing a minimal sequence derived from a non-human immunoglobulin. Humanized antibodies comprise human immunoglobulins (receptor antibodies) in which residues from the complementarity-determining region (CDR) of the receptor are replaced by residues from the CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit, said donor antibody having the desired specificity, affinity, and capability. In some cases, Fv framework residues of human immunoglobulins are replaced by corresponding non-human residues. Humanized antibodies may also contain residues not found in the receptor antibody or in the introduced CDR or framework sequence. Typically, humanized antibodies will contain substantially all of at least one, usually two, variable domains, wherein all or substantially all of the CDR regions correspond to those of non-human immunoglobulins, and all or substantially all of the FR regions are those of the common sequences of human immunoglobulins. Methods for humanizing non-human antibodies are well known in the art.

[0119] An antibody that “binds,” “specifically binds,” or is “specific to” a specific polypeptide or epitope on a specific polypeptide is an antibody that binds to that specific polypeptide or epitope on a specific polypeptide and substantially does not bind to any other polypeptides or polypeptide epitopes. As used herein, the term “specific binding” means that the binder (e.g., an antibody) interacts with a specific antigen, epitope, protein, or target molecule more frequently, more quickly, for a longer duration, with a greater affinity, or in some combination of the above, compared to an alternative substance. A binder (e.g., an antibody) that specifically binds to an antigen can be identified by, for example, immunoassays, ELISA, surface plasmon resonance (SPR) assays (e.g., Biacore), or other techniques known to those skilled in the art. A binder that specifically binds to an antigen binds to a target antigen with a higher affinity than it has for a different antigen. This different antigen may be a related antigen. In some embodiments, the binder that specifically binds to the antigen binds to the target antigen with an affinity at least 20 times greater than its affinity for the different antigens, for example, at least 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, or 100 times greater. In some embodiments, the binder that specifically binds to a particular antigen binds to different antigens with such low affinity that it cannot be detected using assays described herein or otherwise known in the art. In some embodiments, SPR technology is used to measure affinity, for example, in the Biacore system or other systems known to those skilled in the art.

[0120] In some embodiments, the antibody or its antigen-binding fragment is an antibody, such as a full-length antibody comprising an Fc domain containing 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.

[0121] 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-linked 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 monospecific 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 binding agent is a polyclonal antibody. Polyclonal antibodies can be prepared by any method known to those skilled in the art.

[0122] In some embodiments, the binding agent is a monoclonal antibody. Monoclonal antibodies can be prepared by any method known to those skilled in the art. In some embodiments, the binding agent is a humanized antibody. Various methods for generating humanized antibodies are known in the art. In some embodiments, the binding agent is a human antibody. Human antibodies can be prepared using various techniques known in the art.

[0123] In some implementations, the binder is an scFv antibody, Fv, Fab, F(ab')2, F(ab') or a bispecific antibody.

[0124] In some embodiments, bispecific antibodies have reduced toxicity and / or side effects. In some embodiments, bispecific antibodies have 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 have an increased therapeutic index. In some embodiments, bispecific antibodies have an increased therapeutic index compared to a mixture of two individual antibodies or an antibody as a single agent. Several techniques for preparing bispecific antibodies are known to those skilled in the art. In some embodiments, the bispecific antibody comprises a heavy chain constant region that is modified in an amino acid that forms part of the interface between two heavy chains. These modifications are made to enhance heterodimer formation and generally reduce or eliminate homodimer formation. In some embodiments, a knock-in-holes (KIH) strategy is used to generate the bispecific antibody. In some embodiments, the bispecific antibody comprises a variant hinge region that cannot form disulfide bonds between identical heavy chains (e.g., reducing homodimer formation). In some embodiments, the bispecific antibody comprises a heavy chain with amino acid variations that result in altered electrostatic interactions. In some embodiments, the bispecific antibody comprises a heavy chain with amino acid changes that result in altered hydrophobic / hydrophilic interactions. The bispecific antibody can be a complete antibody or an antibody fragment containing an antigen-binding site.

[0125] Binding agents with more than two valence states were also considered. In some embodiments, trispecific or tetraspecific antibodies are generated.

[0126] 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.

[0127] In some cases, the antibody or its antigen-binding fragment is an antibody or combination of antibodies selected from Tables 1, 2, or 3. In some cases, the antibody or its antigen-binding fragment is an IgG antibody selected from Tables 1, 2, or 3. In some cases, the antibody or its antigen-binding fragment is an IgG1 antibody selected from Tables 1, 2, or 3. In some cases, the patient suffers from a condition, and the antibody or its antigen-binding fragment is approved (e.g., by a regulatory agency such as the US Food and Drug Administration or the European Medicines Agency) as a therapeutic agent, as reflected in Tables 1, 2, or 3.

[0128] In some cases, antibodies or their antigen-binding fragments are engineered to enhance the binding of their Fc domain to activating receptors (such as Fcγ receptors), compared to unengineered antibodies.

[0129] Table 1. CD19 and CD20-targeting antibody therapies approved in the US or Europe. Therapeutic monoclonal antibodies approved or under review in the EU or US. (April 17, 2023); antibodysociety.org / resources / approved-antibodies.

[0130]

[0131] Table 2. Commercially sponsored monoclonal antibody therapeutics first approved or under regulatory review outside the EU or US in 2022. Kaplon et al. (2023) Antibodies to watch in 2023, mAbs, 15:1, DOI:10.1080 / 19420862.2022.2153410

[0132]

[0133] Table 3. CD20-targeting antibodies

[0134]

[0135]

[0136]

[0137]

[0138] In some cases, antibodies or their antigen-binding fragments are NK cell engagers, such as bispecific or trispecific antibodies, which bridge NK cell activation receptors (e.g., CD16A, NKG2D, NKp30, or NKp46) with molecules specific to disease cells (e.g., tumor cells). See, for example, Demaria et al., “Natural Killer Cell Engaggers in Cancer Immunotherapy: Next Generation of Immuno-Oncology Treatments,” European Journal of Immunology 51(8):doi.org / 10.1002 / eji.202048953 (2021).

[0139] III. Pharmaceutical Compositions

[0140] This document provides pharmaceutical compositions comprising the natural killer cells described herein and dosage units of the pharmaceutical compositions described herein.

[0141] In some cases, the dosage unit contains between 100 million and 1.5 billion cells, such as 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, or contains approximately the aforementioned number of cells.

[0142] In some cases, the dosage unit contains 100 million to 10 billion cells, such as 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, or 10 billion cells, or contains approximately 100 million to 10 billion cells.

[0143] Pharmaceutical compositions typically include pharmaceutically acceptable carriers. As used herein, the term "pharmaceutically acceptable carrier" includes saline, solvent, dispersion medium, coating, antibacterial and antifungal agents, isotonic agents, and absorption delay agents that are compatible with drug administration.

[0144] In some embodiments, the pharmaceutical composition comprises: a) the natural killer cells described herein; and b) a cryopreservation composition.

[0145] This article describes suitable cryopreservation compositions.

[0146] In some embodiments, the composition is frozen. In some embodiments, the composition has been frozen for at least three months, such as at least six months, at least nine months, at least 12 months, at least 15 months, at least 18 months, at least 24 months, or at least 36 months.

[0147] In some implementations, at least 60%, such as at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% of the natural killer cells are viable after thawing.

[0148] In some embodiments, the pharmaceutical composition comprises: a) the cryopreservation composition described herein; and b) therapeutic cells.

[0149] In some embodiments, the therapeutic cells are animal cells. In some embodiments, the therapeutic cells are human cells.

[0150] 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.

[0151] In some implementations, the immune cells are natural killer (NK) cells. In some implementations, natural killer cells are expanded and stimulated using the methods described herein.

[0152] In some embodiments, the pharmaceutical composition further comprises: c) a buffer solution. Suitable buffer solutions are described herein, such as those used for cryopreservation of the composition.

[0153] In some embodiments, the pharmaceutical composition contains 1×10 7 Or approximately 1×10 7 Up to 1×10 9 Or approximately 1×10 9 Cells / mL. In some embodiments, the pharmaceutical composition contains 1 × 10⁻⁶ cells / mL. 8 Cells / mL. In some embodiments, the pharmaceutical composition contains about 1 × 10⁻⁶ cells / mL. 8 Cells / mL.

[0154] In some embodiments, the pharmaceutical composition contains 1×10 8Or approximately 1×10 8 Up to 1×10 10 Or approximately 1×10 10 Cells / mL.

[0155] In some embodiments, the pharmaceutical composition further comprises an antibody or an antigen-binding fragment thereof, such as the antibody described herein.

[0156] Pharmaceutical compositions are typically formulated to be compatible with their 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.

[0157] Methods for preparing suitable pharmaceutical compositions are known in the art, see, for example, Remington: The Science and Practice of Pharmacy, 21st ed., 2005; and Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY). For example, solutions or suspensions for parenteral, intradermal, or subcutaneous application may include the following components: sterile diluents, such as water for injection, saline solution, fixed oils, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates, or phosphates; and agents for adjusting tension, such as sodium chloride or glucose. pH may be adjusted with acids or bases (such as hydrochloric acid or sodium hydroxide). Parenteral preparations may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0158] Suitable pharmaceutical compositions for injectable use may include sterile aqueous solutions (in the case of water solubility) or dispersions, as well as sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, antibacterial water, Cremophor EL... TM(BASF, Parsippany, NJ) or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and should be a fluid sufficient for easy injection. It should be stable under manufacturing and storage conditions and must be preserved to prevent contamination by microorganisms such as bacteria and fungi. The carrier can 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. For example, appropriate flowability can be maintained by using coatings such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, it will be preferred to include isotonic agents in the composition, such as sugars, polyols such as mannitol, sorbitol, and sodium chloride. Prolonged absorption of the injectable composition can be achieved by including agents that delay absorption (e.g., aluminum monostearate and gelatin) in the composition.

[0159] Sterile injectable solutions can be prepared by mixing the desired amount of the active compound with one or a combination of the ingredients listed above (as needed) in a suitable solvent, followed by filtration and sterilization. Typically, dispersions are prepared by mixing the active compound into a sterile vehicle containing a basic dispersion medium and other desired ingredients from those listed above. In the case of sterile powders used to prepare sterile injectable solutions, preferred methods of preparation include vacuum drying and freeze-drying, which produce powders of the active ingredient and any other desired ingredients from a previously sterile filtered solution.

[0160] IV. Treatment Methods

[0161] The NK cells described in this article can be used to treat autoimmune diseases.

[0162] Therefore, this article also provides a method for treating patients with diseases, such as those related to autoimmunity, such as autoreactive immune cells, such as autoreactive B cells, the method comprising administering NK cells as described herein and CD19 and / or CD20 targeting antibodies as described herein.

[0163] As used herein, the terms "treatment," "treat," and "treating" refer to reversing, alleviating, delaying the onset of, or inhibiting the progression of an autoimmune-related condition, such as the condition described herein, such as systemic lupus erythematosus ("SLE"). In some embodiments, treatment may be administered after more than one symptom has occurred. In other embodiments, treatment may be administered in the absence of symptoms. For example, treatment may be administered to a susceptible individual before the onset of symptoms (e.g., based on a history of symptoms and / or based on genetic or other susceptibility factors). Treatment may also continue after symptoms have subsided, for example, to prevent or delay recurrence.

[0164] As used herein, “delayed” occurrence of a disease or condition or one or more symptoms thereof means delaying, hindering, mitigating, blocking, stabilizing, and / or postponing the onset of a disease, condition, or symptom thereof. Such delay can vary in duration, depending on medical history and / or the subject’s treatment history. As will be apparent to those skilled in the art, sufficient or significant delay may indeed include prevention, as the subject does not develop the disease, condition, or symptoms thereof. For example, a method of “delaying” the onset of an autoimmune condition is a method of reducing the probability of disease occurrence and / or reducing the severity of the condition within a given time frame, compared to not using said method. Such comparison may be based on clinical studies using statistically significant numbers of subjects.

[0165] As used herein, “prevention” refers to a program to prevent the onset of a disease or condition so that the clinical symptoms of the disease do not occur. Therefore, “prevention” involves administering a therapy (e.g., a therapeutic substance) to a subject before and / or before a stage of the disease when signs of the disease are detectable. Subjects may be individuals at risk of developing a disease or condition or at risk of disease progression, such as proliferative lupus nephritis. This includes individuals with more than one known risk factor associated with the onset or progression of a disease or condition. For example, an individual may have a mutation associated with the onset or progression of an autoimmune disease (e.g., SLE). Furthermore, it should be understood that prevention may not result in complete protection against the onset of a disease or condition. In some cases, prevention involves reducing the risk of developing a disease or condition. A reduction in risk may not result in the complete elimination of the risk of developing a disease or condition.

[0166] In some cases, an "increased" or "enhanced" amount means 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) (inclusive of all integers and decimals greater than 1, such as 2.1, 2.2, 2.3, 2.4, etc.). It 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.

[0167] A “decreased” or “lesser” amount means approximately 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) (inclusive of all integers and decimals greater than or equal to 1, such as 1.5, 1.6, 1.7, 1.8, etc.). It 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%, 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.

[0168] This document also provides a method for depleting immune cells (e.g., autoreactive immune cells, such as autoreactive B cells), comprising administering said NK cells (e.g., the NK cells described herein) and CD19 and / or CD20 targeting antibodies (e.g., the antibodies 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 some 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, such as CD19+ / CD20-, CD19+ / CD20+, or CD19- / CD20+ autoreactive immune cells, such as CD19+ / CD20-, CD19+ / CD20+, or CD19- / CD20+ autoreactive B cells.

[0169] In some cases, immune cells (e.g., autoreactive immune cells) comprise or are composed of B cells (e.g., autoreactive B cells). In some cases, B cells are CD19+ / CD20- B cells, CD19+ / CD20+ B cells, CD19- / CD20+ B cells, or combinations thereof.

[0170] In some cases, B cells are selected from B cells of the anus and rectum, B cells of the appendix, B cells of the medullary sinus of the lymph node, B cells of the mantle zone of the lymph node, monocytoid B cells, CD19 positive B cells, and combinations thereof.

[0171] In some cases, CD19-positive B cells are selected from immature B cells, mature B cells, precursor B cells, transitional B cells, and combinations thereof.

[0172] In some cases, immature B cells are selected from CD38-negative immature B cells, E-fraction immature B cells, and combinations thereof.

[0173] In some cases, mature B cells are selected from B-1 B cells, B-2 B cells, Be cells, Peyer's spatch B cells, follicular B cells, F-fraction mature B cells, germinal center B cells, marginal zone B cells of lymph nodes, marginal zone B cells of the spleen, memory B cells, immature B cells, plasmablasts, regulatory B cells, and combinations thereof.

[0174] In some cases, B-1 B cells are selected from B-1a B cells, B-1b B cells, and combinations thereof. In some cases, B-2 B cells are selected from Peyer's node B cells, follicular B cells, F-fraction mature B cells, and combinations thereof. In some cases, follicular B cells are selected from Bm1 B cells, Bm2 B cells, and combinations thereof. In some cases, F-fraction mature B cells are Bm1 B 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, Bm3 B cells, Bm3-delta B cells, Bm4 B cells, centroblasts, centrocytes, tonsillar germinal center B cells, and combinations thereof.

[0175] In some cases, memory B cells are selected from Bm5 B cells, IgD-negative memory B cells, IgM memory B cells, class-switched memory B cells, double-negative memory B cells, unswitched memory B cells, and combinations thereof.

[0176] 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 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.

