TREATMENT OF CANCER WITH NK CELLS AND MULTISPECIFIC ENGAGERS

BACKGROUND

[0001] Targeted therapies, including antibody therapy, have revolutionized cancer treatment. One mechanism of action 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 may render any of the indicated monoclonal antibody therapeutics significantly less effective for these patients, which could prevent these patients from responding or lead to relapse. Thus, a reduced ADCC response could negatively impact their clinical outcomes.

[0002] Despite recent discoveries and developments of several anti-cancer agents, there is still a need for improved methods and therapeutic agents due to poor prognosis for many types of cancers.

[0003] T he present invention addresses these and other deficiencies in the art.

SUMMARY

[0004] NK cells are immune cells that can engage tumor cells through a complex array of receptors on their cell surface, as well as through antibody-dependent cellular cytotoxicity (ADCC). To initiate ADCC, NK cells engage with antibodies via the CD16 receptor on their surface. NK cells may have an advantage over other immune cells, such as the T cells used in CAR-T cell therapy and other cell therapies. In an exemplary advantage, NK cells can be used as allogeneic therapies, meaning that NK cells from one donor can be safely used in one or many patients without the requirement for HLA matching, gene editing, or other genetic manipulations. Allogeneic NK cells with anti-tumor activity can be administered safely to patients without many of the risks associated with T cell therapies, such as severe cytokine release syndrome (CRS), and neurological toxicities or graft versus host disease (GvHD).

[0005] Allogeneic NK cells may provide an important treatment option for cancer patients. In one exemplary/ advantage, NK cells have been well tolerated without evidence of graft-versus-host disease, neurotoxicity or cytokine release syndrome associated with other cell- based therapies. In another exemplary advantage, NK cells do not require prior antigen exposure or expression of a specific antigen to identify and lyse tumor cells. In another exemplary advantage, NK cells have the inherent ability to bridge between innate immunity and engender a multi-clonal adaptive immune response resulting in long-term anticancer immune memory.

[0006] 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. Through direct killing of tumor cells, NK cells also expose tumor antigens for recognition by the adaptive immune system.

[0007] Additionally, cords with preferred characteristics for enhanced clinical activity (e.g., high-affinity CD 16 and Killer cell Immunoglobulin-like Receptor (KIR) B-haplotype) can be selected by utilizing a diverse umbilical cord blood bank as a source for NK cells.

[0008] The administration of the allogenic NK cells, as described herein, can enhance patients’ ADCC responses, e.g., in combination with a multispecific engager, e.g., a multispecific engager described herein.

[0009] Thus, provided herein are pharmaceutical compositions 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 compri sing a first binding domain that specifically binds to CD 16 (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_CDI6A) 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; and 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 wherein the second binding domain that specifically binds to CD30 comprises: 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; 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. [0010] In some embodiments, the CD16 molecule is a CD16A molecule.

[0011] In some embodiments the CD 16 molecule comprises a V/V polymorphism at

Fl 58.

[0012] In some embodiments, the bispecific antibody that specifically binds to CD 16 specifically binds to CD16A.

[0013] In some embodiments, the first binding domain that specifically binds to CD 16 comprises a light chain variable (VL) region comprising SEQ ID NO: 20 and a heavy chain variable (V _H ) region comprising SEQ ID NO: 19.

[0014] In some embodiments, the first binding domain that specifically binds to CD 16 comprises a VL region comprising an amino acid sequence having or having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 20 and a V _H region comprising an amino acid sequence having or having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 19.

[0015] In some embodiments, the second binding domain that specifically binds to CD30 comprises a light chain variable (VL) region comprising SEQ ID NO: 22 and a heavy chain variable (V _H ) region comprising SEQ ID NO: 21.

[0016] In some embodiments the second binding domain that specifically binds to CD30 comprises a VL region comprising an amino acid sequence having or having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 22 and a V _H region comprising an amino acid sequence having or having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 21.

[0017] 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 L 1, L2, and L3 from the N-terminus to the C- terminus in the order: VH_CD30 - L 1 - VL, CD16A - L2 - VH_CD16A - L3 - VL_CD30. In some embodiments each of peptide linkers L 1, L2, and L3 consist of no more than 12 amino acid residues. In some embodiments, linker L,2 of the antibody construct consists of between 3 and 9 amino acid residues, inclusive.

