Treating cancer with NK cells and multispecific engagers

Abstract

Provided herein are methods for treating patients suffering from cancer using natural killer (NK) cells that have a KIR-B haplotype and express the CD16 molecule and a bispecific antibody that binds to CD16 and CD30, as well as methods for producing NK cell populations and pharmaceutical compositions comprising said NK cells and the bispecific antibody. TIFF2024542159000017.tif58159

Description

[Technical field]

[0001] Claiming priority This application claims the benefit of U.S. Provisional Application No. 63 / 275,890, filed November 4, 2021, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] background Targeted therapies, including antibody therapy, have revolutionized cancer treatment. One of the mechanisms by which antibody therapy induces cytotoxicity is through antibody-dependent cell-mediated cytotoxicity (ADCC). Many cancer patients are unable to mount a robust ADCC response. A reduced ADCC response would significantly reduce the efficacy of any applicable monoclonal antibody therapeutic for these patients, which may prevent response or lead to relapse in these patients. Thus, a reduced ADCC response may negatively impact their clinical outcomes.

[0003] Despite several recent discoveries and developments of anti-cancer drugs, many types of cancer have poor prognoses and therefore there remains a need for improved methods and therapeutic agents.

[0004] The present invention addresses these and other deficiencies in the art. Summary of the Invention

[0005] overview NK cells are immune cells that can engage tumor cells through a complex set of receptors on their cell surface and also through antibody-dependent cellular cytotoxicity (ADCC). To initiate ADCC, NK cells engage antibodies through 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. As an exemplary advantage, NK cells can be used for allogeneic therapy. That is, NK cells from one donor can be safely used in one or many patients without the need for HLA matching, gene editing or other genetic manipulation. Allogeneic NK cells with antitumor activity can be safely administered to patients without many of the risks associated with T cell therapy, such as severe cytokine release syndrome (CRS), neurotoxicity or graft-versus-host disease (GvHD).

[0006] Allogeneic NK cells may be an important treatment option for cancer patients. One exemplary advantage is that NK cells are well tolerated and show no evidence of graft-versus-host disease, neurotoxicity, or cytokine release syndrome associated with other cell-based therapies. Another exemplary advantage is that NK cells do not require prior antigen exposure or expression of specific antigens to identify and lyse tumor cells. Another exemplary advantage is that NK cells have the unique ability to bridge innate immunity and generate polyclonal adaptive immune responses that result in long-term anticancer immune memory.

[0007] For example, NK cells can recruit and activate other components of the immune system. Activated NK cells secrete cytokines and chemokines, such as interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα) and macrophage inflammatory protein 1 (MIP1), that signal and recruit T cells to tumors. NK cells directly kill tumor cells, thereby exposing tumor antigens for recognition by the adaptive immune system.

[0008] In addition, by utilizing a diverse cord blood bank as a source of NK cells, cords with favorable characteristics for enhanced clinical activity (e.g., high affinity CD16 and killer cell immunoglobulin-like receptor (KIR) B haplotype) can be selected.

[0009] Administration of allogeneic NK cells as described herein can enhance the ADCC response in a patient, e.g., in combination with a multispecific engager, e.g., a multispecific engager as described herein.

[0010] CD30 is a cell membrane protein of the tumor necrosis factor receptor family that is ubiquitously expressed to varying degrees in classical Hodgkin lymphoma (HL) and several subtypes of peripheral T-cell lymphoma, including anaplastic large cell lymphoma (ALCL), peripheral T-cell lymphoma (PTCL) not otherwise specified (PTCL-NOS), and angioimmunoblastic T-cell lymphoma (AITL).

[0011] In classical HL (cHL), the most common CD30-positive lymphoma, first-line chemotherapy (ABVD or BEACOPP) with or without radiation therapy has shown significant efficacy. In patients with advanced cHL, first-line treatment may also include the CD30-targeted antibody-drug conjugate brentuximab vedotin (AVD) with chemotherapy. However, up to 30% of patients are refractory or relapse after first-line treatment. For patients with relapsed or refractory cHL, high-dose chemotherapy and autologous stem cell transplantation (ASCT) is curative in less than 50% of cases (Majhail NS, Weisdorf DJ, Defor TE, et al. "Long-term results of autologous stem cell transplantation for primary refractory or relapsed Hodgkin's lymphoma." Biol Blood Marrow Transplant. 2006;12(10):1065-1072.doi:10.1016 / j.bbmt.2006.06.006; Josting A, Mueller H, Borchmann P, et al. "Dose intensity of chemotherapy in patients with relapsed Hodgkin's lymphoma." J Clin Oncol. 2010;28(34):5074-5080.doi:10.1200 / JCO.2010.30.5771; Bartlett NL, Herrera AF, Domingo-Domenech E, et al. "A phase 1b study of AFM13 in combination with pembrolizumab in patients with relapsed or refractory Hodgkin lymphoma" Blood.2020;136(21):2401-2409.doi:10.1182 / blood.2019004701).Relapse after ASCT is associated with a poor prognosis, with a median survival of 26 months (Voorhees TJ, Beaven AW. Therapeutic Updates for Relapsed and Refractory Classical Hodgkin Lymphoma. Cancers (Basel). 2020;12(10):2887. Published 2020 Oct 8. doi:10.3390 / cancers12102887). Systemic treatment options for refractory and relapsed patients may include agents such as brentuximab vedotin as monotherapy or in combination with another agent, and / or PD-(L)1 inhibitors. Despite recent advances incorporating promising targeted and immune agents, there remains an unmet medical need in the relapsed / refractory setting for treatments that provide high response levels, longer duration of response, and potential cure, with a clinically acceptable safety profile.

[0012] Regarding peripheral T-cell lymphoma, first-line treatment with CHOP or CHOP-like regimens results in poor outcomes, except in cases of ALK+ALCL. Despite intensified approaches in frontline treatment, such as consolidation with ASCT, these patients remain at significant risk of relapse or early progression. In general, most, if not all, patients undergoing treatment for PTCL, especially in the absence of hematopoietic cell transplantation (HCT), do not achieve remission or relapse, with very poor long-term survival, with median PFS and OS estimates of only 3 and 6 months, respectively (Mak V, Hamm J, Chhanabhai, et al. "Survival of patients with peripheral T-cell lymphoma after first relapse or progression: spectrum of disease and rare long-term survivors" J. Clin. Oncol. 2013;31(16):1970-1976; Biasoli I, Cesaretti M, Bellei M, et al. "Dismal outcome of T-cell lymphoma patients failing first-line treatment: results of a population-based study from the Modena Cancer Registry" Hematol. Oncol. 2015;33(3):147-151; Bellei M, Foss F, Shustov A, et al. "The outcome of “Peripheral T-cell lymphoma patients failing first-line therapy: a report from the prospective, International T-Cell Project” Hematologica. 2018;103(7):1191-1197). Therefore, novel therapeutic agents and treatment regimens are needed to address the unmet medical needs that exist for patients with PTCL.

[0013] AFM13 is a tetravalent bispecific (anti-human CD30 x anti-human CD16A) recombinant antibody construct that is being investigated for the treatment of HL and other CD30-positive malignancies, such as PTCL. AFM13 targets the CD30 antigen expressed on malignant lymphoma cells. At the same time, the anti-CD16A domain binds to CD16A (FcλRIIIA) on NK cells and macrophages. AFM13 forms a bridge between tumor target cells and natural effector cells to trigger the lysis of CD30 antigen-positive cells by NK cells via antibody-dependent cell-mediated cytotoxicity (ADCC).

[0014] It has been shown that NK cell populations are absent in the immunosuppressive tumor microenvironment of HL. Furthermore, NK cells from patients with HL are dysfunctional, in part due to an imbalance between activating and inhibitory receptors (Reiners KS, Kessler J, Sauer M, et al. "Rescue of impaired NK cell activity in Hodgkin lymphoma with bispecific antibodies in vitro and in patients" Mol Ther 2013;21:895-903). Due to these limitations in autologous NK cell function, optimal NK immunotherapy for HL will likely require an allogeneic source.

[0015] Accordingly, the present disclosure provides a method for treating and / or preventing the development of CD30 NK cells, comprising, inter alia, administering to a patient a first pharmaceutical composition comprising natural killer cells (NK cells) comprising a KIR-B haplotype and expression of a CD16 molecule, and administering to a patient a second pharmaceutical composition comprising a bispecific antibody or antigen-binding fragment thereof comprising a first binding domain that specifically binds to CD16 (FcγRIII) and a second binding domain that specifically binds to CD30. +A method for treating a patient suffering from cancer, comprising: a first binding domain that specifically binds to CD16 comprising a light chain variable domain (VL_CD16A) comprising light chain complementarity determining region 1 (CDRL1) comprising SEQ ID NO:9, light chain complementarity determining region 2 (CDRL2) comprising SEQ ID NO:10, light chain complementarity determining region 3 (CDRL3) comprising SEQ ID NO:11, and a heavy chain variable domain (VH_CD16A) comprising heavy chain complementarity determining region 1 (CDRH1) comprising SEQ ID NO:6, heavy chain complementarity determining region 2 (CDRH2) comprising SEQ ID NO:7, and heavy chain complementarity determining region 3 (CDRH3) comprising SEQ ID NO:8; and a second binding domain that specifically binds to CD30 comprising light chain complementarity determining region 1 (CDRL1) comprising SEQ ID NO:15, light chain complementarity determining region 2 (CDRL2) comprising SEQ ID NO:16, heavy chain complementarity determining region 3 (CDRH3) comprising SEQ ID NO:9, and a heavy chain variable domain (VH_CD16A) comprising heavy chain complementarity determining region 1 (CDRH1) comprising SEQ ID NO:17, heavy chain complementarity determining region 2 (CDRH2) comprising SEQ ID NO:18, and heavy chain complementarity determining region 3 (CDRH3) comprising SEQ ID NO:19. and a heavy chain variable domain (VH_CD30) comprising a heavy chain complementarity determining region 1 (CDRH1) comprising SEQ ID NO:12, a heavy chain complementarity determining region 2 (CDRH2) comprising SEQ ID NO:13, and a heavy chain complementarity determining region 3 (CDRH3) comprising SEQ ID NO:14.

[0016] In some embodiments, the NK cells are cord blood-derived NK cells.

[0017] In some embodiments, the cord blood-derived NK cells are produced by a method comprising the steps of: (a) providing a sample of cord blood cells comprising natural killer cells; (b) enriching the seed cells for NK cells by depleting CD3(+) cells from the cells or by positive selection; and (c) expanding the natural killer cells by culturing the seed cells with a first plurality of cells from an inactivated CD4(+) T cell line in a medium comprising IL-2, to produce cord blood-derived natural killer cells.

[0018] In some embodiments, the inactivated CD4(+) T cell line expresses at least one gene selected from the group consisting of the 4-1BBL gene, the membrane-bound IL-21 (mbIL-21) gene, the OX40L gene, and the mutant TNF-α gene. In some embodiments, the inactivated CD4(+) T cell line expresses the 4-1BBL gene, the membrane-bound IL-21 (mbIL-21) gene, and the mutant TNF-α gene.

[0019] In some embodiments, the medium comprising IL-2 further comprises a T cell stimulating antibody selected from the group consisting of OKT3, UCHT1, HTa, or a combination thereof.

[0020] In some embodiments, the CD16 molecule is a CD16A molecule. In some embodiments, the CD16 molecule comprises a V / V polymorphism at F158. In some embodiments, the first binding domain that specifically binds CD16 specifically binds CD16A.

[0021] In some embodiments, the natural killer cells are a population of natural killer cells. In some embodiments, the population of natural killer cells comprises at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% CD16+ cells. In some embodiments, the population of natural killer cells comprises at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% NKG2D+ cells. In some embodiments, the population of natural killer cells comprises at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% NKp46+ cells. In some embodiments, the population of natural killer cells comprises at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% NKp30+ cells. In some embodiments, the population of natural killer cells comprises at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% DNAM-1+ cells. In some embodiments, the population of natural killer cells comprises at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% NKp44+ cells. In some embodiments, the population of natural killer cells comprises less than 20%, e.g., 10% or less, 5% or less, 1% or less, 0.5% or less, or 0% CD3+ cells. In some embodiments, the population of natural killer cells comprises less than 20% or less, e.g., 10% or less, 5% or less, 1% or less, 0.5% or less, or 0% CD14+ cells. In some embodiments, the population of natural killer cells comprises less than or equal to 20%, e.g., less than or equal to 10%, less than or equal to 5%, less than or equal to 1%, less than or equal to 0.5%, or less than or equal to 0% CD19+ cells. In some embodiments, the population of natural killer cells comprises less than or equal to 20%, e.g., less than or equal to 10%, less than or equal to 5%, less than or equal to 1%, less than or equal to 0.5%, or less than or equal to 0% CD38+ cells.

[0022] In some embodiments, the population of NK cells comprises at least 100 million expanded natural killer cells, e.g., 200 million, 250 million, 300 million, 400 million, 500 million, 600 million, 700 million, 750 million, 800 million, 900 million, 1 billion, 2 billion, 3 billion, 4 billion, 5 billion, 6 billion, 7 billion, 8 billion, 9 billion, 10 billion, 15 billion, 20 billion, 25 billion, 50 billion, 75 billion, 80 billion, 9- 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.

[0023] In some embodiments, the population of NK cells is produced by a method comprising: (a) obtaining seed cells comprising natural killer cells from umbilical cord blood; (b) depleting CD3+ cells from the seed cells; and (c) expanding the natural killer cells by culturing the depleted seed cells with a first plurality of Hut78 cells engineered to express membrane-bound IL-21, mutant TNFα, and the 4-1BBL gene to produce expanded natural killer cells, thereby producing the population of natural killer cells. In some embodiments, the population of NK cells is produced by a method comprising: (a) obtaining seed cells comprising natural killer cells from umbilical cord blood; (b) depleting CD3+ cells from the seed cells; (c) expanding the natural killer cells by culturing the depleted seed cells with a first plurality of Hut78 cells engineered to express membrane-bound IL-21, mutant TNFα, and 4-1BBL genes to produce a master cell bank population of expanded natural killer cells; and (d) expanding the master cell bank population of expanded natural killer cells by culturing a second plurality of Hut78 cells engineered to express membrane-bound IL-21, mutant TNFα, and 4-1BBL genes to produce expanded natural killer cells, thereby producing a population of natural killer cells.

[0024] In some embodiments, after step (c), the population of NK cells is produced by a method further comprising the steps of: (i) freezing the master cell bank population of expanded natural killer cells in a plurality of containers; and (ii) thawing the containers containing an aliquot of the master cell bank population of expanded natural killer cells, wherein expanding the master cell bank population of expanded natural killer cells in step (d) comprises expanding said aliquot of the master cell bank population of expanded natural killer cells.

[0025] In some embodiments, the cord blood is from a donor who has a KIR-B haplotype and is homozygous for the CD16 158V polymorphism.

[0026] In some embodiments, the population of NK cells is produced by a method comprising expanding natural killer cells from umbilical cord blood at least 10,000-fold, e.g., 15,000-fold, 20,000-fold, 25,000-fold, 30,000-fold, 35,000-fold, 40,000-fold, 45,000-fold, 50,000-fold, 55,000-fold, 60,000-fold, 65,000-fold, or 70,000-fold.

[0027] In some embodiments, the population of natural killer cells is not enriched or selected after expansion.

[0028] In some embodiments, the percentage of NK cells expressing CD16 in the population of natural killer cells is the same as or higher than the percentage of natural killer cells in seed cells from umbilical cord blood. In some embodiments, the percentage of NK cells expressing NKG2D in the population of natural killer cells is the same as or higher than the percentage of natural killer cells in seed cells from umbilical cord blood. In some embodiments, the percentage of NK cells expressing NKp30 in the population of natural killer cells is the same as or higher than the percentage of natural killer cells in seed cells from umbilical cord blood. In some embodiments, the percentage of NK cells expressing NKp44 in the population of natural killer cells is the same as or higher than the percentage of natural killer cells in seed cells from umbilical cord blood. In some embodiments, the percentage of NK cells expressing NKp46 in the population of natural killer cells is the same as or higher than the percentage of natural killer cells in seed cells from umbilical cord blood. In some embodiments, the percentage of NK cells expressing DNAM-1 in the population of natural killer cells is the same as or higher than the percentage of natural killer cells in seed cells from umbilical cord blood.

[0029] In some embodiments, the natural killer cells do not comprise a CD16 transgene. In some embodiments, the natural killer cells do not express exogenous CD16 protein.

[0030] In some embodiments, the natural killer cells are not genetically engineered.

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

[0032] In some embodiments, the first pharmaceutical composition further comprises (a) human albumin, (b) dextran, (c) glucose, (d) DMSO, and (e) a buffer. In some embodiments, the first pharmaceutical composition comprises 30-50 mg / mL human albumin. In some embodiments, the first pharmaceutical composition comprises 50 mg / mL human albumin. In some embodiments, the first pharmaceutical composition comprises 20-30 mg / mL dextran. In some embodiments, the first pharmaceutical composition comprises 25 mg / mL dextran. In some embodiments, the dextran is dextran 40. In some embodiments, the first pharmaceutical composition comprises 12-15 mg / mL glucose. In some embodiments, the first pharmaceutical composition comprises 12.5 mg / mL glucose. In some embodiments, the first pharmaceutical composition comprises less than 27.5 g / L glucose. In some embodiments, the first pharmaceutical composition comprises 50-60 ml / mL DMSO. In some embodiments, the first pharmaceutical composition comprises 55 mg / mL DMSO. In some embodiments, the first pharmaceutical composition comprises 40-60% v / v buffer. In some embodiments, the buffer is phosphate buffered saline. In some embodiments, the first pharmaceutical composition further comprises (a) about 40 mg / mL human albumin, (b) about 25 mg / mL dextran 40, (c) about 12.5 mg / mL glucose, (d) about 55 mg / mL DMSO, and (e) about 0.5 mL / mL phosphate buffered saline. In some embodiments, the first pharmaceutical composition further comprises 0.5 mL / mL water. In some embodiments, the first pharmaceutical composition further comprises a pharma- ceutically acceptable excipient.

[0033] In some embodiments, the first binding domain that specifically binds to CD16 comprises a light chain variable (V L ) region and SEQ ID NO:19 HIn some embodiments, the first binding domain that specifically binds to CD16 comprises a V region that comprises an amino acid sequence that has 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:20. L and an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:19. H In some embodiments, the second binding domain that specifically binds to CD30 comprises a light chain variable (V L ) region and SEQ ID NO:21 H In some embodiments, the second binding domain that specifically binds to CD30 comprises a V region that comprises an amino acid sequence that has 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:22. L and an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:21. H and a region.

[0034] In some embodiments, the bispecific antibody or antigen-binding fragment thereof is a bispecific antigen-binding fragment, and the variable domains of the bispecific antigen-binding fragment are linked from N-terminus to C-terminus in the order VH_CD30-L1-VL_CD16A-L2-VH_CD16A-L3-VL_CD30 by peptide linkers L1, L2 and L3. In some embodiments, the bispecific antibody or antigen-binding fragment thereof is a bispecific antigen-binding fragment, and the variable domains of the bispecific antigen-binding fragment are linked from N-terminus to C-terminus in the order VH_CD16A-L1-VL_CD30-L2-VH_CD30-L3-VL_CD16A by peptide linkers L1, L2 and L3.

[0035] In some embodiments, each of the peptide linkers L1, L2 and L3 consists of 12 or less amino acid residues. In some embodiments, the linker L2 of the antibody construct consists of 3 to 9 amino acid residues, inclusive.

[0036] In some embodiments, the bispecific antibody or antigen-binding fragment thereof comprises an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 18. In some embodiments, the bispecific antibody or antigen-binding fragment thereof is a bispecific antigen-binding fragment comprising the amino acid sequence set forth in SEQ ID NO:18.

[0037] In some embodiments, the dose of the bispecific antibody or antigen-binding fragment thereof is administered to the patient at 0.01, 0.04, 0.15, 0.5, 1.5, 3.0, 4.5 or 7.0 mg / kg. In some embodiments, the dose of the bispecific antibody or antigen-binding fragment thereof comprises 200 mg of the bispecific antibody or antigen-binding fragment thereof.

[0038] In some embodiments, the cancer is Hodgkin's lymphoma, non-Hodgkin's lymphoma, peripheral T-cell lymphoma, cutaneous T-cell lymphoma, anaplastic large cell lymphoma, CD30 + The cancer is selected from the group consisting of B cell lymphoma, multiple myeloma, and leukemia. In some embodiments, the cancer is Hodgkin's lymphoma. In some embodiments, the cancer is peripheral T cell lymphoma. In some embodiments, the patient relapses after treatment with an anti-CD30 antibody or is refractory to an anti-CD30 antibody. In some embodiments, the anti-CD30 antibody is brentuximab vedotin. In some embodiments, the patient experiences disease progression after treatment with an autologous stem cell transplant or chimeric antigen receptor T cell therapy (CAR-T).

[0039] In some embodiments, the patient is administered 1×10 per dose of NK cells. 8 ~1×10 10 In some embodiments, patients are administered 1×10 NK cells per dose of NK cells. 9 ~8×10 9 In some embodiments, patients are administered 4×10 NK cells per dose of NK cells. 8 , 1×10 9 , 4×10 9 , 8×10 9 NK, or 1.6 x 10 10 cells are administered.

[0040] In some embodiments, the patient is subjected to lymphodepleting chemotherapy prior to treatment. In some embodiments, the lymphodepleting chemotherapy is non-myeloablative chemotherapy. In some embodiments, the lymphodepleting chemotherapy comprises treatment with at least one of cyclophosphamide and fludarabine. In some embodiments, the lymphodepleting chemotherapy comprises treatment with cyclophosphamide and fludarabine. In some embodiments, the lymphodepleting chemotherapy comprises treatment with 100-500 mg / m 2 Cyclophosphamide is administered at a dose of 250 mg / m 2 Cyclophosphamide is administered at a dose of 500 mg / m 2 Cyclophosphamide is administered at a dose of 10-50 mg / m 2 In some embodiments, 30 mg / m 2 / day of fludarabine is administered.

[0041] In some embodiments, the method further comprises administering IL-2 to the patient. In some embodiments, the patient is administered 1×10 IL-2 per dose. 6 IU / m 2 In some embodiments, the patient is administered 1 million or 6 million IU of IL-2 per dose. In some embodiments, administration of IL-2 occurs within 1-4 hours of administration of the NK cells.

[0042] In some embodiments, the administration of a dose of the first pharmaceutical composition comprising the NK cells and a dose of the second pharmaceutical composition comprising the bispecific antibody or antigen-binding fragment thereof is performed weekly. In some embodiments, the administration of the first pharmaceutical composition comprising the NK cells and the second pharmaceutical composition comprising the bispecific antibody or antigen-binding fragment thereof is performed weekly for 4-8 weeks. In some embodiments, the administration of the first pharmaceutical composition comprising the NK cells is performed weekly for 3 weeks, and the administration of the second pharmaceutical composition comprising the bispecific antibody or antigen-binding fragment thereof is performed weekly for 6 weeks. In some embodiments, the administration of the first pharmaceutical composition comprising the NK cells is performed every other week for 6 weeks, and the administration of the second pharmaceutical composition comprising the bispecific antibody or antigen-binding fragment thereof is performed weekly for 6 weeks.

[0043] As used herein, CD30 + Also provided is a method for treating a patient suffering from cancer, comprising administering to the patient a first treatment cycle comprising any one of the treatment methods described herein, and administering to the patient a second treatment cycle comprising any one of the treatment methods described herein, wherein the first treatment cycle and the second treatment cycle are the same or different.

[0044] In some embodiments, the method further comprises administering to the patient a third treatment cycle comprising any one of the methods described herein.

[0045] In some embodiments, the methods include at least two weeks of treatment cessation between each cycle.

[0046] In some embodiments, the CD30 + Treatment continues until the cancer progresses, or until dosing is discontinued due to patient intolerance to the NK cells, bispecific antibody or antigen-binding fragment thereof, or both, or until the patient experiences toxicity of the NK cells, bispecific antibody or antigen-binding fragment thereof, or both.

[0047] In some embodiments, the NK cells are not genetically modified.

[0048] 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, 1% or less of the NK cells are CD3+, 1% or less of the NK cells are CD19+, and 1% or less of the NK cells are CD14+.

[0049] Provided herein is a pharmaceutical composition comprising: (a) a natural killer cell (NK cell) comprising a KIR-B haplotype and expression of a CD16 molecule; and (b) a bispecific antibody or antigen-binding fragment thereof comprising a first binding domain that specifically binds to CD16 (FcγRIII) and a second binding domain that specifically binds to CD30, wherein the first binding domain that specifically binds to CD16 comprises a light chain variable domain (VL_CD16A) comprising a light chain complementarity determining region 1 (CDRL1) comprising SEQ ID NO:9, a light chain complementarity determining region 2 (CDRL2) comprising SEQ ID NO:10, a light chain complementarity determining region 3 (CDRL3) comprising SEQ ID NO:11, a heavy chain complementarity determining region 1 (CDRH1) comprising SEQ ID NO:6, a heavy chain complementarity determining region 2 (CDRH2) comprising SEQ ID NO:7, and a light chain variable domain (VL_CD16A) comprising a light chain complementarity determining region 1 (CDRL1) comprising SEQ ID NO:8, a heavy chain complementarity determining region 2 (CDRH2) comprising SEQ ID NO:9, and a light chain variable domain (VL_CD16A) comprising a light chain complementarity determining region 3 (CDRL3) comprising SEQ ID NO:10, a light chain complementarity determining region 3 (CDRL3) comprising SEQ ID NO:11, a light chain variable domain (VL_CD16A) comprising a light chain complementarity determining region 1 (CDRH1) comprising SEQ ID NO:11, a heavy chain complementarity determining region 2 (CDRH2) comprising SEQ ID NO:12, and a light chain variable domain (VL_CD16A) comprising a light chain complementarity determining region 1 (CDRH1) comprising SEQ ID NO:13, a heavy chain complementarity determining region 2 (CDRH2) comprising SEQ ID NO:14, and a light chain variable domain (VL_CD16A) comprising a light chain complementarity determining region 1 (CDRH1) comprising SEQ ID NO:15 and a second binding domain that specifically binds to CD30 comprising a light chain variable domain (VL_CD30) comprising a light chain complementarity determining region 1 (CDRL1) comprising SEQ ID NO:15, a light chain complementarity determining region 2 (CDRL2) comprising SEQ ID NO:16, and a light chain complementarity determining region 3 (CDRL3) comprising SEQ ID NO:17; and a heavy chain variable domain (VH_CD30) comprising a heavy chain complementarity determining region 1 (CDRH1) comprising SEQ ID NO:12, a heavy chain complementarity determining region 2 (CDRH2) comprising SEQ ID NO:13, and a heavy chain complementarity determining region 3 (CDRH3) comprising SEQ ID NO:14.

[0050] In some embodiments, the CD16 molecule is a CD16A molecule. In some embodiments, the CD16 molecule comprises a V / V polymorphism at F158. In some embodiments, a bispecific antibody that specifically binds to CD16 specifically binds to CD16A.

[0051] In some embodiments, the first binding domain that specifically binds to CD16 comprises a light chain variable (V L ) region and SEQ ID NO:19 H In some embodiments, the first binding domain that specifically binds to CD16 comprises a V region that comprises an amino acid sequence that has 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:20. L and an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:19. H In some embodiments, the second binding domain that specifically binds to CD30 comprises a light chain variable (V L ) region and SEQ ID NO:21 HIn some embodiments, the second binding domain that specifically binds to CD30 comprises a V region that comprises an amino acid sequence that has 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:22. L and an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:21. H and a region.

[0052] In some embodiments, the bispecific antibody or antigen-binding fragment thereof is a bispecific antigen-binding fragment, and the variable domains of the bispecific antigen-binding fragment are linked from N-terminus to C-terminus in the order VH_CD30-L1-VL_CD16A-L2-VH_CD16A-L3-VL_CD30 by peptide linkers L1, L2 and L3. In some embodiments, the bispecific antibody or antigen-binding fragment thereof is a bispecific antigen-binding fragment, and the variable domains of the bispecific antigen-binding fragment are linked from N-terminus to C-terminus in the order VH_CD16A-L1-VL_CD30-L2-VH_CD30-L3-VL_CD16A by peptide linkers L1, L2 and L3.

[0053] In some embodiments, each of the peptide linkers L1, L2 and L3 consists of 12 or less amino acid residues. In some embodiments, the linker L2 of the antibody construct consists of 3 to 9 amino acid residues, inclusive.

[0054] In some embodiments, the bispecific antibody or antigen-binding fragment thereof comprises an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 18. In some embodiments, the bispecific antibody or antigen-binding fragment thereof is a bispecific antigen-binding fragment comprising the amino acid sequence set forth in SEQ ID NO:18.

[0055] In some embodiments, the NK cells are cord blood-derived NK cells.

[0056] In some embodiments, the cord blood-derived NK cells are produced by a method comprising: (a) providing a sample of cord blood cells comprising natural killer cells; (b) removing CD3(+) cells from the cells; and (b) expanding the natural killer cells by culturing the seed cells with a first plurality of cells from an inactivated CD4(+) T cell line in a medium comprising a T cell stimulating antibody selected from the group consisting of OKT3, UCHT1, HTa, or a combination thereof, and IL-2, to produce cord blood-derived natural killer cells.

[0057] In some embodiments, the inactivated CD4(+) T cell line expresses at least one gene selected from the group consisting of the 4-1BBL gene, the membrane-bound IL-21 (mbIL-21) gene, the OX40L gene, and the mouse TNF-α gene. In some embodiments, the inactivated CD4(+) T cell line expresses the 4-1BBL gene, the membrane-bound IL-21 (mbIL-21) gene, and the mouse TNF-α gene.

[0058] In some embodiments, the first binding domain of the bispecific antibody or antigen-binding fragment thereof that specifically binds to CD16 binds to a CD16 molecule on an NK cell.

[0059] In some embodiments, the pharmaceutical composition further comprises (a) human albumin, (b) dextran, (c) glucose, (d) DMSO, and (e) a buffer. In some embodiments, the pharmaceutical composition comprises 30-50 mg / mL human albumin. In some embodiments, the pharmaceutical composition comprises 50 mg / mL human albumin. In some embodiments, the pharmaceutical composition comprises 20-30 mg / mL dextran. In some embodiments, the pharmaceutical composition comprises 25 mg / mL dextran. In some embodiments, the dextran is dextran 40. In some embodiments, the pharmaceutical composition comprises 12-15 mg / mL glucose. In some embodiments, the pharmaceutical composition comprises 12.5 mg / mL glucose. In some embodiments, the pharmaceutical composition comprises less than 27.5 g / L glucose. In some embodiments, the pharmaceutical composition comprises 50-60 ml / mL DMSO. In some embodiments, the pharmaceutical composition comprises 55 mg / mL DMSO. In some embodiments, the pharmaceutical composition comprises 40-60% v / v buffer. In some embodiments, the buffer is phosphate buffered saline. In some embodiments, the pharmaceutical composition comprises (a) about 40 mg / mL human albumin, (b) about 25 mg / mL dextran 40, (c) about 12.5 mg / mL glucose, (d) about 55 mg / mL DMSO, and (e) about 0.5 mL / mL phosphate buffered saline. In some embodiments, the pharmaceutical composition further comprises 0.5 mL / mL water. In some embodiments, the pharmaceutical composition further comprises a pharma- ceutically acceptable excipient.

[0060] Also provided herein is a cryovial containing the pharmaceutical composition described herein.

[0061] As used herein, CD30 + Also provided is a method for treating a patient suffering from cancer comprising administering a pharmaceutical composition described herein.

[0062] Provided herein is a pharmaceutical composition comprising: (a) a natural killer cell (NK cell) comprising a KIR-B haplotype and expression of a CD16 molecule; and (b) a bispecific antibody or antigen-binding fragment thereof comprising a first binding domain that specifically binds to CD16 (FcγRIII) and a second binding domain that specifically binds to CD30, wherein the first binding domain that specifically binds to CD16 comprises a light chain variable domain (VL_CD16A) comprising a light chain complementarity determining region 1 (CDRL1) comprising SEQ ID NO:9, a light chain complementarity determining region 2 (CDRL2) comprising SEQ ID NO:10, a light chain complementarity determining region 3 (CDRL3) comprising SEQ ID NO:11, a heavy chain complementarity determining region 1 (CDRH1) comprising SEQ ID NO:6, a heavy chain complementarity determining region 2 (CDRH2) comprising SEQ ID NO:7, and a light chain variable domain (VL_CD16A) comprising a light chain complementarity determining region 1 (CDRL1) comprising SEQ ID NO:8, a heavy chain complementarity determining region 2 (CDRH2) comprising SEQ ID NO:9, and a light chain variable domain (VL_CD16A) comprising a light chain complementarity determining region 3 (CDRL3) comprising SEQ ID NO:10, a light chain complementarity determining region 3 (CDRL3) comprising SEQ ID NO:11, a light chain variable domain (VL_CD16A) comprising a light chain complementarity determining region 1 (CDRH1) comprising SEQ ID NO:11, a heavy chain complementarity determining region 2 (CDRH2) comprising SEQ ID NO:12, and a light chain variable domain (VL_CD16A) comprising a light chain complementarity determining region 1 (CDRH1) comprising SEQ ID NO:13, a heavy chain complementarity determining region 2 (CDRH2) comprising SEQ ID NO:14, and a light chain variable domain (VL_CD16A) comprising a light chain complementarity determining region 1 (CDRH1) comprising SEQ ID NO:15 and a second binding domain that specifically binds to CD30 comprising a light chain variable domain (VL_CD30) comprising a light chain complementarity determining region 1 (CDRL1) comprising SEQ ID NO:15, a light chain complementarity determining region 2 (CDRL2) comprising SEQ ID NO:16, and a light chain complementarity determining region 3 (CDRL3) comprising SEQ ID NO:17; and a heavy chain variable domain (VH_CD30) comprising a heavy chain complementarity determining region 1 (CDRH1) comprising SEQ ID NO:12, a heavy chain complementarity determining region 2 (CDRH2) comprising SEQ ID NO:13, and a heavy chain complementarity determining region 3 (CDRH3) comprising SEQ ID NO:14.

[0063] In some embodiments, the CD16 molecule is a CD16A molecule.

[0064] In some embodiments, the CD16 molecule comprises a V / V polymorphism at F158.

[0065] In some embodiments, a bispecific antibody that specifically binds CD16 specifically binds CD16A.

[0066] In some embodiments, the first binding domain that specifically binds to CD16 comprises a light chain variable (V L ) region and SEQ ID NO:19 H ) area.

[0067] In some embodiments, the first binding domain that specifically binds to CD16 comprises an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:20. L and an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:19. H and a region.

[0068] In some embodiments, the second binding domain that specifically binds to CD30 comprises a light chain variable (V L ) region and SEQ ID NO:21 H ) area.

[0069] In some embodiments, the second binding domain that specifically binds to CD30 comprises an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:22. Land an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:21. H and a region.

[0070] In some embodiments, the bispecific antibody or antigen-binding fragment thereof is a bispecific antigen-binding fragment, and the variable domains of the bispecific antigen-binding fragment are linked by peptide linkers L1, L2 and L3 in the order VH_CD30-L1-VL_CD16A-L2-VH_CD16A-L3-VL_CD30 from the N-terminus to the C-terminus. In some embodiments, each of the peptide linkers L1, L2 and L3 consists of 12 or less amino acid residues. In some embodiments, the linker L2 of the antibody construct consists of 3 to 9 amino acid residues inclusive.

[0071] In some embodiments, the bispecific antibody or antigen-binding fragment thereof is a bispecific antigen-binding fragment, and the variable domains of the bispecific antigen-binding fragment are linked by peptide linkers L1, L2 and L3 in the order VH_CD16A-L1-VL_CD30-L2-VH_CD30-L3-VL_CD16A from the N-terminus to the C-terminus. In some embodiments, each of the peptide linkers L1, L2 and L3 consists of 12 or less amino acid residues. In some embodiments, the linker L2 of the antibody construct consists of 3 to 9 amino acid residues inclusive.