[0177] In some cases, class-switching memory B cells are selected from IgA memory B cells, IgE memory B cells, IgG memory B cells, IgG-negative class-switching 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-switching memory B cells are selected from CD38-positive IgG-negative class-switching memory B cells, CD38-positive IgG-negative class-switching memory B cells, and combinations thereof. In some cases, CD38-negative IgG-negative class-switching memory B cells are selected from CD24-negative CD38-negative IgG-negative class-switching memory B cells, CD24-positive CD38-negative IgG-negative class-switching memory B cells, and combinations thereof. In some cases, CD38-positive IgG-negative class-converting memory B cells are selected from B220-low CD38-positive IgG-negative class-converting memory B cells, B220-positive CD38-positive IgG-negative class-converting memory B cells, and combinations thereof. In some cases, B220-positive CD38-positive IgG-negative class-converting memory B cells are B220-low CD38-positive IgG-negative class-converting 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, unconverted memory B cells are selected from CD38-negative unconverted memory B cells, CD38-positive unconverted memory B cells, and combinations thereof. In some cases, CD38-negative unconverted memory B cells are selected from B220-low CD38-negative unconverted memory B cells, B220-positive CD38-negative unconverted memory B cells, and combinations thereof. In some cases, B220-positive CD38-negative unconverted memory B cells are B220-low CD38-negative unconverted memory B cells. In some cases, CD38-positive unconverted memory B cells are selected from B220-low CD38-positive unconverted memory B cells, B220-positive CD38-positive unconverted memory B cells, and combinations thereof. In some cases, B220-positive CD38-positive unconverted memory B cells are B220-low CD38-positive unconverted 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 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.

[0178] In some cases, precursor B cells are selected from B / C fraction precursor B cells, C' fraction precursor B cells, D fraction precursor B cells, late pro-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.

[0179] In some cases, transitional B cells are selected from T1 B cells, T2 B cells, T3 B cells, and combinations thereof.

[0180] A. Symptoms

[0181] The methods and compositions disclosed herein can be used to target a variety of conditions, such as autoimmune diseases. The advantage of the methods described herein is that they combine allogeneic cells with exogenous antibodies to specifically target immune cells such as B cells.

[0182] In some cases, autoimmune diseases are driven by autoantibodies and / or autoreactive B cells.

[0183] 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) | catastrophic antiphospholipid syndrome (CAPS) / Asherson syndrome, arteriosclerosis, autoimmune Addison's disease (AAD), autoimmune autonomic ganglionosis (AAG) / autoimmune autonomic dysfunction | 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), type I, II, and III autoimmune polyglandular syndromes (Type 1 APS, Type 2 APS, Type 3 APS, APECED), autoimmune progesterone dermatitis, autoimmune retinopathy (AIR), autoimmune sudden sensorineural hearing loss (SNHL), Barlow's disease, Behcet's disease, shotgun-like chorioretinopathy / shotgun-like uveitis, bullous pemphigoid, Kassman's disease, celiac disease, and chalcogenide. Gast's disease, chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), chronic urticaria (CU), Chag-Strauss syndrome / eosinophilic granulomatous polyangiitis (EGPA), Cogan syndrome, cold agglutinin disease, CREST syndrome | cutaneous systemic scleroderma, Crohn's disease (CD), Cronkhite-Canada syndrome (CSS), cryptogenic organizing pneumonia (COP), herpetiform dermatitis, dermatomyositis, type 1 diabetes mellitus, discoid lupus, Dressler syndrome / post-myocardial infarction / post-pericardiotomy syndrome, eczema / atopic dermatitis, endometriosis, eosinophilic esophagitis, eosinophilic fasciitis, erythema nodosum, primary mixed Cryoglobulinemia, Evans syndrome, fibrotic alveolitis / idiopathic pulmonary fibrosis (IPF), giant cell arteritis / temporal arteritis / Houghton's disease, giant cell myocarditis, glomerulonephritis, Goodbast syndrome / anti-GBM / anti-TBM disease, granulomatous polyangiitis (GPA) / Wegener's granulomatosis, Graves' disease / thyroid ophthalmopathy, Guillain-Barré syndrome (GBS), Hashimoto's thyroiditis / chronic lymphocytic thyroiditis / autoimmune thyroiditis, Henno-Schlan purpura / IgA vasculitis, hidradenitis suppurativa, Hurst's disease / acute hemorrhagic leukoencephalitis (AHLE), hypogammaglobulinemia, IgA nephropathy / Beger's disease, immune-mediated necrotizing myopathy (IMNM)Immune thrombocytopenic purpura (ITP), inclusion body myositis, IgG4-associated sclerotic disease (ISD), interstitial cystitis, juvenile idiopathic arthritis, adult-onset Still's disease, juvenile polymyositis, juvenile dermatomyositis, juvenile myositis, Kawasaki disease, Lambert-Eton myasthenic syndrome (LEMS), leukocytic clotting 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, lymphocytes Hypophysitis / Autoimmune Hypophysitis, Meniere's Disease, Microscopic Polyangiitis (MPA) / ANCA-associated vasculitis, Mixed Connective Tissue Disease (MCTD), Mollen's Ulcer, Mueller-Hacker Disease, Multifocal Motor Neuropathy, Multiple Sclerosis (MS), Myalgic Encephalomyelitis (ME) / Chronic Fatigue Syndrome (CFS), Myasthenia Gravis (MG), Narcolepsy, Neuromyelitis Optic / Devik's Disease, Ocular Cicatricial Pemphigoid, Ocular Clonic-Myoclonic Syndrome (OMS), Recurrent Rheumatism, Paraneoplastic Cerebellar Degeneration, Paraneoplastic Pemphigus, Parry-Romberg Syndrome (PRS) / Hypoplastic Facial Atrophy (HFA) / Progressive Hemifacial Atrophy, Paroxysmal Nocturnal Hemoglobinuria (PNH) Peripheral uveitis / spasm, PANS / PANDAS, Parson-Turner syndrome, pemphigoid of pregnancy / herpes gestationis, 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 gangrenosa, Rasmussen encephalitis, Raynaud's syndrome / phenomenon, reactive arthritis / Reiter's syndrome, reflex sympathetic dystrophy. Complex regional pain syndrome (RSD) / complex regional pain syndrome (CRPS), recurrent polychondritis, restless legs syndrome (RLS) / Willis-Eckbond disease, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome / type II autoimmune polyendocrine syndrome, scleritis, scleroderma, sclerosing mesenteric / mesenteric panniculitis, creeping choroidal lesions, Sjögren's syndrome, stiff-person syndrome (SPS), small fiber sensory neuropathy, systemic lupus erythematosus (SLE), subacute bacterial endocarditis (SBE), subacute cutaneous lupus, Sussac syndrome, Sidnum chorea, sympathetic ophthalmia, Goran's arteritis (vasculitis), testicular autoimmune disease (vasculitis, orchitis), Tolosa-Hunter syndrome,Transverse myelitis (TM), tubulointerstitial nephritis-uveitis syndrome (TINU), ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), pre- / middle / post-uveitis, vasculitis, VEXAS syndrome, vitiligo, Vogt-Koyanagi-Harada syndrome (VKH), and combinations thereof.

[0184] In some implementations, treatment includes improvement of symptoms in a patient or patient group (e.g., as described herein). In some cases, the improvement is compared to a baseline or threshold value. In some cases, the baseline or threshold value is a value generally considered to be within the normal (e.g., healthy) range. In some cases, the baseline or threshold value is based on pre-treatment and post-treatment values ​​(e.g., a patient's own pre-treatment baseline score or a patient group's pre-treatment baseline score).

[0185] In some cases, improvement is measured immediately after application, such as within a treatment cycle. In some cases, the response is measured within one week after application, such as 1, 2, 3, 4, 5, 6, or 7 days. In some cases, the response is measured after the last application of the first treatment cycle (as described herein). In some cases, the response is measured after the last application of the second treatment cycle (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 application, such as the last application of a treatment cycle.

[0186] 1. Lupus

[0187] In some cases, the autoimmune disease is systemic lupus erythematosus (SLE). In other cases, it is lupus nephritis.

[0188] 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 leads to systemic autoimmunity and can cause damage to various tissues and organs (Pisetsky 2001, Anders 2020).

[0189] SLE can lead to arthritis, kidney failure, cardiopulmonary complications, central nervous system (CNS) changes, vasculitis, severe skin rashes, and blood dyscrasias such as anemia, leukopenia, and thrombocytopenia. The presentation of SLE varies from patient to patient and may take many years to arrive at a correct diagnosis.

[0190] Initial treatment for SLE typically consists of glucocorticoids combined with mycophenolate mofetil (MMF) or cyclophosphamide. Reasonable alternatives to initial treatment include MMF combined with a calcineurin inhibitor (vorticol or tacrolimus) or belimumab, or cyclophosphamide combined with belimumab. Patients who fail initial treatment should be switched to alternative therapies, including intravenous cyclophosphamide and / or anti-CD20 monoclonal antibodies (mAbs).

[0191] Lupus nephritis (LN) is a form of glomerulonephritis and constitutes one of the most severe organ manifestations of SLE. In many cases, LN is the initial presentation leading to the 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 the progression of CKD, reduce morbidity and mortality, minimize treatment-related toxicities, and maintain fertility. Standard care involves the use of immunosuppressive agents, such as glucocorticoids, and cyclophosphamide (CYC) or mycophenolate mofetil (MMF), as well as adjuvant therapy, and is based on histological disease subtype. Immunosuppressive therapy is primarily used to treat type III and IV LN and is divided into two phases: an induction phase, in which intensive immunosuppression is administered, typically lasting 3 to 6 months; and a maintenance phase, in which prolonged, lower-intensity treatment is administered to prevent renal flare.

[0192] However, traditional immunosuppressive therapy is not consistently effective. The Lupus Nephritis Collaborative Study Group illustrates the importance of achieving complete or partial remission. Patients achieving complete remission had better 10-year patient survival and renal survival rates (95% and 94%, respectively) compared to those achieving partial remission (76% and 45%, respectively) (Korbert SM 2000). For patients who did not respond to treatment, the 10-year patient survival rates were 46% and 13%, respectively (Chen YE 2008) (Houssiau FA 2004, Houssiau F and Ginzler, 2008).

[0193] The management of patients with refractory disease varies depending on the first-line drug used for induction therapy, clinical factors, and local practice. Switching to another first-line induction drug is the recommended initial approach for patients with refractory LN, and is recommended by the EULAR / ERA-EDTA and American College of Rheumatology guidelines (Yo JH 2019). Typically, CYC-resistant patients are treated with MMF, and MMF-resistant patients are treated with CYC. In addition to switching immunosuppressants, some patients also receive a 3-day intravenous glucocorticoid pulse (Hahn BH 2012).

[0194] LN is histologically classified into six distinct categories, representing different manifestations and severities of renal involvement in SLE (Bajema IM 2018). The classification of LN is based on prognostic differences. Type VI LN essentially presents as renal atrophy in end-stage renal disease (ESKD), while patients with type III, IV, or V LN, but not type I or II, face a direct risk of progression to chronic kidney disease (CKD), which shortens kidney lifespan (Romagnani P 2017, Mackay M 2019). Understanding of the genetics and pathogenesis of LN has greatly improved over the past few decades. However, despite increasing awareness and improved treatment options, LN remains a substantial cause of morbidity and death in patients with SLE. The 5-year mortality rate for LN declined between 1975 and 1995 but has since remained stable, with the rate of progression to ESKD remaining constant (Croca SC 2011, Anders 2020).

[0195] B cells play a major role in the pathogenesis of lymphoma (LN) and are therefore an attractive therapeutic target. Rituximab (RTX) is a chimeric mouse-human monoclonal antibody targeting the B cell surface molecule CD20. Several uncontrolled studies have reported the efficacy of RTX in patients with refractory LN (Lan 2012). However, RTX failed to show benefit in the EXPLORER trial, which excluded patients with severe active LN (Merril JY 2010). Furthermore, the randomized, double-blind, placebo-controlled LUNAR trial (LUNAR) comparing RTX with placebo added to standard of care using prednisolone and MMF in newly diagnosed LN failed to demonstrate significant therapeutic efficacy (Rovin BH 2012). However, a higher complete remission rate regarding proteinuria was observed in patients receiving RTX (32 vs 9%). This reduction in proteinuria persisted until week 78, suggesting that a longer follow-up period may yield significant differences. The study also found higher partial response rates in all patients in the RTX group and in a pre-specified subgroup of African American patients; however, the trial was insufficient to detect differences in partial response rates (YoJH 2019). A recent post-hoc analysis of the LUNAR trial showed significant variability in peripheral blood B-cell depletion in patients with LN treated with RTX. The rate and duration of achieving complete peripheral depletion (0 peripheral B cells / µL) were associated with complete remission at week 78 (Gomez Mendez LM 2018). This was hypothesized to be due to the persistent presence of autoreactive pathogenic B cells in lymphoid structures and the renal tubulointerstitium (Ahuja A 2007).

[0196] Despite the failure of the LUNAR trial to meet its primary endpoint, advocacy for the use of RTX in refractory LN continues and is primarily based on observational evidence, particularly in patients with inadequate responses to first-line induction regimens. In a systematic review of published case reports and case series on the efficacy of RTX in patients with refractory LN, among 300 patients followed for 60 weeks, sustained complete and partial remission rates were 87%, 76%, 67%, and 76% in patients with type III, IV, V, and mixed types, respectively (Weidenbusch M 2013).

[0197] Oxtuzumab is a glycoengineered anti-CD20 monoclonal antibody designed to enhance antibody-dependent cell cytotoxicity (ADCC). It has a type II binding conformation, resulting in a stronger direct cell death effect and more limited monoclonal antibody internalization. These properties lead to more significant and sustained B-cell exhaustion compared to rituximab [Reddy V 2017]. Because better B-cell exhaustion, particularly in the kidneys themselves, could potentially improve CRR rates, oxtuzumab was tested in a small phase II trial called NOBILITY [Furie 2020]. Oxtuzumab was compared to placebo in the context of MMF and moderate-dose rapid tapering of glucocorticoids. The percentage of patients achieving complete renal response (CRR) was significantly higher for those receiving oxtuzumab compared to those receiving placebo, reaching 35% (vs. 23%) at week 76 and 41% (vs. 23%) at week 104. Almost all patients still had very low peripheral B-cell counts (CD19+ count ≤ 5 cells / µL) at week 52, a finding quite different from the LUNAR trial, in which only half of the RTX-treated patients had undetectable peripheral B-cell counts one year after treatment [Rovin BH 2012]. This may be confirmed by a global phase III trial (NCT04221477).

[0198] The roles of pathogenic B and T cells in SLE have been well studied, but the role of innate immune cells has not been fully established. Natural killer (NK) cells are a key component of the innate immune system, providing immune surveillance by recognizing healthy cells and eliminating stress cells, including virus-infected cells and cancer cells.

[0199] NK cell dysfunction has been reported in SLE. NK cells are reduced in number in the peripheral blood of SLE patients, exhibiting decreased cytotoxicity, impaired differentiation, and altered cytokine production (Humbel, 2021, Liu, 2021, Lu, 2022). Interestingly, the reduction in NK cell numbers is associated with disease activity, with patients with higher disease activity showing a greater reduction in NK cell numbers (Humbel, 2021). NK cell dysfunction in SLE may contribute to the pathogenesis of the disease by impairing the clearance of apoptotic cells and immune complexes, leading to the release of self-antigens and the activation of self-reactive B and T cells. Furthermore, altered cytokine production by NK cells may promote the chronic inflammation observed in SLE.

[0200] In addition to reduced NK cell numbers and dysfunction, certain subsets of peripheral SLE NK cells have been reported to exhibit increased IFNγ production and an activated phenotype (Liu, 2021). However, data from different studies are inconsistent and may be related to disease stage and treatment agents. Furthermore, much of our knowledge about the characterization of NK cells in SLE comes from peripheral blood; little is known about the function of tissue-resident NK cells. Recently, single-cell RNA sequencing evaluated the renal immune cell profile of SLE patients, identifying two distinct NK cell clusters (Azari, 2019). Further research is needed to determine how specific NK cell subsets contribute to the pathogenesis of SLE.