[0018] In some embodiments, the bispecific antibody or antigen binding fragment thereof is a bi specific antigen binding fragment; and the variable domains of the bispecific antigen binding fragment are linked by peptide linkers L 1, L2, and L3 from the N-terminus to the C- terminus in the order: VH_CD16A - L1 - VL_CD30 - L2 - VH_CD30 - L3 - VL_CD16A. In some embodiments each of peptide linkers L 1, L2, and L3 consist of no more than 12 amino acid residues. In some embodiments, linker L2 of the antibody construct consists of between 3 and 9 amino acid residues, inclusive.

[0019] In some embodiments the bispecific antibody or antigen binding fragment thereof is a comprises an amino acid sequence having or having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 18.

[0020] In some embodiments the bispecific antibody or antigen binding fragment thereof is a bispecific antigen binding fragment comprising an amino acid sequence set forth in SEQ ID NO: 18.

[0021] In some embodiments the NK cell is a cord blood-derived NK cell.

[0022] In some embodiments the cord blood-derived NK cell has been produced by a method comprising: (a) providing a sample of cord blood cells comprising natural killer cells, (b) depleting the cells of CD3(+) cells; (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 the cord blood-derived natural killer cells.

[0023] In some embodiments the inactivated CD4(+) T cell line expresses at least one gene selected from the group consi sting of a 4-1 BBL gene, a membrane-bound IL-21 (mbIL-21) gene, an OX40L gene, and a mouse TNF-α gene.

[0024] In some embodiments the inactivated CD4(+) T cell line expresses a 4-1BBL gene, a membrane-bound IL-21 (mb IL-21) gene, and a mouse TNF-α gene.

[0025] In some embodiments the first binding domain that specifically binds to CD 16 of the bispecific antibody or antigen binding fragment thereof is bound to the CD16 molecule of the NK cell.

[0026] In some embodiments, the pharmaceutical composition further comprises: (a) human albumin; (b) dextran; (c) glucose; (d) DMSO; and (e) a buffer.

[0027] In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable excipient.

[0028] Also described herein are frozen vials comprising any of the pharmaceutical compositions described herein.

[0029] Also described herein are methods for treating a patient suffering from a CD30 ⁺ cancer comprising administering any of the pharmaceutical compositions described herein.

[0030] Also described herein are methods for treating a patient suffering from a CD30 ⁺ cancer comprising: administering a first pharmaceutical composition comprising a natural killer cell (NK cell) comprising a KIR-B haplotype and expression of a CD16 molecule; and administering 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, 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 (CDRL 1 ) 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; and a heavy chain variable domain ( VH_CD 16 A) 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 wherein the second binding domain that specifically binds to CD30 comprises: 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; 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.

[0031] In some embodiments, the NK cell is a cord blood-derived NK cell.

[0032] In some embodiments, the cord blood-derived NK cell has been produced by a method comprising: (a) providing a sample of cord blood cells comprising natural killer cells; (b) depleting the cells of CD3(+) cells; (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, UCHTI, HTa, or a combination thereof; and IL-2, to produce the cord blood-derived natural killer cells.

[0033] In some embodiments, the inactivated CD4(+) T cell line expresses at least one gene selected from the group consisting of a 4-1BBL gene, a membrane-bound IL-21 (mbIL-21) gene, an OX40L gene, and a mouse TNF-α gene.

[0034] In some embodiments, the inactivated CD4(+) T cell line expresses a 4-1BBL gene, a membrane-bound IL -21 (mbIL-21) gene, and a mouse TNF-α gene.

[0035] In some embodiments, the CD16 molecule is a CD16A molecule. [0036] In some embodiments, the CD 16 molecule comprises a V/V polymorphism at

F158.

[0037] In some embodiments, the first binding domain that specifically binds to CD16 specifically binds to CD 16 A.

[0038] In some embodiments, the pharmaceutical composition further comprises: (a) human albumin; (b) dextran; (c) glucose; (d) DMSO; and (e) a buffer.