[0072] In some embodiments, the bispecific antibody or antigen-binding fragment thereof comprises an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:18.

[0073] In some embodiments, the bispecific antibody or antigen-binding fragment thereof is a bispecific antigen-binding fragment comprising the amino acid sequence shown in SEQ ID NO:18.

[0074] In some embodiments, the NK cells are cord blood-derived NK cells.

[0075] In some embodiments, the cord blood-derived NK cells are produced by a method comprising: (a) providing a sample of cord blood cells comprising natural killer cells; (b) removing CD3(+) cells from the cells; and (b) expanding the natural killer cells by culturing the seed cells with a first plurality of cells from an inactivated CD4(+) T cell line in a medium comprising a T cell stimulating antibody selected from the group consisting of OKT3, UCHT1, HTa, or a combination thereof, and IL-2, to produce cord blood-derived natural killer cells.

[0076] In some embodiments, the inactivated CD4(+) T cell line expresses at least one gene selected from the group consisting of the 4-1BBL gene, the membrane-bound IL-21 (mbIL-21) gene, the OX40L gene, and the mouse TNF-α gene.

[0077] In some embodiments, the inactivated CD4(+) T cell line expresses the 4-1BBL gene, the membrane-bound IL-21 (mbIL-21) gene, and the mouse TNF-α gene.

[0078] In some embodiments, the first binding domain of the bispecific antibody or antigen-binding fragment thereof that specifically binds to CD16 binds to a CD16 molecule on an NK cell.

[0079] In some embodiments, the pharmaceutical composition further comprises (a) human albumin, (b) dextran, (c) glucose, (d) DMSO, and (e) a buffer. In some embodiments, the pharmaceutical composition comprises 30-50 mg / mL human albumin. In some embodiments, the pharmaceutical composition comprises 50 mg / mL human albumin. In some embodiments, the pharmaceutical composition comprises 20-30 mg / mL dextran. In some embodiments, the pharmaceutical composition comprises 25 mg / mL dextran. In some embodiments, the dextran is dextran 40. In some embodiments, the pharmaceutical composition comprises 12-15 mg / mL glucose. In some embodiments, the pharmaceutical composition comprises 12.5 mg / mL glucose. In some embodiments, the pharmaceutical composition comprises less than 27.5 g / L glucose. In some embodiments, the pharmaceutical composition comprises 50-60 ml / mL DMSO. In some embodiments, the pharmaceutical composition comprises 55 mg / mL DMSO. In some embodiments, the pharmaceutical composition comprises 40-60% v / v buffer. In some embodiments, the buffer is phosphate buffered saline. In some embodiments, the pharmaceutical composition comprises (a) about 40 mg / mL human albumin, (b) about 25 mg / mL dextran 40, (c) about 12.5 mg / mL glucose, (d) about 55 mg / mL DMSO, and (e) about 0.5 mL / mL phosphate buffered saline. In some embodiments, the pharmaceutical composition comprises 0.5 mL / mL water.

[0080] In some embodiments, the pharmaceutical composition comprises a pharma- ceutically acceptable excipient.

[0081] Also described herein are cryovials containing any of the pharmaceutical compositions described herein.

[0082] As used herein, CD30+ Also described are methods for treating a patient suffering from cancer comprising administering any of the pharmaceutical compositions described herein.

[0083] The present disclosure provides a method for administering a first pharmaceutical composition comprising natural killer cells (NK cells) comprising a KIR-B haplotype and expression of a CD16 molecule, and a second pharmaceutical composition comprising a bispecific antibody or an antigen-binding fragment thereof comprising a first binding domain that specifically binds to CD16 (FcγRIII) and a second binding domain that specifically binds to CD30. + A method for treating a patient suffering from cancer, comprising: a first binding domain that specifically binds to CD16 comprising a light chain variable domain (VL_CD16A) comprising light chain complementarity determining region 1 (CDRL1) comprising SEQ ID NO:9, light chain complementarity determining region 2 (CDRL2) comprising SEQ ID NO:10, light chain complementarity determining region 3 (CDRL3) comprising SEQ ID NO:11, and a heavy chain variable domain (VH_CD16A) comprising heavy chain complementarity determining region 1 (CDRH1) comprising SEQ ID NO:6, heavy chain complementarity determining region 2 (CDRH2) comprising SEQ ID NO:7, and heavy chain complementarity determining region 3 (CDRH3) comprising SEQ ID NO:8; and a second binding domain that specifically binds to CD30 comprising light chain complementarity determining region 1 (CDRL1) comprising SEQ ID NO:15, light chain complementarity determining region 2 (CDRL2) comprising SEQ ID NO:16, heavy chain complementarity determining region 3 (CDRH3) comprising SEQ ID NO:9, and a heavy chain variable domain (VH_CD16A) comprising heavy chain complementarity determining region 1 (CDRH1) comprising SEQ ID NO:17, heavy chain complementarity determining region 2 (CDRH2) comprising SEQ ID NO:18, and heavy chain complementarity determining region 3 (CDRH3) comprising SEQ ID NO:19. Also described are methods comprising a light chain variable domain (VL_CD30) comprising a light chain complementarity determining region 3 (CDRL3) comprising SEQ ID NO:17, and a heavy chain variable domain (VH_CD30) comprising a heavy chain complementarity determining region 1 (CDRH1) comprising SEQ ID NO:12, a heavy chain complementarity determining region 2 (CDRH2) comprising SEQ ID NO:13, and a heavy chain complementarity determining region 3 (CDRH3) comprising SEQ ID NO:14.

[0084] In some embodiments, the NK cells are cord blood-derived NK cells.

[0085] In some embodiments, the cord blood-derived NK cells are produced by a method comprising: (a) providing a sample of cord blood cells comprising natural killer cells; (b) removing CD3(+) cells from the cells; and (b) expanding the natural killer cells by culturing the seed cells with a first plurality of cells from an inactivated CD4(+) T cell line in a medium comprising a T cell stimulating antibody selected from the group consisting of OKT3, UCHT1, HTa, or a combination thereof, and IL-2, to produce cord blood-derived natural killer cells.

[0086] In some embodiments, the inactivated CD4(+) T cell line expresses at least one gene selected from the group consisting of the 4-1BBL gene, the membrane-bound IL-21 (mbIL-21) gene, the OX40L gene, and the mouse TNF-α gene.

[0087] In some embodiments, the inactivated CD4(+) T cell line expresses the 4-1BBL gene, the membrane-bound IL-21 (mbIL-21) gene, and the mouse TNF-α gene.

[0088] In some embodiments, the CD16 molecule is a CD16A molecule.

[0089] In some embodiments, the CD16 molecule comprises a V / V polymorphism at F158.

[0090] In some embodiments, the first binding domain that specifically binds CD16 specifically binds CD16A.

[0091] In some embodiments, the first pharmaceutical composition further comprises (a) human albumin, (b) dextran, (c) glucose, (d) DMSO, and (e) a buffer. In some embodiments, the first pharmaceutical composition comprises 30-50 mg / mL human albumin. In some embodiments, the first pharmaceutical composition comprises 50 mg / mL human albumin. In some embodiments, the first pharmaceutical composition comprises 20-30 mg / mL dextran. In some embodiments, the first pharmaceutical composition comprises 25 mg / mL dextran. In some embodiments, the dextran is dextran 40. In some embodiments, the first pharmaceutical composition comprises 12-15 mg / mL glucose. In some embodiments, the first pharmaceutical composition comprises 12.5 mg / mL glucose. In some embodiments, the first pharmaceutical composition comprises less than 27.5 g / L glucose. In some embodiments, the first pharmaceutical composition comprises 50-60 ml / mL DMSO. In some embodiments, the first pharmaceutical composition comprises 55 mg / mL DMSO. In some embodiments, the first pharmaceutical composition comprises 40-60% v / v buffer. In some embodiments, the buffer is phosphate buffered saline. In some embodiments, the first pharmaceutical composition further comprises (a) about 40 mg / mL human albumin, (b) about 25 mg / mL dextran 40, (c) about 12.5 mg / mL glucose, (d) about 55 mg / mL DMSO, and (e) about 0.5 mL / mL phosphate buffered saline. In some embodiments, the pharmaceutical composition further comprises 0.5 mL / mL water.

[0092] In some embodiments, the first pharmaceutical composition further comprises a pharma- ceutically acceptable excipient.

[0093] In some embodiments, the first binding domain that specifically binds to CD16 comprises a light chain variable (V L ) region and SEQ ID NO:19 H ) area.

[0094] In some embodiments, the first binding domain that specifically binds to CD16 comprises an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:20. L and an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:19. H and a region.

[0095] In some embodiments, the second binding domain that specifically binds to CD30 comprises a light chain variable (V L ) region and SEQ ID NO:21 H ) area.

[0096] In some embodiments, the second binding domain that specifically binds to CD30 comprises an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:22. Land an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:21. H and a region.

[0097] In some embodiments, the bispecific antibody or antigen-binding fragment thereof is a bispecific antigen-binding fragment, and the variable domains of the bispecific antigen-binding fragment are linked by peptide linkers L1, L2 and L3 in the order VH_CD30-L1-VL_CD16A-L2-VH_CD16A-L3-VL_CD30 from the N-terminus to the C-terminus. In some embodiments, the bispecific antibody or antigen-binding fragment thereof is a bispecific antigen-binding fragment, and the variable domains of the bispecific antigen-binding fragment are linked by peptide linkers L1, L2 and L3 in the order VH_CD16A-L1-VL_CD30-L2-VH_CD30-L3-VL_CD16A from the N-terminus to the C-terminus. In some embodiments, each of the peptide linkers L1, L2 and L3 consists of 12 or less amino acid residues. In some embodiments, the linker L2 of the antibody construct consists of 3 to 9 amino acid residues, inclusive.

[0098] In some embodiments, the bispecific antibody or antigen-binding fragment thereof comprises an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:18.

[0099] In some embodiments, the bispecific antibody or antigen-binding fragment thereof is a bispecific antigen-binding fragment comprising the amino acid sequence shown in SEQ ID NO:18.

[0100] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Methods and materials for use in this invention are described herein, but other suitable methods and materials known in the art can also be used.Materials, methods and examples are merely illustrative and are not intended to be limiting.Publications, patent applications, patents, sequences, database entries and other references mentioned herein are incorporated herein by reference in their entirety.In case of discrepancy, the present specification, including definitions, will prevail.

[0101] Other features and advantages of the invention will become apparent from the following detailed description and drawings, and from the claims.

[0102] INCORPORATION BY REFERENCE All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. [Brief description of the drawings]

[0103] The novel features of the invention are set forth with particularity in the appended claims. The features and advantages of the present invention will be better understood by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0104] [Figure 1] FIG. 1 depicts an exemplary embodiment of a method for the expansion and stimulation of NK cells. [Diagram 2]FIG. 2 shows that in preclinical studies, cord blood-derived NK cells (CB-NK) have approximately 10-fold greater ability to expand in culture than peripheral blood-derived NK cells (PB-NK). [Diagram 3] FIG. 3 shows that expression of tumor-engaging NK activating immune receptors was higher and more consistent in cord blood-derived preparations compared to those generated from peripheral blood. [Figure 4] FIG. 4 depicts the phenotype of expanded stimulated NK cell populations. [Diagram 5] FIG. 5 depicts the key steps in the manufacture of AB-101 formulation, an example of an expanded NK cell population derived from umbilical cord blood. [Figure 6] FIG. 6 shows the purity of AB-101 (n=9). [Figure 7] FIG. 7 depicts the purity of CD3 depleted cells, MCB and DP produced under GMP conditions. [Figure 8] FIG. 8 depicts the expression of NK cell receptors on CD3 depleted cells, MCB and DP produced under GMP conditions. [Figure 9] FIG. 9 depicts NK purity (CD56+ / CD3-) by flow cytometry. [Figure 10] FIG. 10 depicts CD38+ expression of expanded NK cells from three different cord blood donors. [Figure 11] FIG. 11 depicts the CD38+ mean fluorescence intensity of CD38+ NK cells from three different cord blood donors. [Figure 12] FIG. 12 depicts the differences in surface protein expression of the starting NK cell source compared to AB-101 cells. [Figure 13] FIG. 13 represents the cytotoxic activity of AB-101 NK cells from AFM13 and MCB2 in a 4-hour calcein release assay on KARPAS-299 target cells, starting at 10:1 and followed by two-fold serial dilutions to decrease the effector to target (E:T) ratio. [Figure 14]FIG. 14 represents the cytotoxic activity of AB-101 NK cells from AFM13 and MCB1 in a 4-hour calcein release assay on KARPAS-299 target cells, starting at 10:1 and followed by two-fold serial dilutions to decrease the effector to target (E:T) ratio. [Figure 15] FIG. 15 depicts a bar graph of the cytotoxic activity of AFM13 and AB-101 NK cells from MCB1 (right, AB-101 MCB1) and MCB2 (left, AB-101 MCB2) at an effector to target (E:T) ratio of 5:1 in a 4-hour calcein release assay on KARPAS-299 target cells. [Figure 16] Figure 16 shows the retention of bound AFM13 after thawing in preloaded cryopreserved AB-101 cells from MCB2, where filled histograms represent anti-AFM13 (rat anti-AFM13 antibody) + secondary antibody (goat anti-rat FITC antibody) and open histograms represent secondary antibody only. From top to bottom: non-preloaded, non-preloaded + fresh excess AFM, AFM-preloaded, AFM-preloaded + fresh excess AFM. [Figure 17] Figure 17 shows the retention of bound AFM13 after thawing in preloaded cryopreserved AB-101 cells from MCB1, where filled histograms represent anti-AFM13 (rat anti-AFM13 antibody) + secondary antibody (goat anti-rat FITC antibody) and open histograms represent secondary antibody only. From top to bottom: non-preloaded, non-preloaded + fresh excess AFM, AFM-preloaded, AFM-preloaded + fresh excess AFM. [Figure 18] Figure 18 shows the fluorescence intensity of CD16 expression on preloaded AB-101 cells (left: MCB2, right: MCB1). The different conditions show uniform expression of CD16 on AB-101 cells. From top to bottom: no preload, no preload + fresh excess AFM, AFM-preload, AFM-preload + fresh excess AFM. [Figure 19]FIG. 19 depicts NK fratricide (NK-NK cell lysis) by AFM13 on AB-101 NK cells from MCB2 at an effector to target (E:T) ratio of 1:1 in a 4-hour calcein release assay. [Figure 20] FIG. 20 depicts NK fratricide (NK-NK cell lysis) by AFM13 on AB-101 NK cells from MCB1 at an effector to target (E:T) ratio of 1:1 in a 4-hour calcein release assay. [Figure 21] Figure 21 depicts the upregulation of CD107a in response to Karpas-299 target cells and AFM13, where AB-101 NK cells from MCB2 were cultured with and without target cells at a 1:1 cell ratio, and %CD107a+NK cells were determined by flow cytometry. [Figure 22] Figure 22 depicts the upregulation of CD107a in response to Karpas-299 target cells and AFM13, where AB-101 NK cells from MCB1 were cultured with and without target cells at a 1:1 cell ratio, and %CD107a+NK cells were determined by flow cytometry. [Diagram 23] Figure 23 depicts the increased production of intracellular IFNγ in response to Karpas-299 target cells and AFM13, where AB-101 NK cells from MCB2 were cultured with and without target cells at a 1:1 cell ratio, and % IFNγ+ NK cells were determined by flow cytometry. [Figure 24] Figure 24 depicts the increased production of intracellular IFNγ in response to Karpas-299 target cells and AFM13, where AB-101 NK cells from MCB1 were cultured with and without target cells at a 1:1 cell ratio, and % IFNγ+ NK cells were determined by flow cytometry. [Diagram 25]Figure 25 depicts the viability analysis of cryopreserved AFM13-preloaded AB-101 NK cells, in which AFM13 preloaded or empty AB-101 NK cells were evaluated against MDA-MB-231-Luc cells in an intraperitoneal xenograft tumor model in female hIL15-NOG mice. [Figure 26] FIG. 26 depicts the experimental design for an in vivo efficacy study of AFM13 in combination with AB-101 in the Karpas-299 / Luc human tumor xenograft model. [Figure 27] FIG. 27 depicts the results of an in vivo efficacy study combining AFM13 and AB-101 in the Karpas-299 / Luc human tumor xenograft model. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0105] Detailed Description Provided herein, inter alia, are pharmaceutical compositions comprising NK cells, such as those described herein, and multispecific engagers, such as those described herein, as well as frozen vials comprising said pharmaceutical compositions, and methods for treating patients with said pharmaceutical compositions.

[0106] I. Expansion and Stimulation of Natural Killer Cells In some embodiments, the natural killer cells are expanded and stimulated, for example, by culture and stimulation with feeder cells.

[0107] NK cells can be expanded and stimulated, for example, as described in US 2020 / 0108096 or WO 2020 / 101361, both of which are incorporated by reference in their entireties. Briefly, source cells can be cultured on modified HuT-78 (ATCC® TIB-161™) cells engineered to express 4-1BBL, membrane-bound IL-21 and mutant TNFα, as described in US 2020 / 0108096.

[0108] Suitable NK cells can also be expanded and stimulated as described herein.

[0109] In some embodiments, NK cells are expanded and stimulated by a method comprising: (a) providing a composition comprising NK cells, e.g., NK cells, such as CD3(+) depleted NK cells; and (b) culturing in medium comprising feeder cells and / or stimulatory factors, thereby producing a population of expanded and stimulated NK cells.

[0110] A. Natural Killer Cell Sources In some embodiments, the NK cell source is selected from the group consisting of peripheral blood, peripheral blood lymphocytes (PBL), peripheral blood mononuclear cells (PBMC), bone marrow, umbilical cord blood, isolated NK cells, induced pluripotent stem cell derived NK cells, embryonic stem cell derived NK cells, and combinations thereof.

[0111] In some embodiments, the NK cell source is a single unit of umbilical cord blood.

[0112] In some embodiments, the natural killer cell source, e.g., a single unit of umbilical cord blood, is 1×10 7 or about 1 x 10 7 From 1 × 10 9 or about 1 x 10 9 In some embodiments, the natural killer cell source, e.g., a single unit of umbilical cord blood, comprises 1×10 total nucleated cells. 8 or about 1 x 10 8 So, 1.5 x 10 8 or about 1.5 x 10 8 In some embodiments, the natural killer cell source, e.g., a single unit of umbilical cord blood, comprises 1×10 total nucleated cells. 8 In some embodiments, a natural killer cell source, such as a single unit of umbilical cord blood, comprises about 1×10 total nucleated cells. 8In some embodiments, the natural killer cell source, e.g., a single unit of umbilical cord blood, comprises 1×10 total nucleated cells. 9 In some embodiments, a natural killer cell source, such as a single unit of umbilical cord blood, comprises about 1×10 total nucleated cells. 9 Contains total nucleated cells.

[0113] In some embodiments, the NK cell source, e.g., cord blood unit, comprises about 20% to about 80% CD16+ cells. In some embodiments, the NK cell source, e.g., cord blood unit, comprises about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 20% to about 30%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, about 30% to about 50%, about 30% to about 50%, about 30% to about 6 ... %, about 30% to 40%, about 40% to 80%, about 40% to 70%, about 40% to 60%, about 40% to 50%, about 50% to 80%, about 50% to 70%, about 50% to 60%, about 60% to 80%, about 60% to 70%, or about 70% to 80%. In some embodiments, the NK cell source, e.g., the cord blood unit, comprises 80% or less CD16+ cells. Alternatively, some NK cell sources may comprise CD16+ cells at a concentration greater than 80%.

[0114] In some embodiments, the NK cell source, e.g., an umbilical cord blood unit, comprises no more than 40%, such as no more than 30%, such as no more than 20%, such as no more than 10%, e.g., no more than 5% MLG2A+ cells.

[0115] In some embodiments, the NK cell source, e.g., an umbilical cord blood unit, comprises no more than 40%, such as no more than 30%, such as no more than 20%, such as no more than 10%, e.g., no more than 5% NKG2C+ cells.

[0116] In some embodiments, the NK cell source, e.g., an umbilical cord blood unit, comprises no more than 40%, such as no more than 30%, such as no more than 20%, such as no more than 10%, e.g., no more than 5% NKG2D+ cells.

[0117] In some embodiments, the NK cell source, e.g., an umbilical cord blood unit, comprises no more than 40%, such as no more than 30%, such as no more than 20%, such as no more than 10%, e.g., no more than 5% NKp46+ cells.

[0118] In some embodiments, the NK cell source, e.g., an umbilical cord blood unit, comprises no more than 40%, such as no more than 30%, such as no more than 20%, such as no more than 10%, e.g., no more than 5% NKp30+ cells.

[0119] In some embodiments, the NK cell source, e.g., an umbilical cord blood unit, comprises no more than 40%, such as no more than 30%, such as no more than 20%, such as no more than 10%, e.g., no more than 5% DNAM-1+ cells.

[0120] In some embodiments, the NK cell source, e.g., an umbilical cord blood unit, comprises no more than 40%, such as no more than 30%, such as no more than 20%, such as no more than 10%, e.g., no more than 5% NKp44+ cells.

[0121] In some embodiments, the NK cell source, e.g., an umbilical cord blood unit, comprises no more than 40%, such as no more than 30%, such as no more than 20%, such as no more than 10%, e.g., no more than 5% CD25+ cells.

[0122] In some embodiments, the NK cell source, e.g., an umbilical cord blood unit, comprises no more than 40%, such as no more than 30%, such as no more than 20%, such as no more than 10%, e.g., no more than 5% CD62L+ cells.

[0123] In some embodiments, the NK cell source, e.g., an umbilical cord blood unit, comprises no more than 40%, such as no more than 30%, such as no more than 20%, such as no more than 10%, e.g., no more than 5% CD69+ cells.

[0124] In some embodiments, the NK cell source, e.g., an umbilical cord blood unit, comprises no more than 40%, such as no more than 30%, such as no more than 20%, such as no more than 10%, e.g., no more than 5% CXCR3+ cells.

[0125] In some embodiments, the NK cell source, e.g., an umbilical cord blood unit, comprises no more than 40%, such as no more than 30%, such as no more than 20%, such as no more than 10%, e.g., no more than 5% CD57+ cells.

[0126] In some embodiments, the NK cells in the NK cell source comprise a KIR B allele of the KIR receptor family. See, e.g., Hsu et al., "The Killer Cell Immunoglobulin-Like Receptor (KIR) Genomic Region: Gene-Order, Haplotypes and Allelic Polymorphism," Immunological Review 190:40-52 (2002) and Pyo et al., "Different Patterns of Evolution in the Centromeric and Telomeric Regions of Group A and B Haplotypes of the Human Killer Cell Ig-like Receptor Locus," PLoS One 5:e15115 (2010).

[0127] In some embodiments, the NK cells in the NK cell source comprise the 158V / V variant of CD16 (i.e., homozygous CD16 158V polymorphism). See, e.g., Koene et al., "FcγRIIIa-158V / F Polymorphism Influences the Binding of IgG by Natural Killer Cell FcgammaRIIIa, Independently of the FcgammaRIIIa-48L / R / H Phenotype," Blood 90:1109-14 (1997).

[0128] In some embodiments, the NK cells in the cell source comprise both a KIR B allele of the KIR receptor family and the 158 V / V variant of CD16.

[0129] In some embodiments, the NK cells in the cell source are not genetically engineered.

[0130] In some embodiments, the NK cells in the cell source do not contain a CD16 transgene.

[0131] In some embodiments, the NK cells in the cell source do not express exogenous CD16 protein.

[0132] In some embodiments, the NK cell source is depleted of CD3(+). In some embodiments, the method comprises depleting CD3(+) cells from the NK cell source. In some embodiments, depleting CD3(+) cells from the NK cell source comprises contacting the NK cell source with a CD3-binding antibody or antigen-binding fragment thereof. In some embodiments, the CD3-binding antibody or antigen-binding fragment thereof is selected from the group consisting of OKT3, UCHT1, and HIT3a, and fragments thereof. In some embodiments, the CD3-binding antibody or antigen-binding fragment thereof is OKT3 or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof is attached to beads, e.g., magnetic beads. In some embodiments, depleting CD3(+) cells from the composition comprises contacting the composition with a CD3-targeting antibody or antigen-binding fragment thereof attached to beads, and removing the bead-bound CD3(+) cells from the composition. The composition can be depleted of CD3 cells by immunomagnetic selection, for example using the CliniMACS T cell depletion set ((LS depletion set (162-01) Miltenyi Biotec).

[0133] In some embodiments, the NK cell source is enriched for CD56+, such as by gating on CD56 expression.

[0134] In some embodiments, the NK cell source is enriched for CD56+ and depleted of CD3(+), such as by selecting for cells with CD56+CD3- expression.

[0135] In some embodiments, the NK cell source contains both the KIR B allele of the KIR receptor family and the 158 V / V variant of CD16, and is enriched for + and depleted for CD3(+), e.g., by selecting for cells with CD56+CD3- expression.

[0136] B. Feeder cells Disclosed herein are feeder cells for the expansion of NK cells. These feeder cells conveniently allow NK cells to expand to a number suitable for the preparation of pharmaceutical compositions discussed herein. Optionally, these feeder cells allow NK cells to expand without the loss of CD16 expression that is often associated with cell expansion with other types of feeder cells or using other methods. Optionally, these feeder cells increase the tolerance of expanded NK cells to freezing, increasing the proportion of NK cells that are viable after freeze / thaw cycles or increasing the period that cells remain viable in the frozen state. Optionally, these feeder cells allow NK cells to maintain high levels of cytotoxicity, including ADCC, extend survival time, increase persistence, and enhance or maintain high levels of CD16. Optionally, these feeder cells allow NK cells to expand without causing significant levels of exhaustion or senescence.

[0137] Feeder cells can be used to stimulate the NK cells, for example by providing substrate, growth factors and / or cytokines, and can help them expand more rapidly.

[0138] NK cells can be stimulated using various types of feeder cells, including, but not limited to, peripheral blood mononuclear cells (PBMCs), Epstein-Barr virus transformed B lymphoblastoid cells (e.g., EBV-LCL), myeloid leukemia cells (e.g., K562), and CD4(+) T cells (e.g., HuT), and their derivatives.

[0139] In some embodiments, the feeder cells are inactivated, for example, by gamma irradiation or mitomycin c treatment.

[0140] Suitable feeder cells for use in the methods described herein are described, for example, in US 2020 / 0108096, which is incorporated by reference in its entirety.

[0141] In some embodiments, the feeder cells are inactivated CD4(+) T cells. In some embodiments, the inactivated CD4(+) T cells are HuT-78 cells (ATCC® TIB-161™) or variants or derivatives thereof. In some embodiments, the HuT-78 derivative is H9 (ATCC® HTB-176™).

[0142] In some embodiments, the inactivated CD4(+) T cells express OX40L. In some embodiments, the inactivated CD4(+) T cells are HuT-78 cells or variants or derivatives thereof that express OX40L (SEQ ID NO:4) or variants thereof.

[0143] In some embodiments, the feeder cells are HuT-78 cells ("eHut-78 cells") engineered to express at least one gene or a variant thereof selected from the group consisting of 4-1BBL (UniProtKB P41273, SEQ ID NO:1), membrane-bound IL-21 (SEQ ID NO:2) and mutant TNF alpha (SEQ ID NO:3).

[0144] In some embodiments, the inactivated CD4(+) T cells are HuT-78 (ATCC® TIB-161™) cells or variants or derivatives thereof expressing an ortholog of OX40L or a variant thereof. In some embodiments, the feeder cells are HuT-78 cells engineered to express at least one gene selected from the group consisting of a 4-1BBL ortholog or a variant thereof, a membrane-bound IL-21 ortholog or a variant thereof, and a mutant TNF alpha ortholog or a variant thereof.

[0145] In some embodiments, the feeder cells are HuT-78 cells ("eHut-78 cells") that express OX40L (SEQ ID NO:4) and have been engineered to express 4-1BBL (SEQ ID NO:1), membrane-bound IL-21 (SEQ ID NO:2) and mutant TNF alpha (SEQ ID NO:3) or a variant or derivative thereof.

[0146] In some embodiments, the feeder cells are expanded, e.g., from frozen stocks, prior to culturing with the NK cells, e.g., as described in Example 2.

[0147] C. Stimulating factor In addition to or instead of feeder cells, the NK cells can also be stimulated with one or more stimulatory factors other than feeder cells, eg, signaling factors.

[0148] In some embodiments, the stimulatory factors, e.g., signaling factors, are a component of the culture medium, as described herein. In some embodiments, the stimulatory factors, e.g., signaling factors, are a supplement to the culture medium, as described herein.

[0149] In some embodiments, the stimulatory factor is a cytokine. In some embodiments, the cytokine is selected from the group consisting of IL-2, IL-12, IL-15, IL-18, IL-21, IL-23, IL-27, IFN-α, IFNβ, and combinations thereof.

[0150] In some embodiments, the cytokine is IL-2.

[0151] In some embodiments, the cytokine is a combination of IL-2 and IL-15.

[0152] In some embodiments, the cytokines are a combination of IL-2, IL-15 and IL-18.

[0153] In some embodiments, the cytokines are a combination of IL-2, IL-18 and IL-21.

[0154] D. Culture NK cells can be expanded and stimulated by co-culturing an NK cell source with feeder cells and / or other stimulatory factors. Suitable NK cell sources, feeder cells and stimulatory factors are described herein.

[0155] Optionally, the resulting expanded population of natural killer cells is enriched and / or selected after expansion. Optionally, the resulting expanded population of natural killer cells is not enriched and / or selected after expansion.

[0156] Also described herein are compositions comprising various culture compositions described herein, e.g., compositions comprising NK cells, e.g., a population of expanded cord blood-derived natural killer cells that comprise a KIR-B haplotype and are homozygous for the CD16 158V polymorphism, and a plurality of engineered HuT78 cells.

[0157] Also described herein are containers, e.g., vials, freezer bags, etc., that contain the resulting expanded population of natural killer cells. Optionally, a plurality of containers, e.g., at least 10, e.g., 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 containers, that contain a portion of the resulting expanded population of natural killer cells.

[0158] Also described herein are bioreactors that contain various culture compositions described herein, such as bioreactors that contain NK cells. For example, cultures that contain natural killer cells from a natural killer cell source, such as those described herein, and feeder cells, such as those described herein. Also described herein are bioreactors that contain the resulting expanded population of natural killer cells.

[0159] 1. Culture medium Disclosed herein is a culture medium for expanding NK cells.These culture media can conveniently allow NK cells to expand to a suitable number for preparing pharmaceutical compositions discussed herein.In some cases, these culture media can allow NK cells to expand without the loss of CD16 expression that is often associated with cell expansion in other helper cells or other media.

[0160] In some embodiments, the culture medium is a basal culture medium, which may be supplemented with additional components, e.g., as described herein.

[0161] In some embodiments, the culture medium, e.g., the basal culture medium, is a serum-free culture medium. In some embodiments, the culture medium, e.g., the basal culture medium, is a serum-free culture medium supplemented with human plasma and / or serum.

[0162] Suitable basal culture media include DMEM, RPMI1640, MEM, DMEM / F12, SCGM (CellGenix®, 20802-0500 or 20806-0500), LGM-3(TM) (Lonza, CC-3211), TexMACS(TM) (Miltenyi Biotec, 130-097-196), ALyS(TM) 505NK-AC(Cell Science and Technology Institute, Inc., 01600P02), ALyS(TM) 505NK-EX(Cell Science and Technology Institute, Inc., 01400P10), CTS(TM) AIM-V(TM) SFM(ThermoFisher Scientific, A3830801), CTS(TM) OpTmizer(TM)(ThermoFisher Scientific, A1048501, ABS-001, StemXxVivo, and combinations thereof.

[0163] The culture medium may contain or be supplemented with additional components, such as growth factors, signaling factors, nutrients, antigen-binding substances, etc. The culture medium may be supplemented by adding each of one or more additional components to the culture vessel before, at the same time, or after the medium is added to the culture vessel. The one or more additional components may be added together or separately. If added separately, the additional components do not need to be added at the same time.

[0164] In some embodiments, the culture medium comprises plasma, e.g., human plasma. In some embodiments, the culture medium is supplemented with plasma, e.g., human plasma. In some embodiments, the plasma, e.g., human plasma, comprises an anticoagulant, such as trisodium citrate.

[0165] In some embodiments, the medium comprises and / or is supplemented with 0.5% or about 0.5% to 10% or about 10% v / v plasma, e.g., human plasma. In some embodiments, the medium comprises 0.5% or about 0.5% to 9% or about 9%, 0.5% or about 0.5% to 8% or about 8%, 0.5% or about 0.5% to 7% or about 7%, 0.5% or about 0.5% to 6% or about 6%, 0.5% or about 0.5% to 5% or about 5%, 0.5% or about 0.5% to 4% or about 4%, 0.5% or about 0.5% to 3% or about 3%, 0.5% or about 0.5% to 2% or about 2%, 0.5% or from about 0.5%, 1% or about 1%, 1% or about 1% to 10% or about 10%, 1% or about 1% to 9% or about 9%, 1% or about 1% to 8% or about 8%, 1% or about 1% to 7% or about 7%, 1% or about 1% to 6% or about 6%, 1% or about 1% to 5% or about 5%, 1% or about 1% to 4% or about 4%, 1% or about 1% to 3% or about 3%, 1% or about 1% to 2% or about 2%, 2% or about 2% %, 10% or about 10%, 2% or about 2% to 9% or about 9%, 2% or about 2% to 8% or about 8%, 2% or about 2% to 7% or about 7%, 2% or about 2% to 6% or about 6%, 2% or about 2% to 5% or about 5%, 2% or about 2% to 4% or about 4%, 2% or about 2% to 3% or about 3%, 3% or about 3% to 10% or about 10%, 3% or about 3% to 9% or about 9%, 3% or about 3% to 8% or about 8%, 3% or about 3% to, 7% or about 7%, 3% or about 3% to, 6% or about 6%, 3% or about 3% to, 5% or about 5%, 3% or about 3% to, 4% or about 4%, 4% or about 4% to, 10% or about 10%, 4% or about 4% to, 9% or about 9%, 4% or about 4% to, 8% or about 8%, 4% or about 4% to, 7% or about 7%, 4% or about 4% to, 6% or about 6%, 4% or about 4% to, 5% or about 5%,from 5% or about 5%, 10% or about 10%, from 5% or about 5%, from 9% or about 9%, from 4% or about 4%, from 8% or about 8%, from 5% or about 5%, from 7% or about 7%, from 5% or about 5%, from 6% or about 6%, from 6% or about 6%, from 10% or about 10%, from 6% or about 6%, from 9% or about 9%, from 6% or about 6%, from 8% or about 8%, from 6% or about 6%, from 7% or about 7%, from 7% or about 7%, from 9% or about 9%, from 7% or about 7%, from 8% or about 8%, from 8% or about 8%, from 10% or about 10%, from 8% or about 8%, from 9% or about 9%, or from 9% or about 9% to 10% or about 10% In some embodiments, the culture medium comprises and / or is supplemented with 0.8% to 1.2% v / v human plasma. In some embodiments, the culture medium comprises and / or is supplemented with 1.0% v / v human plasma. In some embodiments, the culture medium comprises and / or is supplemented with about 1.0% v / v human plasma.

[0166] In some embodiments, the culture medium comprises serum, e.g., human serum. In some embodiments, the culture medium is supplemented with serum, e.g., human serum. In some embodiments, the serum is inactivated, e.g., heat inactivated. In some embodiments, the serum is filtered, e.g., sterile filtered.