[0201] In some cases, treatment of SLE includes an improvement in overall renal response rate (ORR) (e.g., as described above). In some cases, the improvement is compared to the patient's pre-treatment ORR. In some cases, the improvement is compared to a baseline or threshold value. In some cases, the baseline or threshold value is a value generally considered to be within the normal (e.g., healthy) range. In some cases, the baseline or threshold value is based on the patient's own level, such as pre-treatment levels. In some cases, the improvement is a complete renal response (CRR). In some cases, CRR is a urine protein to creatinine ratio (UPCR) <0.5 and / or normal renal function (serum creatinine less than or equal to ULN) with a decline in baseline serum creatinine not exceeding 15%. In some cases, the improvement is a partial renal response (PRR). In some cases, PRR is a decrease in UPCR from baseline of more than 50% to a value <1 (or a value to <3 if the baseline UPCR is more than 3) and / or an increase in serum creatinine from baseline of not more than 15%. In some cases, the therapeutic response is measured after administration, such as during a treatment cycle. In some cases, the therapeutic response is measured within one week of administration, for example, within 1, 2, 3, 4, 5, 6, or 7 days. In some cases, the therapeutic response is measured after the last administration of the first treatment cycle (as described herein). In some cases, the therapeutic response is measured after the last administration of the second treatment cycle (as described herein). In some cases, the therapeutic response is measured within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 12 months after administration, for example, the last administration of a treatment cycle. In some cases, the therapeutic response (e.g., CRR or PRR) is measured at weeks 12, 22, 52, 76, and / or 104.

[0202] In some cases, SLE treatment includes improvements in one or more of the following (e.g., as described above): the SLE Disease Activity Index (SLEDAI), the combined SELENA-SLEDAI (Systemic Lupus Erythematosus Disease Activity Index), the Systemic Lupus Erythematosus Activity Measurement (SLAM), C3 complement levels, and C4 complement levels. In some cases, improvements occur at 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.

[0203] 2. Rheumatoid Arthritis (RA)

[0204] In some cases, an autoimmune disease is rheumatoid arthritis (RA).

[0205] Rheumatoid arthritis (RA) is a systemic autoimmune disease with an annual incidence of 41 per 100,000 population in the United States and Northern European countries (Myasoedova, 2021). It is characterized by symmetrical inflammatory polyarthritis, but extra-articular features are also common and often associated with poor prognosis. Refractory RA represents an unmet need area. Most patients achieve adequate control with methotrexate and other first-line disease-modifying antirheumatic drugs (DMARDs, such as sulfasalazine, hydroxychloroquine, and leflunomide). Some patients require biologic DMARDs (bDMARDs, such as infliximab, rituximab, etanercept, and tocilizumab) and / or targeted synthetic DMARDs (tsDMARDs, such as baricitinib and tofacitinib), and a portion still fail with multiple treatments. Recent observational studies have used the working definition of refractory RA based on the number of failed DMARDs, with the prevalence of refractory RA estimated to be 6 to 21% of all patients treated with conventional DMARDs (Melville, 2020).

[0206] In some cases, treatment for RA includes improvements in more than one of the following (e.g., as described above): the Rheumatoid Arthritis Disease Activity Score (DAS28), the Clinical Disease Activity Index (CDAI), and participant response to treatment as assessed by the EULAR criteria. In some cases, improvements occur at 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.

[0207] 3. Pemphigus vulgaris (PV)

[0208] In some cases, the autoimmune disease is pemphigus vulgaris (PV).

[0209] Pemphigus is a group of autoimmune bullous diseases involving 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 autoantibodies specific to cadherin desmosome core glycoprotein (Dsg), which bind to epidermal desmosomes, leading to blisters and erosions of the mucous membranes or skin. Despite various approaches explored, treatment of this disease remains challenging. Treatment of PV has explored targeting pathogenic immune pathways with anti-CD20 monoclonal antibodies, BAFF inhibitors, IL-17 blockers, mTOR pathway inhibitors, p38 MAPK, BTK inhibitors, and TNF-α inhibitors (Abulikemu, 2023). However, treatment of this disease remains challenging, with patients experiencing relapses and developing resistance or intolerance to existing treatments.

[0210] In some cases, treatment for PV includes improvements in one or more of the following (e.g., as described above): 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 quality of life as assessed by the Dermatology Quality of Life Index (DLQI), and blood DSG 1 and DSG 3 levels. In some cases, improvements occur at 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.

[0211] 4. Granulomatous polyangiitis (GPA) and microscopic polyangiitis (MPA)

[0212] In some cases, the autoimmune condition is granulomatous polyangiitis (GPA) and / or microscopic polyangiitis (MPA).

[0213] Granulomatous polyangiitis (GPA) and microscopic polyangiitis (MPA) are forms of small- to medium-sized vessel vasculitis. Both GPA and MPA are rare diseases, with prevalences of 24–160 patients per million and 39–94 patients per million, respectively. While there are currently no validated diagnostic criteria, the 1990 American College of Rheumatology (ACR) classification criteria (for GPA) and the 2012 Chapel Hill Consensus nomenclature have helped define these diseases for clinical trial purposes, as their clinical presentations make them suitable for novel treatment approaches. Both GPA and MPA typically present with pulmonary and / or renal manifestations, with GPA frequently involving the upper respiratory tract. Due to their similar clinical presentations, GPA and MPA are often studied together in clinical trials. Because of the systemic nature of vasculitis, the diagnosis and treatment of vasculitis require a multidisciplinary approach (Koike, 2022).

[0214] In some cases, treatment of GPA and / or MPA includes improvements in one or more of the following (e.g., as described above): Birmingham Vasculitis Activity Score, time to disease relapse, number of disease relapses, time to first complete remission, and changes in health-related quality of life as assessed by the Dermatology Quality of Life Index (DLQI). In some cases, improvements occur at 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.

[0215] B. Patient

[0216] Suitable patients for use in the compositions and methods described herein include those who have, have been diagnosed with, or are suspected of having an autoimmune disease (such as that described herein).

[0217] In some embodiments, the treatment methods described herein can be used to treat subjects (e.g., humans, monkeys, dogs, cats, mice) who have been diagnosed with or are suspected of having an autoimmune disease (e.g., as described herein). In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0218] In some implementations, the patient has been diagnosed with or has been diagnosed with an autoimmune disease, such as the autoimmune diseases described herein. In some cases, the patient is resistant, relapsed, or refractory to treatment after initial therapy for the disease. In some cases, the patient has failed at least two lines of prior therapy that meet the standard of care (SOC).

[0219] In some cases, the patient has systemic lupus erythematosus (SLE).

[0220] In some cases, patients’ SLE was diagnosed according to the 2010 American College of Rheumatology (ACR) / European League Against Rheumatism (EULAR) RA classification criteria (Kay, 2012).

[0221] In some cases, the patient's pre-treatment Systemic Lupus Erythematosus Disease Activity Index (SLEDAI-2K) total score was 6 or higher. In other cases, the patient's pre-treatment SLEDAI-2K total score was 8 or higher, excluding alopecia, mucosal ulcers, and fever.

[0222] In some cases, patients have attempted conventional SLE therapy (e.g., at least 12 weeks of two conventional therapies) but failed, including, for example, antimalarial drugs, corticosteroids, immunosuppressants such as mycophenolate mofetil, methotrexate, azathioprine, and / or biologics such as belimumab, anifrolumab, and rituximab. In some cases, patients have received antimalarial drugs (e.g., hydroxychloroquine, chloroquine, quinacrine) at a stable dose, for example, for more than 12 weeks prior to initial treatment and optionally at least 6 weeks prior to the first administration of NK cells. In some cases, patients have received immunomodulatory drugs (e.g., mycophenolate mofetil (MMF) / mycophenolate, azathioprine / 6-mercaptopurine, leflunomide, methotrexate with or without folic acid, calcineurin inhibitors, and / or cyclosporin A) at a stable dose, for example, for at least 12 weeks prior to the first administration of NK cells.

[0223] In some cases, the patient has lupus nephritis. In some cases, the patient has type I, II, III, IV, V, and / or VI lupus nephritis. In some cases, the patient has mild mesangial lupus nephritis, mesangial proliferative lupus nephritis, focal lupus nephritis, diffuse lupus nephritis, membranous lupus nephritis, and / or advanced sclerosing lupus nephritis. In some cases, lupus nephritis is refractory, for example, if the patient has not improved after 3 to 4 months of previous treatment, has not achieved partial remission after 6 to 12 months, and / or has not achieved complete remission after 2 years of previous treatment.

[0224] In some cases, based on the modified NIH Lupus Nephritis Activity and Chronicity Index (Bajema IM 2018), patients have evidence of active disease on renal biopsy. In some cases, patients have detectable anti-double-stranded DNA antibody titers.

[0225] In some cases, the patient is a subject (e.g., an adult subject) with type III or IV lupus nephritis according to the 2018 ISN / RPS criteria (Bajema IM 2018), with or without type V. In some cases, the patient is relapsed or refractory after initial treatment for SLE. In some cases, initial treatment includes one or more of the following: glucocorticoids in combination with mycophenolate mofetil (MMF) or cyclophosphamide; MMF in combination with a calcineurin inhibitor (e.g., vorticol or tacrolimus) or belimumab; cyclophosphamide in combination with belimumab; intravenous cyclophosphamide; anti-CD20 monoclonal antibody (mAb); or intravenous cyclophosphamide in combination with anti-CD20 monoclonal antibody (mAb). In some cases, the patient has not improved within 3 to 4 months of prior treatment, has not achieved partial remission after 6 to 12 months of prior treatment, or has not achieved complete remission after 2 years of prior treatment. In some cases, patients have failed at least two lines of prior treatment that meet the standard of care (SOC) for subjects with lupus nephritis.

[0226] In some cases, the patient has rheumatoid arthritis (RA).

[0227] In some cases, a patient's RA was diagnosed according to the 2010 American College of Rheumatology (ACR) / European League Against Rheumatism (EULAR) RA classification criteria (Kay, 2012). In some cases, the patient had previously received treatment with biological disease-modifying antirheumatic drugs (“bDMARDs”, such as infliximab, rituximab, etanercept, tocilizumab) and / or targeted synthetic disease-modifying antirheumatic drugs (“tsDMARDs”, such as baricitinib, tofacitinib) and was considered refractory, for example by one or more of the following: lack of benefit from bDMARDs or tsDMARDs; lack of benefit from at least two bDMARDs; lack of benefit from at least one bDMARD and one tsDMARD; intolerance to prior therapy beyond one line (e.g., first-, second-, or third-line prior therapy), including bDMARDs and / or tsDMARDs. In some cases, lack of benefit includes inadequate improvement in one or more of the following: joint counts, physical function, and disease activity. In some cases, the patient's swollen joint count (SJC) is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and / or tender joint count (TJC) is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some cases, the patient's swollen joint count (SJC) is at least 6 and the tender joint count (TJC) is at least 6.

[0228] In some cases, the patient has pemphigus vulgaris (PV).

[0229] In some cases, patients have one or more of the following: active lesions; positive anti-desmosome core glycoprotein Dsg1 or Dsg3; and a pemphigus disease area index score (Shimizu, 2014) >80%.

[0230] In some cases, patients have granulomatous polyangiitis (GPA) and / or microscopic polyangiitis (MPA). In some cases, patients have one or more of the following: ≥1 “major” item, ≥3 “other” items, and ≥2 kidney items on the Birmingham Vasculitis Activity Scale, Version 3 (BVASv3).

[0231] C. Lymphocyte depletion

[0232] In some implementations, the patient is lymphocyte depleted before treatment.

[0233] Illustrative lymphocyte depletion chemotherapy regimens, together with relevant beneficial biomarkers, are described in WO 2016 / 191756 and WO 2019 / 079564, which are incorporated herein by reference in their entirety. In some embodiments, the lymphocyte depletion chemotherapy regimen includes administering a dose of cyclophosphamide (200 mg / m²) to the patient. 2 / day up to 2000mg / m 2 ( / day) and a certain dose of fludarabine (20mg / m²) 2 / day up to 900mg / m 2 / sky).

[0234] In some implementations, lymphocyte depletion includes administering 100 or about 100 to 1500 or about 1500 mg / m². 2 Cyclophosphamide, for example, 250 or about 250 to 500 or about 500 mg / m³ 2 Cyclophosphamide, for example, 250 or about 250 to 500 or about 500, 250, 400, 500, about 250, about 400 or about 500 mg / m³ 2 Cyclophosphamide. In some embodiments, lymphocyte depletion includes administration of 500 or about 500 mg / m². 2 Cyclophosphamide. In some embodiments, lymphocyte depletion includes administration of 100 mg / m². 2 or approximately 100 mg / m 2 Cyclophosphamide.

[0235] In some implementations, lymphocyte depletion includes administering 20 or about 20 mg / m². 2 / day up to 40 or approximately 40mg / m 2 Fludarabine per day, for example 30 or about 30 mg / m² 2 sky.

[0236] In some implementations, lymphocyte depletion includes the administration of both cyclophosphamide and fludarabine.

[0237] In some implementations, cyclophosphamide (250 mg / m²) is administered intravenously. 2 / day) and fludarabine (30mg / m²) 2 / day) to deplete the patient's lymphocytes.

[0238] In some implementation methods, cyclophosphamide (500 mg / m²) is administered intravenously. 2 / day) and fludarabine (30mg / m²) 2 / day) to deplete the patient's lymphocytes.

[0239] In some implementations, lymphocyte depletion is performed no more than 5 days before the first dose of NK cells. In other implementations, lymphocyte depletion is performed no more than 7 days before the first dose of NK cells.

[0240] In some implementations, lymphocyte depletion begins 5 days before the first dose of NK cells, is performed daily for 3 consecutive days.

[0241] In some implementations, the first dose of NK cells is given on day 6, and lymphocyte depletion is performed on days 1, 2, and 3.

[0242] D. Application

[0243] This article describes methods including the administration of NK cells (e.g., as described herein). In some cases, NK cells are administered as part of a treatment that further includes the administration of one or more additional agents, including, for example, antibodies (e.g., as described herein), cytokines (e.g., as described herein), lymphocyte depletion agents (e.g., as described herein), corticosteroids (e.g., prednisone, prednisolone, dexamethasone, methylprednisolone), analgesics, antipyretics, and / or antihistamines. For example, patients may undergo pre-medication prior to NK cell infusion, as described herein, for example, as shown in Table 13 or Table 14.

[0244] 1. NK cells

[0245] In some embodiments, NK cells are administered as part of a pharmaceutical composition, such as those described herein. The cells are administered after thawing, and in some cases, where their cryoprotectant is compatible with immediate administration, no further action is required. For a given individual, the treatment regimen typically involves administering multiple aliquots or doses of NK cells retrievable from a common batch or donor over time. In some embodiments, NK cells, such as those described herein, are administered at a dose of 1 × 10⁻⁶ cells per dose.8 Or approximately 1×10 8 Up to 8×10 9 Or approximately 8×10 9 Administer NK cells per dose. In some implementations, NK cells are administered at a dose of 1 × 10⁻⁶ cells per dose. 8 Or approximately 1×10 8 1×10 9 Or approximately 1×10 9 4×10 9 Or approximately 4×10 9 Or 8×10 9 Or approximately 8×10 9 NK cells are administered per dose. In some cases, NK cells are administered at 4 billion or approximately 4 billion cells per dose. In other cases, NK cells are administered at 2 billion or approximately 2 billion cells per dose.

[0246] In some embodiments, NK cells are administered weekly. In some embodiments, NK cells are administered for 3 to 8 weeks or about 3 to 8 weeks. In some embodiments, NK cells are administered for 4 to 8 weeks or about 4 to 8 weeks. In some embodiments, NK cells are administered weekly for 4 weeks or about 4 weeks. In some embodiments, NK cells are administered weekly for 8 weeks or about 8 weeks.