[0039] In some embodiments, the first pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0040] In some embodiments, the first binding domain that specifically binds to CD 16 comprises a light chain variable (V _L ) region comprising SEQ ID NO: 20 and a heavy chain variable (V _H ) region comprising SEQ ID NO: 19.

[0041] In some embodiments, the first binding domain that specifically binds to CD16 comprises a VL region comprising an amino acid sequence having or having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 20 and a V _H region comprising an amino acid sequence having or having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 19.

[0042] In some embodiments, the second binding domain that specifically binds to CD30 comprises a light chain variable (V _L ) region comprising SEQ ID NO: 22 and a heavy chain variable (V _H ) region comprising SEQ ID NO: 21.

[0043] In some embodiments, the second binding domain that specifically binds to CD30 comprises a VL region comprising an amino acid sequence having or having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 22 and a V _H region comprising an amino acid sequence having or having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 21.

[0044] In some embodiments, the bispecific antibody or antigen binding fragment thereof is a bi specific antigen binding fragment; and the variable domains of the bispecific antigen binding fragment are linked by peptide linkers L 1 , L2, and L.3 from the N-tenninus to the C- terminus in the order: VH_CD30 -- L 1 - VL_CD16A - L2 - VH_CD16A - L3 - VL_CD30. 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 L 1, L2, and L3 from the N-terminus to the C-terminus in the order: VH_CD16A - L 1- VL_CD30 - L2 - VH_CD30 - L3 - VL_CD 16 A. In some embodiments, each of peptide linkers L 1, L2, and L3 consist of no more than 12 amino acid residues. In some embodiments, linker L2 of the antibody construct consists of between 3 and 9 amino acid residues, inclusive.

[0045] In some embodiments, the bispecific antibody or antigen binding fragment thereof is a comprises an amino acid sequence having or having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 18.

[0046] In some embodiments, the bispecific antibody or antigen binding fragment thereof is a bispecific antigen binding fragment comprising an amino acid sequence set forth in SEQ ID NO: 18.

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary’ skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0048] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

INCORPORATION BY REFERENCE

[0049] 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

[0050] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained 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:

[0051] FIG. 1 shows an exemplary embodiment of a method for NK cell expansion and stimulation. [0052] FIG. 2 shows cytotoxic activity of AFM13 and AB-101 NK cells from MCB2, in 4 hour calcein-release assays on KARPAS-299 target cells at decreasing effector-to-target (E:T) ratios starting at 10: 1 followed by two-fold serial dilutions.

[0053] FIG. 3 shows cytotoxic activity of AFM13 and AB- 101 NK cells from MCB 1, in 4 hour calcein-release assays on KARPAS-299 target cells at decreasing effector-to-target (E:T) ratios starting at 10: 1 followed by two-fold serial dilutions.

[0054] FIG. 4 shows a bar graph of cytotoxic activity of AFM13 and AB-101 NK cells from MCB1 (right, AB-101 MCB1) and of MCB2 (left, AB-101 MCB2), in 4 hour calcein- release assays on KARPAS-299 target cells at an effector-to-target (E:T) ratio of 5 : 1.

[0055] FIG. 5 show's retention of bound AFM13 on preloaded cryopreserved AB-101 cells from MCB2 after thawing, wherein 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.

[0056] FIG. 6 show's retention of bound AFM13 on preloaded cryopreserved AB- 101 cells from MCB1 after thawing, wherein 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.

[0057] FIG. 7 shows fluorescence intensity on CD 16 expression on preloaded AB-101 cells (left: MCB2, right: MCB1). The various conditions show uniform expression of CD 16 on AB-101 cells. From top to bottom: non-preloaded; non-preloaded + fresh excess AFM; AFM- preloaded; AFM-preloaded + fresh excess AFM.

[0058] FIG. 8 shows NK fratricide (NK-NK cell lysis) by AFM13 on AB-101 NK cells from MCB2, in 4 hour calcein-release assays at an effector-to-target (E:T) ratio of 1 : 1.