[0167] In some embodiments, the culture medium comprises glutamine. In some embodiments, the culture medium is supplemented with glutamine. In some embodiments, the culture medium comprises and / or is supplemented with 2.0 or about 2.0 to 6.0 or about 6.0 mM glutamine.In some embodiments, the culture medium is from 2.0 or about 2.0, 5.5 or about 5.5, from 2.0 or about 2.0, 5.0 or about 5.0, from 2.0 or about 2.0, 4.5 or about 4.5, from 2.0 or about 2.0, 4.0 or about 4.0, from 2.0 or about 2.0, 3.5 or about 3.5, from 2.0 or about 2.0, 3.0 or about 3.0, from 2.0 or about 2.0, 2.5 or about 2.5, from 2.5 or about 2.5, from 6.0 or about 6.0, from 2.5 or about 2.5 , 5.5 or about 5.5, from 2.5 or about 2.5, 5.0 or about 5.0, from 2.5 or about 2.5, 4.5 or about 4.5, from 2.5 or about 2.5, 4.0 or about 4.0, from 2.5 or about 2.5, 3.5 or about 3.5, from 2.5 or about 2.5, 3.0 or about 3.0, from 3.0 or about 3.0, 6.0 or about 6.0, from 3.0 or about 3.0, 5.5 or about 5.5, from 3.0 or about 3.0, 5.0 or about 5.0, from 3.0 or about 3.0, 4.5 or or from about 4.5, 3.0 or about 3.0, 4.0 or about 4.0, 3.0 or about 3.0, 3.5 or about 3.5, 3.5 or about 3.5, 6.0 or about 6.0, 3.5 or about 3.5, 5.5 or about 5.5, 3.5 or about 3.5, 5.0 or about 5.0, 3.5 or about 3.5, 4.5 or about 4.5, 3.5 or about 3.5, 4.0 or about 4.0, 4.0 or about 4.0, 6.0 or about 6.0, 4.0 or about 4.0, 5.5 or about 5. 5, 4.0 or about 4.0 to, 5.0 or about 5.0, 4.0 or about 4.0 to, 4.5 or about 4.5, 4.5 or about 4.5 to, 6.0 or about 6.0, 4.5 or about 4.5 to, 5.5 or about 5.5, 4.5 or about 4.5 to, 5.0 or about 5.0, 5.0 or about 5.0 to, 6.0 or about 6.0, 5.0 or about 5.0 to, 5.5 or about 5.5, or 5.5 or about 5.5 to, 6.0 or about 6.0 mM glutamine.In some embodiments, the culture medium comprises and / or is supplemented with 3.2 mM to 4.8 mM glutamine. In some embodiments, the culture medium comprises and / or is supplemented with 4.0 mM glutamine. In some embodiments, the culture medium comprises and / or is supplemented with about 4.0 mM glutamine.

[0168] In some embodiments, the culture medium comprises one or more cytokines. In some embodiments, the culture medium is supplemented with one or more cytokines.

[0169] In some embodiments, the cytokine is selected from IL-2, IL-12, IL-15, IL-18, and combinations thereof.

[0170] In some embodiments, the culture medium comprises and / or is supplemented with IL-2. In some embodiments, the culture medium comprises and / or is supplemented with IL-2 at or about 150 to 2,500 or about 2,500 IU / mL. In some embodiments, the culture medium comprises and / or is supplemented with IL-2 at or about 200 to 2,250 or about 2,250, 200 or about 200 to 2,000 or about 2,000, 200 or about 200 to 1,750 or about 1,750, 200 or about 200 to 1,500 or about 1,500, 200 or about 200 to 1,250 or about 1,250, 200 or about 200 to 1,000 or about 1,000, 200 or about 200 to 750 or about 750, 200 or about 200 to, 500 or about 500, 200 or about 200 to, 250 or about 250, 250 or about 250 to, 2,500 or about 2,500, 250 or about 250 to, 2,250 or about 2,250, 250 or about 250 to, 2,000 or about 2,000, 250 or about 250 to, 1,750 or about 1,750, 250 or about 250 to, 1,500 or about 1,500, 250 or about 250 to, 1,250 or about 1,250, 250 or about 250 to, 1,000 or about 1,000, 250 or about 250 to, 750 or about 750, 250 or about 250 to, 500 or about 500, 500 or about 500 to, 2,500 or about 2,500, 500 or about 500 to, 2,250 or about 2,250, 500 or about 500 to, 2,000 or about 2,000, 500 or about 500 to, 1,750 or about 1,750, 50 0 or about 500, 1,500 or about 1,500, 500 or about 500, 1,250 or about 1,250, 500 or about 500, 1,000 or about 1,000, 500 or about 500, 750 or about 750, 750 or about 750, 2,250 or about 2,250, 750 or about 750, 2,000 or about 2,000, 750 or about 750, 1,750 or about 1,750, 750 or about 750,500 or about 1,500, 750 or about 750 to, 1,250 or about 1,250, 750 or about 750 to, 1,000 or about 1,000, 1,000 or about 1,000 to, 2,500 or about 2,500, 1,000 or about 1,000 to, 2,250 or about 2,250, 1,000 or about 1,000 to, 2,000 or about 2,000, 1,000 or about 1,000 to, 1,750 or about 1,750, 1,000 or about 1,000 to, 1,500 or about 1,500, 1,000 or about 1,000 to, 1,250 or about 1,250, 1,250 or about 1,250 to, 2,500 or about 2,500, 1,250 or about 1,250 to, 2,250 or about 2,250, 1,250 or about 1,250 to, 2,000 or about 2,000, 1,250 or about 1,250 to, 1,750 or about 1 ,750, 1,250 or about 1,250 to, 1,500 or about 1,500, 1,500 or about 1,500 to, 2,500 or about 2,500, 1,500 or about 1,500 to, 2,250 or about 2,250, 1,500 or about 1,500 to, 2,000 or about 2,000, 1,500 or about 1,500 to, 1,750 or about 1,750, 1,750 or about 1,750 to, 2,500 or about 2, 500, 1,750 or about 1,750 to 2,250 or about 2,250, 1,750 or about 1,750 to 2,000 or about 2,000, 2,000 or about 2,000 to 2,500 or about 2,500, 2,000 or about 2,000 to 2,250 or about 2,250, or 2,250 or about 2,250 to 2,500 or about 2,500 IU / mL of IL-2.

[0171] In some embodiments, the culture medium comprises and / or is supplemented with 64 μg / L to 96 μg / L of IL-2. In some embodiments, the culture medium comprises and / or is supplemented with 80 μg / L of IL-2 (approximately 1,333 IU / mL). In some embodiments, the culture medium comprises and / or is supplemented with about 80 μg / L.

[0172] In some embodiments, the culture medium comprises and / or is supplemented with a combination of IL-2 and IL-15.

[0173] In some embodiments, the culture medium comprises and / or is supplemented with a combination of IL-2, IL-15 and IL-18.

[0174] In some embodiments, the culture medium comprises and / or is supplemented with a combination of IL-2, IL-18 and IL-21.

[0175] In some embodiments, the culture medium comprises and / or is supplemented with glucose. In some embodiments, the culture medium comprises and / or is supplemented with 0.5 or about 0.5 to 3.5 or about 3.5 g / L of glucose. In some embodiments, the culture medium comprises and / or is supplemented with 0.5 or about 0.5 to 3.0 or about 3.0, 0.5 or about 0.5 to 2.5 or about 2.5, 0.5 or about 0.5 to 2.0 or about 2.0, 0.5 or about 0.5 to 1.5 or about 1.5, 0.5 or about 0.5 to 1.0 or about 1.0, 1.0 or about 1.0 to 3.0 or about 3.0, 1.0 or about 1.0 to 2.5 or about 2.5, 1.0 or about 1.0 to 2.0 or about 3.0, In some embodiments, the culture medium comprises and / or is supplemented with 1.6 to 2.4 g / L of glucose. In some embodiments, the culture medium comprises and / or is supplemented with 2.0 g / L of glucose. In some embodiments, the culture medium comprises and / or is supplemented with 2.0 g / L of glucose. In some embodiments, the culture medium comprises and / or is supplemented with 2.0 g / L of glucose. In some embodiments, the culture medium comprises and / or is supplemented with about 2.0 g / L of glucose.

[0176] In some embodiments, the culture medium comprises and / or is supplemented with sodium pyruvate. In some embodiments, the culture medium comprises and / or is supplemented with 0.1 or about 0.1 to 2.0 or about 2.0 mM sodium pyruvate. In some embodiments, the culture medium comprises and / or is supplemented with 0.1 or about 0.1 to 1.8 or about 1.8, 0.1 or about 0.1 to 1.6 or about 1.6, 0.1 or about 0.1 to 1.4 or about 1.4, 0.1 or about 0.1 to 1.2 or about 1.2, 0.1 or about 0.1 to 1.0 or about 1.0, 0.1 or about 0.1 to 0.8 or about 0.8, 0.1 or about 0.1 to 0.6 or about 0.6, 0.1 or about 0.1 to 0.4 or about 0.6, or about 0.4, 0.1 or about 0.1 to, 0.2 or about 0.2, 0.2 or about 0.2 to, 2.0 or about 2.0, 0.2 or about 0.2 to, 1.8 or about 1.8, 0.2 or about 0.2 to, 1.6 or about 1.6, 0.2 or about 0.2 to, 1.4 or about 1.4, 0.2 or about 0.2 to, 1.2 or about 1.2, 0.2 or about 0.2 to, 1.0 or about 1.0, 0.2 or about 0.2 to, 0.8 or about 0.8, 0.2 or about or about 0.2 to, 0.6 or about 0.6, 0.2 or about 0.2 to, 0.4 or about 0.4, 0.4 or about 0.4 to, 2.0 or about 2.0, 0.4 or about 0.4 to, 1.8 or about 1.8, 0.4 or about 0.4 to, 1.6 or about 1.6, 0.4 or about 0.4 to, 1.4 or about 1.4, 0.4 or about 0.4 to, 1.2 or about 1.2, 0.4 or about 0.4 to, 1.0 or about 1.0, 0.4 or about 0.4 to, 8 or about 0.8, 0.4 or about 0.4 to, 0.6 or about 0.6, 0.6 or about 0.6 to, 2.0 or about 2.0, 0.6 or about 0.6 to, 1.8 or about 1.8, 0.6 or about 0.6 to, 1.6 or about 1.6, 0.6 or about 0.6 to, 1.4 or about 1.4, 0.6 or about 0.6 to, 1.2 or about 1.2, 0.6 or about 0.6 to, 1.0 or about 1.0, 0.6 or about 0.6 to, 0.8 or about 0.8, 0.8 or about 0.8 to, 2.0 or about 2.0, 0.8 or about 0.8 to, 1.8 or about 1.8, 0.8 or about 0.8 to, 1.6 or about 1.6, 0.8 or about 0.8 to, 1.4 or about 1.4, 0.8 or about 0.8 to, 1.4 or about 1.4, 0.8 or about 0.8 to, 1.2 or about 1.2, 0.8 or about 0.8 to, 1.0 or about 1.0, 1.0 or about 1.0 to, 2.0 or about 2.0, 1.0 or about 1.0 to, 1.8 or about 1.8, 1.0 or about 1.0 to, 1.6 or about 1.6, 1.0 or about 1.0 to, 1.4 or about 1.4, 1.0 or about 1.0 to, 1.2 or or about 1.2, 1.2 or about 1.2 to 2.0 or about 2.0, 1.2 or about 1.2 to 1.8 or about 1.8, 1.2 or about 1.2 to 1.6 or about 1.6, 1.2 or about 1.2 to 1.4 or about 1.4, 1.4 or about 1.4 to 2.0 or about 2.0, 1.4 or about 1.4 to 1.8 or about 1.8, 1.4 or about 1.4 to 1.6 or about 1.6, 1.6 or about 1.6 to 2.0 or about 2.0, 1.6 or about 1.6 to 1.8 or about 1.8, or 1.8 or about 1.8 to 2.0 or about 2.0 mM sodium pyruvate. In some embodiments, the culture medium comprises 0.8 to 1.2 mM sodium pyruvate. In some embodiments, the culture medium comprises 1.0 mM sodium pyruvate. In some embodiments, the culture medium comprises about 1.0 mM sodium pyruvate.

[0177] In some embodiments, the culture medium comprises and / or is supplemented with sodium bicarbonate. In some embodiments, the culture medium comprises and / or is supplemented with 0.5 or about 0.5 to 3.5 or about 3.5 g / L of sodium bicarbonate. In some embodiments, the culture medium comprises and / or is supplemented with 0.5 or about 0.5 to 3.0 or about 3.0, 0.5 or about 0.5 to 2.5 or about 2.5, 0.5 or about 0.5 to 2.0 or about 2.0, 0.5 or about 0.5 to 1.5 or about 1.5, 0.5 or about 0.5 to 1.0 or about 1.0, 1.0 or about 1.0 to 3.0 or about 3.0, 1.0 or about 1.0 to 2.5 or about 2.5, 1.0 or about 1.0 to 2.0 or The culture medium comprises and / or is supplemented with about 2.0, 1.0 or about 1.0, 1.5 or about 1.5, 1.5 or about 1.5, 3.0 or about 3.0, 1.5 or about 1.5, 2.5 or about 2.5, 1.5 or about 1.5, 2.0 or about 2.0, 2.0 or about 2.0, 3.0 or about 3.0, 2.0 or about 2.0, 2.5 or about 2.5, or 2.5 or about 2.5, 3.0 or about 3.0 g / L of sodium bicarbonate. In some embodiments, the culture medium comprises and / or is supplemented with 1.6 to 2.4 g / L of sodium bicarbonate. In some embodiments, the culture medium comprises and / or is supplemented with 2.0 g / L of sodium bicarbonate. In some embodiments, the culture medium comprises about 2.0 g / L of sodium bicarbonate.

[0178] In some embodiments, the culture medium comprises and / or is supplemented with albumin, e.g., human albumin, e.g., a human albumin solution as described herein. In some embodiments, the culture medium comprises and / or is supplemented with 0.5% or about 0.5% to 3.5% or about 3.5% v / v of a 20% albumin solution, e.g., a 20% human albumin solution. In some embodiments, the culture medium comprises from 0.5% or about 0.5% to 3.0% or about 3.0%, 0.5% or about 0.5% to 2.5% or about 2.5%, 0.5% or about 0.5% to 2.0% or about 2.0%, 0.5% or about 0.5% to 1.5% or about 1.5%, 0.5% or about 0.5% to 1.0% or about 1.0%, 1.0% or about 1.0% to 3.0% or about 3.0%, 1.0% or about 1.0% to 2.5% or about 2.5%, 1.0% or comprises and / or is supplemented with from about 1.0%, 2.0% or about 2.0%, 1.0% or about 1.0% to 1.5% or about 1.5%, 1.5% or about 1.5% to 3.0% or about 3.0%, 1.5% or about 1.5% to 2.5% or about 2.5%, 1.5% or about 1.5% to 2.0% or about 2.0%, 2.0% or about 2.0% to 3.0% or about 3.0%, 2.0% or about 2.0% to 2.5% or about 2.5%, or 2.5% or about 2.5% to 3.0% or about 3.0% v / v of a 20% albumin solution, e.g. a 20% human albumin solution. In some embodiments, the culture medium comprises and / or is supplemented with 1.6% to 2.4% v / v of a 20% albumin solution, such as a 20% human albumin solution. In some embodiments, the culture medium comprises and / or is supplemented with 2.0% v / v of a 20% albumin solution, such as a 20% human albumin solution. In some embodiments, the culture medium comprises about 2.0% v / v of a 20% albumin solution, such as a 20% human albumin solution.

[0179] In some embodiments, the culture medium comprises and / or is supplemented with 2 or about 2 to 6 or about 6 g / L of albumin, e.g., human albumin. In some embodiments, the culture medium comprises 2 or about 2 to 5.5 or about 5.5, 2 or about 2 to 5.0 or about 5.0, 2 or about 2 to 4.5 or about 4.5, 2 or about 2 to 4 or about 4, 2 or about 2 to 3.5 or about 3.5, 2 or about 2 to 3 or about 3, 2 or about 2 to 2.5 or about 2.5, 2.5 or about 2.5 to 6 or about 6, 2.5 or about 2.5 to 5.5 or about 5.5, 2.5 or from about 2.5, 5.5 or about 5.5, 2.5 or about 2.5, 5.0 or about 5.0, 2.5 or about 2.5, 4.5 or about 4.5, 2.5 or about 2.5, 4.0 or about 4.0, 2.5 or about 2.5, 3.5 or about 3.5, 2.5 or about 2.5, 3.0 or about 3.0, 3 or about 3, 6 or about 6, 3 or about 3, 5.5 or about 5.5, 3 or about 3, 5 or about 5, 3 or is from about 3 to 4.5 or about 4.5, 3 or about 3 to 4 or about 4, 3 or about 3 to 3.5 or about 3.5, 3.5 or about 3.5 to 6 or about 6, 3.5 or about 3.5 to 5.5 or about 5.5, 3.5 or about 3.5 to 5 or about 5, 3.5 or about 3.5 to 4.5 or about 4.5, 3.5 or about 3.5 to 4 or about 4, 4 or about 4 to 6 or about 6, 4 or about 4 to 5.5 or about 5.5 , 4 or about 4, 5 or about 5, 4 or about 4, 4.5 or about 4.5, 4.5 or about 4.5, 6 or about 6, 4.5 or about 4.5, 5.5 or about 5.5, 4.5 or about 4.5, 5 or about 5, 5 or about 5, 6 or about 6, 5 or about 5, 5.5 or about 5.5, or 5.5 or about 5.5, 6 or about 6 g / L of albumin, e.g., human albumin. In some embodiments, the culture medium comprises and / or is supplemented with 3.2 to 4.8 g / L of albumin, e.g., human albumin.In some embodiments, the culture medium comprises 4 g / L of albumin, e.g., human albumin. In some embodiments, the culture medium comprises about 4 g / L of albumin, e.g., human albumin.

[0180] In some embodiments, the culture medium is supplemented with poloxamer 188. In some embodiments, the culture medium comprises and / or is supplemented with 0.1 or about 0.1 to 2.0 or about 2.0 g / L of poloxamer 188. In some embodiments, the culture medium comprises and / or is supplemented with 0.1 or about 0.1 to 1.8 or about 1.8, 0.1 or about 0.1 to 1.6 or about 1.6, 0.1 or about 0.1 to 1.4 or about 1.4, 0.1 or about 0.1 to 1.2 or about 1.2, 0.1 or about 0.1 to 1.0 or about 1.0, 0.1 or about 0.1 to 0.8 or about 0.8, 0.1 or about 0.1 to 0.6 or about 0.6, 0.1 or about 0.1 to 0.4 or about 0.8, 0.4, 0.1 or about 0.1 to, 0.2 or about 0.2, 0.2 or about 0.2 to, 2.0 or about 2.0, 0.2 or about 0.2 to, 1.8 or about 1.8, 0.2 or about 0.2 to, 1.6 or about 1.6, 0.2 or about 0.2 to, 1.4 or about 1.4, 0.2 or about 0.2 to, 1.2 or about 1.2, 0.2 or about 0.2 to, 1.0 or about 1.0, 0.2 or about 0.2 to, 0.8 or about 0.8, 0.2 or about 0.2 to from 0.6 or about 0.6, from 0.2 or about 0.2, from 0.4 or about 0.4, from 0.4 or about 0.4, from 2.0 or about 2.0, from 0.4 or about 0.4, from 1.8 or about 1.8, from 0.4 or about 0.4, from 1.6 or about 1.6, from 0.4 or about 0.4, from 1.4 or about 1.4, from 0.4 or about 0.4, from 1.2 or about 1.2, from 0.4 or about 0.4, from 1.0 or about 1.0, from 0.4 or about 0.4 to 0.8 or about 0.8, 0.4 or about 0.4 to, 0.6 or about 0.6 to, 0.6 or about 0.6 to, 2.0 or about 2.0 to, 0.6 or about 0.6 to, 1.8 or about 1.8 to, 0.6 or about 0.6 to, 1.6 or about 1.6 to, 0.6 or about 0.6 to, 1.4 or about 1.4 to, 0.6 or about 0.6 to, 1.2 or about 1.2 to, 0.6 or about 0.6 to, 1.0 or about 1.0 to, 0.6 or about 0.6 to, 0.8 or about 0.8 to, 0.8 or about 0.8 to, 2.0 or about 2.0, 0.8 or about 0.8 to, 1.8 or about 1.8, 0.8 or about 0.8 to, 1.6 or about 1.6, 0.8 or about 0.8 to, 1.4 or about 1.4, 0.8 or about 0.8 to, 1.4 or about 1.4, 0.8 or about 0.8 to, 1.2 or about 1.2, 0.8 or about 0.8 to, 1.0 or about 1.0, 1.0 or about 1.0 to, 2.0 or about 2.0, 1.0 or about 1.0 to, 1.8 or about 1.8, 1.0 or about 1.0 to, 1.6 or about 1.6, 1.0 or about 1.0 to, 1.4 or about 1.4, 1.0 or about 1.0 to, 1.2 or about 1.2 , 1.2 or about 1.2 to 2.0 or about 2.0, 1.2 or about 1.2 to 1.8 or about 1.8, 1.2 or about 1.2 to 1.6 or about 1.6, 1.2 or about 1.2 to 1.4 or about 1.4, 1.4 or about 1.4 to 2.0 or about 2.0, 1.4 or about 1.4 to 1.8 or about 1.8, 1.4 or about 1.4 to 1.6 or about 1.6, 1.6 or about 1.6 to 2.0 or about 2.0, 1.6 or about 1.6 to 1.8 or about 1.8, or 1.8 or about 1.8 to 2.0 or about 2.0 g / L of poloxamer 188. In some embodiments, the culture medium comprises 0.8 to 1.2 g / L of poloxamer 188. In some embodiments, the culture medium comprises 1.0 g / L of poloxamer 188. In some embodiments, the culture medium comprises about 1.0 g / L of poloxamer 188.

[0181] In some embodiments, the culture medium contains and / or is supplemented with one or more antibiotics.

[0182] A first exemplary culture medium is shown in Table 1.

[0183] (Table 1) Example of culture medium No. 1 TIFF2024542159000002.tif34156

[0184] A second exemplary culture medium is shown in Table 2.

[0185] (Table 2) Example of second culture medium TIFF2024542159000003.tif57156

[0186] 2. CD3 binding antibody In some embodiments, the culture medium comprises and / or is supplemented with a CD3 binding antibody or an antigen-binding fragment thereof. In some embodiments, the CD3 binding antibody or an antigen-binding fragment thereof is selected from the group consisting of OKT3, UCHT1, and HIT3a, or variants thereof. In some embodiments, the CD3 binding antibody or an antigen-binding fragment thereof is OKT3 or an antigen-binding fragment thereof.

[0187] In some embodiments, a CD3 binding antibody or antigen-binding fragment thereof and feeder cells are added to the culture vessel prior to adding the NK cells and / or culture medium.

[0188] In some embodiments, the culture medium comprises and / or is supplemented with 5 or about 5 ng / mL to 15 or about 15 ng / mL of OKT3. In some embodiments, the culture medium comprises and / or is supplemented with 5 or about 5 to 12.5 or about 12.5, 5 or about 5 to 10 or about 10, 5 or about 5 to 7.5 or about 7.5, 7.5 or about 7.5 to 15 or about 15, 7.5 or about 7.5 to 12.5 or about 12.5, 7.5 or about 7.5 to 10 or about 10, 10 or about 10 to 15 or about 15, 10 or about 10 to 12.5 or about 12.5, or 12.5 or about 12.5 to 15 or about 15 ng / mL of OKT3. In some embodiments, the culture medium comprises and / or is supplemented with 10 ng / mL of OKT3. In some embodiments, the culture medium contains and / or is supplemented with about 10 ng / mL OKT3.

[0189] 3. Culture vessel Some vessels are consistent with the disclosure herein. In some embodiments, the culture vessel is selected from the group consisting of a flask, a bottle, a dish, a multi-well plate, a roller bottle, a bag, and a bioreactor.

[0190] In some embodiments, the culture vessel is treated to make it hydrophilic. In some embodiments, the culture vessel is treated to promote attachment and / or proliferation. In some embodiments, the culture vessel surface is coated with serum, collagen, laminin, gelatin, poly-L-lysine, fibronectin, extracellular matrix proteins, and combinations thereof.

[0191] In some embodiments, different types of culture vessels are used for different stages of culture.

[0192] In some embodiments, the culture vessel has a volume of at or about 100 mL to at or about 1,000 L. In some embodiments, the culture vessel has a volume of at or about 125 mL, 250 mL, 500 mL, 1 L, 5 L, 10 L, or 20 L.

[0193] In some embodiments, the culture vessel is a bioreactor.

[0194] In some embodiments, the bioreactor is a rocking bed (wave) bioreactor. In some embodiments, the bioreactor is a stirred tank bioreactor. In some embodiments, the bioreactor is a rotating wall vessel. In some embodiments, the bioreactor is a perfusion bioreactor. In some embodiments, the bioreactor is an isolation / expansion automated system. In some embodiments, the bioreactor is an automated or semi-automated bioreactor. In some embodiments, the bioreactor is a disposable bag bioreactor.

[0195] In some embodiments, the bioreactor has a volume of about 100 mL to about 1,000 L. In some embodiments, the bioreactor has a volume of about 10 L to about 1,000 L. In some embodiments, the bioreactor has a volume of about 100 L to about 900 L. In some embodiments, the bioreactor has a volume of about 10 L to about 800 L. In some embodiments, the bioreactor is about 10 L to about 700 L, about 10 L to about 600 L, about 10 L to about 500 L, about 10 L to about 400 L, about 10 L to about 300 L, about 10 L to about 200 L, about 10 L to about 100 L, about 10 L to about 90 L, about 10 L to about 80 L, about 10 L to about 70 L, about 10 L to about 60 L, about 10 L to about 50 L, about 10 L to about 40 L, about 10 L to about 30 L, about 10 L to about 20L, approximately 20L to approximately 1,000L, approximately 20L to approximately 900L, approximately 20L to approximately 800L, approximately 20L to approximately 700L, approximately 20L to approximately 600L, approximately 20L to approximately 500L, approximately 20L to approximately 400L, approximately 20L to approximately Approximately 300L, approximately 20L to approximately 200L, approximately 20L to approximately 100L, approximately 20L to approximately 90L, approximately 20L to approximately 80L, approximately 20L to approximately 70L, approximately 20L to approximately 60L, approximately 20L to approximately 50L, approximately 20L to approximately 40L, Approximately 20L to approximately 30L, approximately 30L to approximately 1,000L, approximately 30L to approximately 900L, approximately 30L to approximately 800L, approximately 30L to approximately 700L, approximately 30L to approximately 600L, approximately 30L to approximately 500L, approximately 30L to approximately 400L , about 30L to about 300L, about 30L to about 200L, about 30L to about 100L, about 30L to about 90L, about 30L to about 80L, about 30L to about 70L, about 30L to about 60L, about 30L to about 50L, about 30L ~40L, 40L~1,000L, 40L~900L, 40L~800L, 40L~700L, 40L~600L, 40L~500L, 40L~400L, 40 L~300L, 40L~200L, 40L~100L, 40L~90L, 40L~80L, 40L~70L, 40L~60L, 40L~50L, 50L~1,000L, about 50L to about 900L, about 50L to about 800L, about 50L to about 700L, about 50L to about 600L, about 50L to about 500L, about 50L to about 400L, about 50L to about 300L, about 50L to about 200L, about 50L to about 100L, about 50L to about 90L, about 50L to about 80L, about 50L to about 70L, about 50L to about 60L, about 60L to about 1,000L, about 60L to about 900L, about 60L to about 800L, about 60L to about 700L, about 60L to about 600L, about 60L to about 500L, about 60L to about 400L, about 60L to about 300L, about 60L to about 200L, about 60L to about 100L, about 60L to about 90L, about 60L to about 80L, about 60L to about 70L, about 70L to about 1,000L, about 70L to about 900L, about 70L to about 800L, about 70L to about 700L, about 70L to about 600L, about 70L to about 500L, about 70L to about 400L, about 70L to about 300L, about 70L to about 200L, about 70L to about 100L, about 70L to about 90L, about 70L to about 80L, about 80L to about 1,000L, about 80L to about 900L, about 80L to about 800L, about 80L to about 700L, about 80L to about 600L, about 80L to about 500L, about 80L to about 400L, about 80L to about 300L, about 80L to about 200L, about 80L to about 100L, about 80L to about 90L, about 90L to about 1,000L, about 90L to about 900L, about 90L to about 800L, about 90L to about 700L, about 90L to about 600L, about 90L to about 500L, about 90L to about 400L, about 90L to about 300L, about 90L to about 200L, about 90L to about 100L, about 100L to about 1,000L, about 100L to about 900L, about 100L to about 800L, about 100L to about 700L, about 100L to about 600L, about 100L to about 500L, about 100L to about 400L, about 100L to about 300L, about 100L to about 200L, about 200L to about 1,000L, about 200L to about 900L, about 200L to about 800L, about 200L to about 700L, about 200L to about 600L, about 200L to about 500L, about 200L to about 400L, about 200L to about 300L, about 300L to about 1,000L, about 300L to about 900L, about 300L to about 800L, about 300L to about 700L, about 300L to about 600L, about 300L to about 500L, about 300L to about 400L, about 400L to about 1,000L, approximately 400L to approximately 900L, approximately 400L to approximately 800L, approximately 400L to approximately 700L, approximately 400L to approximately 600L, approximately 400L to approximately 500L, approximately 500L~Approx. 1,000L, Approx. 500L~Approx. 900L, Approx. 500L~Approx. 800L, Approx. 500L~Approx. 700L, Approx. 500L~Approx. 600L, Approx. 600L The bioreactor has a volume of about 1,000 L, about 600 L to about 900 L, about 600 L to about 800 L, about 600 L to about 700 L, about 700 L to about 1,000 L, about 700 L to about 900 L, about 700 L to about 800 L, about 800 L to about 1,000 L, about 800 L to about 900 L, or about 900 L to about 1,000 L. In some embodiments, the bioreactor has a volume of about 50 L.

[0196] In some embodiments, the bioreactor has a volume between 100 mL and 1,000 L. In some embodiments, the bioreactor has a volume between 10 L and 1,000 L. In some embodiments, the bioreactor has a volume between 100 L and 900 L. In some embodiments, the bioreactor has a volume between 10 L and 800 L. In some embodiments, the bioreactor is configured to accommodate a volume of 10L to 700L, 10L to 600L, 10L to 500L, 10L to 400L, 10L to 300L, 10L to 200L, 10L to 100L, 10L to 90L, 10L to 80L, 10L to 70L, 10L to 60L, 10L to 50L, 10L to 40L, 10L to 30L, 10L to 20L, 20L to 1,000L, 20L to 900L, 20L to 800L, 20L to 700L, 20L to 600L, 20L to 500L, L, 20L~400L, 20L~300L, 20L~200L, 20L~100L, 20L~90L, 20L~80L, 20L~70L, 20L~60L, 20L~50L, 20L~40L, 20L~30L, 30L~1,00 0L, 30L~900L, 30L~800L, 30L~700L, 30L~600L, 30L~500L, 30L~400L, 30L~300L, 30L~200L, 30L~100L, 30L~90L, 30L~80L, 30 L~70L, 30L~60L, 30L~50L, 30L~40L, 40L~1,000L, 40L~900L, 40L~800L, 40L~700L, 40L~600L, 40L~500L, 40L~400L, 40L~300 L, 40L~200L, 40L~100L, 40L~90L, 40L~80L, 40L~70L, 40L~60L, 40L~50L, 50L~1,000L, 50L~900L, 50L~800L, 50L~700L, 50L~ 600L, 50L~500L, 50L~400L, 50L~300L, 50L~200L, 50L~100L, 50L~90L, 50L~80L, 50L~70L, 50L~60L, 60L~1,000L, 60L~900L, 60L~800L, 60L~700L, 60L~600L, 60L~500L, 60L~400L, 60L~300L, 60L~200L, 60L~100L, 60L~90L, 60L~80L, 60L~70L, 70L~1,000L, 70L~900L, 70L~800L, 70L~700L, 70L~600L, 70L~500L, 70L~400L, 70L~300L, 70L~200L, 70L~100L, 70L~90L, 70 L~80L, 80L~1,000L, 80L~900L, 80L~800L, 80L~700L, 80L~600L, 80L~500L, 80L~400L, 80L~300L, 80L~200L, 80L~100L , 80L~90L, 90L~1,000L, 90L~900L, 90L~800L, 90L~700L, 90L~600L, 90L~500L, 90L~400L, 90L~300L, 90L~200L, 90L~ 100L, 100L~1,000L, 100L~900L, 100L~800L, 100L~700L, 100L~600L, 100L~500L, 100L~400L, 100L~300L, 100L~200L, 200L~1,000L, 200L~900L, 200L~800L, 200L~700L, 200L~600L, 200L~500L, 200L~400L, 200L~300L, 300L~1,000L, 30 0L~900L, 300L~800L, 300L~700L, 300L~600L, 300L~500L, 300L~400L, 400L~1,000L, 400L~900L, 400L~800L, 400L~70 In some embodiments, the bioreactor has a volume of 0 L, 400 L to 600 L, 400 L to 500 L, 500 L to 1,000 L, 500 L to 900 L, 500 L to 800 L, 500 L to 700 L, 500 L to 600 L, 600 L to 1,000 L, 600 L to 900 L, 600 L to 800 L, 600 L to 700 L, 700 L to 1,000 L, 700 L to 900 L, 700 L to 800 L, 800 L to 1,000 L, 800 L to 900 L, or 900 L to 1,000 L. In some embodiments, the bioreactor has a volume of 50 L.

[0197] 4. Cell expansion and stimulation In some embodiments, a natural killer cell source, such as a single unit of umbilical cord blood, is co-cultured with feeder cells to produce expanded and stimulated NK cells.

[0198] In some embodiments, the co-culture is carried out in a culture medium described herein, such as the first exemplary culture medium (Table 1) or the second exemplary culture medium (Table 2).

[0199] In some embodiments, a natural killer cell source, e.g., a single unit of umbilical cord blood, is cultured at 1×10 7 or about 1 x 10 7 From 1 × 10 9 or about 1 x 10 9 In some embodiments, the natural killer cell source, e.g., a single unit of umbilical cord blood, prior to expansion, comprises 1×10 total nucleated cells. 8 or about 1 x 10 8 So, 1.5 x 10 8 or about 1.5 x 10 8 In some embodiments, the natural killer cell source, e.g., a single unit of umbilical cord blood, prior to expansion, comprises 1×10 total nucleated cells. 8 In some embodiments, the natural killer cell source, e.g., a single unit of umbilical cord blood, prior to expansion, comprises about 1×10 total nucleated cells. 8 In some embodiments, the natural killer cell source, e.g., a single unit of umbilical cord blood, prior to expansion, comprises 1×10 total nucleated cells. 9 In some embodiments, the natural killer cell source, e.g., a single unit of umbilical cord blood, prior to expansion, comprises about 1×10 total nucleated cells. 9 Contains total nucleated cells.

[0200] In some embodiments, cells from a natural killer cell source, e.g., a single unit of umbilical cord blood, co-cultured with feeder cells are harvested and frozen, e.g., in a cryopreservation composition as described herein. In some embodiments, the frozen cells from the co-culture are an infusion-ready formulation. In some embodiments, the frozen cells from the co-culture are used as a master cell bank (MCB) from which an infusion-ready formulation is produced, e.g., by one or more additional co-culture steps as described herein. Thus, for example, the natural killer cell source can be expanded and stimulated as described herein to produce expanded and stimulated NK cells suitable for use in an infusion-ready formulation without producing any intermediate product. The natural killer cell source can also be expanded and stimulated as described herein to produce an intermediate product, e.g., a first master cell bank (MCB). The first MCB can be used to produce expanded and stimulated NK cells suitable for use in an infusion-ready formulation, or can be used to produce another intermediate product, e.g., a second MCB. The second MCB can be used to produce expanded stimulated NK cells suitable for an infusion-ready formulation, or can be used to produce another intermediate product, such as a third MCB, and so on.