[0247] In some implementations, NK cells are administered on days 6, 9, 13, and 16 of the treatment cycle. In other implementations, NK cells are administered on days 6, 13, and 20 of the treatment cycle.

[0248] In some implementations, the application is repeated for more than one additional treatment cycle, for example, according to the application protocol described above. In some implementations, the additional treatment cycles are 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 after the previous treatment cycle, or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months after the previous treatment cycle.

[0249] In some embodiments, NK cells are cryopreserved in infusion-ready media, such as cryopreservation compositions suitable for intravenous administration, as described herein.

[0250] In some implementations, NK cells are cryopreserved in vials, each containing 1 × 10⁶ cells. 8 Or approximately 1×10 8 Up to 8×10 9 Or approximately 8×10 9 Individual cells. In some implementations, NK cells are cryopreserved in vials containing a single dose.

[0251] In some implementations, the cells are thawed before application, for example, in a 37°C water bath.

[0252] In some implementations, the thawed NK cell vials are aseptically transferred to a single administration container, such as an administration bag using a vial adapter and a sterile syringe. NK cells can be administered to the patient via intravenous infusion from a blood vessel through a Y-type transfusion / infusion device with a filter, by gravity.

[0253] In some embodiments, NK cells are administered as soon as possible, preferably less than 90 minutes after thawing, for example less than 80, 70, 60, 50, 40, 30, 20, or 10 minutes. In some embodiments, NK cells are administered within 30 minutes of thawing.

[0254] In some embodiments, the pharmaceutical composition is administered intravenously via a syringe.

[0255] In some implementations, 1 mL, 4 mL, or 10 mL of the drug product is administered intravenously to the patient via a syringe.

[0256] 2. Antibodies

[0257] In some embodiments, the NK cells described herein (e.g., pharmaceutical compositions comprising the NK cells described herein) are administered in combination with antibodies (e.g., antibodies described herein, such as B-cell depletion antibodies, such as CD19 and / or CD20 antibodies, such as rituximab and / or oxutuzumab). In some embodiments, the antibodies are administered together with the NK cells as part of a pharmaceutical composition. In some embodiments, the antibodies and NK cells are administered separately, for example as part of separate pharmaceutical compositions. The antibodies may be administered before, after, or simultaneously with the administration of the NK cells.

[0258] In some implementations, the antibody is administered before NK cells. In other implementations, the antibody is administered after NK cells.

[0259] In some implementations, NK cells are administered at least 30, 60, 90, 120, 150, 180, 210, or 240 minutes after antibody administration.

[0260] In some implementation schemes, NK cells are administered the day after antibody administration.

[0261] In some implementations, NK cells are administered with each application, while antibodies are administered in partial doses. For example, in some implementations, NK cells are administered weekly, while antibodies are administered monthly.

[0262] In some implementations, the antibody is administered weekly for 8 weeks. In other implementations, the antibody is administered every two weeks for 8 weeks.

[0263] In some implementations, an antibody dose is administered prior to the first dose of cells. In some implementations, an ebulking dose of antibody is administered prior to the first dose of cells.

[0264] In some implementations, the antibody is administered on days 2 and 13 of the treatment cycle. In other implementations, the antibody is administered on days 1 and 15 of the treatment cycle.

[0265] 3. Cytokines

[0266] In some implementations, cytokines are administered to the patient.

[0267] In some embodiments, the cytokine is administered together with NK cells as part of a pharmaceutical composition. In some embodiments, the cytokine is administered separately from NK cells, for example, as part of a separate pharmaceutical composition. In some embodiments, the cytokine is IL-2.

[0268] In some implementation schemes, cytokines are not administered to patients.

[0269] E. Administration

[0270] "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 preventive effective amount, which is the amount necessary to prevent the onset of disease or disease symptoms. An effective amount may be administered in more than one application, administration, or dose. The therapeutically effective amount (i.e., effective dose) of a therapeutic compound depends on the selected therapeutic compound. The composition may be administered more than once daily to more than once weekly; including every other day. Those skilled in the art will understand that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or condition, prior treatment, the subject's general health and / or age, and any other pre-existing conditions. Furthermore, treatment of a subject with a therapeutically effective amount of the therapeutic compound described herein may include single treatment or a series of treatments.

[0271] The dosage, toxicity, and therapeutic efficacy of therapeutic compounds can be determined in cell cultures or laboratory animals using standard pharmaceutical methods, such as determining the LD50 (the dose that is 50% lethal to the population) and ED50 (the dose that is 50% therapeutically effective to the population). The dose ratio between toxicity and therapeutic efficacy is the therapeutic index, which can be expressed as the LD50 / ED50 ratio. Compounds exhibiting a high therapeutic index are preferred. Although compounds exhibiting toxic side effects can be used, delivery systems for targeting such compounds to affected tissue sites should be carefully designed to minimize potential damage to uninfected cells and thereby reduce side effects.

[0272] Data obtained from cell culture experiments and animal studies can be used to determine dosage ranges for human use. Doses of such compounds can be within a range of circulating concentrations, including the ED50, and with low or no toxicity. Doses can vary within this range depending on the dosage form and route of administration used. For any compound used in the methods of this invention, the therapeutically effective dose can first be estimated from cell culture experiments. Doses can be formulated in animal models to achieve a range of circulating blood concentrations, including the IC50 (i.e., the concentration of the test compound that achieves half-maximal inhibition of symptoms) as determined in cell cultures. This information can be used to more accurately determine the useful dose for humans. Plasma levels can be determined by, for example, high-performance liquid chromatography, flow cytometry, or molecular detection.

[0273] F. Treatment cycle

[0274] In some cases, treatment includes the administration of NK cells and antibodies during a treatment cycle (as described herein). For example, in some cases, the treatment cycle includes lymphocyte depletion, followed by the administration of NK cells and B-cell depletion antibodies. In some cases, B-cell depletion antibodies are administered before the first administration of NK cells. In some cases, B-cell depletion antibodies are administered both before and after the first administration of NK cells (e.g., at intervals of approximately two weeks, such as on days 2 and 13). In some cases, NK cells are administered more than once during a treatment cycle (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times), such as weekly. In some cases, NK cells are administered approximately weekly after lymphocyte depletion (e.g., on days 6, 13, 20, and so on). In some cases, NK cells are administered on days 6, 13, and 20. In some cases, the treatment cycle is repeated more than once, such as 1, 2, 3, 4, 5, or 6 times. In some cases, treatment cycles are spaced more than a month apart, for example, intervals of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months.

[0275] For example, an exemplary treatment cycle (e.g., for SLE) includes lymphocyte depletion at the start of the treatment cycle (e.g., before administration of NK cells). In some cases, lymphocyte depletion includes a 3-day (days 1-3) lymphocyte depletion regimen to induce lymphocyte depletion and create an optimal environment for in vivo NK cell expansion. In this example, a B-cell depletion antibody (e.g., rituximab or octotuzumab) is administered twice intravenously (e.g., 1000 mg) during the treatment cycle (approximately 2 weeks apart), optionally after pre-treatment with methylprednisolone to reduce the risk of infusion-related reactions. In some cases, NK cells are administered intravenously after administration of the lymphocyte depletion regimen and after the first dose of antibody. In some cases, the treatment cycle includes administration of NK cells according to one of the dosing levels in Table 4. Lymphocyte depletion regimens may include, for example, a fludarabine and cyclophosphamide regimen, such as fludarabine administered at 30 mg / m² on days 1, 2, and 3 at the start of each treatment cycle. 2 Cyclophosphamide was administered at 1000 mg / m² on day 3 of each treatment cycle. 2 Administration. In one instance, the antibody was administered at 1000 mg on days 2 and 13 of each treatment cycle. In some cases, the first dose of AB-101 was administered at least 48 hours after the last infusion of the lymphocyte depletion regimen in each treatment cycle, using the dosing regimen shown in Table 4. In another instance, a second treatment cycle was administered, for example, approximately 24 weeks after the first dose of NK cells in the first treatment cycle.

[0276] Table 4. Example dose levels and dosing days for AB-101

[0277]

[0278] In another example, a treatment cycle (e.g., 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 (days 1-3) lymphocyte depletion regimen, and a B-cell depletion antibody (e.g., rituximab or octotuzumab) is administered twice intravenously (e.g., 1000 mg) during the treatment cycle (approximately 2 weeks apart, e.g., on days 2 and 13), optionally after pre-treatment to reduce the risk of infusion-related reactions (e.g., as described herein). In some cases, NK cells are administered via intravenous infusion after the lymphocyte depletion regimen and after the first dose of antibody. In some cases, the treatment cycle includes administration of NK cells according to one of the dosing levels in Table 5. Lymphocyte depletion regimens may include, for example, a fludarabine and cyclophosphamide regimen, e.g., in which fludarabine is administered at 30 mg / m² on days 1, 2, and 3 at the start of each treatment cycle. 2Cyclophosphamide was administered at 1000 mg / m² on day 3 of each treatment cycle. 2 Administration. In one instance, the antibody was administered at 1000 mg on days 2 and 13 of each treatment cycle. In some cases, the first dose of AB-101 was administered at least 48 hours after the last infusion of the lymphocyte depletion regimen in each treatment cycle, using the dosing regimen shown in Table 5. In some cases, AB-101 was administered on days 6, 13, and 20 of the treatment cycle. In some cases, a second treatment cycle was administered, for example, approximately 24 weeks after the first dose of NK cells in the first treatment cycle.

[0279] Table 5. Example dose levels of AB-101 and B-cell depletion antibodies

[0280]

[0281] V. Variant

[0282] In some embodiments, the fusion protein or its components described herein, or the NK cell genotype described herein, is at least 80%, for example, at least 85%, 90%, 95%, 98%, or 100% identical in amino acid sequence to exemplary sequences (e.g., as provided herein), for example, with differences where at most 1%, 2%, 5%, 10%, 15%, or 20% of residues in the exemplary sequence are replaced by, for example, conserved mutations, including or other than those described herein. In a preferred embodiment, the variant retains the desired activity of the parent.

[0283] To determine the percentage of identity between two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., for optimal alignment, vacancies may be introduced in one or both of the first and second amino acid or nucleic acid sequences, and non-homologous sequences may be ignored for comparison purposes). The length of the reference sequence aligned for comparison purposes is at least 80% of the length of a reference sequence, and in some embodiments at least 90% or 100%. Nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, the 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 vacancies that need to be introduced for optimal alignment of the two sequences and the length of each vacancies.

[0284] The percentage of identity between the test peptide or nucleic acid sequence (i.e., the query) and the second peptide or nucleic acid sequence (i.e., the target) is determined by various methods known to those skilled in the art, such as using publicly available computer software, such as Smith Waterman Alignment (Smith, TF and MS Waterman (1981) J MolBiol 147:195-7); “BestFit” (Smith and Waterman, Advances in Applied Mathematics, 482-489 (1981)), as incorporated in GeneMatcher Plus™, Schwarz and Dayhof (1979) Atlas of Protein Sequence and Structure, Dayhof, MO, Ed, pp 353-358; BLAST program (Basic Local Alignment Search Tool; (Altschul, SF, W. Gish, et al. (1990) J Mol Biol 215: Software such as BLAST-2, BLAST-P, BLAST-N, BLAST-X, WU-BLAST-2, ALIGN, ALIGN-2, CLUSTAL, or Megalign (DNASTAR) can be used. Furthermore, those skilled in the art can determine suitable parameters for measuring alignment, including any algorithm required to achieve maximum alignment over the length of the compared sequences. Typically, for the target protein or nucleic acid, the length of the comparison can be any length, up to and including the full target length (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 relative to the full length of the query sequence.

[0285] For the purposes of this disclosure, the comparison of sequences and the determination of the percentage identity between two sequences can be accomplished using a Blossum 62 scoring matrix, where the void penalty is 12, the void extension penalty is 4, and the shifted void penalty is 5.

[0286] 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.

[0287] VI. Definition

[0288] Unless otherwise defined, all technical terms, symbols, and other technical and scientific terms or terminology used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be construed as indicating a material difference from the commonly understood meaning in the art.

[0289] In this application, various embodiments may be presented in scope form. It should be understood that the scope form is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of this disclosure. Therefore, the scope description should be considered to specifically disclose all possible sub-scopes and individual numerical values ​​within those scopes. For example, a description of a scope such as 1 to 6 should be considered to specifically disclose sub-scopes such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and individual numerical values ​​within those scopes such as 1, 2, 3, 4, 5, and 6. This is unrelated to the width of the scope.

[0290] As used in the specification and claims, the singular forms “a,” “an,” and “the” include a plural referent unless the context clearly indicates otherwise. For example, the term “a sample” includes multiple samples, including mixtures thereof.

[0291] The terms “determine,” “measure,” “evaluate,” “assess,” “determine,” and “analyze” are often used interchangeably herein to refer to a form of measurement. These terms include determining the presence of an element (e.g., detection). These terms may include quantitative, qualitative, or both quantitative and qualitative determinations. Assessments may be relative or absolute. “Detecting the presence of…” may include determining the quantity of something present, in addition to determining whether something is present or not, depending on the context.

[0292] The terms “subject,” “individual,” or “patient” are often used interchangeably in this article.

[0293] The term "in vivo" is used to describe events that occur inside a subject's body.

[0294] The term "ex vivo" is used to describe events that occur outside the subject's body. Ex vivo assays are not performed on the subject, but rather on a sample separated from the subject. An example of an ex vivo assay performed on a sample is an "in vitro" assay.

[0295] The term "in vitro" is used to describe events that occur in a container used to contain laboratory reagents, such that the reagents are separated from the biological source from which the material was obtained. In vitro assays can encompass cell-based assays in which live or dead cells are used. In vitro assays can also encompass cell-free assays in which intact cells are not used.

[0296] As used herein, the term “about” for a number means adding or subtracting 10% of that number. The term “about” for a range means subtracting 10% of its lowest value and adding 10% of its highest value.

[0297] As used herein, the term "buffer solution" refers to an aqueous solution consisting of a mixture (or vice versa) of a weak acid and its conjugate base.

[0298] As used herein, the term "cell culture medium" refers to a mixture used for the in vitro growth and proliferation of cells, which contains essential elements for cell growth and proliferation, such as sugars, amino acids, various nutrients, and inorganic substances.

[0299] As used in this article, the buffer solution is not a cell culture medium.

[0300] As used herein, the term "bioreactor" refers to a culture device capable of continuously controlling a series of conditions affecting cell culture, such as dissolved oxygen concentration, dissolved carbon dioxide concentration, pH, and temperature.

[0301] As used herein, the term "vector" refers to a nucleic acid molecule capable of replicating another nucleic acid linked to it. This term includes vectors as self-replicating nucleic acid structures as well as vectors integrated into the genome of a host cell into which they have been introduced. Some vectors are suitable for delivering the nucleic acid molecules or polynucleotides of this application. Certain vectors are capable of directing the expression of nucleic acids operatively linked to them. Such vectors are referred to herein as expression vectors.

[0302] The term "operably linked" refers to two or more nucleic acid sequences or polypeptide elements that are typically physically linked and functionally related to each other. For example, if a promoter is capable of initiating or regulating the transcription or expression of a coding sequence, then the promoter is operably linked to the coding sequence, in which case the coding sequence should be understood as being "under the control" of the promoter.

[0303] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells that have been introduced with exogenous nucleic acids, including progeny of such cells. Host cells include “engineered cells,” “transformers,” and “transformed cells,” which include primary engineered (e.g., transformed) cells and their derived progeny, regardless of passage number. Progeny cells may not be identical to parent cells in terms of nucleic acid content but may contain mutations. This article includes mutant progeny with the same function or biological activity as those screened or selected in the original transformed cells.

[0304] Depending on the circumstances, host cells can be stabilized or transiently transfected with polynucleotides encoding fusion proteins as described herein.

[0305] The chapter titles used in this article are for organizational purposes only and should not be construed as limiting the topics described.