[0059] FIG. 9 shows NK fratricide (NK-NK cell lysis) by AFM13 on AB-101 NK cells from MCB1, in 4 hour calcein-release assays at an effector-to-target (E:T) ratio of 1:1.

[0060] FIG. 10 show's up-regulation of CD 107a in response to Karpas-299 target cells and AFM13, wherein the AB-101 NK cells from MCB2 were co-cultured with and without the target cells at a 1 : 1 cell ratio, wherein the %CD107a+ NK cells was determined by flow cytometry/.

[0061] FIG. 11 show's up-regulation of CD107a in response to Karpas-299 target cells and AFM13, wherein the AB-101 NK cells from MCB1 were co-cultured with and without the target cells at a 1 : 1 cell ratio, wherein the %CD107a+ NK cells was determined by flow cytometry.

[0062] FIG. 12 shows increased production of intracel lular IFNy in response to Karpas- 299 target cells and AI M 13. wherein the AB-101 NK cells from MCB2 were co-cultured with and without the target cells at a 1 : 1 cell ratio, wherein the %IFNγ+ NK cells was determined by flow cytometry/.

[0063] FIG. 13 shows increased production of intracellular IFNy in response to Karpas- 299 target cells and AFM13, wherein the AB-101 NK cells from MCB1 were co-cultured with and without the target cells at a 1 : 1 cell ratio, wherein the %IFNγ+ NK cells was determined by flow cytometry.

[0064] FIG. 14 shows viability analysis of cryopreserved AFM 13 -preloaded AB-101 NK cells, wherein the efficacy of AFM 13 -preloaded or empty AB-101 NK cells were evaluated on MDA-MB-23 1-Luc cells in an intraperitoneal xenograft tumor model in female hILl 5-NOG mice.

DETAILED DESCRIPTION

[0065] Provided herein are, among other things, pharmaceutical compositions comprising NK cell(s), e.g., as described herein, and multispecific engager(s), e.g., as described herein as well as frozen vial(s) comprising the pharmaceutical composition(s), and methods for treating patients with the pharmaceutical composition(s).

I. EXPANSION AND STIMULATION OF NATURAL KILLER CELLS

[0066] In some embodiments, natural killer cells are expanded and stimulated, e.g., by culturing and stimulation with feeder cells.

[0067] NK cells can be expanded and stimulated as described, for example, in US 2020/0108096 or WO 2020/101361 , both of winch are incorporated herein by reference in their entirety. Briefly, the source cells can be cultured on modified HuT-78 (ATCC® TIB-161™) cells that, have been engineered to express 4-1 BBL, membrane bound IL-21, and a mutant TNFa as described in US 2020/0108096.

[0068] Suitable NK cells can also be expanded and stimulated as described herein.

[0069] In some embodiments, NK cells are expanded and stimulated by a method comprising: (a) providing NK cells, e.g., a composition comprising NK cells, e.g., CD3(+) depleted NK cells; and (b) culturing in a medium comprising feeder cells and/or stimulation factors, thereby producing a population of expanded and stimulated NK cells. A. Natural Killer Cell Sources

[0070] In some embodiments, the NK cell source is selected from the group consisting of peripheral blood, peripheral blood lymphocytes (PBLs), peripheral blood mononuclear cells (PBMCs), bone marrow, umbilical cord blood, isolated NK cells, NK cells derived from induced pluripotent stem cells, NK cells derived from embryonic stem cells, and combinations thereof.

[0071] In some embodiments, the NK cell source is a single unit of cord blood.

[0072] In some embodiments, the natural killer cell source, e.g., single unit of cord blood, comprises from or from about 1 x 10 ⁷ to or to about 1 x 10 ⁹ total nucleated cells. In some embodiments, the natural killer cell source, e.g., single unit of cord blood, comprises from or from about 1 x 10 ⁸ to or to about 1.5 x 10 ⁸ total nucleated cells. In some embodiments, the natural killer cell source, e.g., single unit of cord blood, comprises 1 x 10 ⁸ total nucleated cells. In some embodiments, the natural killer cell source, e.g., single unit, of cord blood, comprises about 1 x 10 ⁸ total nucleated cells. In some embodiments, the natural killer cell source, e.g., single unit of cord blood, comprises 1 x 10 ⁹ total nucleated cells. In some embodiments, the natural killer cell source, e.g., single unit of cord blood, comprises about 1 x 10 ⁹ total nucleated cells.