[0201] In some embodiments, the ratio of feeder cells to natural killer cell source cells or MCB cells inoculated into the co-culture is from at or about 1:1 to at or about 4:1. In some embodiments, the ratio of feeder cells to natural killer cell source cells or MCB cells is from 1:1 or about 1:1, 3.5:1 or about 3.5:1, 1:1 or about 1:1, 3:1 or about 3:1, 1:1 or about 1:1, 2.5:1 or about 2.5:1, 1.1 or about 1.1, 2:1 or about 2:1, 1:1 or about 1:1, 1.5:1 or about 1.5:1, 1.5:1 or about 1.5:1, 4:1 or about 4:1, 1.5:1 or about 1.5:1, 3.5:1 or about 3.5:1, 1.5:1 or about 1.5:1, 3:1 or about 3:1, 1.5:1 or about 1.5:1, 2.5:1 or about 2.5:1, 1.5: from 1 or about 1.5:1, 2:1 or about 2:1, 2:1 or about 2:1 to 4:1 or about 4:1, 2:1 or about 2:1 to 3.5:1 or about 3.5:1, 2:1 or about 2:1 to 3:1 or about 3:1, 2:1 or about 2:1 to 2.5:1 or about 2.5:1, 2.5:1 or about 2.5:1 to 4:1 or about 4:1, 2.5:1 or about 2.5:1 to 3.5:1 or about 3.5:1, 2.5:1 or about 2.5:1 to 3:1 or about 3:1, 3:1 or about 3:1 to 4:1 or about 4:1, 3:1 or about 3:1 to 3.5:1 or about 3.5:1, or 3.5:1 or about 3.5:1 to 4:1 or about 4:1. In some embodiments, the ratio of feeder cells to cells of the natural killer cell source or MCB inoculated into the co-culture is 2.5: 1. In some embodiments, the ratio of feeder cells to cells of the natural killer cell source or MCB inoculated into the co-culture is about 2.5: 1.

[0202] In some embodiments, the co-culture is carried out in a disposable culture bag, such as a 1 L disposable culture bag. In some embodiments, the co-culture is carried out in a bioreactor, such as a 50 L bioreactor. In some embodiments, the culture medium is added to the co-culture after the initial inoculation.

[0203] In some embodiments, the co-culture is carried out for 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 days or more. In some embodiments, the co-culture is carried out for up to 16 days.

[0204] In some embodiments, the co-culture is carried out at or about 37°C.

[0205] In some embodiments, the co-culture is carried out at or about pH 7.9.

[0206] In some embodiments, the co-cultivation is carried out at a dissolved oxygen (DO) level of 50% or greater.

[0207] In some embodiments, a first example culture medium (Table 1) is used to produce MCBs and a second example culture medium (Table 2) is used to produce cells suitable for an injection-ready formulation.

[0208] In some embodiments, the co-culture of a natural killer cell source, e.g., a single unit of umbilical cord blood, with feeder cells is performed at 50×10 8 or about 50 x 10 8 From, 50 x 10 12 or about 50 x 10 12 In some embodiments, the expansion produces 50×10 cells, e.g., MCB cells or infusion-ready formulation cells. 8 Or about 50 x 10 8 From, 25 × 10 10 Or about 25 x 10 10 , 10×10 8 Or about 10 x 108 From 1 × 10 10 Or about 1 x 10 10 , 50×10 8 Or about 50 x 10 8 From, 75 × 10 9 Or about 75 x 10 9 , 50×10 8 Or about 50 x 10 8 From, 50 x 10 9 Or about 50 x 10 9 , 50×10 8 Or about 50 x 10 8 From, 25 × 10 9 Or about 25 x 10 9 , 50×10 8 Or about 50 x 10 8 From 1 × 10 9 Or about 1 x 10 9 , 50×10 8 Or about 50 x 10 8 From, 75 × 10 8 Or about 75 x 10 8 , 75×10 8 Or about 75 x 10 8 From, 50 x 10 10 Or about 50 x 10 10 , 75×10 8 Or about 75 x 10 8 From, 25 × 10 10 Or about 25 x 10 10 , 75×10 8 Or about 75 x 10 8 From 1 × 10 10 Or about 1 x 10 10 , 75×10 8 Or about 75 x 10 8 From, 75 × 10 9 Or about 75 x 10 9 , 75×10 8 Or about 75 x 10 8 From, 50 x 10 9 Or about 50 x 10 9 , 75×10 8 Or about 75 x 10 8 From, 25 × 10 9 Or about 25 x 10 9 , 75×108 Or about 75 x 10 8 From 1 × 10 9 Or about 1 x 10 9 , 1×10 9 Or about 1 x 10 9 From, 50 x 10 10 Or about 50 x 10 10 , 1×10 9 Or about 1 x 10 9 From, 25 × 10 10 Or about 25 x 10 10 , 1×10 9 Or about 1 x 10 9 From 1 × 10 10 Or about 1 x 10 10 , 1×10 9 Or about 1 x 10 9 From, 75 × 10 9 Or about 75 x 10 9 , 1×10 9 Or about 1 x 10 9 From, 50 x 10 9 Or about 50 x 10 9 , 1×10 9 Or about 1 x 10 9 From, 25 × 10 9 Or about 25 x 10 9 , 25×10 9 Or about 25 x 10 9 From, 50 x 10 10 Or about 50 x 10 10 , 25×10 9 Or about 25 x 10 9 From, 25 × 10 10 Or about 25 x 10 10 , 25×10 9 Or about 25 x 10 9 From 1 × 10 10 Or about 1 x 10 10 , 25×10 9 Or about 25 x 10 9 From, 75 × 10 9 Or about 75 x 10 9 , 25×10 9 Or about 25 x 10 9 From, 50 x 10 9 Or about 50 x 109 , 50×10 9 Or about 50 x 10 9 From, 50 x 10 10 Or about 50 x 10 10 , 50×10 9 Or about 50 x 10 9 From, 25 × 10 10 Or about 25 x 10 10 , 50×10 9 Or about 50 x 10 9 From 1 × 10 10 Or about 1 x 10 10 , 50×10 9 Or about 50 x 10 9 From, 75 × 10 9 Or about 75 x 10 9 , 75×10 9 Or about 75 x 10 9 From, 50 x 10 10 Or about 50 x 10 10 , 75×10 9 Or about 75 x 10 9 From, 25 × 10 10 Or about 25 x 10 10 , 75×10 9 Or about 75 x 10 9 From 1 × 10 10 Or about 1 x 10 10 , 1×10 10 Or about 1 x 10 10 From, 50 x 10 10 Or about 50 x 10 10 , 1×10 10 Or about 1 x 10 10 From, 25 × 10 10 Or about 25 x 10 10 , or 25 × 10 10 Or about 25 x 10 10 From, 50 x 10 10 Or about 50 x 10 10 Generate individual cells, such as MCB cells or injection-ready formulation cells.

[0209] In some embodiments, the expansion results in 60 or about 60 to 100 or about 100 vials, each containing at or about 600 million to at or about 1 billion cells, e.g., MCB cells or infusion ready formulation cells. In some embodiments, the expansion results in 80 or about 80 vials, each containing or consisting of at or about 800 million cells, e.g., MCB cells or infusion ready formulation cells.

[0210] In some embodiments, the expansion results in an increase in the number of cells, e.g., the number of MCB cells, from at or about 100 to at or about 500 fold compared to the number of cells, e.g., NK cells, in the natural killer cell source. In some embodiments, the expansion is from 100 or about 100 to 500 or about 500, 100 or about 100 to 400 or about 400, 100 or about 100 to 300 or about 300, 100 or about 100 to 200 or about 200, 200 or about 200 to 450 or about 450, 200 or about 200 to 400 or about 400, 100 or about 100 to 350 or about 350, 200 or about 200 to 300 or about 300, 200 or about 200 to 250 or about 250, 250 or about from 250, 500 or about 500, from 250 or about 250, 450 or about 450, from 200 or about 200, 400 or about 400, from 250 or about 250, 350 or about 350, from 250 or about 250, 300 or about 300, from 300 or about 300, 500 or about 500, from 300 or about 300, 450 or about 450, from 300 or about 300, 400 or about 400, from 300 or about 300, 350 or about 350, from 350 or about 350, from 500 or about 500, from 350 or about 350, from 450 or about 450, from 350 or about 350, from 400 or about 400 fold increase.

[0211] In some embodiments, the expansion results in a 100 or about 100 to 70,000 or about 70,000-fold increase in the number of cells, e.g., the number of MCB cells, compared to the number of cells, e.g., the number of cells, e.g., NK cells, in the natural killer cell source. In some embodiments, the expansion results in at least a 10,000-fold, e.g., 15,000-fold, 20,000-fold, 25,000-fold, 30,000-fold, 35,000-fold, 40,000-fold, 45,000-fold, 50,000-fold, 55,000-fold, 60,000-fold, 65,000-fold, or 70,000-fold increase in the number of cells, e.g., the number of MCB cells, compared to the number of cells, e.g., the number of cells, e.g., the number of cells, in the natural killer cell source,

[0212] In some embodiments, co-culture of MCB cells with feeder cells produces 500 or about 500 million to 1.5 billion or about 1.5 billion cells, e.g., NK cells suitable for use in MCB and / or infusion-ready formulations. In some embodiments, co-culture of MCB cells with feeder cells produces 500 or about 500 million to 1.5 billion or about 1.5 billion, 500 or about 500 million to 1.25 billion or about 1.25 billion, 500 or about 500 million to 1 billion or about 1 billion, 500 or about 500 million to 750 million or about 750 million, 750 million or about 750 million to 1.5 billion or about 1.5 billion, 500 or about 500 million to 1.25 billion. 0 million or about 1.25 billion, 750 million or about 750 million to 1 billion or about 1 billion, 1 billion or about 1 billion to 1.5 billion or about 1.5 billion, 1 billion or about 1 billion to 1.25 billion or about 1.25 billion, or 1.25 billion or about 1.25 billion to 1.5 billion or about 1.5 billion cells, e.g., NK cells suitable for use in an MCB and / or infusion ready formulation.

[0213] In some embodiments, co-culture of MCB cells with feeder cells results in 50 or about 50 to 150 or about 150 vials of cells, e.g., vials of infusion-ready formulation cells, each containing at or about 750 million to at or about 1.25 billion cells, e.g., NK cells suitable for use in MCB and / or infusion-ready formulations. In some embodiments, co-culture of MCB cells with feeder cells results in 100 or about 100 vials, each containing or consisting of at or about 1 billion cells, e.g., NK cells suitable for use in MCB and / or infusion-ready formulations.

[0214] In some embodiments, expansion results in a 100 or about 100 to 500 or about 500-fold increase in the number of cells, e.g., the number of NK cells suitable for use in the MCB and / or infusion-ready formulations, compared to the number of starting MCB cells. In some embodiments, the expansion increases the number of cells, e.g., the number of NK cells suitable for use in an MCB and / or infusion ready formulation, compared to the number of starting MCB cells, by 100 or about 100, 500 or about 500, 100 or about 100, 400 or about 400, 100 or about 100, 300 or about 300, 100 or about 100, 200 or about 200, 200 or about 200, 450 or about 450, 200 or about 200, 400 or about 400, 100 or about 100, 350 or about 350, 200 or about 200, 300 or about 300, 200 or about 200, 250 or about 250, 250 or about 250, 300 or about 200, 350 or about 350, 300 or about 20 ... from 0 or about 250, 500 or about 500, from 250 or about 250, 450 or about 450, from 200 or about 200, 400 or about 400, from 250 or about 250, 350 or about 350, from 250 or about 250, 300 or about 300, from 300 or about 300, 500 or about 500, from 300 or about 300, 450 or about 450, from 300 or about 300, 400 or about 400, from 300 or about 300, 350 or about 350, from 350 or about 350, from 500 or about 500, from 350 or about 350, from 450 or about 450, from 350 or about 350, from 400 or about 400 fold increase.

[0215] In some embodiments, the expansion results in a 100 or about 100 to 70,000 or about 70,000-fold increase in the number of cells, e.g., the number of NK cells suitable for use in an MCB and / or infusion ready formulation, compared to the number of starting MCB cells. In some embodiments, the expansion results in at least a 10,000-fold, e.g., 15,000-fold, 20,000-fold, 25,000-fold, 30,000-fold, 35,000-fold, 40,000-fold, 45,000-fold, 50,000-fold, 55,000-fold, 60,000-fold, 65,000-fold, or 70,000-fold increase in the number of cells, e.g., the number of NK cells suitable for use in an MCB and / or infusion ready formulation, compared to the number of starting MCB cells.

[0216] In embodiments in which cells are engineered during expansion and stimulation as described herein, not all of the expanded and stimulated cells will necessarily be successfully engineered, e.g., successfully transduced, e.g., successfully transduced with a vector comprising a heterologous protein, e.g., a heterologous protein comprising a CAR and / or IL-15 as described herein. Thus, the methods described herein can further include a step of sorting engineered cells, e.g., engineered cells as described herein, from non-engineered cells.

[0217] In some embodiments, engineered cells, e.g., transduced cells, are selected from non-engineered cells, e.g., non-transduced cells, using a reagent specific for an antigen of the engineered cells, e.g., an antibody that targets an antigen of the engineered cells but not of the non-engineered cells. In some embodiments, the antigen of the engineered cells is a component of a CAR, e.g., a CAR described herein.

[0218] Systems for antigen-based cell separation are commercially available, such as the CliniMACS® sorting system (Miltenyi Biotec).

[0219] In some embodiments, engineered cells, e.g., transduced cells, are sorted from non-engineered cells, e.g., non-transduced cells, using flow cytometry.

[0220] In some embodiments, the selected engineered cells are used as an MCB, hi some embodiments, the selected engineered cells are used as a component in a ready-to-inject formulation.

[0221] In some embodiments, engineered cells, e.g., transduced cells, are sorted from non-engineered cells, e.g., non-transduced cells, using microfluidic cell sorting methods, e.g., as described in Dalili et al., "A Review of Sorting, Separation and Isolation of Cells and Microbeads for Biomedical Applications: Microfluidic Approaches," Analyst 144:87 (2019).

[0222] In some embodiments, 1% or about 1% to 99% or about 99% of the expanded stimulated cells are successfully engineered, e.g., successfully transduced, e.g., successfully transduced with a vector comprising a heterologous protein, e.g., a heterologous protein comprising a CAR and / or IL-15 as described herein. In some embodiments, 1% or about 1% to 90% or about 90%, 1% or about 1% to 80% or about 80%, 1% or about 1% to 70% or about 70%, 1% or about 1% to 60% or about 60%, 1% or about 1% to 50% or about 50%, 1% or about 1% to 40% or about 40%, 1% or about 1% to 30% or about 30%, 1% or about 1% to 20% or about 30%, 1% or about 1% to 35% or about 35%, 1% or about 1% to 40% or about 40%, 1% or about 1% to 50% or about 5 ...60% or about 60%, 1% or about 1% to 70% or about 70%, 1% or about 1% to 80% or about 80%, 1% or about 1% to 70% or about 70%, 1% or about 1% to 60% or about 60%, 1% or about 1% to 50% or about 50%, 1% or about 1% to 40% or about 40%, from about 20%, 1% or about 1%, 10% or about 10%, 1% or about 1%, 5% or about 5%, 5% or about 5%, 99% or about 99%, 5% or about 5%, 90% or about 90%, 5% or about 5%, 80% or about 80%, 5% or about 5%, 70% or about 70%, 5% or about 5%, 60% or about 60%, 5% or about 5%, 50% or about 50%, 5% or about 5% to 40% or from about 40%, 5% or about 5%, 30% or about 30%, 5% or about 5%, 20% or about 20%, 5% or about 5%, 10% or about 10%, 10% or about 10%, 99% or about 99%, 10% or about 10%, 90% or about 90%, 10% or about 10%, 80% or about 80%, 10% or about 10%, 70% or about 70%, 10% or about 10%, 60% or about 60%, 10% or from about 10%, 50% or about 50%, from 10% or about 10%, 40% or about 40%, from 10% or about 10%, 30% or about 30%, from 10% or about 10%, 20% or about 20%, from 20% or about 20%, 99% or about 99%, from 20% or about 20%, 90% or about 90%, from 20% or about 20%, 80% or about 80%, from 20% or about 20%, 70% or about 70%, from 20% or about 20%,60% or about 60%, from 20% or about 20%, 50% or about 50%, from 20% or about 20%, 40% or about 40%, from 20% or about 20%, from 30% or about 30%, from 30% or about 30%, 99% or about 99%, from 30% or about 30%, 90% or about 90%, from 30% or about 30%, 80% or about 80%, from 30% or about 30%, 70% or about 70%, from 30% or about 30%, 60% or about 60%, from 30% or about 30%, 0% or about 50%, 30% or about 30%, 40% or about 40%, 40% or about 40%, 99% or about 99%, 40% or about 40%, 90% or about 90%, 40% or about 40%, 80% or about 80%, 40% or about 40%, 70% or about 70%, 40% or about 40%, 70% or about 70%, 40% or about 40%, 60% or about 60%, 40% or about 40%, 50% or about 50%, 50% or about 50% to 99 % or about 99%, from 50% or about 50%, 90% or about 90%, from 50% or about 50%, 80% or about 80%, from 50% or about 50%, 70% or about 70%, from 50% or about 50%, 60% or about 60%, from 60% or about 60%, from 99% or about 99%, from 60% or about 60%, from 90% or about 90%, from 60% or about 60%, 80% or about 80%, from 60% or about 60%, from 70% or about 70%, from 70% or about 70% to 99% or about 99%, 70% or about 70% to 90%, 70% or about 70% to 80%, 80% or about 80% to 99%, 80% or about 80% to 90%, or 90% or about 90% to 99% or about 99% are successfully engineered, e.g., successfully transduced, e.g., successfully transduced with a vector comprising a heterologous protein, e.g., a heterologous protein comprising a CAR and / or IL-15 as described herein.

[0223] In some embodiments, the frozen cells of the first or second MCB are thawed and cultured. In some embodiments, a single vial of frozen cells of the first or second MCB, such as a single vial containing 800 million or about 800 million cells, such as the first or second MCB cells, is thawed and cultured. In some embodiments, the frozen first or second MCB cells are cultured with additional feeder cells to produce cells suitable for use as the second or third MCB or for use in a ready-to-inject formulation. In some embodiments, cells are harvested and frozen from the co-culture of the first or second MCB.

[0224] In some embodiments, cells are harvested from the co-culture of the natural killer cell source, the first MCB, or the second MCB, and frozen in a cryopreservation composition, e.g., a cryopreservation composition described herein. In some embodiments, the cells are washed after harvesting. Thus, provided herein is a pharmaceutical composition comprising activated and stimulated NK cells, e.g., activated and stimulated NK cells produced by the methods described herein, e.g., harvested and washed activated and stimulated NK cells produced by the methods described herein, and a cryopreservation composition, e.g., a cryopreservation composition described herein.

[0225] In some embodiments, prior to freezing, the cells are mixed with a cryopreservation composition, such as those described herein. In some embodiments, the cells are frozen in a freezer bag. In some embodiments, the cells are frozen in a freezer vial.

[0226] In some embodiments, the method further comprises isolating NK cells from the expanded stimulated population of NK cells.

[0227] An exemplary process for expanding and stimulating NK cells is shown in FIG.

[0228] In some embodiments, the natural killer cells are not genetically engineered.

[0229] E. Properties of Expanded and Stimulated NK Cells The expanded and stimulated NK cell population, once expanded ex vivo and stimulated, e.g., as described herein, not only have numbers / densities (e.g., as described above) that would not naturally occur in the human body, but also have phenotypic characteristics (e.g., gene expression and / or surface protein expression) that differ from the starting source material or other naturally occurring NK cell populations.

[0230] In some cases, the starting NK cell source is a sample derived from a single individual, e.g., a single umbilical cord blood unit that has not been expanded ex vivo. In some cases, therefore, the expanded and stimulated NK cells share a common lineage; that is, they all result from the expansion of the starting NK cell source, and therefore share a certain genotype, due to the clonal expansion of a cell population that is itself from a single organism. Nevertheless, they do not naturally occur at the densities achieved by ex vivo expansion, and they also have different phenotypic characteristics from the starting NK cell source.

[0231] In some cases, the population of expanded stimulated NK cells comprises at least 100 million expanded natural killer cells, e.g., 200 million, 250 million, 300 million, 400 million, 500 million, 600 million, 700 million, 750 million, 800 million, 900 million, 1 billion, 2 billion, 3 billion, 4 billion, 5 billion, 6 billion, 7 billion, 8 billion, 9 billion, 10 billion, 15 billion, 20 billion, 25 billion, 50 billion, 75 billion, 80 billion, 9- 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.

[0232] In some embodiments, the expanded stimulated NK cells comprise at least 80%, e.g., at least 90%, at least 95%, at least 99%, or 100% CD56+CD3- cells.

[0233] In some embodiments, the expanded and stimulated NK cells are not genetically engineered.

[0234] In some embodiments, the expanded stimulated NK cells do not comprise a CD16 transgene.

[0235] In some embodiments, the expanded stimulated NK cells do not express exogenous CD16 protein.

[0236] Expanded and stimulated NK cells can be characterized, for example, by surface expression of one or more of CD16, CD56, CD3, CD38, CD14, CD19, NKG2D, NKp46, NKp30, DNAM-1, and NKp44.

[0237] The surface protein expression levels stated herein are optionally achieved without positive selection for the particular surface protein mentioned. For example, optionally, the NK cell source, e.g., a single umbilical cord unit, contains both the KIR B allele of the KIR receptor family and the 158 V / V variant of CD16, and is enriched for + and depleted for CD3(+), e.g., by gating on CD56+CD3- expression or using magnetic beads, including immunoaffinity magnetic beads (e.g., the CliniMACS Prodigy® system), but no other surface protein expression selection is performed during expansion and stimulation.

[0238] In some embodiments, the expanded and stimulated NK cells, e.g., those obtained from expansion and stimulation of a single cord blood unit as described above, comprise at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100%, NKG2D+ cells.

[0239] In some embodiments, the expanded and stimulated NK cells, e.g., those obtained from expansion and stimulation of a single cord blood unit as described above, comprise at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100%, NKp46+ cells.

[0240] In some embodiments, the expanded and stimulated NK cells, e.g., those obtained from expansion and stimulation of a single cord blood unit as described above, comprise at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100%, NKp30+ cells.

[0241] In some embodiments, the expanded and stimulated NK cells, e.g., those obtained from expansion and stimulation of a single cord blood unit as described above, contain at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100%, DNAM-1+ cells.

[0242] In some embodiments, the expanded and stimulated NK cells, e.g., those obtained from expansion and stimulation of a single cord blood unit as described above, comprise at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100%, NKp44+ cells.

[0243] In some embodiments, the expanded and stimulated NK cells, e.g., those obtained from expansion and stimulation of a single cord blood unit as described above, comprise at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100%, CD94+(KLRD1) cells.

[0244] In some embodiments, the expanded and stimulated NK cells, e.g., those obtained from expansion and stimulation of a single cord blood unit as described above, contain 20% or less, e.g., 10% or less, 5% or less, 1% or less, or 0% CD3+ cells.

[0245] In some embodiments, the expanded and stimulated NK cells, e.g., those obtained from expansion and stimulation of a single cord blood unit as described above, contain 20% or less, e.g., 10% or less, 5% or less, 1% or less, or 0% CD14+ cells.

[0246] In some embodiments, the expanded and stimulated NK cells, e.g., those obtained from expansion and stimulation of a single cord blood unit as described above, contain 20% or less, e.g., 10% or less, 5% or less, 1% or less, or 0% CD19+ cells.

[0247] In some embodiments, the expanded and stimulated NK cells, e.g., those obtained from expansion and stimulation of a single cord blood unit as described above, contain 20% or less, e.g., 10% or less, 5% or less, 1% or less, or 0% CXCR+ cells.

[0248] In some embodiments, the expanded and stimulated NK cells, e.g., those obtained from expansion and stimulation of a single cord blood unit as described above, contain 20% or less, e.g., 10% or less, 5% or less, 1% or less, or 0% CD122+(IL2RB) cells.

[0249] As described herein, the inventors have demonstrated that NK cells expanded and stimulated by the methods described herein surprisingly express high levels of CD16 throughout the expansion and stimulation process, resulting in a cell population with high CD16 expression. High CD16 expression obviates the need to engineer the expanded cells to express CD16. This is important for initiating ADCC and is therefore a surprising and unexpected advantage of the expansion and stimulation methods described herein. Thus, in some embodiments, expanded and stimulated NK cells, such as those obtained from the expansion and stimulation of, e.g., a single cord blood unit as described above, comprise 50% or more, e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of CD16+ NK cells.

[0250] In some embodiments, the expanded and stimulated NK cells, e.g., those obtained from expansion and stimulation of a single cord blood unit as described above, contain both a KIR B allele of the KIR receptor family and the 158 V / V variant of CD16, and comprise 50% or more, e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, CD16+ NK cells.

[0251] In some embodiments, the percentage of expanded and stimulated NK cells expressing CD16, e.g., those obtained from expansion and stimulation of a single cord blood unit as described above, is the same as or higher than the percentage of natural killer cells in seed cells from cord blood.

[0252] In some embodiments, the percentage of expanded and stimulated NK cells that express NKG2D, e.g., those obtained from expansion and stimulation of a single cord blood unit, e.g., as described above, is the same as or higher than the percentage of natural killer cells in seed cells from cord blood.

[0253] In some embodiments, the percentage of expanded and stimulated NK cells that express NKp30, e.g., those obtained from expansion and stimulation of a single cord blood unit, e.g., as described above, is the same as or higher than the percentage of natural killer cells in seed cells from cord blood.

[0254] In some embodiments, the percentage of expanded and stimulated NK cells expressing DNAM-1, e.g., those obtained from expansion and stimulation of a single cord blood unit as described above, is the same as or higher than the percentage of natural killer cells in seed cells from cord blood.

[0255] In some embodiments, the percentage of expanded and stimulated NK cells that express NKp44, e.g., those obtained from expansion and stimulation of a single cord blood unit as described above, is the same as or higher than the percentage of natural killer cells in seed cells from cord blood.

[0256] In some embodiments, the percentage of expanded and stimulated NK cells that express NKp46, e.g., those obtained from expansion and stimulation of a single cord blood unit as described above, is the same as or higher than the percentage of natural killer cells in seed cells from cord blood.

[0257] As described herein, the inventors have also demonstrated that NK cells expanded and stimulated by the methods described herein surprisingly express low levels of CD38. CD38 is an effective target for certain cancer therapies (e.g., multiple myeloma and acute myeloid leukemia). See, e.g., Jiao et al., "CD38: Targeted Therapy in Multiple Myeloma and Therapeutic Potential for Solid Cancerrs," Expert Opinion on Investigational Drugs 29(11):1295-1308 (2020). Nevertheless, when anti-CD38 antibodies are administered with NK cells, there is a risk of increased fratricide, since NK cells naturally express CD38. However, NK cells expanded and stimulated by the methods described herein express low levels of CD38, thus overcoming the expected fratricide. While other groups have relied on engineering methods such as genome editing to reduce CD38 expression (see, e.g., Gurney et al., "CD38 Knockout Natural Killer Cells Expressing an Affinity Optimized CD38 Chimeric Antigen Receptor Successfully Target Acute Myeloid Leukemia with Reduced Effector Cell Fratricide," Haematologica doi:10.3324 / haematol.2020.271908 (2020)), NK cells expanded and stimulated by the methods described herein express low levels of CD38 without the need for genetic engineering, which provides a surprising and unexpected advantage for treating CD38+ cancers with NK cells expanded and stimulated as described herein, e.g., in combination with a CD38 antibody.

[0258] Thus, in some embodiments, expanded and stimulated NK cells, e.g., those obtained from expansion and stimulation of a single cord blood unit as described above, contain 80% or less CD38+ cells, e.g., 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or 20% or less CD38+ cells.

[0259] In some embodiments, expanded and stimulated NK cells, e.g., those obtained from expansion and stimulation of a single cord blood unit as described above, contain both a KIR B allele of the KIR receptor family and the 158 V / V variant of CD16, and contain 80% or less CD38+ cells, e.g., 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or 20% or less CD38+ cells.

[0260] In some embodiments, the expanded and stimulated NK cells, e.g., those obtained from expansion and stimulation of a single cord blood unit as described above, contain both a KIR B allele of the KIR receptor family and the 158 V / V variant of CD16, and comprise 80% or less CD38+ cells, e.g., 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or 20% or less CD38+ cells, and 50% or more, e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% CD16+ NK cells.

[0261] In some embodiments, expanded and stimulated NK cells, e.g., those obtained from expansion and stimulation of a single umbilical cord blood unit as described above, contain a KIR B allele of the KIR receptor family and a 158 allele of CD16.and V / V variants, and i) 50% or more, e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of CD16+ NK cells, and / or ii) 80% or less, e.g., 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or 20% of CD38+ cells, and / or iii) at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99%. %, or 100% NKG2D+ cells, and / or iv) at least 60%, e.g. at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% NKp46+ cells, and / or v) at least 60%, e.g. at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% NKp30+ cells, and / or vi) at least 60%, e.g. at least 70%, at least 80%, at least 90% , at least 95%, at least 99%, or 100% DNAM-1+ cells, and / or vii) at least 60%, e.g. at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% NKp44+ cells, and / or viii) at least 60%, e.g. at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% CD94+ (KLRD1) cells, and / or ix) no more than 20%, e.g. and / or x) 20% or less, such as 10% or less, 5% or less, 1% or less or 0% CD14+ cells, and / or xi) 20% or less, such as 10% or less, 5% or less, 1% or less or 0% CD19+ cells, and / or xii) 20% or less, such as 10% or less, 5% or less, 1% or less or 0% CXCR+ cells, and / or xiii) 20% or less, such as 10% or less, 5% or less, 1% or less or 0% CD122+ (IL2RB) cells.

[0262] In some embodiments, the feeder cells do not persist in the expanded stimulated NK cells, but a residual signature of the feeder cells can be detected, e.g., by the presence of residual cells (e.g., by detecting cells with specific surface protein expression) or by the presence of residual nucleic acids and / or proteins expressed by the feeder cells.

[0263] For example, in some cases, the methods described herein include expanding and stimulating natural killer cells using engineered feeder cells, such as the eHuT-78 cells described above, engineered to express a sequence not expressed by cells in a natural killer cell source that contains natural killer cells. For example, the engineered feeder cells can be engineered to express at least one gene selected from the group consisting of 4-1BBL (UniProtKB P41273, SEQ ID NO:1), membrane-bound IL-21 (SEQ ID NO:2), and mutant TNF alpha (SEQ ID NO:3), or a variant thereof ("eHut-78 cells").

[0264] Although these feeder cells will not persist in the expanded stimulated NK cells, the expanded stimulated NK cells may retain detectable residual amounts of cells, proteins and / or nucleic acids from the feeder cells, and thus their remaining presence in the expanded stimulated NK cells can be detected, for example, by detecting the cells themselves (e.g., by flow cytometry) or by detecting the proteins they express and / or the nucleic acids they express.

[0265] Thus, also described herein is a population of expanded stimulated NK cells that contains residual feeder cells (live or dead) or cellular impurities of residual feeder cells (e.g., residual feeder cell proteins or portions thereof and / or genetic material, e.g., nucleic acids or portions thereof). Optionally, the expanded stimulated NK cells contain more than 0% but not more than 0.3% residual feeder cells, e.g., eHuT-78 feeder cells.

[0266] Optionally, the expanded stimulated NK cells comprise residual feeder cell nucleic acid, such as that encoding residual 4-1BBL (UniProtKB P41273, SEQ ID NO:1), membrane bound IL-21 (SEQ ID NO:2) and / or mutant TNF alpha (SEQ ID NO:3), or a portion thereof. Optionally, the membrane bound IL-21 comprises the CD8 transmembrane domain.

[0267] Optionally, the expanded stimulated NK cells contain greater than 0% and less than or equal to 0.2% % remaining feeder cells, e.g., as measured by the relative proportion of feeder cell-specific proteins or nucleic acid sequences (i.e., proteins or nucleic acid sequences not expressed by natural killer cells) in the sample, e.g., by qPCR, e.g., as described herein.

[0268] In some embodiments, the remaining feeder cells are CD4(+) T cells. In some embodiments, the remaining feeder cells are engineered CD4(+) T cells. In some embodiments, the remaining feeder cells are engineered to express at least one gene or variant thereof selected from the group consisting of 4-1BBL (UniProtKB P41273, SEQ ID NO:1), membrane-bound IL-21 (SEQ ID NO:2), and mutant TNF alpha (SEQ ID NO:3) ("eHut-78 cells"). Thus, optionally, the feeder cell-specific protein is 4-1BBL (UniProtKB P41273, SEQ ID NO:1), membrane-bound IL-21 (SEQ ID NO:2), and / or mutant TNF alpha (SEQ ID NO:3). And thus, the feeder cell-specific nucleic acid is a nucleic acid encoding 4-1BBL (UniProtKB P41273, SEQ ID NO:1), membrane-bound IL-21 (SEQ ID NO:2), and / or mutant TNF alpha (SEQ ID NO:3), or a portion thereof. Optionally, the membrane-bound IL-21 comprises the CD8 transmembrane domain.

[0269] A wide variety of methods can be used to analyze and detect the presence of nucleic acid or protein gene products in biological samples. As used herein, "detection" can refer to a method used to discover, determine, or confirm the presence or existence of a compound and / or substance (e.g., a cell, a protein, and / or a nucleic acid). In some embodiments, the detection method can be used to detect proteins. In some embodiments, detection can include chemiluminescence or fluorescence techniques. In some embodiments, detection can include immunological methods (e.g., quantitative enzyme-linked immunosorbent assay (ELISA), Western blotting, or dot blotting) that use antibodies to specifically react with whole proteins or specific epitopes on proteins. In some embodiments, detection can include immunoprecipitation of proteins (Jungblut et al., J Biotechnol. 31; 41(2-3): 111-20 (1995); Franco et al., Eur J Morphol. 39(1): 3-25 (2001)). In some embodiments, the detection method can be used to detect nucleic acids (e.g., DNA and / or RNA). In some embodiments, detection can include Northern blot analysis, nuclease protection assay (NPA), in situ hybridization, or reverse transcription-polymerase chain reaction (RT-PCR) (Raj et al., Nat. Methods 5, 877-879 (2008); Jin et al., J Clin Lab Anal. 11(1):2-9 (1997); Ahmed, J Environ Sci Health C Environ Carcinog Ecotoxicol Rev. 20(2):77-116 (2002)).

[0270] Accordingly, also described herein are methods for detecting a population of NK cells that have been co-cultured with engineered feeder cells, e.g., eHuT-78 feeder cells described herein, and expanded and stimulated, e.g., expanded and stimulated using the methods described herein.

[0271] II. Cryopreservation A. Freeze preservation composition Provided herein are cryopreservation compositions, e.g., cryopreservation compositions suitable for intravenous administration, e.g., of NK cells, e.g., NK cells described herein. In some embodiments, a pharmaceutical composition comprises the cryopreservation composition and cells, e.g., NK cells described herein.

[0272] 1. Albumin In some embodiments, the cryopreservation composition comprises an albumin protein, such as human albumin protein (UniProtKB Accession P0278, SEQ ID NO:5) or a variant thereof. In some embodiments, the cryopreservation composition comprises an albumin protein, such as an orthologue of human albumin protein, or a variant thereof. In some embodiments, the cryopreservation composition comprises an albumin protein, such as a biologically active portion of human albumin, or a variant thereof.

[0273] In some embodiments, albumin, for example human albumin, is provided as a solution, which is also referred to herein as albumin solution or human albumin solution.Thus, in some embodiments, the cryopreservation composition is or comprises an albumin solution, for example a human albumin solution.In some embodiments, the albumin solution is a serum-free albumin solution.