[0306] VII. References

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[0387] VIII. Examples

[0388] The following examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0389] Example 1: AB-101

[0390] AB-101 is a generic, off-the-shelf, cryopreserved allogeneic umbilical cord blood-derived NK cell therapy product containing ex vivo expanded and activated effector cells designed to enhance ADCC antitumor responses in patients, such as those treated with monoclonal antibodies or NK cell conjugates. AB-101 is prepared as described, for example, in WO2022 / 133056.

[0391] Example 2: AB-101 combined with anti-CD20 antibody mediates in vitro killing of healthy donor B cells

[0392] In a 4-hour kill assay, AB-101 was combined with healthy donor PBMCs (0.2:1 or 1:1 ratio) with or without antibodies. Donor B cells (CD19+CD20+) were analyzed by flow cytometry, and the percentage of apoptotic cells was detected using caspase 3 / 7 dye. The numbers in the figure show the percentage of caspase-positive B cells. The results showed that, compared with oxutuzumab alone or AB-101 plus PBMCs (without antibody) in the above figure, the combination of AB-101 with anti-CD20 antibody (below figure) resulted in ADCC and increased B cell death. Figure 1 As shown, this combination mediates the killing of healthy donor B cells.

[0393] Example 3: AB-101 combined with anti-CD19 or anti-CD20 antibodies mediates in vitro killing of healthy donor B cells.

[0394] In a 4-hour killing assay, AB-101 was mixed with healthy donor PBMCs (0.2:1 or 1:1 ratio) with or without 0.01 or 0.1 μg / mL of antibody. Donor B cells (CD19+CD20+) were analyzed by flow cytometry, and the percentage of apoptotic cells was detected using caspase 3 / 7 dye. Results showed that AB-101 combined with antibody resulted in ADCC and increased B cell death (symbol on the y-axis) compared to PBMCs + antibody lacking AB-101. Oxtuzumab combined with AB-101 is demonstrated to be the most effective combination for B cell killing. Figure 2 As shown, AB-101, in combination with anti-CD19 (tancituzumab) or anti-CD20 antibody (rituximab or oxotuzumab), mediates in vitro killing of healthy donor B cells.

[0395] The ADCC of AB-101 against peripheral blood mononuclear cells (PBMCs) from healthy individuals and PBMCs isolated from SLE patients was evaluated in combination with anti-CD20 (rituximab and octotuzumab) or anti-CD19 (tancituzumab) monoclonal antibodies. PBMCs were incubated with or without antibodies at different effector (NK cell) to target (PBMC) ratios. Apoptosis (cysteine-positive) cells were quantified by flow cytometry.

[0396] Whole blood from healthy or SLE donors was collected in ACD-A anticoagulant and transported overnight at room temperature. PBMCs were isolated and viable cell counts were determined. PBMCs were resuspended in assay medium and cultured at 2 × 10⁻⁶. 5 101 cells / well plate. Thaw AB-101 in a 37°C water bath. Once thawed, wash AB-101 in culture medium, centrifuge, and count cells in aliquots. Adjust the AB-101 cell count to 5 × 10⁻⁶ cells / well. 6 Cells / mL. AB-101 was further diluted in the culture medium and administered at 4 × 10⁻⁶ cells / mL. 4 1 cell / well or 2 × 10 5Cells / well were added to PBMCs to produce E:T ratios of 1:1 and 0.2:1. Antibodies (0.01, 0.1, or 1 μg / mL of rituximab, octotuzumab, or tancituzumab) were added to the co-culture, and the plates were incubated at 37°C with 5% CO2 for 3.5 h. Caspase 3 / 7 green was added to each well, and the plates were incubated at 37°C for an additional 30 min. After the incubation period, the co-cultures were prepared for flow cytometry analysis. Due to antibody interference in flow cytometry detection, B cells in rituximab and octotuzumab-treated cells were identified as CD19+ cells, and B cells in tancituzumab-treated cells were identified as CD20+ cells. Target cell specific lysis (%) was calculated as follows: Specific lysis (%) = (sample wells - spontaneous) / (100 - spontaneous) 100.

[0397] Cytotoxicity assays using healthy human PBMCs co-cultured with AB-101 showed enhanced AB-101-mediated B-cell killing at different E:T ratios in the presence of anti-CD20 or anti-CD19 antibodies (Table 6). Oxtuzumab, in combination with AB-101, induced B-cell apoptosis more effectively at lower E:T ratios than rituximab or tancituzumab, presumably through enhanced antibody glycoengineering. It was noted that B-cell apoptosis was minimal to no increase when PBMCs and AB-101 were used alone (Table 6). PBMCs isolated from SLE patient samples (n=3) were tested with AB-101 in the same co-culture system as healthy human PBMCs. Anti-CD20 or anti-CD19 antibodies were added to the co-cultures, and B-cell apoptosis was measured. Enhanced ADCC against SLE B cells was observed in both antibody concentration-dependent and E:T ratio-dependent manner when AB-101 was combined with anti-CD20 or anti-CD19 antibodies. Figure 3 and Figure 4 At a 1:1 E:T ratio and 1 μg / mL antibody, enhanced cytotoxicity against SLE B cells was observed with oxutuzumab (range 78.6 to 95.4%), tancituzumab (range 24.2 to 76.9%), and rituximab (range 19.2 to 62.2%), compared to minimal B cell killing (range 2.3 to 8.3%) with human IgG1 control antibody (Table 7, representative donors). The specificity of cell killing in SLE PBMC samples was evaluated by examining the effect of this combination on SLE T cells. In the presence of AB-101 and any of the three antibodies tested (Table 7, representative donors), little to no off-target apoptosis was observed in this cell population.

[0398] In summary, AB-101, in combination with anti-CD19 and anti-CD20 monoclonal antibodies, has been shown to kill SLE B cells via an ADCC mechanism. This killing is specific to B cells, as no significant effect on SLE T cells was observed under the same co-culture conditions.

[0399] Table 6. Effects of AB-101 and anti-CD19 or anti-CD20 treatment on the percentage of B cells and T cells in healthy human donors (summary of all donors)

[0400]

[0401] Figure 3 Representative FACS plots show PBMCs alone (top left), PBMCs plus AB-101 (bottom left), PBMCs with anti-CD20 antibody (center), or PBMCs with AB-101 plus anti-CD20 antibody at a 1:1 E:T ratio (right) for dead / dying caspase 3 / 7. + CD19 + SLE is gated by B cells. Lymphocytes are first gated via forward scattering (FSC), then by side scattering (SSC), and finally by single-cell gating. B cells use CD45... + CD14 - CD3 - CD56 - CD16 - and CD19 + Implement gating.

[0402] PBMCs from SLE patients were isolated from peripheral blood and mixed with thawed AB-101, with or without anti-CD20 (rituximab or oxutuzumab) (top image), or with anti-CD19 (tancituzumab) or human IgG1 isotype control (bottom image), for 4 hours. The percentage of caspase-positive B cells was determined by flow cytometry. Figure 4 Data are expressed as the average value of repeated wells ± SD.

[0403] Table 7. Effects of AB-101 and anti-CD19 or anti-CD20 treatments on the percentage of B cells and T cells in SLE.

[0404]

[0405] Example 4: AB-101 combined with anti-CD20 antibody resulted in minimal T cell killing.

[0406] AB-101 was cultured with healthy donor PBMCs, with or without antibodies. After 4 hours, the percentage of caspase-positive T cells (CD3+) was determined by flow cytometry. Compared with PBMCs alone, the addition of AB-101 and oxutuzumab resulted in a minimal increase in T cell killing. Figure 5 As shown, AB-101 combined with anti-CD20 antibody (Octuzumab) resulted in minimal killing of T cells (CD3+).

[0407] Example 5: AB-101 combined with anti-CD20 antibody mediates the killing of SLE donor B cells in a 4-hour cytotoxicity assay. hurt

[0408] AB-101 was cultured with SLE donor PBMCs, with or without antibody. After 4 hours, the percentage of caspase-positive B cells (CD19+) was determined by flow cytometry. The combination of AB-101 and oxutuzumab (0.1 or 1 μg / mL) resulted in increased killing of SLE donor B cells. In the presence of the antibody, a higher proportion of AB-101 resulted in more B cell killing. Figure 6 As shown, in a 4-hour cytotoxicity assay, AB-101 combined with anti-CD20 antibody (Octuzumab) mediated the killing of SLE donor B cells.

[0409] Example 6: Humanized NSG Mouse Model

[0410] To evaluate the in vivo ADCC effect of the combination of AB-101 and the anti-CD20 monoclonal antibody oxotuzumab against human B cells, a CD34+ humanized NSG (huNSG) mouse model was used. The CD34+ huNSG mouse model is specifically designed for studying human hematopoietic stem cells and the immune system. In short, the model consists of NSG mice that have undergone whole-body irradiation conditioned and then engrafted with CD34+ hematopoietic stem cells derived from human umbilical cord blood (Ishikawa 2005). Approximately 12 to 16 weeks post-engraftment, these huNSG mice exhibited high levels of human B cells and moderate levels of T cells in their peripheral blood, along with a small number of myeloid cells but very few NK cells. Given the high levels of human B cells, this model is considered suitable for assessing the effects of treatment on B cell levels and general animal health.

[0411] Use 19- to 23-week-old female CD34 + Humanized NSG (NOD.Cg-Prkdc) scid Il2rg tm1Wjl / SzJ) mice were used as experimental recipients. After receiving the mice, they were allowed a 7-day acclimatization period before the start of the study. Animals were placed on day 0 based on human CD45... + CD19 + B-cell implantation levels were allocated to the treatment groups to ensure that the mean values ​​of each group were similar.

[0412] The selected dosage and regimen for administering AB-101 and oxutuzumab were based on previously published preclinical studies using oxutuzumab in similar humanized models (Bacac M, 2018), as well as internal studies using B-cell lymphoma xenografts and preliminary studies using huNSG mice. The selected dose of 150 μg / kg oxutuzumab was considered a dose level for partially depleted human B cells, allowing a window for observing ADCC when using the combination.

[0413] Dosing regimen such as Figure 10 As shown. In the single agent group, AB-101 (1×10⁻⁶) 7 AB-101 was administered to huNSG mice via a single slow bolus intravenous injection (IV) on days 0 and 7, and oxutuzumab was administered via intraperitoneal (IP) injection on day 0, or on days 0 and 7. For the combination group, oxutuzumab was administered first on day 0, or on days 0 and 7, followed by AB-101. Hydrolyzed animals were treated similarly to the combination group, receiving both IP and IV injections on days 0 and 7. Peripheral blood was collected on day -2 (baseline), 7, 14, 21, and 28. Blood collection on day 7 was performed prior to the second administration of any test substance. Depletion of human B cells in peripheral blood and tissues was assessed following treatment with AB-101, oxutuzumab, or a combination of AB-101 and oxutuzumab. Toxicity was assessed by mortality / cage-side observation, clinical observation, body weight measurement, clinical chemistry (alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, blood urea nitrogen, creatinine, calcium, total bilirubin, phosphorus, total protein, lipemia index, hemolysis index), and gross and microscopic evaluation of organs and selected tissues.

[0414] The efficacy of AB-101, oxotuzumab, and the combination of AB-101 and oxotuzumab was assessed by weekly monitoring of changes in peripheral blood CD19+ B cell levels from baseline (day -2) after treatment. Figure 11On day 7, the combination of single-dose oxutuzumab and AB-101 resulted in comparable CD19+ B cell exhaustion (mean 28.15% ± 4, groups 5 and 6, n=8) compared to oxutuzumab alone (mean 29.65% ± 3, groups 2 and 3, n=8). However, when comparing the percentage change in CD19+ B cells on day 21 with either two-dose oxutuzumab (mean 49.3% ± 11, n=5) or the combination of AB-101 and oxutuzumab (mean 33.6% ± 3, n=5), there was a trend toward enhanced B cell exhaustion with the combination (Table 8). For comparison, the mean percentage change in CD19+ B cells from baseline in the solvent and AB-101 alone groups were 59.67 ± 17.6 and 72.3% ± 11.1, respectively. In the presence of AB-101 alone or in combination with oxotuzumab, few to no off-target apoptosis (T cells, myeloid cells) was observed.

[0415] Table 8. Effects of AB-101 and oxotuzumab on the percentage change in CD19+ B cells from baseline (day -2).

[0416]

[0417] Data for day 7 were obtained after a single dose of obin, AB-101, or obin + AB-101, and data for day 14 were obtained after two doses of obin, AB-101, or obin + AB-101 for the specified groups. In this text, day 7 data for groups 2 and 3, and groups 5 and 6, are averaged because the test sample was administered only once at that time point. Comparable CD19+ B cell exhaustion was observed with obinuzumab alone (mean 29.65% ± 3, n=8) and obinuzumab + AB-101 (mean 28.15% ± 4, n=8). Obin = obinuzumab, AVG = mean, SEM = standard error of the mean.

[0418] The effects of treatment on toxicity endpoints were evaluated. No deaths or clinical observations were observed in any treatment group. There were no significant differences in the percentage change in body weight among the solvent, oxotuzumab, AB-101, or AB-101 and oxotuzumab combination treatment groups. Figure 12 In all groups, no AB-101 or oxutuzumab-related effects were observed in clinical chemistry parameters, whether as monotherapy or in combination. Individual and mean fluctuations were considered incidental, consistent with biological variation, and / or negligible in magnitude, and unrelated to test item administration.

[0419] No test-related macroscopic findings were found in organs from any treatment group during autopsies. No microscopic findings were reported related to AB-101 or oxutuzumab administration.

[0420] In summary, the potency and tolerability of the combination of AB-101 and the anti-CD20 monoclonal antibody olotuzumab were evaluated in a CD34+ humanized NSG mouse model. Given the high levels of human B cells, this model was considered suitable for assessing the effects of treatment on B cell levels and general animal health. AB-101 in combination with olotuzumab showed a trend toward enhanced B cell depletion compared to AB-101 or olotuzumab alone. Little to no off-target apoptosis (T cells, myeloid cells) was observed in vivo, and no combination-related toxicities were observed.

[0421] The NSG mouse model was used to determine the biodistribution and pharmacokinetics (PK) of AB-101. Solvents (PBS, dextran, albumin (human), DMSO) and AB-101 cells (0.5 × 10⁻⁶) were used. 7 1 cell / mouse, 2×10 7 Administered intravenously at 0.25 mL / mouse (cells / mouse), for a total of 8 doses. Animals in the solvent and AB-101 groups were sacrificed at 4 hours, 1, 3, 7, 14 and 78 days after the last administration (n = 3 male mice and n = 3 female mice at each time point).

[0422] The pharmacokinetics of AB-101 were determined using qPCR. A set of primers / probes specific to the human β-globin gene was used to detect AB-101 DNA against an NSG mouse matrix DNA background.

[0423] From 4 hours post-administration until 3 days after the last dose of AB-101 (day 53), AB-101 cells were primarily detected in highly perfused tissues (lung, spleen, heart, and liver) and at the injection site. 7 days post-administration (day 57), AB-101 cells were detected in the lungs (3 of 6 samples), spleen (5 of 6 samples), and injection site (5 of 6 samples). At 14 and 28 days post-administration (days 64 and 78, respectively), AB-101 cells were detected in two and one injection site samples, respectively. The occasional occurrences and low concentrations observed in the injection site samples on days 64 and 78 do not indicate systemic persistence of AB-101.

[0424] Biodistribution studies showed that the in vivo distribution of AB-101 cells was consistent with the intravenous administration route of the cell product. The cells lacked long-term persistence potential and were cleared within 7 days after administration, with no evidence of permanent engraftment. The pharmacokinetic profile of AB-101 is as follows: Figure 13 As shown.

[0425] Toxicological studies were conducted using dose range exploration (DRF) and gastrointestinal toxicology (GLP) studies. The purpose of the DRF studies was to identify the safe dose range of AB-101 cells in NSG mice after repeated intravenous administration. The purpose of the GLP studies was to evaluate the toxicity of AB-101 in NSG mice after repeated intravenous administration of the doses identified in the DRF studies.

[0426] Mice carry two mutations in the NOD / ShiLtJ genetic background: severe combined immunodeficiency (scid) and a completely null allele (IL2rgnull) in the common γ chain of the IL2 receptor. The scid mutation is located in the DNA repair complex protein Prkdc and confers defects in mouse B cells and T cells. The IL2rgnull mutation blocks cytokine signaling through multiple receptors, leading to defects in functional NK cells.

[0427] The aim of the DRF study was to determine the safe dose range of AB-101 cells in NSG mice after multiple intravenous administrations. The test protocol involved eight weekly intravenous administrations via the tail vein. The test dose range proposed in the preclinical toxicology assessment was designed to deliver a dose 4 × 10⁻⁶ of the equivalent human dose. 9 Higher AB-101 product exposure per cell / dose. 0.1 × 10⁻⁶ cells / dose tested in the DRF study. 7 Up to 2.5×10 7 The dosage range per cell / dose / animal provides an adequate safety margin for intended clinical administration. Mouse and human dosages converted to allometric proportions (Nair 2016): intermediate dose level 0.5 × 10⁻⁶. 7 One cell / animal is equivalent to 14 × 10 9 Human equivalent dose per cell, high dose level 2.5 × 10⁻⁶ 7 One cell / animal is equivalent to 70 × 10 9 The human equivalent dose per cell was determined for a 70kg patient. The results of the DRF study were used to guide dosing in GLP toxicology studies. The experimental design of the DRF study is shown in Table 9.

[0428] Table 9. Experimental Design

[0429]

[0430] The dosing formulation was freshly prepared from frozen test sample vials on each dosing day. Cell test samples and solvents were thawed by immersion in a water bath set to 37°C. Once thawed, the test sample was diluted in the solvent to achieve the target cell count (±15%) for animal dosing. A small-scale pilot pretest run was conducted to test the stability of the cell test sample in the formulation and its syringe compatibility during benchtop storage.

[0431] The solvent control or cell assay was administered to groups 1 through 3 on days 1 and 3, followed by weekly administration starting on day 12 (days 1, 3, 12, 19, 26, 33, 40, 47, and 54). The cell assay was administered to group 4 only on days 1 and 3, and weekly to group 5 throughout the course of administration (days 1, 8, 15, 22, 29, 36, 43, and 50). Administration was done via slow IV bolus injection (over a 30-40 second time interval) via the tail vein. The dose levels for groups 1 through 3 were 0, 0.1 × 10⁻⁶, and 0.1 × 10⁻⁶, respectively. 7 1 cell and 0.5 × 10 7 10 cells, with a dose level of 2.5 × 10⁻⁶ in groups 4 and 5. 7 One cell and administered at a dose of 0.25 mL.

[0432] Following the first dose, animals in group 4 exhibited reduced activity and / or cold skin to the touch. These observations subsided before the next dose. After the second dose (day 3), animals in group 4 exhibited severe reduced activity, arched back posture, piloerection, partial eye closure, pallor, loss of skin elastin, cold skin to the touch, and irregular breathing—both clinical and / or veterinary findings. Due to the severity of these findings, these animals were euthanized prematurely on day 3.

[0433] Mild swelling (foot / limb) and / or impaired limb function and / or limb abduction were observed in all groups, including the solvent control group. These findings were present in most animals and were considered to be solvent-related, but were not considered adverse due to their mild nature and lack of pain response on palpation.

[0434] Test-item-related findings of tremor and untidy appearance were observed in both groups 3 and 5. Additional test-item-related clinical and / or veterinary findings in group 5 included moderate decreased activity, ataxia, slow breathing, partial / complete eye closure, cold skin to the touch, and loss of skin elastin. These findings in group 5 were primarily observed after the sixth dose, and animals generally recovered by the second day. After the eighth dose, animals in group 5 exhibited severe decreased activity, dehydration, weakness, ataxia, partial eye closure, irregular breathing, untidy appearance, low posture, limb abduction, and cold skin to the touch. Although the severity observed after the eighth dose was noteworthy, all animals recovered by the second day. The findings in group 5 were considered undesirable because the severity of the observed results increased with increasing dose number. The findings in group 3 were not considered undesirable because of their low incidence and mild nature. No test-item-related findings were observed in group 2.

[0435] Conclusion. AB-101 was administered weekly by injection for 8 weeks, at a dose level ranging from 0.1 × 10⁻⁶. 7 Up to 2.5×10 7 Cells / dose / animal. Among the different dose levels tested in the DRF study, the 2.5 × 10⁻⁶ dose was observed after the 8th administration. 7 At a dose level of 0.5 × 10⁻⁶ cells, adverse clinical findings were observed, including severe reduced activity, dehydration, weakness, ataxia, partial eye closure, irregular breathing, unkempt appearance, low posture, limb abduction, and cold skin to the touch. Despite recovery, these findings were considered undesirable because the severity and number of those observed in these animals increased after the 8th dose. 7 and 2.5×10 7 Non-adverse test sample-related changes were observed at a dose level of 0.5 × 10⁻⁶ cells. 7 At a dose level of 1.5 × 10⁻⁶ cells, non-adverse clinical outcomes were observed, including tremor, unkempt appearance, and piloerection. 7 At individual cell dose levels, non-adverse testant-related changes were observed, such as decreased weight gain and altered clinicopathological endpoints, including decreased erythrocyte mass and reticulocyte counts, and increased leukocytes and neutrophils. No anatomical pathological findings were observed in the animals in the DRF study. In summary, NSG mice will be administered 2.5 × 10⁻⁶ cells intravenously once weekly for 8 weeks. 7 The dose level for each cell was determined as the maximum tolerated dose (MTD).

[0436] The purpose of the GLP toxicity study was to evaluate the acute and delayed toxicity of AB-101 in NSG mice following repeated intravenous administration. The doses used in the GLP toxicity study were selected based on results obtained from the DRF study. In the DRF study, the dose was 2.5 × 10⁻⁶ after the 8th dose. 7 At doses of 100 cells, adverse clinical observations were observed. Therefore, in GLP toxicity studies, 2×10⁻⁶ cells / day was not recommended. 7 The cell count was used as the maximum dose. Mice were administered AB-101 once a week for 8 weeks. The dose used in the GLP toxicity studies was 0.5 × 10⁻⁶ cells / mL. 6 One cell / animal and 2×10 7 Each cell / animal provides an adequate safety margin (0.5 × 10⁻⁶) for the intended clinical dosing. 7 One cell / animal is equivalent to 14 × 10 9 Equivalent human cell dose per cell, 2 × 10⁻⁶ cells 7 One cell / animal is equivalent to 56 × 10⁶ 9 The equivalent human cell dose per cell (for a 70kg patient). Mouse and human doses were converted according to allometric growth ratios. The experimental design of the study is shown in Table 10.

[0437] Table 10. Experimental Design for Polar or Delayed Toxicity Studies

[0438]

[0439] Similar to the DRF study, the dosing formulation was freshly prepared from frozen test sample vials on each dosing day.

[0440] The cell assay and solvent control were administered via intravenous injection through the tail vein. The administration was carried out by slow bolus injection (over a period of 30 to 40 seconds). Administration was performed once weekly for 8 weeks.

[0441] In mice, at a concentration of 0.5 × 10⁻⁶ 7 and 2×10 7 Weekly intravenous administration of AB-101 at dose levels of surviving cells did not result in test-related death, weight change, or changes in ophthalmic, clinicopathological, or anatomical pathological endpoints. Based on the lack of adverse findings, the No-Observed-Effect Level (NOEL) was 2 × 10⁻⁶. 7 Dosage level of each surviving cell.

[0442] Example 7: Treatment of lupus nephritis with AB-101 and B-cell depleted monoclonal antibodies

[0443] B cells are recognized as key mediators in the pathogenesis of SLE, and anti-CD20 monoclonal antibodies promote direct B cell killing via antibody-dependent cell cytotoxicity (ADCC) and apoptosis induction. However, studies have shown that B cell depletion in some patients' tissues is incomplete after rituximab treatment, leading to the escape of pathogenic B cells and hindering effective immune system resetting. Reduced numbers of NK cells from SLE patients in peripheral blood, accompanied by decreased cytotoxicity and ADCC defects, have been shown, potentially contributing to incomplete B cell depletion with rituximab. Various non-clinical studies have demonstrated that AB-101 enhances anti-CD20-mediated ADCC against CD20-expressing malignant B lymphocytes in vitro and in vivo, unaffected by the negative effects of glucocorticoids. Furthermore, AB-101 in combination with rituximab has been shown to induce B cell apoptosis in PBMCs isolated from SLE patients. Preliminary clinical data from subjects with advanced B-cell malignancies using rituximab and AB-101 have shown that this treatment is generally well-tolerated and can induce a deep therapeutic response in malignant B-cell disease.

[0444] AB-101 is a non-engineered, allogeneic, off-the-shelf, cryopreserved cord blood-derived natural killer (NK) cell therapy product. Cord blood units (CBUs) are pre-screened for the KIR-B haplotype and a natural high-affinity variant of CD16 (158V / V), the latter associated with ADCC enhancement, thereby producing a highly active NK cell product without additional engineering.

[0445] AB-101 is a commercially available, cryopreserved allogeneic NK cell product in a ready-to-use infusion medium. The starting material is FDA-approved cord blood units with the following properties: selected killer cell immunoglobulin-like receptor B (KIR B) haplotype: exhibiting a more active phenotype (Cooley, 2009); selected V / V CD16 polymorphism at F158: exhibiting higher binding affinity to the Fc domain of monoclonal antibodies to enhance antibody-dependent cell cytotoxicity [ADCC] (Musolino, 2008).

[0446] In preclinical studies, AB-101, when combined with tumor-targeting monoclonal antibodies, has demonstrated direct, specific, and potent killing of various tumor cell lines in vitro and in vivo. AB-101 has also shown the ability to secrete cytokines such as tumor necrosis factor alpha (TNFα) and interferon gamma (IFNγ) upon activation. AB-101 is currently being investigated in Phase 1 / 2 clinical trials in patients with B-cell-derived r / r NHL as monotherapy and in combination with rituximab (ClinicalTrials.gov:NCT04673617), and in patients with r / r HL and CD30+ PTCL in combination with AFM13 (a CD30 / CD16 bispecific antibody designed to redirect and enhance the cytotoxicity of natural killer (NK) cell-mediated antibody-dependent cells) (NCT05883449).

[0447] What makes NK cells unique is their ability to induce a rapid cytolytic response without antigen presentation or pre-sensitization (Miller 2013; Malmberg 2017). This is achieved in part by integrating signals from both inhibitory and activating NK cell receptors; this combined regulation of activity allows for the recognition of a wide range of malignant or virally 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 also exert their effects through antibody-dependent cytotoxicity (ADCC), whereby NK cells bind to the crystallizable fragment (Fc) portion of tumor-targeting antibodies coated on the surface of target cells and lyse the target cells by releasing cytotoxic factors such as perforin and granzymes.

[0448] In SLE, NK cell dysfunction includes ADCC deficiency (Kozlowski, 1982; Green, 2005). Anti-CD20 antibodies utilize ADCC as part of their mechanism of action to kill malignant B cells in cancer (Manches, 2003; Boross, 2012) and deplete pathogenic B cells in SLE (Albert, 2008). As mentioned above, rituximab treatment in SLE / LN patients has failed to demonstrate significant therapeutic efficacy and has varied with the degree of B cell depletion (Rovin, 2012). Oxtuzumab treatment in SLE / LN patients has shown improved efficacy and more rapid, deeper, and more durable B cell depletion compared to that observed with rituximab (Furie, 2020); however, not all patients have a response, indicating an unmet need in this area. Replacing endogenous SLE NK cells with allogeneic NK cell products would provide capable effector cells to enhance the activity of anti-CD20 antibodies. AB-101, in combination with these antibodies, enhances B cell exhaustion via ADCC. ADCC is mediated through the expression of the Fcγ receptor (FcγR), or CD16 (or FcγRIII), on the surface of NK cells. CD16 is expressed on ≥80% of AB-101, making it an ideal cell therapy candidate for combination with anti-CD20 antibodies for SLE / LN.

[0449] Non-clinical pharmacology of AB-101 has been investigated in in vitro and in vivo studies to evaluate its cytotoxicity, biodistribution, potency, and safety against tumor cell lines.

[0450] Related to the ADCC mechanism of action, the cytotoxic activity of AB-101 against B-cell lymphoma or leukemia cell lines, normal human peripheral blood mononuclear cells (PBMCs), and PBMCs isolated from SLE patients was evaluated in combination with anti-CD20 (rituximab and octotuzumab) or anti-CD19 (tancituzumab) monoclonal antibodies. Tumor cells or PBMCs were incubated with or without antibodies at different effector (NK cell) to target (tumor cell or PBMC) ratios (E:T ratio). Apoptosis (cysteine-positive) cells were quantified by flow cytometry.

[0451] All three antibodies enhanced the ADCC activity of AB-101 against B-cell lymphoma or leukemia cell lines. Cytotoxicity assays using normal human PBMCs co-cultured with AB-101 demonstrated enhanced AB-101-mediated B-cell killing at different E:T ratios in the presence of anti-CD20 or anti-CD19 antibodies. When AB-101 was combined with rituximab… Figure 7 ) or Oxtuzumab ( Figure 9Enhanced ADCC against SLE B cells was observed in both antibody concentration-dependent and E:T ratio-dependent manner. The specificity of cell killing in SLE PBMC samples was evaluated by examining the effect of this combination 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.

[0452] PBMCs from SLE patients were isolated from peripheral blood and mixed with thawed AB-101 (with or without CD20 antibody rituximab) for 4 hours. The percentage of caspase-positive B cells was determined by flow cytometry. Data are presented as mean + / - SD of replicate wells. Representative data from a single SLE patient sample are shown.

[0453] The combination of AB-101 with B-cell depletion monoclonal antibodies (such as rituximab or oxotuzumab) may be an effective means of inducing substantial and deep B-cell depletion, inhibiting the replenishment of short-lived plasmablasts, and allowing the immune system to be reset in these patients. This has the potential to provide meaningful and durable clinical remission in patients with advanced proliferative lupus nephritis.

[0454] Subjects may also receive antihypertensive and antiproteinuria therapy, which involves blocking the renin-angiotensin system (e.g., angiotensin-converting enzyme [ACE] inhibitors or angiotensin II receptor blockers).

[0455] Example A

[0456] In this case, adult subjects with type III or IV lupus nephritis (with or without type V) who had relapsed or were unresponsive to prior standard of care were treated with AB-101 plus a B-cell depleting monoclonal antibody (e.g., rituximab) following a lymphocyte depletion regimen. Patients were administered AB-101 and rituximab for a maximum of two treatment cycles, spaced 24 weeks apart.

[0457] Subjects will receive a lymphocyte depletion regimen for three consecutive days (days 1-3) at the start of each treatment cycle to induce lymphocyte depletion and create an optimal environment for in vivo expansion of AB-101. Rituximab will be administered twice intravenously at 1000 mg per treatment cycle (approximately 2 weeks apart), following pre-dose with methylprednisolone to reduce the risk of infusion-related reactions. AB-101 will be administered at one of three levels after the lymphocyte depletion regimen and after the first dose of rituximab (Table 11).

[0458] Table 11. Dosage levels and administration dates for AB-101

[0459]

[0460] The lymphocyte depletion regimen is a fludarabine and cyclophosphamide regimen, in which fludarabine is administered at a dose of 30 mg / m² on days 1, 2, and 3 at the start of each treatment cycle. 2 Cyclophosphamide was administered at 1000 mg / m² on day 3 of each treatment cycle. 2 Apply.

[0461] Rituximab was administered at 1000 mg on days 2 and 13 of each treatment cycle.