[0274] In some embodiments, the albumin solution is suitable for intravenous use.

[0275] In some embodiments, the albumin solution comprises at or about 40 to at or about 200 g / L of albumin. In some embodiments, the albumin solution comprises at or about 40 to at or about 50 g / L of albumin, e.g., human albumin. In some embodiments, the albumin solution comprises at or about 200 g / L of albumin, e.g., human albumin. In some embodiments, the albumin solution comprises at or about 200 g / L of albumin, e.g., human albumin.

[0276] In some embodiments, the albumin solution comprises a protein composition, of which 95% or more is albumin protein, e.g., human albumin protein, in some embodiments, 96%, 97%, 98%, or 99% or more of the protein is albumin, e.g., human albumin.

[0277] In some embodiments, the albumin solution further comprises sodium. In some embodiments, the albumin solution comprises from at or about 100 to at or about 200 mmol of sodium. In some embodiments, the albumin solution comprises from at or about 130 to at or about 160 mmol of sodium.

[0278] In some embodiments, the albumin solution further comprises potassium. In some embodiments, the albumin solution further comprises 3 mmol or less of potassium. In some embodiments, the albumin solution further comprises 2 mmol or less of potassium.

[0279] In some embodiments, the albumin solution further comprises one or more stabilizers. In some embodiments, the stabilizers are selected from the group consisting of sodium caprylate, caprylic acid, (2S)-2-acetamido-3-(1H-indol-3-yl)propanoic acid (also referred to as acetyltryptophan, N-acetyl-L-tryptophan and acetyl-L-tryptophan), 2-acetamido-3-(1H-indol-3-yl)propanoic acid (also referred to as N-acetyltryptophan, DL-acetyltroptohan and N-acetyl-DL-tryptophan). In some embodiments, the solution comprises less than 0.1 mmol of each of the one or more stabilizers per gram of protein in the solution. In some embodiments, the solution comprises from 0.05 or about 0.05 to 0.1 or about 0.1, for example 0.064 or about 0.064 to 0.096 or about 0.096 mmol of each stabilizer per gram of protein in the solution. In some embodiments, the solution comprises less than 0.1 mmol of total stabilizer per gram of protein in the solution, in some embodiments, the solution comprises from 0.05 or about 0.05 to 0.1 or about 0.1, such as 0.064 or about 0.064 to 0.096 or about 0.096 mmol of total stabilizer per gram of protein in the solution.

[0280] In some embodiments, the albumin solution consists of a protein composition that is 95% or more albumin protein, sodium, potassium, and one or more stabilizers selected from the group consisting of sodium caprylate, caprylic acid, (2S)-2-acetamido-3-(1H-indol-3-yl)propanoic acid (also referred to as acetyltryptophan, N-acetyl-L-tryptophan, and acetyl-L-tryptophan), 2-acetamido-3-(1H-indol-3-yl)propanoic acid (also referred to as N-acetyltryptophan, DL-acetyltroptohan, and N-acetyl-DL-tryptophan), in water.

[0281] In some embodiments, the cryopreservation composition comprises from at or about 10% v / v to at or about 50% v / v albumin solution, such as an albumin solution described herein.In some embodiments, the cryopreserved composition is from 10% or about 10% to 50% or about 50%, from 10% or about 10% to 45% or about 45%, from 10% or about 10% to 40% or about 40%, from 10% or about 10% to 35% or about 35%, from 10% or about 10% to 30% or about 30%, from 10% or about 10% to 25% or about 25%, from 10% or about 10% to 20% or about 20%, from 10% or about 10% to 15% or about 15%, from 15% or about 15%, %, 50% or about 50%, 15% or about 15% to 45% or about 45%, 15% or about 15% to 40% or about 40%, 15% or about 15% to 35% or about 35%, 15% or about 15% to 30% or about 30%, 15% or about 15% to 25% or about 25%, 15% or about 15% to 20% or about 20%, 20% or about 20% to 50% or about 50%, 20% or about 20% to 45% or about 45%, 20% or about 20% to 40 % or about 40%, from 20% or about 20%, 35% or about 35%, from 20% or about 20%, from 30% or about 30%, from 20% or about 20%, from 25% or about 25%, from 25% or about 25%, from 50% or about 50%, from 25% or about 25%, from 45% or about 45%, from 25% or about 25%, from 40% or about 40%, from 25% or about 25%, from 35% or about 35%, from 25% or about 25%, from 30% or about 30%, from 30% or about 30% to 50% or about The albumin solution may comprise from 50%, 30% or about 30%, 45% or about 45%, 30% or about 30%, 40% or about 40%, 30% or about 30%, 35% or about 35%, 35% or about 35%, 50% or about 50%, 35% or about 35%, 45% or about 45%, 35% or about 35%, 40% or about 40%, 40% or about 40%, 50% or about 50%, 40% or about 40%, 45% or about 45%, or 45% or about 45% to 50% or about 50% v / v of an albumin solution as described herein.In some embodiments, the cryopreservation composition comprises about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% v / v of an albumin solution described herein.In some embodiments, the cryopreservation composition comprises 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% v / v of an albumin solution described herein.

[0282] In some embodiments, the cryopreservation composition comprises from 20 or about 20 to 100 or about 100 g / L of albumin, e.g., human albumin. In some embodiments, the cryopreservation composition comprises from 20 or about 20 to 100 or about 100, 20 or about 20 to 90 or about 90, 20 or about 20 to 80 or about 80, 20 or about 20 to 70 or about 70, 20 or about 20 to 60 or about 60, 20 or about 20 to 50 or about 50, 20 or about 20 to 40 or about 40, 20 or about 20 to 30 or about 30, 30 or about 30 to 10, 0 or about 100, 30 or about 30 to, 90 or about 90, 30 or about 30 to, 80 or about 80, 30 or about 30 to, 70 or about 70, 30 or about 30 to, 60 or about 60, 30 or about 30 to, 50 or about 50, 30 or about 30 to, 40 or about 40, 40 or about 40 to, 100 or about 100, 40 or about 40 to, 90 or about 90, 40 or about 40 to, 80 or about 80, 40 or about 50 or from about 40, 70 or about 70, 40 or about 40, 60 or about 60, 40 or about 40, 50 or about 50, 50 or about 50 to, 100 or about 100, 50 or about 50 to, 90 or about 90, 50 or about 50 to, 80 or about 80, 50 or about 50 to, 70 or about 70, 50 or about 50 to, 60 or about 60, 60 or about 60 to, 100 or about 100, 60 or about 60 to, 90 or about or from about 90, 60 or about 60, 80 or about 80, 60 or about 60, 70 or about 70, 70 or about 70, 100 or about 100, 70 or about 70, 90 or about 90, 70 or about 70, 80 or about 80, 80 or about 80, 100 or about 100, 80 or about 80, 90 or about 90, or 90 or about 90, 100 or about 100 g / L of albumin, e.g., human albumin.

[0283] In some embodiments, the cryopreservation composition comprises 20 g / L of albumin, e.g., human albumin. In some embodiments, the cryopreservation composition comprises 40 g / L of albumin, e.g., human albumin. In some embodiments, the cryopreservation composition comprises 70 g / L of albumin, e.g., human albumin. In some embodiments, the cryopreservation composition comprises 100 g / L of albumin, e.g., human albumin.

[0284] In some embodiments, the cryopreservation composition comprises about 20 g / L of albumin, e.g., human albumin. In some embodiments, the cryopreservation composition comprises about 40 g / L of albumin, e.g., human albumin. In some embodiments, the cryopreservation composition comprises about 70 g / L of albumin, e.g., human albumin. In some embodiments, the cryopreservation composition comprises about 100 g / L of albumin, e.g., human albumin.

[0285] In some embodiments, the cryopreservation composition further comprises a stabilizer, for example, an albumin stabilizer. In some embodiments, the stabilizer is selected from the group consisting of sodium caprylate, caprylic acid, (2S)-2-acetamido-3-(1H-indol-3-yl)propanoic acid (also called acetyltryptophan, N-acetyl-L-tryptophan and acetyl-L-tryptophan), 2-acetamido-3-(1H-indol-3-yl)propanoic acid (also called N-acetyltryptophan, DL-acetyltroptohan and N-acetyl-DL-tryptophan). In some embodiments, the cryopreservation composition comprises less than 0.1 mmol of each of one or more stabilizers per gram of protein, for example per gram of albumin protein, in the composition. In some embodiments, the cryopreservation composition comprises 0.05 or about 0.05 to 0.1 or about 0.1, for example 0.064 or about 0.064 to 0.096 or about 0.096mmol of each stabilizer per gram of protein, for example per gram of albumin protein, in the composition. In some embodiments, the cryopreservation composition comprises less than 0.1mmol of total stabilizer per gram of protein, for example per gram of albumin protein, in the cryopreservation composition. In some embodiments, the cryopreservation composition comprises 0.05 or about 0.05 to 0.1 or about 0.1, for example 0.064 or about 0.064 to 0.096 or about 0.096mmol of total stabilizer per gram of protein, for example per gram of albumin protein, in the cryopreservation composition.

[0286] 2. Dextran In some embodiments, the cryopreservation composition comprises dextran or a derivative thereof.

[0287] Dextran is a polymer of anhydroglucose ((CH)) composed of approximately 95% α-D-(1-6) bonds. 10 O5) nDextran fractions are supplied in molecular weights ranging from about 1,000 Daltons to about 2,000,000 Daltons. They are designated numerically (Dextran X), e.g., Dextran 1, Dextran 10, Dextran 40, Dextran 70, etc., where X corresponds to the average molecular weight divided by 1,000 Daltons. Thus, for example, Dextran 40 has an average molecular weight of 40,000 or about 40,000 Daltons.

[0288] In some embodiments, the average molecular weight of the dextran is at or about 1,000 Daltons to at or about 2,000,000 Daltons. In some embodiments, the average molecular weight of the dextran is at or about 40,000 Daltons. In some embodiments, the average molecular weight of the dextran is at or about 70,000 Daltons.

[0289] In some embodiments, the dextran is selected from the group consisting of dextran 40, dextran 70, and combinations thereof. In some embodiments, the dextran is dextran 40.

[0290] In some embodiments, the dextran, e.g., Dextran 40, is provided in a solution, also referred to herein as a dextran solution or a Dextran 40 solution. Thus, in some embodiments, the composition comprises a dextran solution, e.g., a Dextran 40 solution.

[0291] In some embodiments, the dextran solution is suitable for intravenous use.

[0292] In some embodiments, the dextran solution comprises about 5% to about 50% w / w dextran, e.g., dextran 40. In some embodiments, the dextran solution comprises from 5% or about 5%, 50% or about 50%, 5% or about 5% to 45% or about 45%, 5% or about 5% to 40% or about 40%, 5% or about 5% to 35% or about 35%, 5% or about 5% to 30% or about 30%, 5% or about 5% to 25% or about 25%, 5% or about 5% to 20% or about 20%, 5% or about 5% to 15% or about 15%, 5% or about 5% to 10% or about 10%, From 0%, 10% or about 10%, to 50% or about 50%, from 10% or about 10%, to 45% or about 45%, from 10% or about 10%, to 40% or about 40%, from 10% or about 10%, to 35% or about 35%, from 10% or about 10%, to 30% or about 30%, from 10% or about 10%, to 25% or about 25%, from 10% or about 10%, to 20% or about 20%, from 10% or about 10%, to 15% or about 15%, from 15% or about 15%, to 50% or about 50 %, from 15% or about 15%, to 45% or about 45%, from 15% or about 15%, to 40% or about 40%, from 15% or about 15%, to 35% or about 35%, from 15% or about 15%, to 30% or about 30%, from 15% or about 15%, to 25% or about 25%, from 15% or about 15%, to 20% or about 20%, from 20% or about 20%, to 50% or about 50%, from 20% or about 20%, to 45% or about 45%, from 20% or about 20% to 40% or about 40% , from 20% or about 20%, 35% or about 35%, from 20% or about 20%, 30% or about 30%, from 20% or about 20%, 25% or about 25%, from 25% or about 25%, from 50% or about 50%, from 25% or about 25%, 45% or about 45%, from 25% or about 25%, from 40% or about 40%, from 25% or about 25%, 35% or about 35%, from 25% or about 25%, from 30% or about 30%, from 30% or about 30% to 50% or about 50%,30% or about 30%, 45% or about 45%, 30% or about 30%, 40% or about 40%, 30% or about 30%, 35% or about 35%, 35% or about 35%, 50% or about 50%, 35% or about 35%, 45% or about 45%, 35% or about 35%, 40% or about 40%, 40% or about 40%, 50% or about 50%, 40% or about 40%, 45% or about 45%, or 45% or about 45%, 50% or about 50% w / w dextran, e.g., dextran 40. In some embodiments, the dextran solution comprises 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% w / w dextran, such as dextran 40. In some embodiments, the dextran solution comprises about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% w / w dextran, such as dextran 40.

[0293] In some embodiments, the dextran solution comprises from 25 or about 25 g / L to 200 or about 200 g / L of dextran, such as dextran 40. In some embodiments, the dextran solution comprises from 35 or about 35 to 200 or about 200, 25 or about 25 to 175 or about 175, 25 or about 25 to 150 or about 150, 25 or about 25 to 125 or about 125, 25 or about 25 to 100 or about 100, 25 or about 25 to 75 or about 75, 25 or about 25 to 50 or about 50, From 0, 50 or about 50, 200 or about 200, 50 or about 50, 175 or about 175, 50 or about 50, 150 or about 150, 50 or about 50, 125 or about 125, 50 or about 50, 100 or about 100, 50 or about 50, 75 or about 75, 75 or about 75, 200 or about 200, 75 or about 75, or 175 or from about 175, 75 or about 75, 150 or about 150, 75 or about 75, 125 or about 125, 75 or about 75, 100 or about 100, 100 or about 100, 200 or about 200, 100 or about 100, 175 or about 175, 100 or about 100, 150 or about 150, 100 or about 100, 125 or about 125, The dextran solution comprises from 125 or about 125 to 200 or about 200, from 125 or about 125 to 175 or about 175, from 125 or about 125 to 150 or about 150, from 150 or about 150 to 200 or about 200, from 150 or about 150 to 175 or about 175, or from 175 or about 175 to 200 or about 200 g / L of dextran, such as dextran 40. In some embodiments, the dextran solution comprises 25, 50, 75, 100, 125, 150, 175, or 200 g / L of dextran, such as dextran 40. In some embodiments, the dextran solution comprises 100 g / L of dextran, such as dextran 40.In some embodiments, the dextran solution comprises about 25, about 50, about 75, about 100, about 125, about 150, about 175, or about 200 g / L of dextran, such as dextran 40. In some embodiments, the dextran solution comprises about 100 g / L of dextran, such as dextran 40.

[0294] In some embodiments, the dextran solution further comprises glucose (also called dextrose). In some embodiments, the dextran solution comprises from 10 or about 10 g / L to 100 or about 100 g / L of glucose. In some embodiments, the dextran solution comprises from 10 or about 10 to 100 or about 100, from 10 or about 10 to 90 or about 90, from 10 or about 10 to 80 or about 80, from 10 or about 10 to 70 or about 70, from 10 or about 10 to 60 or about 60, from 10 or about 10 to 50 or about 50, from 10 or about 10 to 40 or about 40, from 10 or about 10 to 30 or about 30, from 10 or about 10 to 20 or about 20, from ... From 0 or about 20, 100 or about 100, from 20 or about 20, 90 or about 90, from 20 or about 20, 80 or about 80, from 20 or about 20, 70 or about 70, from 20 or about 20, 60 or about 60, from 20 or about 20, 50 or about 50, from 20 or about 20, 40 or about 40, from 20 or about 20, 30 or about 30, from 30 or about 30, 100 or about 100, from 30 or about 30, 90 or about 90, From 30 or about 30, 80 or about 80, from 30 or about 30, 70 or about 70, from 30 or about 30, 60 or about 60, from 30 or about 30, 50 or about 50, from 30 or about 30, 40 or about 40, from 40 or about 40, from 100 or about 100, from 40 or about 40, 90 or about 90, from 40 or about 40, 80 or about 80, from 40 or about 40, 70 or about 70, from 40 or about 40, 60 or about 60, From 0 or about 40, from 50 or about 50, from 50 or about 50, from 100 or about 100, from 50 or about 50, from 90 or about 90, from 50 or about 50, from 80 or about 80, from 50 or about 50, from 70 or about 70, from 50 or about 50, from 60 or about 60, from 60 or about 60, from 100 or about 100, from 60 or about 60, from 90 or about 90, from 60 or about 60, from 80 or about 80, from 60 or about 60, from 70 or about 70,The dextran solution comprises 70 or about 70 to 100 or about 100, 70 or about 70 to 90 or about 90, 70 or about 70 to 80 or about 80, 80 or about 80 to 90 or about 90, 80 or about 80 to 100 or about 100, 80 or about 80 to 90 or about 90, or 90 or about 90 to 100 or about 100 g / L of glucose. In some embodiments, the dextran solution comprises 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 g / L of glucose. In some embodiments, the dextran solution comprises 50 g / L of glucose. In some embodiments, the dextran solution comprises about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100 g / L of glucose. In some embodiments, the dextran solution contains 50 g / L glucose.

[0295] In some embodiments, the dextran solution consists of dextran, such as dextran 40, and glucose in water.

[0296] In some embodiments, the cryopreservation composition comprises from at or about 10% v / v to at or about 50% v / v of a dextran solution described herein.In some embodiments, the cryopreserved composition is from 10% or about 10% to 50%, 10% or about 10% to 45% or about 45%, 10% or about 10% to 40% or about 40%, 10% or about 10% to 35% or about 35%, 10% or about 10% to 30% or about 30%, 10% or about 10% to 25% or about 25%, 10% or about 10% to 20% or about 20%, 10% or about 10% to 15% or about 15%, 15% or about 15% to 50%, % or about 50%, from 15% or about 15%, 45% or about 45%, from 15% or about 15%, 40% or about 40%, from 15% or about 15%, 35% or about 35%, from 15% or about 15%, 30% or about 30%, from 15% or about 15%, 25% or about 25%, from 15% or about 15%, 20% or about 20%, from 20% or about 20%, 50% or about 50%, from 20% or about 20%, 45% or about 45%, from 20% or about 20% to 40% or is from about 40%, 20% or about 20%, 35% or about 35%, 20% or about 20%, 30% or about 30%, 20% or about 20%, 25% or about 25%, 25% or about 25%, 50% or about 50%, 25% or about 25%, 45% or about 45%, 25% or about 25%, 40% or about 40%, 25% or about 25%, 35% or about 35%, 25% or about 25%, 30% or about 30%, 30% or about 30%, 50% or about 50% %, 30% or about 30%, 45% or about 45%, 30% or about 30%, 40% or about 40%, 30% or about 30%, 35% or about 35%, 35% or about 35%, 50% or about 50%, 35% or about 35%, 45% or about 45%, 35% or about 35%, 40% or about 40%, 40% or about 40%, 50% or about 50%, 40% or about 40%, 45% or about 45%, or 45% or about 45%, 50% or about 50% v / v dextran solution, such as a dextran solution described herein.In some embodiments, the cryopreservation composition comprises a 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% v / v dextran solution, such as a dextran solution described herein. In some embodiments, the cryopreservation composition comprises about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% v / v dextran solution, such as a dextran solution described herein.

[0297] In some embodiments, the cryopreservation composition comprises from 10 or about 10 to 50 or about 50 g / L of dextran, such as dextran 40. In some embodiments, the cryopreservation composition comprises from 10 or about 10 to 50 or about 50, from 10 or about 10 to 45 or about 45, from 10 or about 10 to 40 or about 40, from 10 or about 10 to 35 or about 35, from 10 or about 10 to 30 or about 30, from 10 or about 10 to 25 or about 25, from 10 or about 10 to 20 or about 20, from 10 or about 10 to 15 or about 15, from 15 or about From 15, 50 or about 50, from 15 or about 15, 45 or about 45, from 15 or about 15, 40 or about 40, from 15 or about 15, 35 or about 35, from 15 or about 15, 30 or about 30, from 15 or about 15, 25 or about 25, from 15 or about 15, from 20 or about 20, from 20 or about 20, from 50 or about 50, from 20 or about 20, from 45 or about 45, from 20 or about 20, or about 40, 20 or about 20 to, 30 or about 30, 20 or about 20 to, 25 or about 25, 25 or about 25 to, 50 or about 50, 25 or about 25 to, 45 or about 45, 25 or about 25 to, 40 or about 40, 25 or about 25 to, 35 or about 35, 25 or about 25 to, 30 or about 30, 30 or about 30 to, 50 or about 50, 30 or about 30 to, 45 or about 45 , 30 or about 30, 40 or about 40, 30 or about 30, 35 or about 35, 35 or about 35, 50 or about 50, 35 or about 35, 45 or about 45, 35 or about 35, 40 or about 40, 40 or about 40, 50 or about 50, 40 or about 40, 45 or about 45, or 45 or about 45, 50 or about 50 g / L of dextran, e.g., dextran 40. In some embodiments, the cryopreservation composition comprises 10, 15, 20, 25, 30, 30, 35, 40, 45, or 50 g / L of dextran, e.g., dextran 40.In some embodiments, the cryopreservation composition comprises about 10, about 15, about 20, about 25, about 30, about 30, about 35, about 40, about 45, or about 50 g / L of dextran, e.g., dextran 40.

[0298] 3. Glucose In some embodiments, the cryopreservation composition comprises glucose.

[0299] In some aspects, the cryopreservation composition comprises a dextran solution that includes glucose, as described above.

[0300] In some embodiments, the cryopreservation composition comprises a dextran solution that does not contain glucose. In some embodiments, glucose is added separately to the cryopreservation composition, e.g., when the dextran solution does not contain glucose.

[0301] In some embodiments, the cryopreservation composition comprises 5 or about 5 to 25 or about 25 g / L of glucose. In some embodiments, the cryopreservation composition comprises 5 or about 5 to 25 or about 25, 5 or about 5 to 20 or about 20, 5 or about 5 to 15 or about 15, 5 or about 5 to 10 or about 10, 10 or about 10 to 25 or about 25, 10 or about 10 to 20 or about 20, 10 or about 10 to 15 or about 15, 15 or about 15 to 25 or about 25, 15 or about 15 to 20 or about 20, or 20 or about 20 to 25 or about 25 g / L of glucose. In some embodiments, the cryopreservation composition comprises 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5, or 25 g / L of glucose. In some embodiments, the cryopreservation composition comprises 12.5 g / L of glucose. In some embodiments, the cryopreservation composition comprises about 5, about 7.5, about 10, about 12.5, about 15, about 17.5, about 20, about 22.5, or about 25 g / L of glucose. In some embodiments, the cryopreservation composition comprises about 12.5 g / L of glucose.

[0302] In some embodiments, the cryopreservation composition comprises less than 2.75% w / v glucose. In some embodiments, the cryopreservation composition comprises less than 27.5 g / L glucose. In some embodiments, the cryopreservation composition comprises less than 2% w / v glucose. In some embodiments, the cryopreservation composition comprises less than 1.5% w / v glucose. In some embodiments, the cryopreservation composition comprises about 1.25% w / v glucose or less.

[0303] 4. Dimethyl sulfoxide In some embodiments, the cryopreservation composition comprises dimethyl sulfoxide (DMSO, also known as methyl sulfoxide and methylsulfinylmethane).

[0304] In some embodiments, DMSO is provided as a solution, also referred to herein as a DMSO solution. Thus, in some embodiments, the cryopreservation composition comprises a DMSO solution.

[0305] In some embodiments, the DMSO solution is suitable for intravenous use.

[0306] In some embodiments, the DMSO solution comprises 1.1 g / mL of DMSO. In some embodiments, the DMSO solution comprises about 1.1 g / mL of DMSO.

[0307] In some embodiments, the cryopreservation composition comprises 1% or about 1% to 10% or about 10% v / v DMSO solution. In some embodiments, the cryopreservation composition comprises 1% or about 1% to 10% or about 10%, 1% or about 1% to 9% or about 9%, 1% or about 1% to 8% or about 8%, 1% or about 1% to 7% or about 7%, 1% or about 1% to 6% or about 6%, 1% or about 1% to 5% or about 5%, 1% or about 1% to 4% or about 4%, 1% or about 1% to 3% or about 3%, 1% or about 1% to 2% or about 2%, 2% or about 2% to 1%, 0% or about 10%, 2% or about 2% to 9% or about 9%, 8% or about 8%, 2% or about 2% to 7% or about 7%, 2% or about 2% to 6% or about 6%, 2% or about 2% to 5% or about 5%, 2% or about 2% to 4% or about 4%, 2% or about 2% to 3% or about 3%, 3% or about 3% to 10% or about 10%, 3% or about 3% to 9% or about 9%, 3% or about 3% to 8% or about 8%, 3% or about 3% to 7%, % or about 7%, 3% or about 3% to 6% or about 6%, 3% or about 3% to 5% or about 5%, 3% or about 3% to 4%, 4% or about 4% to 10% or about 10%, 4% or about 4% to 9% or about 9%, 4% or about 4% to 8% or about 8%, 4% or about 4% to 7% or about 7%, 4% or about 4% to 6% or about 6%, 4% or about 4% to 5% or about 5%, 5% or about 5% to 10% or about 10%, 5 % or about 5%, 9% or about 9%, 5% or about 5%, 8% or about 8%, 5% or about 5%, 7% or about 7%, 5% or about 5%, 6% or about 6%, 6% or about 6%, 10% or about 10%, 6% or about 6%, 9% or about 9%, 6% or about 6%, 8% or about 8%, 6% or about 6%, 7% or about 7%, 7% or about 7%, 10% or about 10%, 7% or about 7%, 9% or about 9%, 7% or about 7%,8% or about 8%, 8% or about 8% to 10% or about 10%, 8% or about 8% to 9% or about 9%, or 9% or about 9% to 10% or about 10% v / v DMSO solution. In some embodiments, the cryopreservation composition comprises 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% v / v DMSO solution. In some embodiments, the cryopreservation composition comprises a 5% DMSO solution. In some embodiments, the cryopreservation composition comprises about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or 10% v / v DMSO solution. In some embodiments, the cryopreservation composition comprises about 5% DMSO solution.

[0308] In some embodiments, the cryopreservation composition comprises 11 or about 11 to 110 or about 110 g / L DMSO. In some embodiments, the cryopreservation composition comprises 11 or about 11 to 110 or about 110, 11 or about 11 to 99 or about 99, 11 or about 11 to 88 or about 88, 11 or about 11 to 77 or about 77, 11 or about 11 to 66 or about 66, 11 or about 11 to 55 or about 55, 11 or about 11 to 44 or about 44, 11 or about 11 to 33 or about 33, 11 or about 11 to 22 or about 22, 22 or about 22 to 1, 10 or about 110, from 22 or about 22, 99 or about 99, from 22 or about 22, 88 or about 88, from 22 or about 22, 77 or about 77, from 22 or about 22, 77 or about 77, from 22 or about 22, 66 or about 66, from 22 or about 22, 55 or about 55, from 22 or about 22, 44 or about 44, from 22 or about 22, 33 or about 33, from 33 or about 33, 110 or about 110, from 33 or about 33, 99 or about 99, 3 3 or about 33, 88 or about 88, 33 or about 33, 77 or about 77, 33 or about 33, 66 or about 66, 33 or about 33, 55 or about 55, 33 or about 33, 44 or about 44, 44 or about 44, 110 or about 110, 44 or about 44, 99 or about 99, 44 or about 44, 88 or about 88, 44 or about 44, 77 or about 77, 44 or about 44, 66 or about 66, 44 or about 44, 5 5 or about 55, 55 or about 55 to, 110 or about 110, 55 or about 55 to, 99 or about 99, 55 or about 55 to, 88 or about 88, 55 or about 55 to, 77 or about 77, 55 or about 55 to, 66 or about 66, 66 or about 66 to, 110 or about 110, 66 or about 66 to, 99 or about 99, 66 or about 66 to, 88 or about 88, 66 or about 66 to, 77 or about 77, 77 or about 77 to, 119 or about 119,The cryopreservation composition comprises 77 or about 77, 88 or about 88, 88 or about 88, 110 or about 110, 88 or about 88, 99 or about 99, or 99 or about 99, 110 or about 110 g / L of DMSO. In some embodiments, the cryopreservation composition comprises 11, 22, 33, 44, 55, 66, 77, 88, 99, or 110 g / L of DMSO. In some embodiments, the cryopreservation composition comprises 55 g / L of DMSO. In some embodiments, the cryopreservation composition comprises about 11, about 22, about 33, about 44, about 55, about 66, about 77, about 88, about 99, or about 110 g / L of DMSO. In some embodiments, the cryopreservation composition comprises about 55 g / L of DMSO.

[0309] 5. Buffer In some embodiments, the cryopreservation composition comprises a buffer solution, for example a buffer solution suitable for intravenous administration.

[0310] Buffer solutions include, but are not limited to, phosphate buffered saline (PBS), Ringer's solution, Tyrode's buffer, Hank's balanced salt solution, Earle's balanced salt solution, saline, and Tris.

[0311] In some embodiments, the buffer solution is phosphate buffered saline (PBS).

[0312] 6. Exemplary Cryopreservation Compositions In some embodiments, the cryopreservation composition comprises or consists of 1) albumin, such as human albumin, 2) dextran, such as dextran 40, 3) DMSO, and 4) a buffer solution. In some embodiments, the cryopreservation composition further comprises glucose. In some embodiments, the cryopreservation composition consists of 1) albumin, such as human albumin, 2) dextran, such as dextran 40, 3) glucose, 4) DMSO, and 5) a buffer solution.

[0313] In some embodiments, the cryopreservation composition comprises 1) an albumin solution as described herein, 2) a dextran solution as described herein, 3) a DMSO solution as described herein, and 4) a buffer solution.

[0314] In some embodiments, the cryopreservation composition consists of 1) an albumin solution as described herein, 2) a dextran solution as described herein, 3) a DMSO solution as described herein, and 4) a buffer solution.

[0315] In some embodiments, the cryopreservation composition does not comprise cell culture medium.

[0316] In one embodiment, the cryopreservation composition comprises 40 mg / mL human albumin, 25 mg / mL dextran 40, 12.5 mg / mL glucose, and 55 mg / mL DMSO, or comprises about 40 mg / mL human albumin, 25 mg / mL dextran 40, 12.5 mg / mL glucose, and 55 mg / mL DMSO.

[0317] In one embodiment, the cryopreservation composition comprises 40 mg / mL human albumin, 25 mg / mL dextran 40, 12.5 mg / mL glucose, 55 mg / mL DMSO and 0.5 mL / mL of 100% phosphate buffered saline (PBS) in water or about 40 mg / mL human albumin, 25 mg / mL dextran 40, 12.5 mg / mL glucose, 55 mg / mL DMSO and 0.5 mL / mL of 100% phosphate buffered saline (PBS). PBS) or consisting of 40 mg / mL human albumin, 25 mg / mL dextran 40, 12.5 mg / mL glucose, 55 mg / mL DMSO and 0.5 mL / mL of 100% phosphate buffered saline (PBS), or consisting of about 40 mg / mL human albumin, 25 mg / mL dextran 40, 12.5 mg / mL glucose, 55 mg / mL DMSO and 0.5 mL / mL of 100% phosphate buffered saline (PBS).

[0318] In one embodiment, the cryopreservation composition comprises 32 mg / mL human albumin, 25 mg / mL dextran 40, 12.5 mg / mL glucose, and 55 mg / mL DMSO, or comprises about 32 mg / mL human albumin, 25 mg / mL dextran 40, 12.5 mg / mL glucose, and 55 mg / mL DMSO.

[0319] In one embodiment, the cryopreservation composition comprises or consists of 32 mg / mL human albumin, 25 mg / mL dextran 40, 12.5 mg / mL glucose, 55 mg / mL DMSO and 0.54 mL / mL 100% phosphate buffered saline (PBS) or about 32 mg / mL human albumin, 25 mg / mL dextran 40, 12.5 mg / mL glucose, 55 mg / mL DMSO and 0.54 mL / mL 100% phosphate buffered saline (PBS) in water.

[0320] Exemplary cryopreservation compositions are shown in Table 3.

[0321] Table 3. Exemplary cryopreservation compositions TIFF2024542159000004.tif56156

[0322] (Table 4) First cryopreservation composition example TIFF2024542159000005.tif59156

[0323] (Table 5) Second cryopreservation composition example TIFF2024542159000006.tif59156

[0324] B. Freezing preservation method The cryopreservation compositions described herein can be used to cryopreserve cells, e.g., therapeutic cells, e.g., natural killer (NK) cells, e.g., NK cells described herein.

[0325] In some embodiments, the cell is an animal cell. In some embodiments, the cell is a human cell.

[0326] In some embodiments, the cell is an immune cell. In some embodiments, the immune cell is selected from a basophil, an eosinophil, a neutrophil, a mast cell, a monocyte, a macrophage, a neutrophil, a dendritic cell, a natural killer cell, a B cell, a T cell, and combinations thereof.

[0327] In some embodiments, the immune cells are natural killer (NK) cells. In some embodiments, the natural killer cells are expanded and stimulated by the methods described herein.

[0328] In some embodiments, the step of cryopreserving cells comprises mixing the cells with a cryopreservation composition described herein or a component thereof to produce a composition, e.g., a pharmaceutical composition, and freezing the mixture.

[0329] In some embodiments, the step of cryopreserving the cells comprises mixing a composition comprising the cells with a cryopreservation composition or a component thereof described herein to produce a composition, e.g., a pharmaceutical composition, and freezing the mixture. In some embodiments, the composition comprising the cells comprises the cells and a buffer. Suitable buffers are described herein.

[0330] In some embodiments, the step of cryopreserving the cells comprises mixing a composition comprising the cells and a buffer, e.g., PBS, with a composition comprising albumin, dextran and DMSO, and freezing the mixture.

[0331] In some embodiments, the step of cryopreserving the cells comprises mixing a composition comprising the cells and a buffer, e.g., PBS, 1:1 with a composition comprising 40 mg / mL albumin, e.g., human albumin, 25 mg / mL dextran, e.g., dextran 40, 12.5 mg / mL glucose and 55 mg / mL DMSO.

[0332] In some embodiments, the composition comprising cells and a buffer, e.g., PBS, comprises 2×10 7 Or about 2 x 10 7 So, 2 x 10 9 Or about 2 x 10 9 In some embodiments, the composition comprising cells and a buffer, e.g., PBS, comprises 2×10 cells / mL. 8 In some embodiments, the composition comprising cells and a buffer, e.g., PBS, comprises about 2×10 cells / mL. 8 Contains cells / mL.

[0333] In some embodiments, the step of cryopreserving the cells comprises mixing the cells, a buffer, such as PBS, albumin, such as human albumin, dextran, such as dextran 40, and DMSO, and freezing the mixture.

[0334] In some embodiments, the mixture comprises 1×10 7 Or about 1 x 10 7 From 1 × 10 9 Or about 1 x 10 9 In some embodiments, the mixture contains 1×10 cells / mL. 8 In some embodiments, the mixture contains about 1×10 cells / mL. 8 Contains cells / mL.

[0335] Suitable ranges for albumin, dextran and DMSO are given above.

[0336] In some embodiments, the composition is frozen at -135°C or below.

[0337] In some embodiments, the composition is frozen at a controlled rate.

[0338] In some embodiments, the composition further comprises a multispecific engager, such as a multispecific engager described herein.

[0339] III. Multispecific Engagers As used herein, the term "multispecific engager" refers to an antibody construct that is "at least bispecific". That is, the antibody construct comprises at least a first binding domain and a second binding domain, where the first binding domain binds to one antigen or target (here, the NK cell receptor, e.g., CD16a) and the second binding domain binds to another antigen or target (here, the target cell surface antigen CD30). Thus, an antibody construct as defined in the context of the present disclosure comprises specificity for at least two different antigens or targets. For example, the first domain preferably binds to an extracellular epitope of the NK cell receptor of one or more species selected from human, Macaca species, and rodent species.