[0462] The first dose of AB-101 is administered at least 48 hours after the final infusion of the lymphocyte depletion regimen in each treatment cycle, using the dosing regimen listed in Table 11.

[0463] Example B

[0464] In this example, using the Common Criteria for Adverse Events (CTCAE) v.5.0 criteria, adult subjects with relapsed / refractory lupus nephritis type III or IV (with or without type V) were treated with AB-101 plus a B-cell depleting monoclonal antibody (e.g., rituximab, octotuzumab) following a lymphocyte depletion regimen. Patients were administered AB-101 and the B-cell depleting monoclonal antibody for a maximum of two treatment cycles, spaced 24 weeks apart. Figure 8 As shown. Subjects who achieve complete renal response (CRR) at week 22 will receive only the designated monoclonal antibody (if applicable). Subjects who do not achieve complete renal response will receive a second cycle of the initial treatment regimen.

[0465] All subjects will receive a 3-day (days 1-3) lymphocyte depletion regimen during their first treatment cycle, consisting of fludarabine (days 1-3) and cyclophosphamide (day 3) to induce lymphocyte depletion and create an optimal environment for in vivo expansion of AB-101. Rituximab or oxutuzumab will be administered twice intravenously at 1000 mg intervals (approximately 2 weeks apart) during each treatment cycle, following pre-dose with methylprednisolone to reduce the risk of infusion-related reactions. AB-101 will be administered using two different regimens after the lymphocyte depletion regimen and after the first dose of rituximab (see below).

[0466] Patients in seven cohorts will be treated with AB-101 (with or without B-cell depletion antibodies), as follows:

[0467] Table 12. Cohort and AB-101 Dose Levels

[0468]

[0469] Subjects in the monotherapy DL1 cohort will receive AB-101 as monotherapy on days 6, 13, and 20 of cycle 1.

[0470] Fludarabine (30 mg / m²) 2 Administer on days 1, 2, and 3 at the start of each treatment cycle; dosage adjustments may be made for renal insufficiency or other medical conditions. Cyclophosphamide (1000 mg / m²) 2 AB-101 is administered on day 3 of each treatment cycle. AB-101 is administered via gravity IV infusion at a rate of 5 to 10 mL / min on days 6, 13, and 20 of each treatment cycle. Patients may be pretreated with one or more of the following: acetaminophen (500 to 1,000 mg orally), diphenhydramine 12.5 to 25 mg IV or 25 mg orally (e.g., when required to treat hypersensitivity to AB-101), and / or a second-generation oral antihistamine (e.g., cetirizine, fexofenadine, or loratadine).

[0471] 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.

[0472] Rituximab or oxutuzumab (1000 mg, if administered) is administered via infusion (at a rate of 50 to 400 mg / hour) on days 1 and 15 of each treatment cycle. Patients may be pretreated with one or more of the following: corticosteroids (prednisone, prednisolone, dexamethasone, or methylprednisolone), analgesics / antipyretics, and / or antihistamines, as described in Table 13, for example, to reduce the incidence and severity of infusion-related reactions (IRR). Patients may also be pretreated as described in Table 14. Patient dosage may also be adjusted as described in Table 14.

[0473] Table 13. Examples of pre-treatment medication

[0474]

[0475] Table 14. Examples of pre-treatment medication and optional dosage adjustments

[0476]

[0477] Example 8: Treatment of autoimmune diseases using NK cell and B cell depleted monoclonal antibodies

[0478] This study will evaluate the safety and activity of allogeneic NK cells in combination with a B-cell depleted antibody (rituximab) in adult subjects with one of the following disease conditions that have failed previous treatment regimens and are considered 'resistant / refractory' by the investigator using clinical indication-specific disease criteria (below): rheumatoid arthritis (RA); pemphigus vulgaris (PV); granulomatous polyangiitis (GPA) / microscopic polyangiitis (MPA); and systemic lupus erythematosus (SLE).

[0479] Disease-specific inclusion criteria

[0480] Rheumatoid arthritis:

[0481] i. A documented diagnosis of RA that meets the 2010 American College of Rheumatology (ACR) / European League Against Rheumatism (EULAR) RA classification criteria (Kay, 2012).

[0482] ii. Patients who have previously received treatment with biologic disease-modifying antirheumatic drugs (bDMARDs, such as infliximab, rituximab, etanercept, tocilizumab) and / or targeted synthetic disease-modifying antirheumatic drugs (tsDMARDs, such as baricitinib, tofacitinib) and are considered refractory by any of the following:

[0483] a. According to the researchers, there is a lack of benefit for at least two bDMARDs or one bDMARD and one tsDMARD.

[0484] b. Lack of benefit may include insufficient improvement in joint count, physical function, or disease activity. Intolerance to at least two lines of prior therapy (including bDMARD and / or tsDMARD).

[0485] iii. The minimum number of swollen joint counts (SJC) is 6, and the minimum number of tender joint counts (TJC) is 6.

[0486] pemphigus vulgaris:

[0487] i. Diagnosed pemphigus vulgaris with active lesions.

[0488] ii. Positive for anti-desmosome core glycoprotein Dsg1 or Dsg3.

[0489] iii. The pemphigus disease area index score (Shimizu, 2014) was >80%.

[0490] Granulomatous polyangiitis (GPA) / Microscopic polyangiitis (MPA):

[0491] i. Clinical diagnosis of granulomatous polyangiitis (GPA) or microscopic polyangiitis (MPA).

[0492] ii. Has ≥1 “major” item, or ≥3 “other” items, or ≥2 kidney items on the Birmingham Vasculitis Activity Scale, Version 3 (BVASv3).

[0493] Systemic lupus erythematosus:

[0494] i. SLE diagnosed according to the 2019 European League Against Rheumatism / American College of Rheumatology (EULAR / ACR) classification criteria.

[0495] ii. At the time of screening, the total systemic lupus erythematosus disease activity index (SLEDAI-2K) score is ≥ 6.

[0496] iii. Positive for anti-double-stranded deoxyribonucleic acid (dsDNA) antibody.

[0497] The patient was administered: 1) NK cells; and 2) a B-cell depletion antibody (rituximab). The administered NK cells (AB-101) were a cryopreserved, ready-to-use suspension cell therapy composed of unmodified, ex vivo expanded allogeneic umbilical cord blood-derived NK cells prepared by FDA-approved umbilical cord blood units and pre-screened for: a) V / V CD16 polymorphism at F158 to enhance antibody-dependent cell cytotoxicity (ADCC) and b) killer cell immunoglobulin-like receptor B (KIR-B) haplotype.

[0498] All subjects will receive one treatment cycle. The treatment cycle will begin with a 3-day (day 1 to day 3) lymphocyte depletion regimen consisting of fludarabine (25 mg / m² on days 1, 2, and 3). 2 (Reduce or discontinue the dose for subjects with renal impairment) and cyclophosphamide (1000 mg / m² on day 3). 2 The regimen consists of rituximab, administered twice intravenously (1000 mg on days 2 and 13), following pre-administration with methylprednisolone to reduce the risk of infusion-related reactions. NK cells will be administered at 1B (1×10⁻⁶) doses on days 6, 13, and 20. 9 ) cells per administration. The first dose of NK cells should be administered at least 48 hours (and no more than 7 days) after the last infusion of the lymphocyte depletion regimen. The infusion schedule, cycle duration, and study drug administration regimen may be modified based on newly generated safety, pharmacokinetic, and biomarker data as determined by the investigator.

[0499] Research evaluation / endpoint

[0500] Safety: Safety will be assessed throughout the study by monitoring adverse events (AEs), concomitant medications, physical examinations, vital signs, and laboratory tests. The severity of AEs will be graded according to CTCAE version 5.0, except for the following adverse events: Cytokine release syndrome (CRS); the severity of CRS events will be assessed according to the American Society for Transplantation and Cell Therapy (ASTCT) classification (Lee, 2019); Immune effector cell-associated neurotoxicity syndrome (ICANS); the severity of ICANS events will be assessed according to the American Society for Transplantation and Cell Therapy (ASTCT) classification (Lee, 2019); Graft-versus-host disease (GvHD); the diagnosis and grading of GvHD should follow the Mount Sinai International Consortium for Acute GvHD (MAGIC) criteria (Harris, 2016).

[0501] 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.

[0502] Exploratory endpoints (changes from baseline at weeks 12 and 24). Rheumatoid arthritis: changes in the Clinical Disease Activity Index (CDAI); changes in participant response to treatment as assessed by the EULAR criteria. Pemphigus vulgaris: time and number of disease relapses; time to first complete remission, assessed by PDAI; changes in health-related QoL (via the Dermatology Quality of Life Index (DLQI)); serum DSG 1 and 3 levels. Granulomatous polyangiitis (GPA) and Microscopic polyangiitis (MPA): time and number of disease relapses; time to first complete remission; changes in health-related QoL (via the Dermatology Quality of Life Index (DLQI)). Systemic lupus erythematosus (SLE): changes in the Systemic Lupus Erythematosus Activity Measurement (SLAM); C3 and C4 complement levels.

[0503] Example 9: Characterization of SLE donor cells

[0504] SLE and healthy donor B cells and NK cells were characterized. Figure 14 As shown, SLE donors exhibit altered B-cell subsets, with an increase in transitional B cells and a decrease in activated memory B cells. Figure 15 As shown, SLE donors had reduced total NK cells, CD16+, and NKG2D, but reduced CD56. bright CD16 negIncreased NK cells.

[0505] Other implementation plans

[0506] It should be understood that although the invention has been described in conjunction with a detailed description, the foregoing description is intended to be illustrative and not to limit the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

Claims

1. A method for treating a patient suffering from an autoimmune disease, the method comprising administering a population of natural killer cells (NK cells) and an antibody targeting immune cells, wherein the NK cells are allogeneic to the patient.

2. The method according to claim 1, wherein the immune cells participate in the autoimmune response.

3. The method according to claim 1 or claim 2, wherein the immune cell is a B cell.

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 targeting human CD19 and / or human CD20.

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

7. The method according to any one of claims 1 to 6, wherein the autoimmune disease is selected from: acromegaly, acquired aplastic anemia, acquired hemophilia, primary agammaglobulinemia, alopecia areata, ankylosing spondylitis (AS), anti-NMDA receptor encephalitis, antiphospholipid syndrome (APS) | catastrophic antiphospholipid syndrome (CAPS) / Asherson syndrome, arteriosclerosis, autoimmune Addison's disease (AAD), autoimmune autonomic ganglion disease (AAG) / autoimmune autonomic dysfunction | autoimmune gastrointestinal motility disorder (AGID), autoimmune encephalitis | acute disseminated encephalomyelitis (ADEM), autoimmune gastritis, autoimmune hemolytic anemia (… Autoimmune hepatitis (AIHA), autoimmune hyperlipidemia, autoimmune hypophysitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune myelofibrosis, autoimmune myocarditis, autoimmune oophoritis, autoimmune pancreatitis (AIP), type I, II, and III autoimmune polyglandular syndrome (Type 1 APS, Type 2 APS, Type 3 APS, APECED), autoimmune progesterone dermatitis, autoimmune retinopathy (AIR), autoimmune sudden sensorineural hearing loss (SNHL), Barlow's disease, Behcet's disease, shotgun chorioretinopathy / shotgun uveitis, bullous diseases. Pemphigoid, Kassman's disease, celiac disease, Chagas disease, chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), chronic urticaria (CU), Chag-Strauss syndrome / eosinophilic granulomatous polyangiitis (EGPA), Cogan syndrome, cold agglutinin disease, CREST syndrome | cutaneous systemic scleroderma, Crohn's disease (CD), Cronkhite-Canada syndrome (CSS), cryptogenic organizing pneumonia (COP), herpetiform dermatitis, dermatomyositis, type 1 diabetes mellitus, discoid lupus, Dressler's syndrome / post-myocardial infarction / post-pericardiotomy syndrome, eczema / atopic dermatitis, endometriosis, eosinophilic esophagitis, eosinophilic esophagitis Cystic fasciitis, erythema nodosum, primary mixed cryoglobulinemia, Evans syndrome, fibrotic alveolitis / idiopathic pulmonary fibrosis (IPF), giant cell arteritis / temporal arteritis / Houghton's disease, giant cell myocarditis, glomerulonephritis, Goodbast syndrome / anti-GBM / anti-TBM disease, granulomatous polyangiitis (GPA) / Wegener's granulomatosis, Graves' disease / thyroid ophthalmopathy, Guillain-Barré syndrome (GBS), Hashimoto's thyroiditis / chronic lymphocytic thyroiditis / autoimmune thyroiditis, Henno-Schlan purpura / IgA vasculitis, hidradenitis suppurativa, Hurst's disease / acute hemorrhagic leukoencephalitis (AHLE), hypogammaglobulinemia, IgA nephropathy / Beggar's disease,Immune-mediated necrotizing myopathy (IMNM), immune thrombocytopenic purpura (ITP), inclusion body myositis, IgG4-associated sclerotic disease (ISD), interstitial cystitis, juvenile idiopathic arthritis, adult-onset Still's disease, juvenile polymyositis, juvenile dermatomyositis, Kawasaki disease, Lambert-Eton myasthenic syndrome (LEMS), leukocytoclastic vasculitis, lichen planus, lichen sclerosus, woody conjunctivitis, linear IgA disease (LAD), linear IgA bullous dermatosis (LABD), lupus nephritis, Lyme disease, chronic Lyme disease, post-treatment Lyme disease syndrome (PTLDS), lymphatic Cellular colitis / microscopic colitis, lymphocytic hypophysitis / autoimmune hypophysitis, Meniere's disease, microscopic polyangiitis (MPA) / ANCA-associated vasculitis, mixed connective tissue disease (MCTD), Mollen's ulcer, Mueller-Hacker disease, multifocal motor neuropathy, multiple sclerosis (MS), myalgic encephalomyelitis (ME) / chronic fatigue syndrome (CFS), myasthenia gravis (MG), narcolepsy, neuromyelitis optica / Devik's disease, ocular cicatricial pemphigoid, oculoclonus-myoclonus syndrome (OMS), recurrent rheumatism, paraneoplastic cerebellar degeneration, paraneoplastic pemphigoid, Parry-Romberg syndrome (PRS) / hemifacial atrophy (HFA) / progressive facial asymmetry Lateral atrophy, paroxysmal nocturnal hemoglobinuria (PNH), peripheral uveitis / paresthesia, PANS / PANDAS, Parson-Turner syndrome, pemphigoid of pregnancy / herpes gestationis, 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 gangrenosa, Rasmussen's encephalitis, Raynaud's syndrome / phenomenon, reaction Rheumatoid arthritis / Reiter's syndrome, reflex sympathetic dystrophy syndrome (RSD) / complex regional pain syndrome (CRPS), relapsing polychondritis, restless legs syndrome (RLS) / Willis-Eckbond disease, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt's syndrome / type II autoimmune polyendocrine syndrome, scleritis, scleroderma, sclerosing mesenteric / mesenteric panniculitis, creeping choroidal lesions, Sjögren's syndrome, stiff-person syndrome (SPS), small fiber sensory neuropathy, systemic lupus erythematosus (SLE), subacute bacterial endocarditis (SBE), subacute cutaneous lupus, Sussac syndrome, Sidnum chorea, sympathetic ophthalmia, Gau's arteritis (vasculitis)Testicular autoimmune disease (vasculitis, orchitis), Tolosa-Hunter syndrome, transverse myelitis (TM), tubulointerstitial nephritis-uveitis syndrome (TINU), ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), pre / intermediate / post-uveitis, vasculitis, VEXAS syndrome, vitiligo, Vogt-Koyanagi-Harada syndrome (VKH), and combinations thereof.