[0340] Multispecific antibody constructs include, for example, bispecific and trispecific antibody constructs, or constructs with four or more specificities (e.g., four, five, ...). Examples of multispecific antibody constructs are described, for example, in WO 2006 / 125668, WO 2015 / 158636, WO 2017 / 064221, WO 2019 / 175368, WO 2019 / 198051, WO 2020 / 043670, WO 2021 / 130383, and Ellwanger et al. (MAbs. 2019 Jul; 11(5): 899-918).

[0341] In some embodiments, the multispecific engager is a bispecific antibody or antigen-binding fragment thereof comprising a first binding domain that specifically binds CD16 (FcγRIII) and a second binding domain that specifically binds CD30.

[0342] In some embodiments, the multispecific engager is a bispecific engager, hi some embodiments, the bispecific engager comprises a CD16 binding domain and a CD30 binding domain.

[0343] In some embodiments, the CD16 binding domain comprises a light chain variable domain (VL_CD16A) comprising light chain complementarity determining region 1 (CDRL1) comprising SEQ ID NO:9, light chain complementarity determining region 2 (CDRL2) comprising SEQ ID NO:10, light chain complementarity determining region 3 (CDRL3) comprising SEQ ID NO:11, and a heavy chain variable domain (VH_CD16A) comprising heavy chain complementarity determining region 1 (CDRH1) comprising SEQ ID NO:6, heavy chain complementarity determining region 2 (CDRH2) comprising SEQ ID NO:7, and heavy chain complementarity determining region 3 (CDRH3) comprising SEQ ID NO:8.

[0344] In some embodiments, the CD16 binding domain comprises a light chain variable (VL) region comprising, or consisting of, an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:20; and a heavy chain variable (VH) region comprising, or consisting of, an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to NO:19.

[0345] In some embodiments, the CD16 binding domain comprises a light chain variable (VL) region comprising, or consisting of, SEQ ID NO:20 and a heavy chain variable (VH) region comprising, or consisting of, SEQ ID NO:19.

[0346] In some embodiments, the CD30 binding domain comprises a light chain variable domain (VL_CD30) comprising light chain complementarity determining region 1 (CDRL1) comprising SEQ ID NO:15, light chain complementarity determining region 2 (CDRL2) comprising SEQ ID NO:16, light chain complementarity determining region 3 (CDRL3) comprising SEQ ID NO:17, and a heavy chain variable domain (VH_CD30) comprising heavy chain complementarity determining region 1 (CDRH1) comprising SEQ ID NO:12, heavy chain complementarity determining region 2 (CDRH2) comprising SEQ ID NO:13, and heavy chain complementarity determining region 3 (CDRH3) comprising SEQ ID NO:14.

[0347] In some embodiments, the CD30 binding domain comprises a light chain variable (VL) region comprising, or consisting of, an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:22; and a heavy chain variable (VH) region comprising, or consisting of, an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to NO:21.

[0348] In some embodiments, the CD30 binding domain comprises a light chain variable (VL) region comprising SEQ ID NO:22 and a heavy chain variable (VH) region comprising SEQ ID NO:21.

[0349] In some embodiments, the multispecific engager is a tetravalent homodimer. In some embodiments, the tetravalent homodimer comprises a first polypeptide monomer and a second polypeptide monomer, each comprising or consisting of, from N-terminus to C-terminus, CD16A heavy chain variable domain (VH_CD16A)-first linker (L1)-CD30 light chain variable domain (VL_CD30)-second linker (L2)-CD30 heavy chain variable domain (VH_CD30)-third linker (L3)-and CD16 light chain variable domain (VL_CD16). In some embodiments, the first and second polypeptides dimerize head-to-tail by non-covalent interactions of domains in the Ig variable heavy chain (VH) and Ig variable light chain (VL).

[0350] In some embodiments, the multispecific engager is a tetravalent homodimer. In some embodiments, the tetravalent homodimer comprises a first polypeptide monomer and a second polypeptide monomer, each comprising or consisting of, from N-terminus to C-terminus, CD30 heavy chain variable domain (VH_CD30)-first linker (L1)-CD16A light chain variable domain (VL_CD16A)-second linker (L2)-CD16A heavy chain variable domain (VH_CD16A)-third linker (L3)-and CD30 light chain variable domain (VL_CD30). In some embodiments, the first and second polypeptides dimerize head-to-tail by non-covalent interactions of domains in the Ig variable heavy chain (VH) and Ig variable light chain (VL).

[0351] In some embodiments, VH_CD16 comprises heavy chain complementarity determining region 1 (CDRH1) comprising SEQ ID NO:6, heavy chain complementarity determining region 2 (CDRH2) comprising SEQ ID NO:7, and heavy chain complementarity determining region 3 (CDRH3) comprising SEQ ID NO:8. In some embodiments, VH_CD16 comprises or consists of SEQ ID NO:19.

[0352] In some embodiments, VL_CD16 comprises a light chain complementarity determining region 1 (CDRL1) comprising SEQ ID NO:9, a light chain complementarity determining region 2 (CDRL2) comprising SEQ ID NO:10, and a light chain complementarity determining region 3 (CDRL3) comprising SEQ ID NO:11. In some embodiments, VL_CD16A comprises or consists of SEQ ID NO:20.

[0353] In some embodiments, VH_CD30 comprises heavy chain complementarity determining region 1 (CDRH1) comprising SEQ ID NO: 12, heavy chain complementarity determining region 2 (CDRH2) comprising SEQ ID NO: 13, and heavy chain complementarity determining region 3 (CDRH3) comprising SEQ ID NO: 14. In some embodiments, VH_CD30 comprises or consists of SEQ ID NO: 21.

[0354] In some embodiments, VL_CD30 comprises a light chain complementarity determining region 1 (CDRL1) comprising SEQ ID NO: 15, a light chain complementarity determining region 2 (CDRL2) comprising SEQ ID NO: 16, and a light chain complementarity determining region 3 (CDRL3) comprising SEQ ID NO: 17. In some embodiments, VL_CD16A comprises or consists of SEQ ID NO: 22.

[0355] In some embodiments, L1 comprises or consists of SEQ ID NO:26.

[0356] In some embodiments, L2 comprises or consists of SEQ ID NO:27.

[0357] In some embodiments, L3 comprises or consists of SEQ ID NO:28.

[0358] In some embodiments, the first and second polymer monomers each comprise or consist of SEQ ID NO:18.

[0359] In some embodiments, the multispecific engager comprises a tag, such as a histidine tag, such as a hexa-histidine tag (SEQ ID NO:25).

[0360] In some embodiments, the multispecific engager is AFM13. See, e.g., Wu et al., "AFF13: a first-in-class tetravalent bispecific anti-CD30 / CD16A antibody for NK cell-mediated immunotherapy," J. Hemat & Oncol 8, 96 (2015), which is incorporated by reference in its entirety. See also Reusch et al., "A Novel Tetravalent Bispecific TandAb (CD30 / CD16A) efficiently recruits NK cells for the lysis of CD30+ tumor cells," mAbs 6(3):727-38 (2014), which is incorporated by reference in its entirety; and Rothe et al., "A Phase 1 Study for the Bispecific Anti-CD30 / CD16A Antibody Construct AFM13 in Patients with Relapsed or Refractory Hodgkin Lymphoma," Blood 125(26):4024-31 (2015), which is incorporated by reference in its entirety.

[0361] As used herein, the term "NK cell receptor" defines proteins and protein complexes on the surface of NK cells. Thus, this term defines cell surface molecules that are unique to NK cells, but do not necessarily express exclusively on the surface of NK cells, but are also expressed on other cells, such as macrophages and T cells. Examples of NK cell receptors include, but are not limited to, FcγRIII (CD16a, CD16b), NKp46, and NKG2D.

[0362] As used herein, "CD16a" refers to the activating receptor CD16a, also known as FcγRIIIA, expressed on the cell surface of NK cells. CD16a is an activating receptor that triggers the cytotoxic activity of NK cells. The affinity of antibodies to CD16a positively correlates with their ability to trigger NK cell activation, so that higher affinity to CD16a reduces the antibody dose required for activation. The antigen-binding site of an antigen-binding protein binds to CD16a but not to CD16b. For example, an antigen-binding site that includes heavy (VH) and light (VL) chain variable domains that bind to CD16a but not to CD16B can be provided by an antigen-binding site that specifically binds to an epitope of CD16a that includes amino acid residues of the C-terminal sequence SFFPPGYQ ​​(SEQ ID NO:23) of CD16a (SEQ ID NO:24) that are not present in CD16b, and / or residues Gl30 and / or Y141.

[0363] As used herein, "CD16b" refers to the receptor CD16b expressed on neutrophils and eosinophils, also known as FcγRIIIB. This receptor is glycosylphosphatidylinositol (GPI) anchored and is understood not to trigger any kind of cytotoxic activity of CD16b positive immune cells.

[0364] As used herein, the term "target cell surface antigen" refers to an antigenic structure expressed by a cell and present on the cell surface in a manner accessible to the antibody constructs described herein. In some cases, the "target cell surface antigen" to which the multispecific antibody constructs described herein bind is CD30. CD30, also known as TNFRSF8, is a cell membrane protein of the tumor necrosis factor receptor family and is also a tumor marker.

[0365] Given that the antibody constructs defined in the context of the present invention are (at least) bispecific, they do not occur in nature and are significantly different from natural products. Thus, a "multispecific" antibody construct or immunoglobulin is an artificial hybrid antibody or immunoglobulin that has at least two distinct binding surfaces with different specificities. Multispecific antibody constructs can be produced by a variety of methods, including fusion of hybridomas or linking of Fab' fragments. See, for example, Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990).

[0366] Said at least two binding domains and variable domain (VH / VL) of the antibody construct of the present disclosure may or may not include a peptide linker (spacer or connector peptide). In some embodiments, the term "peptide linker" includes an amino acid sequence that connects the amino acid sequence of one (variable and / or binding) domain with the amino acid sequence of another (variable and / or binding) domain of the antibody construct defined herein. A peptide linker can also be used to fuse a third domain to another domain or Fc portion of the antibody construct defined herein. Preferably, such a peptide linker does not contain any polymerization activity.

[0367] As used herein, the term "binding domain" characterizes a domain that (specifically) binds to / interacts with / recognizes a given target epitope or a given target surface on a target molecule (antigen), for example on the NK cell receptor antigen, e.g. CD16, and on the target cell surface antigen CD30, respectively. The structure and function of the first binding domain (e.g., that which recognizes CD16), and preferably also the structure and / or function of the second binding domain (that which recognizes the target cell surface antigen), are based on the structure and / or function of an antibody, e.g., a full-length immunoglobulin molecule or an entire immunoglobulin molecule, and / or are derived from the variable heavy (VH) and / or variable light (VL) domains of an antibody or a fragment thereof. Preferably, the first binding domain is characterized by the presence of three light chain CDRs (i.e. CDR1, CDR2 and CDR3 of the VL region) and / or three heavy chain CDRs (i.e. CDR1, CDR2 and CDR3 of the VH region). The second binding domain also preferably comprises the minimum structural requirements of an antibody that allow target binding. More preferably, the second binding domain comprises at least three light chain CDRs (i.e. CDR1, CDR2 and CDR3 of the VL region) and / or three heavy chain CDRs (i.e. CDR1, CDR2 and CDR3 of the VH region). Optionally, the first and / or second binding domain may be produced or obtained by phage display or library screening methods rather than by grafting CDR sequences from an existing (monoclonal) antibody onto a scaffold.

[0368] In some embodiments, the binding domain is in the form of one or more polypeptides. Such polypeptides may contain proteinaceous and non-proteinaceous parts (e.g., chemical linkers or cross-linkers such as glutaraldehyde). Proteins (including their fragments, preferably biologically active fragments, and peptides, usually those having less than 30 amino acids) contain two or more amino acids coupled together via a peptide covalent bond (resulting in a chain of amino acids).

[0369] As used herein, the term "polypeptide" refers to a group of molecules, usually consisting of more than 30 amino acids. Polypeptides can further form multimers, such as dimers, trimers and higher oligomers, i.e., consisting of two or more polypeptide molecules. The polypeptide molecules forming such dimers, trimers, etc. may be identical or non-identical. The corresponding higher-order structures of such multimers are therefore called homodimers or heterodimers, homotrimers or heterotrimers, etc. An example of a heteromultimer is an antibody molecule, which in its native form consists of two identical light chain polypeptides and two identical heavy chain polypeptides. The terms "peptide", "polypeptide" and "protein" also refer to naturally modified peptides / polypeptides / proteins, where the modifications are affected by post-translational modifications such as, for example, glycosylation, acetylation, phosphorylation, etc. "Peptide", "polypeptide" or "protein", as referred to herein, can also be chemically modified, such as PEGylation. Such modifications are well known in the art and are described herein below.

[0370] Preferably, the binding domain that binds to the NK cell receptor antigen, e.g., CD16, and / or the binding domain that binds to the target cell surface antigen CD30 is a human binding domain, a humanized binding domain, or a chimeric binding domain derived from a mouse. Antibodies and antibody constructs that include at least one human binding domain avoid some of the problems associated with antibodies or antibody constructs that have non-human variable and / or constant regions, such as those of rodents (e.g., mice, rats, hamsters, or rabbits). The presence of such rodent-derived proteins may lead to rapid clearance of the antibody or antibody construct, or to the generation of an immune response by the patient against the antibody or antibody construct. To avoid the use of rodent-derived antibodies or antibody constructs, human or fully human antibodies / antibody constructs can be generated by introducing human antibody functions into rodents, such that the rodents produce fully human antibodies.

[0371] In some embodiments, this antigen binding site for CD16A does not bind CD16B, but binds with similar affinity to known CD16A allotypes F158 and V158. Two allelic single nucleotide polymorphisms have been identified in human CD16A that change the 158th amino acid, which is important for interaction with the hinge region of IgG. The allele frequencies of the homozygous 158F / F allele and the heterozygous 158V / F allele are similar in the Caucasian population, 35-52% or 38-50%, respectively, while the homozygous 158V / V allele is found in only 10-15% (Lopez-Escamez JA et al.; BMC Med Genet 2011; 12:2). Thus, activation of NK cells in all patients by this anti-CD16A domain due to similar affinity is favorable. Further CD16A antigen binding sites comprising heavy and light chain variable domains that bind to CD16A but not to CD16B are described in WO 2006 / 125668.

[0372] In some embodiments, the heavy and light chain domains incorporate the immunologically active homologs or variants of the CDR or framework sequences described herein.In some embodiments, the CDR variant sequences are modified to change non-critical residues or residues in non-critical regions.Non-critical amino acids can be identified by known methods such as affinity maturation, CDR walking mutagenesis, site-directed mutagenesis, crystallization, nuclear magnetic resonance, photoaffinity labeling, or alanine scanning mutagenesis.

[0373] IV. Pharmaceutical Compositions Provided herein are pharmaceutical compositions comprising the natural killer cells described herein and dosage units of the pharmaceutical compositions described herein.

[0374] In some cases, the dosage unit comprises between 100 million and 1.5 billion cells, e.g., 100 million, 200 million, 300 million, 400 million, 500 million, 600 million, 700 million, 800 million, 900 million, 1 billion, 1.1 billion, 1.2 billion, 1.3 billion, 1.4 billion, or 1.5 billion.

[0375] Pharmaceutical compositions typically include a pharma- ceutically acceptable carrier. As used herein, the term "pharma- ceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration.

[0376] In some embodiments, the pharmaceutical composition comprises a) a natural killer cell described herein and b) a cryopreserved composition.

[0377] Suitable cryopreservation compositions are described herein.

[0378] In some embodiments, the composition is frozen. In some embodiments, the composition is frozen for at least 3 months, e.g., at least 6 months, at least 9 months, at least 12 months, at least 15 months, at least 18 months, at least 24 months, or at least 36 months.

[0379] In some embodiments, at least 60%, e.g., 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 being thawed.

[0380] In some embodiments, the pharmaceutical composition comprises a) a cryopreservation composition described herein and b) therapeutic cells.

[0381] In some embodiments, the therapeutic cells are animal cells, hi some embodiments, the therapeutic cells are human cells.

[0382] In some embodiments, the therapeutic cell is an immune cell. In some embodiments, the immune cell is selected from a basophil, an eosinophil, a neutrophil, a mast cell, a monocyte, a macrophage, a neutrophil, a dendritic cell, a natural killer cell, a B cell, a T cell, and a combination thereof.

[0383] In some embodiments, the immune cells are natural killer (NK) cells. In some embodiments, the natural killer cells are expanded and stimulated by the methods described herein.

[0384] In some embodiments, the pharmaceutical composition further comprises c) a buffer solution. Suitable buffer solutions are described herein, for example, with respect to the cryopreserved composition.

[0385] In some embodiments, the pharmaceutical composition comprises 1×10 7 or about 1 x 10 7 From 1 × 10 9 or about 1 x 10 9 In some embodiments, the pharmaceutical composition comprises 1×10 cells / mL. 8 In some embodiments, the pharmaceutical composition comprises about 1×10 cells / mL. 8 Contains cells / mL.

[0386] In some embodiments, the pharmaceutical composition further comprises a multispecific engager, such as a multispecific engager described herein.

[0387] Pharmaceutical compositions are typically formulated to be compatible with their intended route of administration, which include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.

[0388] Methods for formulating suitable pharmaceutical compositions are known in the art. For example, see "Remington: The Science and Practice of Pharmacy", 21st Edition, 2005, and "Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs" series (Dekker, NY). For example, solutions or suspensions used for parenteral, intradermal or subcutaneous application can contain the following components: a sterile diluent, such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, 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 tonicity, such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be enclosed in glass or plastic ampoules, disposable syringes or multiple dose vials.

[0389] Pharmaceutical compositions suitable for use as injections can include sterile aqueous solutions (if water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or suspensions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and fluid to the extent that easy syringability can be ensured. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it will be preferable to include isotonic agents, such as sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride, in the composition. Prolonged absorption of injectable compositions can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin, in the composition.

[0390] Sterile injectable solution can be prepared by incorporating active compound in the required amount in suitable solvent with one or combination of above listed components as required, followed by filtration sterilization.Generally, dispersion can be prepared by incorporating active compound into a sterile medium that contains basic dispersion medium and other required components selected from above listed.In the case of sterile powder for preparing sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying, which gives powder from the solution of active ingredient and any other desired components that are previously sterilized and filtered.

[0391] V. Treatment Methods The NK cells described herein are useful for treating cancer or other proliferative disorders.

[0392] Accordingly, also provided herein is a method of treating a patient suffering from a disorder, e.g., a disorder associated with cancer, e.g., a CD30+ cancer, comprising administering an NK cell, e.g., an NK cell described herein, and a CD30-targeted multispecific engager, e.g., a multispecific engager described herein.

[0393] Also provided herein is a method of preventing, reducing and / or inhibiting recurrence, growth, proliferation, migration and / or metastasis of a cancer cell or population of cancer cells in a subject in need thereof, the method comprising administering an NK cell, such as an NK cell described herein, and a CD30-targeted multispecific engager, such as a multispecific engager described herein.

[0394] Also provided herein is a method of enhancing, improving and / or increasing a response to cancer treatment in a subject in need thereof, the method comprising administering NK cells, such as NK cells described herein, and a CD30-targeted multispecific engager, such as a multispecific engager described herein.

[0395] Also provided herein is a method for inducing the immune system in a subject in need thereof, the method comprising administering NK cells, such as NK cells described herein, and a CD30-targeted multispecific engager, such as a multispecific engager described herein.

[0396] The methods described herein include methods for treating disorders associated with abnormal apoptotic or differentiation processes, such as cell proliferation or differentiation disorders, such as cancers, including both solid tumors and hematopoietic cancers. Generally, the methods include administering a therapeutically effective amount of a treatment described herein to a subject in need of such treatment or determined to be in need of such treatment. In some embodiments, the methods include administering a therapeutically effective amount of a treatment comprising NK cells, such as NK cells described herein, and a CD30-targeted multispecific engager, such as a multispecific engager described herein.

[0397] As used herein, the terms "treatment", "treat" and "treating" refer to reversing, alleviating, delaying the onset or inhibiting the progression of disorders associated with abnormal apoptotic or differentiation processes. For example, treatment may result in a reduction in tumor size or tumor growth rate. Administration of a therapeutically effective amount of a compound described herein for treating a condition associated with abnormal apoptotic or differentiation processes will result in, among other things, a reduction in tumor size, a decrease in growth rate, a reduction in the risk or frequency of recurrence, a delay in recurrence, a reduction in metastasis, an increase in survival rate, and / or a reduction in morbidity and mortality. In some embodiments, treatment may be administered after one or more symptoms have developed. In another embodiment, treatment may be administered in the absence of symptoms. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., taking into account the history of symptoms and / or taking into account genetic or other susceptibility factors). Treatment may be continued after symptoms have resolved, for example to prevent or delay their recurrence.

[0398] As used herein, the term "inhibition", as it relates to cancer and / or cancer cell proliferation, refers to inhibiting the growth, division, maturation or viability of cancer cells, and / or causing the death of cancer cells, either individually or collectively with other cancer cells, by cytotoxicity, nutrient deprivation, or induction of apoptosis.

[0399] As used herein, "delaying" the onset of a disease or disorder or one or more symptoms means to postpone, impede, slow, retard, stabilize and / or postpone the onset of the disease, disorder or its symptoms. The length of time of this delay can vary depending on the history of the disease and / or the subject being treated. As will be clear to those skilled in the art, a sufficient or significant delay can actually encompass prevention, in that the subject does not develop the disease, disorder or its symptoms. For example, a method of "delaying" the onset of cancer is one that reduces the probability of disease onset in a given time frame and / or reduces the extent of disease in a given time frame when compared to not using the method. Such comparisons can be based on clinical trials using a statistically significant number of subjects.

[0400] As used herein, "prevent" or "preventing" refers to a regimen that protects against the onset of a disease or disorder, such that clinical symptoms of the disease do not develop. Thus, "prevention" refers to administering a treatment (e.g., administering a therapeutic agent) to a subject before symptoms of the disease are detectable in the subject and / or before the disease reaches a certain stage (e.g., administering a therapeutic agent to a subject with cancer that has not yet metastasized). The subject may be an individual at risk of developing a disease or disorder, or an individual at risk of disease progression, e.g., cancer metastasis. Such as an individual with one or more risk factors known to be associated with the onset or onset of a disease or disorder. For example, an individual may have a mutation associated with the onset or progression of cancer. Furthermore, it is understood that prevention may not result in complete protection from the onset of a disease or disorder. In some cases, prevention includes reducing the risk of developing a disease or disorder. Reducing the risk may not result in complete elimination of the risk of developing a disease or disorder.

[0401] An "increased" or "enhanced" amount (e.g., with respect to anti-tumor response, cancer cell metastasis) refers to an increase that is 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 fold or more (e.g., 100, 500, 1000 fold) (including all integers and decimals in between and greater than 1, e.g., 2.1, 2.2, 2.3, 2.4 fold, etc.) of the amounts or levels described herein. This can 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 amounts or levels described herein.

[0402] A "decreased" or "reduced" or "less" amount (e.g., with respect to tumor size, proliferation or growth of cancer cells) refers to a decrease that is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 times or less (e.g., 100, 500, 1000 times) (including all integers and decimals in between and greater than 1, e.g., 1.5, 1.6, 1.7, 1.8 times, etc.) of the amounts or levels described herein. This can include a decrease 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 amounts or levels described herein.

[0403] A. Disability The method disclosed herein and the composition produced are useful for targeting some disorders, such as cell proliferation disorders.The advantage of the approach herein is that allogeneic cells are combined with exogenous antibody administration to specific target proliferation cells that are targeted by the exogenous antibody.Unlike conventional therapies such as chemotherapy and radiotherapy, the approach and pharmaceutical composition herein can be used to specifically target cells that exhibit harmful proliferation activity without administering systemic drugs or toxins that may affect proliferation cells indiscriminately.

[0404] Examples of cell proliferation and / or differentiation disorders include cancer, e.g., carcinoma, sarcoma, metastatic disorders, or hematopoietic neoplastic disorders, e.g., leukemia. Metastatic tumors can arise from a number of primary tumor types, including, but not limited to, those of prostate, colon, lung, breast, and liver origin.

[0405] As used herein, the terms "cancer," "hyperproliferative," and "neoplastic" refer to abnormal conditions or disease states characterized by cells capable of autonomous growth, i.e., rapidly proliferating cell growth. Hyperproliferative and neoplastic disease states may be classified as pathological, i.e., characterizing or constituting a disease state, or non-pathological, i.e., deviations from normal but not associated with a disease state. The term is intended to encompass any type of cancerous growth or oncogenic process, metastatic tissue, or malignantly transformed cells, tissues, or organs, regardless of histopathological type or stage of invasiveness. In disease states characterized by malignant tumor growth, "pathological hyperproliferative" cells exist. Examples of non-pathological hyperproliferative cells include the proliferation of cells associated with wound healing.

[0406] The term "cancer" or "neoplasm" includes malignant diseases of various organ systems, such as those affecting the lung, breast, thyroid, lymphatic system, gastrointestinal and genitourinary tract, as well as adenocarcinomas, including most colorectal cancers, renal cell carcinoma, prostate cancer and / or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine, and cancer of the esophagus.

[0407] The term "cancer" is recognized in the art and refers to malignant diseases of epithelial or endocrine tissues, including respiratory cancer, gastrointestinal cancer, genitourinary cancer, testicular cancer, breast cancer, prostate cancer, endocrine cancer, and melanoma. In some embodiments, the disease is renal cancer or melanoma. Exemplary cancers include those formed from tissues of the cervix, lung, prostate, breast, head and neck, colon, and ovary. The term also includes carcinosarcomas, including malignant tumors composed of carcinomatous and sarcomatous tissues. "Adenocarcinoma" refers to cancers derived from glandular tissue or in which the tumor tissue forms recognizable glandular structures.

[0408] The term "sarcoma" is art-recognized and refers to malignant tumors of mesenchymal derivation.

[0409] Further examples of proliferative disorders include hematopoietic neoplastic disorders. As used herein, the term "hematopoietic neoplastic disorders" includes diseases involving hyperplastic / neoplastic cells of hematopoietic origin, such as those arising from myeloid, lymphoid or erythroid lineages, or precursor cells thereof. Preferably, the disease arises from poorly differentiated acute leukemias, such as erythroblastic leukemia and acute megakaryoblastic leukemia. Further exemplary myeloid disorders include, but are not limited to, acute promyelocytic leukemia (APML), acute myeloid leukemia (AML) and chronic myeloid leukemia (CML) (reviewed in Vaickus, L. (1991) Crit Rev. in Oncol. / Hemotol. 11:267-97). Lymphoid malignancies include, but are not limited to, acute lymphoblastic leukemia (ALL), including B-cell and T-cell ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM). Additional forms of malignant lymphoma include, but are not limited to, non-Hodgkin's lymphoma and its variants, peripheral T-cell lymphoma, adult T-cell leukemia / lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease and Reed-Sternberg disease.

[0410] In some embodiments, the cancer is acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, Kaposi's sarcoma, AIDS-related lymphoma, primary CNS lymphoma, anal cancer, appendix cancer, astrocytoma, typical teratoid / rhabdoid tumor, basal cell carcinoma, bile duct carcinoma, bladder cancer, bone cancer, brain tumor, breast cancer, bronchial tumor, Burkitt's lymphoma, carcinoid, cardiac tumor, medulloblastoma, germ cell tumor, primary CNS lymphoma, cervical cancer, bile duct carcinoma, chordoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloproliferative neoplasm, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, ductal carcinoma in situ, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, Ewing's sarcoma, extracranial germ cell tumor. tumors, extragonadal germ cell tumors, eye cancer (e.g. intraocular melanoma or retinoblastoma), fallopian tube cancer, fibrous histiocytoma of bone, osteosarcoma, gallbladder cancer, stomach cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), germ cell tumors, gestational trophoblastic disease, hairy cell leukemia, head and neck cancer, heart tumors, hepatocellular carcinoma, histiocytosis, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumors, pancreatic neuroendocrine tumors, kidney (renal cell) cancer, Langerhans cell histiocytosis, pharyngeal cancer, leukemia, lip and oral cavity cancer, liver cancer, lung cancer (e.g. non-small cell lung cancer, small cell lung cancer, pleuropulmonary blastoma, and tracheobronchial tumors), lymphoma, male breast cancer, malignant fibrous histiocytoma of bone, melanoma, Merkel cell carcinoma, mesothelioma, metastatic cancer, metastatic cervical squamous cell carcinoma (metastatic squamous neck cancer, midline carcinoma, oral cavity cancer, multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm, myeloproliferative neoplasm, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, oral cavity cancer, lip and oral cavity cancer, oropharyngeal cancer, osteosarcoma, malignant fibrous histiocytoma, ovarian cancer, pancreatic cancer, pancreatic neuroendocrine tumor, papillomatosis, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, plasma cell neoplasm, multiple myeloma, pleuropulmonary blastoma, pregnancy and breast cancercancer), primary central nervous system lymphoma, primary peritoneal cancer, prostate cancer, rectal cancer, recurrent cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma (e.g., childhood rhabdomyosarcoma, childhood vascular tumor, Ewing's sarcoma, Kaposi's sarcoma, osteosarcoma, soft tissue sarcoma, uterine sarcoma), Sezary syndrome, skin cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer, gastric cancer, T-cell lymphoma, testicular cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer, thymoma / thymic carcinoma, thyroid cancer, tracheobronchial tumor, transitional cell carcinoma of the renal pelvis and ureter, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vascular tumor, vulvar cancer, and Wilms' tumor.

[0411] In some embodiments, the cancer is a solid tumor.

[0412] In some embodiments, the cancer is metastatic.

[0413] In some embodiments, the cancer is a CD30+ cancer.

[0414] In some embodiments, the CD30+ cancer is a lymphoma. In some embodiments, the lymphoma is selected from the group consisting of classical Hodgkin's lymphoma (CHL), anaplastic large cell lymphoma (ALCL), gray zone lymphoma (GZL), Epstein-Barr virus positive diffuse large B-cell lymphoma (EBV+DLBCL), and combinations thereof.

[0415] In some embodiments, the cancer is Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell non-Hodgkin's lymphoma, peripheral T-cell lymphoma, peripheral T-cell lymphoma, unspecified type, cutaneous T-cell lymphoma, anaplastic large cell lymphoma, CD30 + The patient is selected from the group consisting of B-cell lymphoma, multiple myeloma, mycosis fungoides, and leukemia. In some embodiments, the patient has relapsed disease. In some embodiments, the patient is refractory to previous therapeutic intervention.

[0416] In some embodiments, the disorder is relapsed or refractory classical Hodgkin's lymphoma (cHL).

[0417] In some embodiments, the disorder is relapsed or refractory peripheral T-cell lymphoma (PTCL). In some embodiments, the PTCL is a PTCL subtype selected from the group consisting of peripheral T-cell lymphoma-not otherwise specified, angioimmunoblastic T-cell lymphoma, anaplastic large cell lymphoma anaplastic lymphoma kinase (ALK) positive, and anaplastic large cell lymphoma ALK negative.

[0418] B.Patient Suitable patients for the compositions and methods herein include those who suffer from, have been diagnosed with, or are suspected of having a cell proliferation and / or differentiation disorder, such as cancer.Patients who are the subject of the technology of the present disclosure generally respond better to the methods and compositions herein.One reason for this is that the pharmaceutical compositions are allogeneic, and rather than targeting proliferating cells in general, the target cells are identified by antibodies.As a result, there is less off-target impact, and patients are more likely to complete treatment regimens without substantial adverse off-target effects.

[0419] In some embodiments, the methods of treatment provided herein can be used to treat subjects (e.g., humans, monkeys, dogs, cats, mice) diagnosed with or suspected of having a cell proliferation and / or differentiation disorder, such as cancer. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0420] As used herein, a subject refers to a mammal, including, for example, a human.

[0421] In some embodiments, the mammal is selected from the group consisting of armadillo, donkey, bat, bear, beaver, cat, chimpanzee, cow, coyote, deer, dog, dolphin, elephant, fox, panda, gibbon, giraffe, goat, gopher, hedgehog, hippopotamus, horse, humpback whale, jaguar, kangaroo, koala, leopard, lion, llama, lynx, mole, monkey, mouse, narwhal, orangutan, killer whale, otter, bull, pig, polar bear, porcupine, puma, rabbit, raccoon, rat, rhinoceros, sheep, squirrel, tiger, walrus, weasel, wolf, zebra, goat, horse, and combinations thereof.

[0422] In some embodiments, the mammal is a human.

[0423] The subject, e.g., a human subject, can be a child, e.g., from at or about 0 to at or about 14 years old. The subject can be an adolescent, e.g., from at or about 15 to at or about 24 years old. The subject can be an adult, e.g., from at or about 25 to at or about 64 years old. The subject can be an elderly person, e.g., 65+ years old.

[0424] In some embodiments, the subject may be a human who exhibits one or more symptoms associated with a cell proliferation and / or differentiation disorder, such as cancer, such as a tumor. The methods of treatment provided herein may be used to treat cancer at various stages. For example, cancer stages include, but are not limited to, early stage, advanced, locally advanced, in remission, refractory, recurrent after remission, and progressive. In some embodiments, the subject is at an early stage of cancer. In another embodiment, the subject is at an advanced stage of cancer. In various embodiments, the subject has stage I, stage II, stage III, or stage IV cancer. The treatment methods described herein can promote tumor reduction or regression, reduce or inhibit tumor growth or cancer cell proliferation, and / or induce, increase, or promote tumor cell death. In some embodiments, the subject is in cancer remission. The treatment methods described herein can prevent or delay cancer metastasis or recurrence.

[0425] In some embodiments, the subject is at risk of, or has a genetic or other predisposition (e.g., risk factor) to, developing a diagnosed or yet to be diagnosed cell proliferation and / or differentiation disorder, e.g., cancer.

[0426] As used herein, an "at risk" individual refers to an individual who is at risk of developing a condition to be treated, such as a cell proliferation and / or differentiation disorder, such as cancer. In general, an "at risk" subject may or may not have detectable disease, and may or may not exhibit detectable disease prior to the treatment methods described herein. "At risk" refers to an individual having one or more so-called risk factors, which are measurable parameters known in the art that correlate with a disease or condition. For example, an at risk subject may have one or more risk factors, which are measurable parameters that correlate with the development of cancer. A subject who has one or more of these risk factors has a higher probability of developing cancer than an individual who does not have these risk factors. In general, risk factors may include, for example, age, sex, race, diet, medical history, the presence of precursor diseases, genetic (e.g., hereditary) considerations, and environmental exposures. In some embodiments, subjects at risk for cancer include, for example, those who have relatives who have suffered from the disease, and those whose risk has been determined by analysis of genetic or biochemical markers.

[0427] In addition, the subject may have undergone one or more standard therapies, such as chemotherapy, radiation therapy, immunotherapy, surgery, or a combination thereof, etc. Thus, the one or more kinase inhibitors may be administered before, during, or after the administration of chemotherapy, radiation therapy, immunotherapy, surgery, or a combination thereof.

[0428] In certain embodiments, the subject may be a human who is (i) substantially refractory to at least one chemotherapy treatment, or (ii) relapsing after chemotherapy treatment, or both (i) and (ii). In some embodiments, the subject is refractory to at least two, at least three, or at least four chemotherapy treatments (including standard or experimental chemotherapy).

[0429] In some embodiments, the subject relapses after treatment with an anti-CD30 antibody or is refractory to an anti-CD30 antibody. In some embodiments, the anti-CD30 antibody is brentuximab bedotin.

[0430] In some embodiments, the subject has experienced disease progression following treatment with autologous stem cell transplant or chimeric antigen receptor T cell therapy (CAR-T).

[0431] In some embodiments, the patient is diagnosed with or has been diagnosed with a CD30+ cancer.

[0432] In some embodiments, the patient is diagnosed with or has been diagnosed with CD30+ cancer by immunohistochemical staining of cancer biopsy or surgical specimen.In some embodiments, the patient is diagnosed with or has been diagnosed with CD30+ cancer by chromogenic in situ hybridization.In some embodiments, the patient is diagnosed with or has been diagnosed with CD30+ cancer by fluorescent in situ hybridization of cancer biopsy or surgical specimen.In some embodiments, the patient is diagnosed with or has been diagnosed with CD30+ cancer by genetic analysis.

[0433] In some embodiments, the patient is refractory to treatment with a CD30 inhibitor or relapses following treatment with a CD30 inhibitor.

[0434] In some embodiments, the patient is refractory to or relapses after treatment with a chemotherapeutic agent.

[0435] In some embodiments, the chemotherapeutic agent is selected from the group consisting of cisplatin, docetaxel, carboplatin, gemcitabine, cisplatin, pemetrexed, or a combination thereof.

[0436] In some embodiments, the patient is refractory to treatment with a tyrosine kinase inhibitor or relapses after treatment with a tyrosine kinase inhibitor. In some embodiments, the tyrosine kinase inhibitor is selected from the group consisting of gefitinib, erlontinib, afatinib, osimertinib, and combinations thereof.

[0437] In some embodiments, the patient has recurrent or refractory classical Hodgkin lymphoma and has received at least two lines of therapy, including one line of prior combination chemotherapy. In some embodiments, the prior therapy includes brentuximab vedotin and a checkpoint inhibitor.

[0438] In some embodiments, the patient has relapsed or refractory peripheral T-cell lymphoma and has received at least one prior line of combination therapy. In some embodiments, the prior therapy comprises brentuximab vedotin. In some embodiments, the patient has relapsed or refractory peripheral T-cell lymphoma and is intolerant or has been intolerant to brentuximab vedotin.

[0439] C. Lymphocyte Depletion In some embodiments, the patient undergoes lymphodepletion prior to treatment.

[0440] Exemplary lymphocyte-depleting chemotherapy regimens, along with associated informative biomarkers, are described in WO 2016 / 191756 and WO 2019 / 079564, which are incorporated by reference in their entireties. In certain embodiments, the lymphocyte-depleting chemotherapy regimen comprises a dose of cyclophosphamide (200 mg / m 2 / day~2000mg / m 2 / day) and the dose of fludarabine (20 mg / m 2 / day~900mg / m 2 / day) to the patient.

[0441] In some embodiments, lymphodepletion is at or about 250 to about 500 mg / m 2 cyclophosphamide, e.g., from at or about 250, to at or about 500, 250, 400, 500, about 250, about 400, or about 500 mg / m 2 This includes administration of cyclophosphamide.

[0442] In some embodiments, lymphodepletion is at or about 20 mg / m 2 / day ~40 or about 40 mg / m 2 / day fludarabine, e.g., 30 or about 30 mg / m 2 / day dosing.

[0443] In some embodiments, lymphodepletion comprises administration of both cyclophosmamide and fludarabine.

[0444] In some embodiments, the patient is treated with cyclophosphamide (250 mg / m 2 / day) and fludarabine (30 mg / m 2 / day) intravenously to lymphocyte depletion.

[0445] In some embodiments, the patient is treated with cyclophosphamide (500 mg / m 2 / day) and fludarabine (30 mg / m 2 / day) intravenously to lymphocyte depletion.

[0446] In some embodiments, lymphodepletion is performed within 5 days prior to the first administration of NK cells, hi some embodiments, lymphodepletion is performed within 7 days prior to the first administration of NK cells.

[0447] In some embodiments, lymphodepletion is performed daily for three consecutive days (ie, from day -5 to day -3) beginning five days prior to the first administration of NK cells.

[0448] In some embodiments, lymphodepletion is performed on days −5, −4 and −3.

[0449] D. Administration 1. NK cells In some embodiments, the NK cells are administered to a patient as part of a pharmaceutical composition, such as a pharmaceutical composition described herein. The cells are administered after thawing, and optionally without any further manipulation if the cryopreserved material is compatible with immediate administration. For a given individual, the treatment regimen often involves administration over time of multiple aliquots or doses of NK cells drawn from a common batch or donor.

[0450] In some embodiments, treatment involves administration of a common batch, master cell bank, or dose of NK cells derived from a donor (e.g., as described herein). In some embodiments, treatment involves administration of different batches, master cell banks, or doses of NK cells derived from donors (e.g., as described herein). For example, if a patient initially administered NK cells produced from a first donor develops immunogenicity to the NK cells produced from the first donor, the patient can be administered NK cells produced from a second donor.

[0451] The NK cells can be administered by any suitable means, such as by bolus injection, or by injection, such as intravenous or subcutaneous injection, intraocular injection, periocular injection, subretinal injection, intravitreal injection, transseptal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjectval injection, subconjunctival injection, subtenon injection, retrobulbar injection, periocular injection, or posterior juxtascleral delivery. In some embodiments, they are administered by parenteral, intrapulmonary and intranasal administration, and, if desired for localized treatment, by intralesional administration. Parenteral injections include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In some embodiments, a given dose is administered by a single bolus administration of cells. In some embodiments, a given dose is administered by multiple bolus administration of cells, for example, over a period of 3 days or less, or by continuous infusion administration of cells. In some embodiments, administration of the cell dose or any additional therapy, such as multispecific engager therapy, lymphocyte depletion therapy, intervention therapy and / or combination therapy, is performed by outpatient delivery.

[0452] In the context of adoptive cell therapy, administration of a given "dose" can include administration of a given amount or number of cells as a single composition and / or administration of a single continuous administration, such as administration of a single injection or continuous infusion. A given "dose" can also include administration of a given amount or number of cells as divided doses or administration of multiple compositions given in multiple separate compositions or infusions over a specified period of time, such as over 3 days or less. Thus, a dose is, depending on the situation, a single administration or continuous administration of a specified number of cells given or initiated at a single time point. However, depending on the situation, a dose is administered by multiple injections or infusions over a period of 3 days or less, such as once a day for 3 or 2 days, or multiple infusions in one day.

[0453] In some embodiments, the NK cells are administered to the patient in a number ranging from at or about 1 million to at or about 100 billion cells. In some embodiments, the NK cells, e.g., the NK cells described herein, are administered in a number ranging from 5×10 per dose. 6 or about 5 x 10 6 From 1 × 10 9 or about 1 x 10 9 In some embodiments, the NK cells are administered at a dose of 5×10 6 Or about 5 x 10 6 , 1×10 7 Or about 1 x 10 7 , 3×10 7 Or about 3 x 10 7 , 1×10 8 Or about 1 x 10 8 , 3×10 8 Or about 3 x 10 8 , or 1 × 10 9 Or about 1 x 10 9In some embodiments, the NK cells are administered at or about 1 million to or about 20 billion cells per dose (e.g., 5 million or about 5 million cells, 25 million or about 25 million cells, 50 million or about 50 million cells, 75 million or about 75 million cells, 100 million or about 100 million cells, 200 million or about 200 million cells, 300 million or about 300 million cells, 400 million or about 400 million cells, 500 million or about 500 million cells, 1 billion or about 1 billion cells, 2 billion or about 20 billion cells, 300 million or about 300 million cells, 400 million or about 400 million cells, 500 million or about 500 million cells, 1 billion or about 1 billion cells, 2 billion or about 20 billion cells, 500 million or about 500 million ... 0 billion cells, 3 billion or about 3 billion cells, 4 billion or about 4 billion cells, 5 billion or about 5 billion cells, 6 billion or about 6 billion cells, 7 billion or about 7 billion cells, 8 billion or about 8 billion cells, 9 billion or about 9 billion cells, 10 billion or about 10 billion cells, or a range defined by any two of the foregoing values), 10 million or about 10 million to 20 billion or about 20 billion cells (e.g., 25 million or about 25 million cells, 50 million or about 50 million cells, or a range defined by any two of the foregoing values). million cells, 75 million or about 75 million cells, 100 million or about 100 million cells, 200 million or about 200 million cells, 300 million or about 300 million cells, 400 million or about 400 million cells, 500 million or about 500 million cells, 1 billion or about 1 billion cells, 2 billion or about 2 billion cells, 3 billion or about 3 billion cells, 4 billion or about 4 billion cells, 5 billion or about 5 billion cells, 6 billion or about 6 billion cells, 7 billion or about 7 billion cells, 8 billion or about 8 billion cells, 9 billion or about 9 billion cells, 10 billion or about 10 billion cells, 20 billion or about 20 billion cells, or a range defined by any two of the foregoing values), and optionally from 100 million or about 100 million cells to 50 billion or about 50 billion cells (e.g., 150 million or about 150 million cells, 200 million or about 200 million cells, 300 million or about 300 million cells, 400 million or about 400 million cells, 500 million or about 500 million cells, 1 billion or about 1 billion cells, 2 billion or about 2 billion cells,3 billion or about 3 billion cells, 4 billion or about 4 billion cells, 5 billion or about 5 billion cells, 6 billion or about 6 billion cells, 7 billion or about 7 billion cells, 8 billion or about 8 billion cells, 9 billion or about 9 billion cells, 10 billion or about 10 billion cells, 20 billion or about 20 billion cells, 30 billion or about 30 billion cells, 40 billion or about 40 billion cells), or any value between any of these ranges.

[0454] Thus, in some embodiments, NK cells are administered at 1 x 10 per dose. 6 , 2×10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 , or 9 x 10 6 or about 1 x 10 6 , 2×10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 , or 9×10 6 cells, 1 x 10 per dose 7 , 2×10 7 , 3×10 7 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 , or 9 x 10 7 or about 1 x 10 7 , 2×10 7 , 3×10 7 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 , or 9 x 10 7 cells, 1 x 10 per dose 8, 2×10 8 , 3×10 8 , 4×10 8 , 5×10 8 , 6×10 8 , 7×10 8 , 8×10 8 , or 9 x 10 8 or about 1 x 10 8 , 2×10 8 , 3×10 8 , 4×10 8 , 5×10 8 , 6×10 8 , 7×10 8 , 8×10 8 , or 9 x 10 8 cells, 1 x 10 per dose 9 , 2×10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , or 9 x 10 9 or about 1 x 10 9 , 2×10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , or 9 x 10 9 cells, 1 x 10 per dose 10 Or 2 x 10 10 or about 1 x 10 10 Or 2 x 10 10 The cell is administered in a dose containing 10 cells.

[0455] Thus, in some embodiments, the NK cells are at least 1 x 10 per dose. 6 , 2×10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 , or 9 x 106 or at least about 1 × 10 6 , 2×10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 , or 9 x 10 6 cells, at least 1 x 10 per dose 7 , 2×10 7 , 3×10 7 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 , or 9 x 10 7 or at least about 1 × 10 7 , 2×10 7 , 3×10 7 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 , or 9 x 10 7 cells, at least 1 x 10 per dose 8 , 2×10 8 , 3×10 8 , 4×10 8 , 5×10 8 , 6×10 8 , 7×10 8 , 8×10 8 , or 9 x 10 8 or at least about 1 × 10 8 , 2×10 8 , 3×10 8 , 4×10 8 , 5×10 8 , 6×10 8 , 7×10 8 , 8×10 8 , or 9 x 10 8 cells, at least 1 x 10 per dose 9 , 2×10 9 , 3×10 9 , 4×10 9 , 5×10 9, 6×10 9 , 7×10 9 , 8×10 9 , or 9 x 10 9 or at least about 1 × 10 9 , 2×10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , or 9 x 10 9 cells, or at least 1 x 10 per dose 10 Or 2 x 10 10 or at least about 1 × 10 10 Or 2 x 10 10 The cell is administered in a dose containing 10 cells.

[0456] In some embodiments, the dose of cells is a flat dose of cells or a fixed dose of cells and is not tied to or based on the patient's body surface area or weight.

[0457] In some embodiments, the dose of cells is administered based on the patient's weight. For example, the dose can be determined per kilogram of the patient's weight. In some embodiments, the dose of cells is 1×10 5 Or about 1 x 10 5 cells / kg to 1 x 10 8 Or about 1 x 10 8 cells / kg, e.g. 1.5 x 10 5 Or about 1.5 x 10 5 cells / kg to 1.5 x 10 7 Or about 1.5 x 10 7 cells / kg, or 4 x 10 5 Or about 4 x 10 5 cells / kg to 4 x 10 6 Or about 4 x 10 6 Contains cells / kg.

[0458] Thus, in some embodiments, NK cells are administered at a concentration of 1×10 5 , 1.5×10 5 , 2×10 5 , 2.5×10 5 , 3×10 5 , 3.5×10 5 , 4×10 5 , 4.5×10 5 , 5×10 5 , 5.5×10 5 , 6×10 5 , 6.5×10 5 , 7×10 5 , 7.5×10 5 , 8×10 5 , 8.5×10 5 , 9×10 5 , or 9.5 × 10 5 or about 1 x 10 5 , 1.5×10 5 , 2×10 5 , 2.5×10 5 , 3×10 5 , 3.5×10 5 , 4×10 5 , 4.5×10 5 , 5×10 5 , 5.5×10 5 , 6×10 5 , 6.5×10 5 , 7×10 5 , 7.5×10 5 , 8×10 5 , 8.5×10 5 , 9×10 5 , or 9.5 × 10 5 Cells / kg, 1 x 10 6 , 1.5×10 6 , 2×10 6 , 2.5×10 6 , 3×10 6 , 3.5×10 6 , 4×10 6 , 4.5×10 6 , 5×10 6 , 5.5×10 6 , 6×10 6 , 6.5×10 6 , 7×10 6 , 7.5×10 6 , 8×106 , 8.5×10 6 , 9×10 6 , or 9.5 × 10 6 cells / kg, or 1 x 10 7 , 1.5×10 7 , 2×10 7 , 2.5×10 7 , 3×10 7 , 3.5×10 7 , 4×10 7 , 4.5×10 7 , 5×10 7 , 5.5×10 7 , 6×10 7 , 6.5×10 7 , 7×10 7 , 7.5×10 7 , 8×10 7 , 8.5×10 7 , 9×10 7 Or about 1 x 10 7 , 1.5×10 7 , 2×10 7 , 2.5×10 7 , 3×10 7 , 3.5×10 7 , 4×10 7 , 4.5×10 7 , 5×10 7 , 5.5×10 7 , 6×10 7 , 6.5×10 7 , 7×10 7 , 7.5×10 7 , 8×10 7 , 8.5×10 7 , 9×10 7 cells / kg, or 9.5 x 10 7 pcs or 1×10 8 , 1.5×10 8 , or 2 × 10 8 or about 1 x 10 8 , 1.5×10 8 , or 2 × 10 8 The cells are administered in a dose containing cells / kg.

[0459] In some embodiments, the NK cells are at least 1 x 10 5 , 1.5×105 , 2×10 5 , 2.5×10 5 , 3×10 5 , 3.5×10 5 , 4×10 5 , 4.5×10 5 , 5×10 5 , 5.5×10 5 , 6×10 5 , 6.5×10 5 , 7×10 5 , 7.5×10 5 , 8×10 5 , 8.5×10 5 , 9×10 5 , or 9.5 × 10 5 or at least about 1 × 10 5 , 1.5×10 5 , 2×10 5 , 2.5×10 5 , 3×10 5 , 3.5×10 5 , 4×10 5 , 4.5×10 5 , 5×10 5 , 5.5×10 5 , 6×10 5 , 6.5×10 5 , 7×10 5 , 7.5×10 5 , 8×10 5 , 8.5×10 5 , 9×10 5 , or 9.5 × 10 5 cells / kg, at least 1 x 10 6 , 1.5×10 6 , 2×10 6 , 2.5×10 6 , 3×10 6 , 3.5×10 6 , 4×10 6 , 4.5×10 6 , 5×10 6 , 5.5×10 6 , 6×10 6 , 6.5×10 6 , 7×10 6 , 7.5×10 6 , 8×10 6 , 8.5×10 6 , 9×106 , or 9.5 × 10 6 or at least about 1 × 10 6 , 1.5×10 6 , 2×10 6 , 2.5×10 6 , 3×10 6 , 3.5×10 6 , 4×10 6 , 4.5×10 6 , 5×10 6 , 5.5×10 6 , 6×10 6 , 6.5×10 6 , 7×10 6 , 7.5×10 6 , 8×10 6 , 8.5×10 6 , 9×10 6 , or 9.5 × 10 6 cells / kg, at least 1 x 10 7 , 1.5×10 7 , 2×10 7 , 2.5×10 7 , 3×10 7 , 3.5×10 7 , 4×10 7 , 4.5×10 7 , 5×10 7 , 5.5×10 7 , 6×10 7 , 6.5×10 7 , 7×10 7 , 7.5×10 7 , 8×10 7 , 8.5×10 7 , 9×10 7 or at least about 1 × 10 7 , 1.5×10 7 , 2×10 7 , 2.5×10 7 , 3×10 7 , 3.5×10 7 , 4×10 7 , 4.5×10 7 , 5×10 7 , 5.5×10 7 , 6×10 7 , 6.5×10 7 , 7×10 7 , 7.5×10 7, 8×10 7 , 8.5×10 7 , 9×10 7 cells / kg, or 9.5 x 10 7 pieces, or at least 1×10 8 Or 1.5 x 10 8 or at least about 1 × 10 8 Or 1.5 x 10 8 The cells are administered in a dose containing cells / kg.

[0460] In some embodiments, a dose of cells, e.g., NK cells, is administered to a subject as a single administration, or is administered only once within a period of two weeks, one month, three months, six months, one year or more.

[0461] The ability to administer multiple doses may allow a patient to obtain a deeper or longer-lasting response from the treatment. Thus, in some embodiments, a patient receives multiple administrations of NK cells, such as two or more administrations or at least one subsequent administration. In some embodiments, the subject is administered two, three, four, five, six, seven, eight, nine, or ten times. In some embodiments, the at least one subsequent administration comprises a second administration. In some embodiments, the at least one subsequent administration comprises a second administration and a third administration. In some embodiments, the at least one subsequent administration comprises a second, third, and fourth administration. In some embodiments, the at least one subsequent administration comprises a second, third, fourth, and fifth administration. In some embodiments, the at least one subsequent administration comprises a second, third, fourth, fifth, and sixth administration. In some embodiments, the at least one subsequent administration comprises a second, third, fourth, fifth, sixth, and seventh administration. In some embodiments, the at least one subsequent administration includes a second, third, fourth, fifth, sixth, seventh, and eighth administration. In some embodiments, the patient can be given a response-based dosing, during which the patient continues to receive the NK cell therapy as long as the patient finds benefit. The number of administrations and the number of cells administered at each administration can also be tailored to the individual patient. In some embodiments, the number of cells administered to the subject in one or more additional or subsequent administrations is the same or similar to the first administration. Thus, the NK cell therapy described herein can be tailored to each patient based on their own response. In some cases, the treatment can be terminated if the patient no longer derives benefit from the NK cell therapy. In some cases, the treatment can be resumed if the patient relapses.

[0462] In some embodiments involving multiple or repeated administrations, the NK cells are administered once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, once every eleven weeks, once every twelve weeks, once every thirteen weeks, once every fourteen weeks, once every fifteen weeks, or once every sixteen weeks. For example, a patient may receive a first administration, a second administration, and a third administration, where each administration is administered one week apart. In some embodiments, the NK cells are administered once a month. In some embodiments, the NK cells are administered once every two or three months. In some embodiments, the NK cells are administered over a period of three weeks or about three weeks. In some embodiments, the NK cells are administered over a period of four weeks or about four weeks. In some embodiments, the NK cells are administered over a period of eight weeks or about eight weeks.

[0463] In some embodiments, the administration schedule can be changed during the course of treatment.Thus, for example, a patient can receive a first series (or cycle) of administration every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks, and a second series of administration every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks, and the time between the administration of the first series and the administration of the second series is different.In some embodiments, a patient can receive a first series of administration 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times, and a second series of administration 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times, and the number of administrations in the first series and the second series can be the same or different. Thus, for example, a patient may receive four doses administered every two weeks in a first series of doses, and four doses administered every 12 weeks in a second series of doses.

[0464] In some embodiments, the NK cells are administered 1-4 times over a 9 month period.

[0465] In some embodiments, the NK cells are cryopreserved in an infusion-ready medium, e.g., a cryopreservation composition suitable for intravenous administration, e.g., those described herein.

[0466] In some embodiments, the NK cells are present at 1 x 10 per vial. 7 or about 1 x 10 7 From 1 × 10 9 or about 1 x 10 9 In some embodiments, the NK cells are cryopreserved in vials containing single doses.

[0467] In some embodiments, the cells are thawed, for example in water at 37° C., prior to administration.

[0468] In some embodiments, a vial of thawed NK cells is aseptically transferred, for example, using a vial adapter and a sterile syringe, into a single administration container, e.g., an administration bag. The NK cells can be administered from the container to the patient by gravity through a Y-type transfusion / infusion set filter as an IV infusion.

[0469] In some embodiments, the NK cells are administered as soon as possible after thawing, preferably in less than 90 minutes, e.g., in less than 80, 70, 60, 50, 40, 30, 20, or 10 minutes, hi some embodiments, the NK cells are administered within 30 minutes of thawing.

[0470] In some embodiments, the pharmaceutical composition is administered intravenously via a syringe.

[0471] In some embodiments, 1 mL, 4 mL, or 10 mL of the formulation is administered intravenously via syringe to the patient.

[0472] In some embodiments, the patient is administered acetaminophen before administering the NK cell infusion. In some embodiments, the patient is administered 100, 200, 250, 300, 400, 500, 600, 700, 750, 800, 900, 1000, 1100, 1200, 1250, 1300, 1400, 1500, 1600, 1700, 1750, 1800, 1900, or 2000 mg of acetaminophen. In some embodiments, the acetaminophen is administered to the patient immediately before the NK cells. In some embodiments, the acetaminophen is administered 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 110, 120, 150, or 180 minutes before the NK cells. In some embodiments, the acetaminophen is administered orally.

[0473] In some embodiments, the patient is administered diphenhydramine before administering the NK cell infusion. In some embodiments, the patient is administered 5, 10, 12.5, 15, 17.5, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, or 100 mg of diphenhydramine. In some embodiments, the diphenhydramine is administered to the patient immediately before the NK cells. In some embodiments, the diphenhydramine is administered 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 110, 120, 150, or 180 minutes before the NK cells. In some embodiments, the acetaminophen is administered orally.

[0474] In some embodiments, the patient is monitored for a period of time before and after administration of NK cells. For example, the patient's vital signs can be monitored. These can include temperature, respiratory rate, heart rate, blood pressure, and oxygen saturation by pulse oximetry (SaO2). Optionally, the measurement of at least one vital sign begins 5, 10, 15 20, 25, or 30 minutes before administration of NK cells. After administration of NK cells, at least one vital sign can be monitored continuously for 1, 2, 3, 4, or 5 hours, or at regular or irregular intervals, such as approximately every 5, 10, 15, 20, 25, or 30 minutes. Optionally, vital signs can be monitored until the patient is in a stable state after administration of NK cells.

[0475] 2. Multispecific Engagers In some embodiments, the NK cells described herein, e.g., a pharmaceutical composition comprising the NK cells described herein, are administered to a patient in combination with a multispecific engager, e.g., a multispecific engager described herein, e.g., a CD30-targeted multispecific engager. In some embodiments, the multispecific engager is administered together with the NK cells as part of a pharmaceutical composition. In some embodiments, the multispecific engager is administered separately from the NK cells, e.g., as part of a separate pharmaceutical composition.

[0476] The multispecific engager can be administered before, after, or simultaneously with the administration of the NK cells.

[0477] In some embodiments, the multispecific engager is administered before the NK cells. In some embodiments, the multispecific engager is administered after the NK cells.

[0478] In some embodiments, the NK cells are administered the day after the multispecific engager is administered.

[0479] In some embodiments, the NK cells are administered at each dosing time, while the multispecific engager is administered on a portion of the dosing day, e.g., in some embodiments, the NK cells are administered once a week and the multispecific engager is administered once a month.

[0480] In some embodiments, a dose of the multispecific engager is given prior to the first administration of cells. In some embodiments, a debulking dose of the multispecific engager is given prior to the first administration of cells.

[0481] In some embodiments, the dose is from 0.01 or about 0.01 to 10 mg / kg, for example, 0.1 to 9, 0.01 to 8, 0.01 to 7, 0.01 to 6, 0.01 to 5, 0.01 to 4, 0.01 to 3, 0.01 to 2, 0.01 to 1.5, 0.01 to 1, 0.01 to 0.5, 0.01 to 0.15, 0.01 to 0.04, 0.04 to 10, 0.04 to 9, 0.04 to 8, 0.04 to 7, 0.04 to 6, 0.04 to 5, 0.04 to 4, 0.04 to 3, 0.04 to 2, 0.04 to 1.5, 0.04 to 1, 0.04 to 0.5, 0.04 to 0.15, 0.15 to 10, 0.15 to 9, 0.15~8, 0.15~7, 0.15~6, 0.15~5, 0.15~4, 0.15~3, 0.15~2, 0.15~1.5, 0.15~1, 0.15~0.5, 0.5~10, 0.5~9, 0.5~8, 0.5~7, 0.5~6, 0.5~5, 0.5~4, 0.5~3, 0. 5~2, 0.5~1.5, 0.5~1, 1~10, 1~9, 1~8, 1~7, 1~6, 1~5, 1~4, 1~3, 1~2, 1~1.5, 1.5~10, 1.5~9, 1.5~8, 1.5~7, 1.5~6, 1.5~5, 1.5~4, 1.5~3, 1.5~2, 2~10, 2~9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10 mg / kg, or about 0.1-9, 0.01-8, 0.01-7, 0.01-6, 0.01-5, 0.01-4, 0.01-3, 0.01-2, 0.01-1.5, 0.01-1, 0.01-0.5, 0.01-0.15, 0.01 ~0.04, 0.04~10, 0.04~9, 0.04~8, 0.04~7, 0.04~6, 0.04~5, 0.04~4, 0.04~3, 0.04~2, 0.04~1.5, 0.04~1, 0.04~0.5, 0.04~0.15, 0.15~10, 0.15~9, 0.15~ 8, 0.15~7, 0.15~6, 0.15~5, 0.15~4, 0.15~3, 0.15~2, 0.15~1.5, 0.15~1, 0.15~0.5, 0.5~10, 0.5~9, 0.5~8, 0.5~7, 0.5~6, 0.5~5, 0.5~4, 0.5~3, 0.5~2, 0.5~1.5, 0.5~1, 1~10, 1~9, 1~8, 1~7, 1~6, 1~5, 1~4, 1~3, 1~2, 1~1.5, 1.5~10, 1.5~9, 1.5~8, 1.5~7, 1.5~6 1.5-5, 1.5-4, 1.5-3, 1.5-2, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10 mg / kg of the multispecific engager is administered to the patient.

[0482] In some embodiments, at or about 0.01 mg / kg, 0.04 mg / kg, 0.015 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 1.5 mg / kg, 3.0 mg / kg, 4.5 mg / kg, or 7.0 mg / kg of a multispecific engager is administered to a patient.

[0483] In some embodiments, the dose of the multispecific engager is from at or about 100 to at or about 300 mg, e.g., 100-275, 100-250, 100-225, 100-200, 100-175, 100-150, 100-125, 125-300, 125-275, 125-250, 125-225, 125-20 0, 125~175, 125~150, 150~300, 150~275, 150~250, 150~225, 150~200, 150~175, 175~300, 175~275, 175~250, 175~225, 175~200, 200~300, 200~275, 200~250, 200~225, 225~300, 225~275, 22 5-250, 250-275, or 275-300 mg, or about 100-275, 100-250, 100-225, 100-200, 100-175, 100-150, 100-125, 125-300, 125-275, 125-250, 125-225, 125-200, 125-175, 125-150, 150-300, 150- Contains 275, 150-250, 150-225, 150-200, 150-175, 175-300, 175-275, 175-250, 175-225, 175-200, 200-300, 200-275, 200-250, 200-225, 225-300, 225-275, 225-250, 250-275, or 275-300 mg.

[0484] The multispecific engager can be administered by any suitable means, such as by bolus injection, or by injection, such as intravenous or subcutaneous injection, intraocular injection, periocular injection, subretinal injection, intravitreal injection, transseptal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjectval injection, subconjunctival injection, subtenon injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral delivery. In some embodiments, the multispecific engager is administered by parenteral, intrapulmonary and intranasal administration, as well as by intralesional administration, if desired for localized treatment. Parenteral injections include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. In some embodiments, a given dose is administered by a single bolus administration of the multispecific engager. In some embodiments, a given dose is administered by multiple bolus administration of the multispecific engager, for example over a period of 3 days or less, or by continuous infusion administration of the cells.

[0485] In some embodiments, the multispecific engager is administered prior to administration of the NK cells. In some embodiments, administration of the multispecific engager is completed at least 15, 30, 45, 60, 90, or 120 minutes before initiating administration of the NK cells. In some embodiments, the NK cells are administered prior to administration of the multispecific engager. In some embodiments, administration of the NK cells is completed at least 15, 30, 45, 60, 90, or 120 minutes before initiating administration of the multispecific engager. In some embodiments, the multispecific engager is administered at a rate such that the doses are administered over 1, 2, 3, 4, 5, 6, 7, or 8 hours. In some embodiments, the multispecific engager is administered at a rate such that the doses are administered over 4 hours.

[0486] In some embodiments, the multispecific engager will be administered once a week for 6 weeks and the NK cells will be administered once a week for 4 weeks. In some embodiments, the multispecific engager will be administered at a fixed dose of 200mg IV QW over 4 hours on days 1, 8, 15, 22, 29, and 36 of each cycle. In some embodiments, the NK cells will be administered at a dose of 1 billion or 4 billion cells on days 1, 8, and 15 of each cycle. In some embodiments, the patient will receive 1, 2, 3, 4, or 5 cycles of treatment. In some embodiments, there will be a 1, 2, 3, 4, or 5 week break between treatment cycles.

[0487] 3. Cytokines In some embodiments, a cytokine is administered to the patient.

[0488] In some embodiments, the cytokines are administered together with the NK cells as part of a pharmaceutical composition. In some embodiments, the cytokines are administered separately from the NK cells, e.g., as part of a separate pharmaceutical composition.

[0489] In some embodiments, the cytokine is IL-2.

[0490] In some embodiments, the IL-2 is administered subcutaneously.

[0491] In some embodiments, IL-2 is administered 1 to 4 or about 1 to about 4 hours following completion of NK cell administration. In some embodiments, IL-2 is administered at least 1 hour after completion of NK cell administration. In some embodiments, IL-2 is administered within 4 hours after completion of NK cell administration. In some embodiments, IL-2 is administered at least 1 hour and within 4 hours after completion of NK cell administration.

[0492] In some embodiments, up to 10 million IU / M 2, for example up to 1 million, 2 million, 3 million, 4 million, 5 million, 6 million, 7 million, 8 million, 9 million, or 10 million IU / m 2 of IL-2 is administered.

[0493] In some embodiments, 1 million or about 1 million, 2 million or about 2 million, 3 million or about 3 million, 4 million or about 4 million, 5 million or about 5 million, 6 million or about 6 million, 7 million or about 7 million, 8 million or about 8 million, 9 million or about 9 million, 10 million or about 10 million IU / M 2 of IL-2 is administered.

[0494] In some embodiments, 1 x 10 6 or about 1 x 10 6 IU / M 2 In some embodiments, 2×10 IL-2 is administered. 6 or about 2 x 10 6 IU / M 2 of IL-2 is administered.

[0495] In some embodiments, 1 x 10 6 IU / M 2 Less than 100 mg of IL-2 is administered to the patient.

[0496] In some embodiments, a flat dose of IL-2 is administered to the patient. In some embodiments, a flat dose of 6 million IU or about 6 million IU is administered to the patient.

[0497] In some embodiments, IL-2 is not administered to the patient.

[0498] 4. Pretreatment In some embodiments, the patient is pretreated with medication. In some embodiments, the medication is selected from the group consisting of H1 antagonists, H2 antagonists, acetaminophen, prophylactic antiemetics, and combinations thereof. In some embodiments, the H1 antagonist is diphenhydramine. In some embodiments, the H2 antagonist is famotidine.

[0499] E. Treatment Cycles and Regimens In some embodiments, the NK cells and multispecific engagers described herein are administered to a patient as part of a treatment cycle spanning multiple days, hi some embodiments, the NK cells and multispecific engagers are administered to a patient as part of a treatment regimen comprising one or more treatment cycles.

[0500] In some embodiments, the treatment regimen is directed to treating a patient's disorder (e.g., CD30 + The treatment is continued until the patient's cancer) progresses, or dosing is discontinued due to patient intolerance to the NK cells, multispecific engager, or both, or the patient experiences toxicity of the NK cells, multispecific engager, or both.

[0501] In some embodiments, the treatment regimen includes a treatment break (e.g., a period during which no administration of NK cells or multispecific engagers occurs) between treatment cycles. In some embodiments, the treatment break is 1-8 weeks, e.g., 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-8, 3-7, 3-6, 3-5, 3-4, 4-8, 4-7, 4-6, 4-5, 5-8, 5-7, 5-6, 6-8, 6-7, or 7-8 weeks. In some embodiments, the treatment break is at least 1, 2, 3, 4, 5, 6, 7, or 8 weeks. In some embodiments, the treatment break is 1, 2, 3, 4, 5, 6, 7, or 8 weeks, or about 1, 2, 3, 4, 5, 6, 7, or 8 weeks.

[0502] In some embodiments, the treatment cycle is from 2 to 60 days, e.g., from 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 10, 2 to 5, 5 to 60, 5 to 50, 5 to 40, 5 to 30, 5 to 20, 5 to 10, 10 to 60, 10 to 50, 10 to 40, 10 to 30, 10 to 20, 20 to 60, 20 to 50, 20 to 40, 20 to 30, 30 to 60, 30 to 50, 30 to 40, 40 to 60, 40 to 50, or 50 to 60 days. In some embodiments, the treatment cycle is 1-8 weeks (e.g., 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-8, 3-7, 3-6, 3-5, 3-4, 4-8, 4-7, 4-6, 4-5, 5-8, 5-7, 5-6, 6-8, 6-7, or 7-8 weeks). In some embodiments, the treatment cycle is at or about 1, 2, 3, 4, 5, 6, 7, or 8 weeks.

[0503] In some embodiments, the treatment regimen comprises 1 to 5 treatment cycles, such as 1 to 4, 1 to 3, 1 to 2, 2 to 5, 2 to 4, 2 to 3, 3 to 5, 3 to 4, or 4 to 5 treatment cycles. In some embodiments, the treatment regimen comprises 1, 2, 3, 4, or 5 treatment cycles.

[0504] In some embodiments, the treatment cycles of the treatment regimens are the same (e.g., follow the same dosing and timing schedule). In some embodiments, the treatment cycles of the treatment regimens are different (e.g., follow different dosing and timing schedules).

[0505] In some embodiments, a treatment cycle comprises multiple administrations of a multispecific engager, e.g., a multispecific engager described herein, and / or one or more administrations of NK cells, e.g., NK cells described herein.

[0506] As part of a treatment cycle, doses of the NK cells and multispecific engager are optionally administered together (e.g., simultaneously or sequentially during a single treatment day). In another example, the NK cells and multispecific engager are administered separately (e.g., on different treatment days).

[0507] In some embodiments, a treatment cycle includes treatment days that are evenly spaced throughout the treatment cycle, for example, if the treatment cycle is 6 weeks long (or thereabouts), then optionally treatment days are provided every week (or thereabouts).

[0508] In some embodiments, a treatment cycle comprises administration of a multispecific engager on each treatment day, hi some embodiments, a treatment cycle comprises administration of a multispecific engager on a portion of the treatment days.

[0509] In some embodiments, a treatment cycle comprises administration of NK cells on each treatment day. In some embodiments, a treatment cycle comprises administration of NK cells on a portion of the treatment days. In some embodiments, NK cells are administered only in the first half of a treatment cycle.