8. The method of claim 7, wherein the autoimmune disease is systemic lupus erythematosus (SLE).

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

10. The method of claim 7, wherein the autoimmune disease is rheumatoid arthritis (RA).

11. The method of claim 7, wherein the autoimmune disease is pemphigus vulgaris (PV).

12. The method of claim 7, wherein the autoimmune disease is granulomatous polyangiitis (GPA).

13. The method of claim 7, wherein the autoimmune disease is microscopic polyangiitis (MPA).

14. The method according to any one of the preceding claims, wherein the patient relapses after treatment with anti-CD19 and / or anti-CD20 antibodies.

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

16. The method according to any one of the preceding claims, wherein the patient is administered 1×10 8 Up to 1×10 10 NK cells.

17. The method according to any one of the preceding claims, wherein the patient is administered 1×10 9 Up to 8×10 9 NK cells.

18. The method according to any one of the preceding claims, wherein the patient is administered 4 × 10 8 1×10 9 4×10 9 Or 8×10 9 NK cells.

19. The method according to any one of the preceding claims, wherein the patient is administered 5 × 10 8 1×10 9 Or 4×10 9 NK cells.

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

21. The method of claim 20, wherein the antibody is rituximab, oxotuzumab, or tancituzumab.

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

23. The method of claim 20, wherein the antibody is oxotuzumab.

24. The method of claim 20, wherein the antibody is tanxituzumab.

25. The method according to any one of the preceding claims, wherein the patient has undergone lymphocyte depletion chemotherapy prior to treatment.

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

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

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

29. The method according to any one of claims 27 to 28, wherein the cyclophosphamide is at a concentration of 100 to 500 mg / m³. 2 Apply daily.

30. The method of claim 29, wherein the cyclophosphamide is at a concentration of 250 or 300 mg / m³. 2 Apply daily.

31. The method of claim 29, wherein the cyclophosphamide is at 500 mg / m³ 2 Apply daily.

32. The method according to any one of claims 27 to 31, wherein the fludarabine is administered at a concentration of 10 to 50 mg / m³. 2 Apply daily.

33. The method of claim 31, wherein the fludarabine is at a concentration of 30 mg / m². 2 Apply daily.

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

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

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

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

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

39. The method according to any one of the preceding 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 preceding claims, wherein lymphocyte depletion is performed on days 1, 2 and 3 of the treatment cycle.

41. The method according to any one of the preceding 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 preceding claims, wherein the NK cells are administered in doses of 2 billion or 4 billion cells, or about 2 billion or 4 billion cells.

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

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

45. The method according to any one of the preceding claims, wherein the NK cells are administered at 5 × 10⁻⁶ doses on days 6, 13, and 20. 8 1×10 9 Or 4×10 9 One NK cell, or approximately 5 × 10 8 1×10 9 Or 4×10 9 Administer one NK cell.

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

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

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

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

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

51. The method according to any one of the preceding claims, wherein 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+.

52. The method according to any one of the preceding claims, wherein each administration of NK cells is an administration of 1 × 10-1 9 Up to 5×10 9 NK cells.

53. The method of claim 34, wherein each administration of NK cells is an administration of 1 × 10⁻⁶ cells. 9 Up to 5×10 9 NK cells.

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

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

56. The method according to any one of the preceding claims, wherein the expanded natural killer cell 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.

57. The method according to any one of the preceding claims, wherein the expanded natural killer cell 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.

58. The method according to any one of the preceding claims, wherein the expanded natural killer cell 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.

59. The method according to any one of the preceding claims, wherein the expanded natural killer cell 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.

60. The method according to any one of the preceding claims, wherein the expanded natural killer cell 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.

61. The method according to any one of the preceding claims, wherein the expanded natural killer cell 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.

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

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

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

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

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

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

68. The method according to any one of the preceding claims, wherein the expanded natural killer cells are not genetically engineered.

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

70. The method according to any one of the preceding claims, wherein the NK cell population comprises at least 100 million expanded natural killer cells, such as 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, 10 billion, 20 billion, 25 billion, 30 billion, 40 billion, 50 billion, 60 billion, 70 billion, 80 billion, 90 billion, 1 trillion, 2 trillion, 3 trillion, 4 trillion, 5 trillion, 6 trillion, 7 trillion, 8 trillion, 9 trillion, 1 trillion, 2 trillion, 3 trillion, 4 trillion, 5 trillion, 6 trillion, 7 trillion, 8 trillion, 9 trillion, or 10 trillion expanded natural killer cells.

71. The method according to any one of the preceding claims, wherein the NK cell population is generated by a method comprising: (a) Obtaining seed cells containing natural killer cells from umbilical cord blood; (b) Deplete the CD3+ cells in the seed cells; (c) Natural killer cells are generated by expanding the natural killer cells by culturing depleted seed cells with a first group of Hut78 cells engineered to express membrane-bound IL-21, mutant TNFα, and 4-1BBL genes. This results in an expanded population of natural killer cells.

72. The method according to any one of the preceding claims, wherein the NK cell population is generated by a method comprising: (a) Obtaining seed cells containing natural killer cells from umbilical cord blood; (b) Deplete the CD3+ cells in the seed cells; (c) To generate an expanded master cell bank population of natural killer cells by culturing depleted seed cells with a first group of Hut78 cells engineered to express membrane-bound IL-21, mutant TNFα, and 4-1BBL genes; and (d) The expanded natural killer cells were generated by expanding the master cell bank population of the expanded natural killer cells by culturing them with a second group of Hut78 cells engineered to express membrane-bound IL-21, mutant TNFα and 4-1BBL genes. This results in an expanded population of natural killer cells.

73. The method of claim 71 or claim 72, wherein the NK cell population is generated by further comprising the following method after step (c): (i) A master cell bank population of natural killer cells cryopreserved and expanded in multiple containers; and (ii) Thawing containers containing aliquots of the master cell bank population of expanded natural killer cells. Step (d) involves amplifying the master cell bank population of the amplified natural killer cells by amplifying equal portions of the master cell bank population of the amplified natural killer cells.

74. The method according to any one of claims 71 to 73, wherein the 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 NK cell population is generated by a method comprising expanding the natural killer cells derived from umbilical cord blood by at least 10,000-fold, such as 15,000-fold, 20,000-fold, 25,000-fold, 30,000-fold, 35,000-fold, 40,000-fold, 45,000-fold, 50,000-fold, 55,000-fold, 60,000-fold, 65,000-fold, or 70,000-fold.

76. The method according to any one of claims 71 to 75, wherein the expanded natural killer cell population is not enriched or sorted after expansion.

77. The method according to any one of claims 71 to 76, wherein the percentage of CD16-expressing NK cells in the expanded natural killer cell population is the same as or higher than the percentage of natural killer cells from 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 natural killer cell population is the same as or higher than the percentage of natural killer cells from 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 natural killer cell population is the same as or higher than the percentage of natural killer cells from 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 natural killer cell population is the same as or higher than the percentage of natural killer cells from the seed cells derived from umbilical cord blood.

81. The method according to any one of claims 71 to 80, wherein 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 from 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 natural killer cell population is the same as or higher than the percentage of natural killer cells from the seed cells derived from umbilical cord blood.

83. A method for treating systemic lupus erythematosus (SLE), the method comprising: Natural killer cell (NK cell) populations and antibodies targeting B cells were administered to a patient diagnosed with SLE, wherein the NK cells were allogeneic to the patient, and wherein the NK cells were KIR-B haplotyped and homozygous for CD16 158V polymorphism.

84. The method of 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 of claim 85, wherein the antibody is rituximab, oxotuzumab, or tancituzumab.

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

88. The method according to any one of claims 83 to 87, wherein the patient is administered 1 × 10 9 Up to 5×10 9 A population of NK cells.

89. The method of claim 88, wherein the patient is administered 2 × 10 9 A population of NK cells, or approximately 2 × 10⁹ cells 9 A population of NK cells.

90. The method of claim 88, wherein the patient is administered 4 × 10 9 A population of NK cells, or approximately 4 × 10⁹ cells 9 A population of NK cells.

91. The method of claim 88, wherein the patient is administered 5 × 10 8 A population of NK cells, or approximately 5 × 10⁶ 8 A population of NK cells.

92. The method of claim 88, wherein the patient is administered 1×10 9 A population of NK cells, or approximately 1 × 10⁹ cells 9 A population of NK cells.

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

94. The method of claim 93, wherein the patient is administered 100 mg of the antibody, or about 100 mg of the antibody.

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

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

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

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

99. The method according to any one of claims 97 to 98, wherein the cyclophosphamide is at a concentration of 100 to 1500 mg / m³. 2 Apply daily.

100. The method of claim 99, wherein the cyclophosphamide is at a concentration of 500 or 1000 mg / m³. 2 Apply daily.

101. The method according to claim 99, wherein the cyclophosphamide is at a concentration of 1000 mg / m³. 2 Apply daily.

102. The method according to any one of claims 97 to 101, wherein the fludarabine is administered at a concentration of 10 to 50 mg / m³. 2 Apply daily.

103. The method according to claim 102, wherein the fludarabine is at a concentration of 30 mg / m². 2 Apply daily.

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

105. The method of claim 104, wherein the first treatment cycle comprises administering lymphocyte-depleting chemotherapy prior to the administration of NK cells.

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

107. The method of claim 106, wherein fludarabine is administered on days 1, 2, and 3 of the treatment cycle.

108. The method according to claim 106 or 107, wherein fludarabine is administered at 30 mg / m². 2 Apply daily.

109. The method according to any one of claims 106 to 108, wherein the cyclophosphamide is administered on day 3 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 administration of the lymphocyte depletion chemotherapy.

111. The method according to any one of claims 106 to 110, wherein the cyclophosphamide is at a concentration of 100 mg / m³. 2 Apply daily.

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

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

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

115. The method according to any one of claims 104 to 114, wherein the NK cells are administered for more than one day during days 6, 9, 13, 16, and 20.

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

117. The method of 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 at a rate of 2 × 10⁻⁶ on days 6 and 13. 9 Administered to cells, and on day 20 at 1×10 9 Apply to individual cells.

119. The method according to any one of claims 104 to 114, wherein the NK cells are administered at a rate of 4 × 10⁻⁶ on days 6 and 13. 9 Administered to individual cells, and on day 20 at 2 × 10⁻⁶. 9 Apply to individual cells.

120. The method according to any one of claims 104 to 114, wherein the NK cells are administered at a rate of 4 × 10⁻⁶ on days 6 and 13. 9 Administered to individual cells, and on days 9 and 16 at 2 × 10⁻⁶. 9 Apply to individual cells.

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

122. The method of claim 121, wherein the second treatment cycle comprises administering lymphocyte-depleting chemotherapy prior to the administration of NK cells.

123. The method of claim 122, wherein the administration of lymphocyte-depleting chemotherapy included in the second treatment cycle comprises administration of cyclophosphamide and fludarabine.

124. The method of claim 123, wherein fludarabine is administered on days 1, 2, and 3 of the second treatment cycle.

125. The method according to claim 123 or 124, wherein the fludarabine is administered at 30 mg / m² during the second treatment cycle. 2 Apply daily.

126. The method according to any one of claims 123 to 125, wherein the cyclophosphamide is administered on day 3 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 administration of the lymphocyte depletion chemotherapy.

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

129. The method according to any one of claims 121 to 128, wherein the antibody is administered on days 2 and 13 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 of claim 130, wherein the antibody is administered at 1000 mg during the second treatment cycle.

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

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

134. The method of 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 at a rate of 2 × 10⁻⁶ on days 6 and 13 of the second treatment cycle. 9 Administered to cells, and on day 20 at 1×10 9 Apply to individual cells.

136. The method according to any one of claims 121 to 132, wherein the NK cells are administered at a rate of 4 × 10⁻⁶ on days 6 and 13 of the second treatment cycle. 9 Administered to individual cells, and on day 20 at 2 × 10⁻⁶. 9 Apply to individual cells.

137. The method according to any one of claims 121 to 132, wherein the NK cells are administered at a rate of 4 × 10⁻⁶ on days 6 and 13 of the second treatment cycle. 9 Administered to individual cells, and on days 9 and 16 at 2 × 10⁻⁶. 9 Apply to individual cells.

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 of 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 suffers from type III or type IV lupus nephritis.

141. The method of claim 140, wherein the patient suffers from lupus nephritis type III.

142. The method of claim 140, wherein the patient suffers from lupus nephritis type IV.

143. The method according to any one of claims 84 to 142, wherein the patient suffers from lupus nephritis type V.

144. The method according to any one of claims 83 to 143, wherein the patient is a patient who has relapsed or is refractory to SLE after prior treatment.

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

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

147. The method of claim 146, wherein the improvement is complete renal remission (CRR).

148. The method of claim 147, wherein the CRR is measured as a urine protein to creatinine ratio (UPCR) <0.5 and / or normal renal function and a decline in baseline serum creatinine of no more than 15%.

149. The method of claim 148, wherein normal renal function is measured as serum creatinine less than or equal to ULN.

150. The method of claim 146, wherein the improvement is partial renal remission (PRR).

151. The method of claim 150, wherein the PRR determination is a UPCR value that is more than 50% lower than the baseline and less than 3.

152. The method of claim 151, wherein the PRR determination is a UPCR value that is more than 50% lower than the baseline and less than 1.

153. The method according to any one of claims 146 to 152, wherein the ORR is determined 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 determined 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 preceding claims, wherein the NK cells are not genetically modified.

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

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

158. The method according to any one of the preceding claims, wherein 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+.

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

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

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

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

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

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

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

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

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

174. The method according to any one of the preceding claims, wherein the NK cell population comprises at least 100 million expanded natural killer cells, such as 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, 10 billion, 20 billion, 25 billion, 30 billion, 40 billion, 50 billion, 60 billion, 70 billion, 80 billion, 90 billion, 1 trillion, 2 trillion, 3 trillion, 4 trillion, 5 trillion, 6 trillion, 7 trillion, 8 trillion, 9 trillion, or 10 trillion expanded natural killer cells.

175. The method according to any one of the preceding claims, wherein the NK cell population is generated by a method comprising: (a) Obtaining seed cells containing natural killer cells from umbilical cord blood; (b) Deplete the CD3+ cells in the seed cells; (c) Natural killer cells are generated by expanding the natural killer cells by culturing depleted seed cells with a first group of Hut78 cells engineered to express membrane-bound IL-21, mutant TNFα, and 4-1BBL genes. This results in an expanded population of natural killer cells.

176. The method according to any one of the preceding claims, wherein the NK cell population is generated by a method comprising: (a) Obtaining seed cells containing natural killer cells from umbilical cord blood; (b) Deplete the CD3+ cells in the seed cells; (c) To generate an expanded master cell bank population of natural killer cells by culturing depleted seed cells with a first group of Hut78 cells engineered to express membrane-bound IL-21, mutant TNFα, and 4-1BBL genes; and (d) Expanded natural killer cells are generated by culturing the second group of Hut78 cells engineered to express membrane-bound IL-21, mutant TNFα and 4-1BBL genes to expand the master cell bank population of the expanded natural killer cells. This results in an expanded population of natural killer cells.

177. The method according to claim 175 or 176, wherein the NK cell population is generated by further comprising the following method after step (c): (i) A master cell pool of natural killer cells cryopreserved and expanded in multiple containers; and (ii) Thawing containers containing aliquots of the master cell bank of expanded natural killer cells. Step (d) involves amplifying the master cell bank population of the amplified natural killer cells by amplifying equal portions of the master cell bank population of the amplified natural killer cells.

178. The method according to any one of claims 175 to 177, wherein the 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 preceding claims, wherein the NK cell population is generated by a method comprising expanding the natural killer cells derived from umbilical cord blood by at least 10,000-fold, such as 15,000-fold, 20,000-fold, 25,000-fold, 30,000-fold, 35,000-fold, 40,000-fold, 45,000-fold, 50,000-fold, 55,000-fold, 60,000-fold, 65,000-fold, or 70,000-fold.

180. The method according to any one of claims 175 to 179, wherein the expanded natural killer cell population is not enriched or sorted after expansion.

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

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

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

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

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

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

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