[0510] In some embodiments, the treatment cycle further comprises administration of a cytokine, e.g., a cytokine described herein, e.g., IL-2. In some embodiments, the cytokine is administered together with the NK cells and / or multispecific engager (e.g., simultaneously or sequentially with the NK cells and / or multispecific engager). In another example, the cytokine and are administered separately from the NK cells and / or multispecific engager (e.g., on different treatment days). In some embodiments, the cytokine (e.g., IL-2) is administered only on days that the NK cells are administered. In some embodiments, the cytokine (e.g., IL-2) is administered on each day that the NK cells are administered. In some embodiments, the cytokine (e.g., IL-2) is administered on some but not all of the days that the NK cells are administered.

[0511] In some embodiments, a treatment cycle comprises administering doses of a multispecific engager evenly spaced (or about) throughout the treatment cycle (e.g., once a week for a 6 week treatment cycle) and administering NK cells during the first half of the treatment cycle (e.g., on the same treatment day as the multispecific engager during the first half of the treatment cycle), optionally with IL-2 administration (e.g., as described above). Thus, optionally, a treatment cycle comprises treatment days where each treatment day comprises administration of NK cells, a multispecific engager, and IL-2 (e.g., 3 days evenly spaced or about the first 3 weeks of a 6 week treatment cycle, e.g., treatment days on days 1, 8, and 15), followed by treatment days where each treatment day comprises administration of the multispecific engager alone (e.g., 3 days evenly spaced or about the last 3 weeks of a 6 week treatment cycle, e.g., treatment days on days 22, 29, and 36). In some embodiments, the treatment regimen includes repeating this treatment cycle up to three times (e.g., 1, 2, or 3 times), and in some embodiments, the treatment regimen includes a treatment break between each treatment cycle (e.g., at or about 2-4 weeks). In some embodiments, the first of these treatment cycles includes lymphocyte depletion prior to the first treatment day (e.g., at or about days -3 and -4 for the first treatment cycle only). In some embodiments, each of these treatment cycles includes lymphocyte depletion prior to the first treatment day (e.g., at or about days -3 and -4 for each treatment cycle).

[0512] In some embodiments, the treatment cycle further comprises lymphodepletion, such as those described herein, hi some embodiments, lymphodepletion is performed prior to any administration of NK cells or multispecific engagers during the cycle.

[0513] Optionally, lymphodepletion for a treatment cycle is performed 1 to 5 days, e.g., 1 to 4, 1 to 3, 1 to 2, 2 to 5, 2 to 4, 2 to 3, 3 to 5, 3 to 4, or 4 to 5 days, before any dose of NK cells or multispecific engager is administered for a treatment cycle. Optionally, lymphodepletion for a treatment cycle is performed 1, 2, 3, 4, or 5 days before any dose of NK cells or multispecific engager is administered for a treatment cycle.

[0514] Optionally, the treatment regimen includes treatment cycles that each include lymphocyte depletion. Optionally, the treatment regimen includes some treatment cycles that include lymphocyte depletion and some treatment cycles that do not. Optionally, the first treatment cycle of a treatment regimen includes lymphocyte depletion and subsequent treatment cycles of the treatment regimen do not include lymphocyte depletion.

[0515] F. Medication An "effective amount" is an amount sufficient to achieve a beneficial or desired result. For example, a therapeutic amount is an amount that achieves a desired therapeutic effect. This amount may be the same as or different from a prophylactically effective amount, which is the amount necessary to prevent the onset of a disease or disease state. An effective amount can be administered in one or more administrations, applications or doses. The therapeutically effective amount (i.e., effective dosage) of a therapeutic compound will depend on the therapeutic compound selected. The composition can be administered one or more times per day to one or more times per week, including once every two days. Those skilled in the art will appreciate that the dosage and timing required to effectively treat a subject may depend on certain factors, including but not limited to the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and the presence of other diseases. Furthermore, treatment of a subject with a therapeutically effective amount of a therapeutic compound described herein can include a single treatment or a series of treatments.

[0516] The dosage, toxicity and therapeutic efficacy of therapeutic compounds can be determined in cell cultures or experimental animals by standard pharmaceutical procedures, for example, to determine the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between the toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio, LD50 / ED50. Compounds that exhibit high therapeutic indices are preferred. Although compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissues in order to minimize potential damage to non-infected cells, thereby reducing side effects.

[0517] Data obtained from cell culture assays and animal studies can be used to formulate a dosage range for use in humans. The dosage of such compounds can be within a range of circulating concentrations that include the ED50 with little or no toxicity. Dosages can vary within this range depending on the dosage form used and the route of administration utilized. For any compound used in the methods of the invention, a therapeutically effective amount can be estimated initially from cell culture assays. Doses can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound that achieves 50% maximal inhibition of symptoms) determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

[0518] VI. Variants In some embodiments, the molecules or components thereof described herein, fusion proteins or components thereof described herein, or NK cell genotypes described herein are at least 80%, e.g., at least 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of an exemplary sequence (e.g., those provided herein), with differences including, e.g., mutations described herein or other differences in addition to the mutations described herein, in up to 1%, 2%, 5%, 10%, 15%, or 20% of the residues of the exemplary sequences replaced, e.g., by conservative mutations. In preferred embodiments, the variants retain the desired activity of the parent.

[0519] To determine the percent identity of two nucleic acid sequences, the sequences are aligned for optimal comparison (e.g., gaps can be introduced into one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). The length of the reference sequence that is aligned for comparison is at least 80% of the full length of the reference sequence, and in some embodiments, at least 90% or 100%. The nucleotides at the corresponding amino acid or nucleotide positions are then compared. If a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, the molecules are identical at that position (as used herein, nucleic acid "identity" is equivalent to nucleic acid "homology"). The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences.

[0520] Percent identity between a subject polypeptide or nucleic acid sequence of interest (i.e., a query) and a second polypeptide or nucleic acid sequence (i.e., a target) can be determined in a variety of ways that are within the skill of the art, for example, using publicly available computer software, such as Smith Waterman alignment (Smith, T F and MS Waterman (1981) J Mol Biol 147:195-7; "BestFit" as incorporated in GeneMatcher Plus™ (Smith and Waterman, Advances in Applied Mathematics, 482-489 (1981)), Schwarz and Dayhof (1979) "Atlas of Protein Sequence and Structure," Dayhof, MO, ed., pp 353-358; BLAST programs (Basic Local Alignment Search Tool; (Altschul, S F, W. Gish, et al. (1990) J Mol Biol 215:403-10), BLAST-2, BLAST-P, BLAST-N, BLAST-X, WU-BLAST-2, ALIGN, ALIGN-2, CLUSTAL, or Megalign (DNASTAR) software. In addition, those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. In general, for a target protein or target nucleic acid, the length of comparison can be any length up to and including the full length of the target (e.g., 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%). For the purposes of this disclosure, percent identity is relative to the full length of the query sequence.

[0521] For the present disclosure, sequence comparisons and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix, with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0522] 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.

[0523] VII. Definitions Unless otherwise defined, all terminology, notations, and other technical and scientific terms and terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. In some cases, terms with commonly understood meanings are defined herein for clarity of description and / or ready reference, but the inclusion of such definitions herein should not necessarily be construed as representing a substantial departure from what is commonly understood in the art.

[0524] Throughout this application, various embodiments may be presented in a range format. Descriptions in range format should be understood to be merely for convenience and brevity, and should not be construed as inflexible limitations on the scope of the disclosure. Thus, the description of a range should be considered to specifically disclose all possible subranges and individual numbers within that range. For example, the description of a range such as 1-6 should be considered to specifically disclose subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, and the individual numbers within that range, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0525] As used in this specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a sample" includes a plurality of samples, including mixtures thereof.

[0526] The terms "determining," "measuring," "evaluating," "assessing," "assaying," and "analyzing" are often used interchangeably herein to refer to forms of measurement. These terms encompass determining whether an element is present (e.g., detecting). These terms can encompass quantitative, qualitative, or both quantitative and qualitative determinations. Evaluating can be relative or absolute. "Detecting the presence of" can encompass determining whether something is present or absent, as well as determining the amount of it present, depending on the context.

[0527] The terms "subject," "individual," or "patient" are often used interchangeably herein.

[0528] The term "in vivo" is used to describe events that take place inside a subject's body.

[0529] The term "ex vivo" is used to describe events that occur outside of a subject's body. An ex vivo assay is not performed on a subject. Instead, it is performed on a sample that is independent of the subject. An example of an ex vivo assay performed on a sample is an "in vitro" assay.

[0530] The term "in vitro" is used to describe events that occur in a container that holds laboratory reagents such that the material is separate from the biological source from which it is obtained. In vitro assays can include cell-based assays in which live or dead cells are used. In vitro assays can also include cell-free assays in which no intact cells are used.

[0531] As used herein, the term "about" with respect to a number refers to that number plus or minus 10% of that number. The term "about" with respect to a range refers to that range minus 10% of the lowest value and plus 10% of the highest value.

[0532] As used herein, the term "buffer solution" refers to an aqueous solution consisting of a mixture of a weak acid and its conjugate base, or vice versa.

[0533] As used herein, the term "cell culture medium" refers to a mixture for the growth and proliferation of cells in vitro that contains essential elements for cell growth and proliferation, such as sugars, amino acids, various nutrients, inorganic substances, etc.

[0534] A buffer solution, as used herein, is not a cell culture medium.

[0535] As used herein, the term "bioreactor" refers to a culture device capable of continuously controlling a range of conditions that affect cell culture, such as dissolved oxygen concentration, dissolved carbon dioxide concentration, pH, and temperature.

[0536] As used herein, the term "vector" refers to a nucleic acid molecule that has the ability to propagate another nucleic acid to which it is linked. This term encompasses both the vector as a self-replicating nucleic acid structure and the vector that is integrated into the genome of the host cell into which it is introduced. Several vectors are suitable for delivering the nucleic acid molecules or polynucleotides of the present application. Certain vectors have the ability to direct the expression of the nucleic acid to which they are functionally linked. Such vectors are referred to herein as "expression vectors".

[0537] The term "operably linked" refers to two or more nucleic acid sequences or polypeptide elements that are usually physically linked and in a functional relationship to each other. For example, a promoter is operably linked to a coding sequence if the promoter is capable of initiating or regulating the transcription or expression of the coding sequence, and the coding sequence is then understood to be "under the control" of the promoter.

[0538] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells encompass "engineered cells," "transformants," and "transformed cells," which include the primary engineered (e.g., transformed) cell and its progeny, regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are encompassed herein.

[0539] Optionally, host cells can be stably or transiently transfected with a polynucleotide encoding a fusion protein as described herein.

[0540] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. EXAMPLES

[0541] VIII. Working Examples The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

[0542] Example 1: Off-the-shelf NK cell therapy platform An example of a method for expanding and stimulating NK cells is shown in FIG. Anti-affinity variants of the receptor CD16 (158V / V variant, see e.g., Koene et al. "FcγRIIIa-158V / F Polymorphism Influences the Binding of IgG by Natural Killer Cell FcgammaRIIIa, Independently of the FcgammaRIIIa-48L / R / H Phenotype," Blood 90:1109-14 (1997)) and KIR-B genotype (KIR B alleles of the KIR receptor family, see e.g., Hsu et al. "The Killer Cell Immunoglobulin-Like Receptor (KIR) Genomic Region: Gene-Order, Haplotypes and Allelic Polymorphism," Immunological Review 190:40-52 (2002) and Pyo et al. "Different Patterns of Evolution in the Centromeric and Telomeric Regions of Group A and B Haplotypes of the Human Killer Cell A single unit of FDA-approved frozen umbilical cord blood bearing a marker gene (Ig-like Receptor Locus, see PLoS One 5:e15115 (2010)) was selected as the source of NK cells.

[0543] The cord blood units were thawed and the freezing medium was removed by centrifugation. The cell preparation was then depleted of T cells using the QuadroMACS cell selection system (Miltenyi) and CD3 (T cell) MicroBeads. 6 × 10 8Total nucleated cell (TNC) populations were labeled with microbeads and separated using a QuadroMACS device and buffer. After T cell depletion, the remaining cells, primarily monocytes and NK cells, were washed and collected in antibiotic-free medium (CellgroSCGM). The cell preparations were then assessed for total nucleated cell count, viability, and %CD3+ cells. Cord blood NK cells were CD3 depleted as shown in Figure 1.

[0544] CD3- cell preparations were inoculated into gas-permeable cell expansion bags containing growth medium. To enhance expansion for master cell bank (MCB) production, cells were co-cultured with replication-incompetent engineered HuT-78 (eHUT-78) feeder cells. CellgroSCGM growth medium was initially supplemented with 10 ng / mL anti-CD3 antibody (OKT3), human plasma, glutamine, and IL-2.

[0545] As shown in FIG. 1, the NK cells are optionally engineered during one of the co-culture steps, e.g., with a lentiviral vector to introduce, e.g., a CAR, into the NK cells.

[0546] The cells were incubated as static cultures at 37°C in a 5% CO2 balanced air environment for 12-16 days with additional medium changes every 2-4 days. After the cultures expanded more than 100-fold, the cultured cells were harvested and then suspended in freezing medium and packed into freezing bags. In this example, 10 cells were cultured per bag or vial during co-culture. 8 80 bags or vials of cells were produced. The cryobags were frozen using a controlled rate freezer and stored in vapor phase liquid nitrogen (LN2) tanks at <-150°C. These cryopreserved NK cells obtained from an FDA-approved cord blood unit constituted the Master Cell Bank (MCB).

[0547] To produce the product, a bag of frozen cells from the MCB was thawed and the freezing medium was removed. The thawed cells were inoculated into disposable culture bags and co-cultured with feeder cells, e.g., eHUT78 feeder cells, to produce the product. In this example, several thousand lots of product (e.g., 10 9 Cells are cultured in a 50 L bioreactor, mixed with a cryopreservation composition, and frozen in multiple storage containers such as cryovials to produce a total volume of 4,000-8,000 frozen vials at cells / vial. This formulation is a ready-to-use, injection-ready product that can be used for direct injection. Each lot of the formulation is designed to be administered to hundreds to thousands of patients (e.g., to reach a target dose of 4×10 9 As individual cells, they can be used for injection into individual patients (e.g., 100 to 1,000 patients).

[0548] Example 2: Feeder cell expansion As an example, suitable feeder cells, e.g., eHut-78 cells, were thawed from frozen stocks and expanded and cultured in 125 mL flasks in RPMI 1640 (Life Technologies) 89% v / v, inactivated fetal bovine serum (FBS) (Life Technologies) (10% v / v) and glutamine (hyclone) (2 mM) at or about 37°C and 3-7 or about 3-7% CO2 for or about 18-24 days. Every 2-3 days, cells were split into 125 mL to 2 L flasks. Cells were harvested by centrifugation and gamma irradiated. Harvested and irradiated cells were mixed with cryopreservation medium (Cryostor CS10) in 2 mL cryovials and frozen in a controlled rate freezer with a temperature drop of about 15°C every 5 minutes to a final temperature of at or about -90°C, then transferred to a liquid nitrogen tank or freezer to a final temperature of at or about -150°C.

[0549] After freezing, cell viability is approximately equal to the original cell number (here, at least 1.0 × 10 8≥70% of cells expressed mTNF-α, ≥85% of cells expressed mbIL-21+, or ≥85% of cells expressed 4-1BBL.

[0550] Example 3: Expansion and stimulation of NK cells As an example, suitable NK cells can be prepared using HuT-78 cells transduced to express 4-1BBL, membrane-bound IL-21 and mutant TNF alpha ("eHut-78P cells") as feeder cells as follows: The feeder cells are suspended in 1% (v / v) CellGro medium and irradiated with 20,000 cGy in a gamma irradiator. Seed cells (e.g. CD3-depleted PBMCs or CD3-depleted umbilical cord blood cells) are grown on the feeder cells in CellGro medium containing human plasma, glutamine, IL-2 and OKT-3 in static culture at 37°C. Split the cells every 2-4 days. The total culture period was 19 days. The NK cells are harvested by centrifugation and stored frozen. Thawed NK was dissolved in phosphate-buffered saline (PBS 1×, FujiFilm Irvine) (50% v / v), albumin (human) (20% v / v OctaPharma albumin solution, which is 200 g / L protein with ≥96% human albumin, 130–160 mmol sodium, ≤2 mmol potassium, 0.064–0.096 mmol / g protein N-acetyl-DL-tryptophan, 0.064–0.096 mmol / g protein caprylic acid, water to a total volume of 1000 ml), and dextran 40 in Dextrose (25% Hospira Dextran 40 Dextrose Injection, USP, v / v, which contains 10 g / 100 mL of dextran 40 and 5 g / 100 mL of dextrose hydrate in water) and dimethyl sulfoxide (DMSO) (1.101 g / cm at 20 °C) 3 The antibody is administered to the patient in an infusion medium consisting of a 5% v / v solution of Avantor DMSL having a density of 100 μg / ml.

[0551] In some examples, the seed cells are CD3-depleted cord blood cells. The cell fraction can be depleted of CD3 cells by immunomagnetic selection using, for example, the CliniMACS T cell depletion set (LS depletion set (162-01) Miltenyi Biotec).

[0552] Preferably, the cord blood seed cells are selected to express CD16 with a V / V polymorphism at F158 (Fc gamma RIIIa-158V / V genotype) (Musolino et al. 2008 J Clin Oncol 26:1789). Preferably, the cord blood seed cells are KIR-B haplotype.

[0553] Example 4: Umbilical cord blood as a source of NK cells NK cells comprise 5-15% of peripheral blood lymphocytes. Traditionally, peripheral blood has been used as a source of NK cells for therapeutic use. However, as shown herein, NK cells derived from umbilical cord blood have an almost 10-fold higher expansion potential than cells derived from peripheral blood in the culture systems described herein, without premature cell exhaustion or senescence. Expression of receptors of interest on the NK cell surface, such as those involved in NK cell activation upon tumor cell engagement, appeared to be more consistent between donors in umbilical cord blood NK cells than in peripheral blood NK cells. Use of the manufacturing process described herein consistently activated NK cells in umbilical cord blood in a donor-independent manner, resulting in a highly scaled, active and consistent NK cell product.

[0554] In preclinical studies, cord blood-derived NK cells (CB-NK) have been shown to have approximately 10-fold greater ability to expand in culture than peripheral blood-derived NK cells (PB-NK), as shown in Figure 2. Cord blood-derived preparations showed higher and more consistent expression of tumor-engaging NK-activating immune receptors compared to those generated from peripheral blood, as shown in Figure 3.

[0555] Example 5: Expanded and stimulated NK cell phenotype In one example, NK cells from a cord blood unit are expanded and stimulated with eHut-78 cells according to the expansion and stimulation process described in Example 1. As shown in Figure 4, the resulting population of expanded and stimulated NK cells consistently have high CD16(158V) activating NK cell receptor expression.

[0556] Example 6: AB-101 AB-101 is a versatile, pre-made, cryopreserved allogeneic cord blood-derived NK cell therapy product containing ex vivo expanded and activated effector cells designed to enhance ADCC anti-tumor responses in patients, e.g., those treated with monoclonal antibodies or NK cell engagers. AB-101 is composed of cord blood-derived mononuclear cells (CBMCs) that have been enriched for NK cells by T lymphocyte depletion and cultured with a replication-incompetent engineered T cell feeder line supplemented with IL-2 and anti-CD3 antibody (OKT3).

[0557] AB-101 is an FDA-approved cord blood derived allogeneic NK cell line specifically designed to treat hematological and solid tumors in combination with therapeutic monoclonal antibodies (mAbs). The AB-101 manufacturing process results in an NK cell product with the following attributes: Consistent NK cell profile. High surface receptor expression of antibody-engaging CD16 and tumor antigen-engaging / activating receptors, e.g. NKG2D, NKp46, Nkp30 and NKp44. KIR-B haplotypes. KIR-B haplotypes are associated with improved clinical outcomes in the haploidentical transplant setting and increased therapeutic potential in the allogeneic setting. · CD16 F158V polymorphism. Binding of the high affinity CD16 F158V variant to mAb Fc domains has been recognized to promote enhanced antibody-dependent cellular cytotoxicity (ADCC). Unmodified NK cells. Genetic augmentation or gene editing is not required for and is not included in the AB-101 formulation.

[0558] The components and composition of AB-101 are listed in Table 6. AB-101 inhibits NK cells (CD16 + , CD56 + ). AB-101 contains negligible T cells, B cells, and macrophages (≤0.2% CD3 + , ≤1.0% CD19 + , ≤1.0% CD14 + ). Residual eHuT-78P feeder cells used in the culture of AB-101 represent ≦0.2% of the preparation.

[0559] Table 6. Components and composition of AB-101 TIFF2024542159000007.tif107156

[0560] Initial stability studies indicate that AB-101 is stable in the vapor phase of liquid nitrogen for up to six months. Long-term stability studies to evaluate product stability beyond six months are currently underway, and updated stability information will be incorporated into the Certificate of Analysis.

[0561] The manufacture of AB-101 formulation consists of the following key steps (Figure 5): Thawing of FDA approved cord blood units (Hemacord, BLA125937) Removal of cryopreservation medium from cord blood units (CBUs) ·CD3 depletion using the FDA cleared Vario MACS Cell Selection System (Miltenyi) Expansion and co-culture in bags using an engineered feeder cell line (eHuT-78 cells) · Testing and cryopreservation of AB-101 master cell bank (approximately 200 bags) Thawing (single bag), expansion, and co-culture with engineered HuT-78 cells Further expansion in bioreactors Collection and filling (1 x 10 per vial 9 NK cells) - Freeze storage of AB-101 formulation (approximately 150 vials) Extensive characterization to determine consistency, purity, potency and safety.

[0562] This manufacturing process reproducibly produces extremely large quantities of highly pure and active AB-101 formulated NK cells, as shown in Table 7. Data points represent product generated from three independent cord blood units.

[0563] (Table 7) AB-101 product characteristics analysis TIFF2024542159000008.tif79160

[0564] Identity (CD3-, CD56+) The frequency of CD3-, CD56+ cells is used to assess the identity of the AB-101 formulation. A sample of the AB-101 formulation is thawed and resuspended in staining buffer. The resuspended sample is added to a fluorochrome-labeled ...

Claims

1. CD30 ＋ A pharmaceutical for use in a method for treating a patient suffering from cancer, comprising as active ingredients, in the same or separate compositions, natural killer cells (NK cells) comprising a KIR-B haplotype and expression of a CD16 molecule, and a bispecific antibody or antigen-binding fragment thereof comprising a first binding domain that specifically binds to CD16 (FcγRIII) and a second binding domain that specifically binds to CD30; the method comprises administering to the patient simultaneously or separately the natural killer cells and the bispecific antibody or antigen-binding fragment thereof; The first binding domain that specifically binds to CD16 is a light chain variable domain (VL_CD16A) comprising a light chain complementarity determining region 1 (CDRL1) comprising SEQ ID NO:9, a light chain complementarity determining region 2 (CDRL2) comprising SEQ ID NO:10, and a light chain complementarity determining region 3 (CDRL3) comprising SEQ ID NO:11; a heavy chain variable domain (VH_CD16A) comprising a heavy chain complementarity determining region 1 (CDRH1) comprising SEQ ID NO:6, a heavy chain complementarity determining region 2 (CDRH2) comprising SEQ ID NO:7, and a heavy chain complementarity determining region 3 (CDRH3) comprising SEQ ID NO:8; and The second binding domain that specifically binds to CD30 is a light chain variable domain (VL_CD30) comprising a light chain complementarity determining region 1 (CDRL1) comprising SEQ ID NO: 15, a light chain complementarity determining region 2 (CDRL2) comprising SEQ ID NO: 16, and a light chain complementarity determining region 3 (CDRL3) comprising SEQ ID NO: 17; a heavy chain variable domain (VH_CD30) comprising a heavy chain complementarity determining region 1 (CDRH1) comprising SEQ ID NO: 12, a heavy chain complementarity determining region 2 (CDRH2) comprising SEQ ID NO: 13, and a heavy chain complementarity determining region 3 (CDRH3) comprising SEQ ID NO: 14; Including, The medicine.

2. A pharmaceutical for use in a method for treating a patient suffering from CD30 + cancer, comprising natural killer cells (NK cells) comprising a KIR-B haplotype and expression of a CD16 molecule as an active ingredient; the method comprises administering to the patient the natural killer cells in combination with a bispecific antibody or antigen-binding fragment thereof, the bispecific antibody comprising a first binding domain that specifically binds to CD16 (FcγRIII) and a second binding domain that specifically binds to CD30; The first binding domain that specifically binds to CD16 is a light chain variable domain (VL_CD16A) comprising a light chain complementarity determining region 1 (CDRL1) comprising SEQ ID NO:9, a light chain complementarity determining region 2 (CDRL2) comprising SEQ ID NO:10, and a light chain complementarity determining region 3 (CDRL3) comprising SEQ ID NO:11; a heavy chain variable domain (VH_CD16A) comprising a heavy chain complementarity determining region 1 (CDRH1) comprising SEQ ID NO:6, a heavy chain complementarity determining region 2 (CDRH2) comprising SEQ ID NO:7, and a heavy chain complementarity determining region 3 (CDRH3) comprising SEQ ID NO:8; and The second binding domain that specifically binds to CD30 is a light chain variable domain (VL_CD30) comprising a light chain complementarity determining region 1 (CDRL1) comprising SEQ ID NO: 15, a light chain complementarity determining region 2 (CDRL2) comprising SEQ ID NO: 16, and a light chain complementarity determining region 3 (CDRL3) comprising SEQ ID NO: 17; a heavy chain variable domain (VH_CD30) comprising a heavy chain complementarity determining region 1 (CDRH1) comprising SEQ ID NO: 12, a heavy chain complementarity determining region 2 (CDRH2) comprising SEQ ID NO: 13, and a heavy chain complementarity determining region 3 (CDRH3) comprising SEQ ID NO: 14; Including, The medicine.

3. A pharmaceutical for use in a method for treating a patient suffering from a CD30+ cancer, the pharmaceutical comprising, as an active ingredient, a bispecific antibody or antigen-binding fragment thereof, the bispecific antibody comprising a first binding domain that specifically binds to CD16 (FcγRIII) and a second binding domain that specifically binds to CD30; the method comprises administering to the patient the bispecific antibody or antigen-binding fragment thereof in combination with natural killer cells (NK cells) comprising a KIR-B haplotype and expression of a CD16 molecule; The first binding domain that specifically binds to CD16 is a light chain variable domain (VL_CD16A) comprising a light chain complementarity determining region 1 (CDRL1) comprising SEQ ID NO:9, a light chain complementarity determining region 2 (CDRL2) comprising SEQ ID NO:10, and a light chain complementarity determining region 3 (CDRL3) comprising SEQ ID NO:11; a heavy chain variable domain (VH_CD16A) comprising a heavy chain complementarity determining region 1 (CDRH1) comprising SEQ ID NO:6, a heavy chain complementarity determining region 2 (CDRH2) comprising SEQ ID NO:7, and a heavy chain complementarity determining region 3 (CDRH3) comprising SEQ ID NO:8; and The second binding domain that specifically binds to CD30 is a light chain variable domain (VL_CD30) comprising a light chain complementarity determining region 1 (CDRL1) comprising SEQ ID NO: 15, a light chain complementarity determining region 2 (CDRL2) comprising SEQ ID NO: 16, and a light chain complementarity determining region 3 (CDRL3) comprising SEQ ID NO: 17; a heavy chain variable domain (VH_CD30) comprising a heavy chain complementarity determining region 1 (CDRH1) comprising SEQ ID NO: 12, a heavy chain complementarity determining region 2 (CDRH2) comprising SEQ ID NO: 13, and a heavy chain complementarity determining region 3 (CDRH3) comprising SEQ ID NO: 14; Including, The medicine.

4. A pharmaceutical described in any one of claims 1 to 3, wherein the method includes a first treatment cycle and a second treatment cycle, and the first treatment cycle and the second treatment cycle are the same or different.

5. (a) natural killer cells (NK cells) containing a KIR-B haplotype and expression of the CD16 molecule; and (b) a bispecific antibody or an antigen-binding fragment thereof, comprising a first binding domain that specifically binds to CD16 (FcγRIII) and a second binding domain that specifically binds to CD30; 1. A pharmaceutical composition comprising: The first binding domain that specifically binds to CD16 is a light chain variable domain (VL_CD16A) comprising a light chain complementarity determining region 1 (CDRL1) comprising SEQ ID NO:9, a light chain complementarity determining region 2 (CDRL2) comprising SEQ ID NO:10, and a light chain complementarity determining region 3 (CDRL3) comprising SEQ ID NO:11; a heavy chain variable domain (VH_CD16A) comprising a heavy chain complementarity determining region 1 (CDRH1) comprising SEQ ID NO:6, a heavy chain complementarity determining region 2 (CDRH2) comprising SEQ ID NO:7, and a heavy chain complementarity determining region 3 (CDRH3) comprising SEQ ID NO:8; and The second binding domain that specifically binds to CD30 is a light chain variable domain (VL_CD30) comprising a light chain complementarity determining region 1 (CDRL1) comprising SEQ ID NO: 15, a light chain complementarity determining region 2 (CDRL2) comprising SEQ ID NO: 16, and a light chain complementarity determining region 3 (CDRL3) comprising SEQ ID NO: 17; a heavy chain variable domain (VH_CD30) comprising a heavy chain complementarity determining region 1 (CDRH1) comprising SEQ ID NO: 12, a heavy chain complementarity determining region 2 (CDRH2) comprising SEQ ID NO: 13, and a heavy chain complementarity determining region 3 (CDRH3) comprising SEQ ID NO: 14; Including, The pharmaceutical composition.

6. The pharmaceutical composition of claim 5, wherein the CD16 molecule is a CD16A molecule.

7. 7. The pharmaceutical composition of claim 5 or 6, wherein the CD16 molecule comprises a V / V polymorphism at F158.

8. The pharmaceutical composition of claim 5, wherein the bispecific antibody that specifically binds to CD16 specifically binds to CD16A.

9. The first binding domain that specifically binds to CD16 is a light chain variable (V) comprising SEQ ID NO:20 L ) region and heavy chain variable (V H 6. The pharmaceutical composition of claim 5, comprising a .alpha.-.beta ...

10. The first binding domain that specifically binds to CD16 comprises an amino acid sequence that has 85% identity or at least 85% identity to SEQ ID NO:

20. L and a V region comprising an amino acid sequence having 85% identity or at least 85% identity to SEQ ID NO:

19. H 6. The pharmaceutical composition of claim 5, comprising a region.

11. The second binding domain that specifically binds to CD30 is a light chain variable (V) comprising SEQ ID NO:

22. L ) region and the heavy chain variable (V H 6. The pharmaceutical composition of claim 5, comprising a .alpha.-.beta ...

12. The second binding domain that specifically binds to CD30 comprises an amino acid sequence that has 85% identity or at least 85% identity to SEQ ID NO:

22. L and a V region comprising an amino acid sequence having 85% identity or at least 85% identity to SEQ ID NO:

21. H 6. The pharmaceutical composition of claim 5, comprising a region.

13. the bispecific antibody or antigen-binding fragment thereof is a bispecific antigen-binding fragment, and the variable domains of the bispecific antigen-binding fragment are linked by peptide linkers L1, L2 and L3 in the following order from the N-terminus to the C-terminus: VH_CD30-L1-VL_CD16A-L2-VH_CD16A-L3-VL_CD30; 6. The pharmaceutical composition of claim 5.

14. the bispecific antibody or antigen-binding fragment thereof is a bispecific antigen-binding fragment, the variable domains of the bispecific antigen-binding fragment are linked by peptide linkers L1, L2 and L3 in the following order from the N-terminus to the C-terminus: VH_CD16A-L1-VL_CD30-L2-VH_CD30-L3-VL_CD16A; 6. The pharmaceutical composition of claim 5.

15. 15. The pharmaceutical composition of claim 13 or 14, wherein each of the peptide linkers L1, L2 and L3 consists of 12 or fewer amino acid residues.

16. The pharmaceutical composition according to claim 13 or 14, wherein the linker L2 of the antibody construct consists of 3 to 9 amino acid residues inclusive.

17. 6. The pharmaceutical composition of claim 5, wherein the bispecific antibody or antigen-binding fragment thereof comprises an amino acid sequence having 85% identity or at least 85% identity to SEQ ID NO:

18.

18. 6. The pharmaceutical composition of claim 5, wherein the bispecific antibody or antigen-binding fragment thereof is a bispecific antigen-binding fragment comprising the amino acid sequence shown in SEQ ID NO:

18.

19. 6. The pharmaceutical composition of claim 5, wherein the NK cells are cord blood-derived NK cells.

20. To produce cord blood-derived natural killer cells, (a) providing a sample of cord blood cells containing natural killer cells; (b) removing CD3(+) cells from the cells; (c) expanding the natural killer cells by culturing the seed cells with a first plurality of cells from an inactivated CD4(+) T cell line in a medium containing a T cell stimulatory antibody selected from the group consisting of OKT3, UCHT1, HTa, or a combination thereof, and IL-2.

20. The pharmaceutical composition of claim 19, wherein the cord blood-derived NK cells are produced by a method comprising:

21. The pharmaceutical composition of claim 20, wherein the inactivated CD4(+) T cell line expresses at least one gene selected from the group consisting of the 4-1BBL gene, the membrane-bound IL-21 (mbIL-21) gene, the OX40L gene, and the mouse TNF-α gene.

22. The pharmaceutical composition of claim 21, wherein the inactivated CD4(+) T cell line expresses the 4-1BBL gene, the membrane-bound IL-21 (mbIL-21) gene, and the mouse TNF-α gene.

23. The pharmaceutical composition of claim 5, wherein the first binding domain of the bispecific antibody or antigen-binding fragment thereof that specifically binds to CD16 binds to a CD16 molecule on an NK cell.

24. (a) human albumin, (b) dextran, (c) glucose, (d) DMSO, and (e) Buffer solution 6. The pharmaceutical composition of claim 5, further comprising:

25. 25. The pharmaceutical composition of claim 24, comprising 30 to 50 mg / mL of human albumin.

26. 26. The pharmaceutical composition of claim 25, comprising 50 mg / mL human albumin.

27. 25. The pharmaceutical composition of claim 24, comprising 20 to 30 mg / mL of dextran.

28. 28. The pharmaceutical composition of claim 27, comprising 25 mg / mL of dextran.

29. 25. The pharmaceutical composition of claim 24, wherein the dextran is dextran 40.

30. 25. The pharmaceutical composition of claim 24, comprising 12 to 15 mg / mL of glucose.

31. 31. The pharmaceutical composition of claim 30, comprising 12.5 mg / mL glucose.

32. 25. The pharmaceutical composition of claim 24, comprising less than 27.5 g / L of glucose.

33. 25. The pharmaceutical composition of claim 24, comprising 50-60 ml / mL of DMSO.

34. 34. The pharmaceutical composition of claim 33, comprising 55 mg / mL of DMSO.

35. 25. The pharmaceutical composition of claim 24, comprising 40-60% v / v of a buffer.

36. 25. The pharmaceutical composition of claim 24, wherein the buffer is phosphate buffered saline.

37. (a) about 40 mg / mL human albumin; (b) Dextran 40 at approximately 25 mg / mL; (c) approximately 12.5 mg / mL glucose; (d) about 55 mg / mL DMSO, and (e) approximately 0.5 mL / mL of phosphate buffered saline 25. The pharmaceutical composition of claim 24, comprising:

38. 25. The pharmaceutical composition of claim 24, further comprising 0.5 mL / mL of water.

39. 6. The pharmaceutical composition of claim 5, further comprising a pharmaceutically acceptable excipient.

40. 6. A frozen vial containing the pharmaceutical composition of claim 5.

41. CD30 ＋ 6. The pharmaceutical composition of claim 5 for treating a patient suffering from cancer.

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