[0073] In some embodiments, the NK cell source, e.g., the cord blood unit, comprises from about 20% to about 80% CD16+ cells. In some embodiments, the NK cell source, e.g., the cord blood unit, comprises from or from about 20% to or to about 80%, from about 20% to or to about 70%, from about 20% to or to about 60%, from about 20% to or to about 50%, from about 20% to or to about. 40%, from about 20% to or to about 30%, from about 30% to or to about 80%, from about 30% to or to about 70%, from about 30% to or to about 60%, from about 30% to or to about 50%, from about 30% to or to about 40%, from about. 40% to or to about. 80%, from about 40% to or to about 70%, from about 40% to or to about 60%, from about 40% to or to about 50%, from about 50% to or to about 80%, from about 50% to or to about 70%, from about 50% to or to about 60%, from about 60% to or to about 80%, from about 60% to or to about 70%, or from about 70% to or to about 80% CD 16+ cells. In some embodiments, the NK cell source, e.g., the cord blood unit, comprises less than or equal to 80% CD16+ cells.

[0074] In some embodiments, the NK cell source, e.g., the cord blood unit, comprises less than or equal to 40%, e.g., less than or equal to 30%, e.g., less than or equal to 20%, e.g., less than or equal to 10%, e.g., less than or equal to 5% Ml. (12 A cells.

[0075] In some embodiments, the NK cell source, e.g., the cord blood unit, comprises less than or equal to 40%, e.g., less than or equal to 30%, e.g., less than or equal to 20%, e.g., less than or equal to 10%, e.g., less than or equal to 5% NKG2C+ cells. [0076] In some embodiments, the NK cell source, e.g., the cord blood unit, comprises less than or equal to 40%, e.g., less than or equal to 30%, e.g., less than or equal to 20%, e.g., less than or equal to 10%, e.g., less than or equal to 5% NKG2D+ cells.

[0077] In some embodiments, the NK cell source, e.g., the cord blood unit, comprises less than or equal to 40%, e.g., less than or equal to 30%, e.g., less than or equal to 20%, e.g., less than or equal to 10%, e.g., less than or equal to 5% NKp46+ cells.

[0078] In some embodiments, the NK cell source, e.g., the cord blood unit, comprises less than or equal to 40%, e.g., less than or equal to 30%, e.g., less than or equal to 20%, e.g., less than or equal to 10%, e.g., less than or equal to 5% NKp30+ cells.

[0079] In some embodiments, the NK cell source, e.g., the cord blood unit, comprises less than or equal to 40%, e.g., less than or equal to 30%, e.g., less than or equal to 20%, e.g., less than or equal to 10%, e.g., less than or equal to 5% DNAM-1 + cells.

[0080] In some embodiments, the NK cell source, e.g., the cord blood unit, comprises less than or equal to 40%, e.g., less than or equal to 30%, e.g., less than or equal to 20%, e.g., less than or equal to 10%, e.g., less than or equal to 5% NKp44+ cells.

[0081] In some embodiments, the NK cell source, e.g., the cord blood unit, comprises less than or equal to 40%, e.g., less than or equal to 30%, e.g., less than or equal to 20%, e.g., less than or equal to 10%, e.g., less than or equal to 5% CD25+ cells.

[0082] In some embodiments, the NK cell source, e.g., the cord blood unit, comprises less than or equal to 40%, e.g., less than or equal to 30%, e.g., less than or equal to 20%, e.g., less than or equal to 10%, e.g., less than or equal to 5% CD62L+ cells.

[0083] In some embodiments, the NK cell source, e.g., the cord blood unit, comprises less than or equal to 40%, e.g., less than or equal to 30%, e.g., less than or equal to 20%, e.g., less than or equal to 10%, e.g., less than or equal to 5% CD69+ cells.

[0084] In some embodiments, the NK cell source, e.g., the cord blood unit, comprises less than or equal to 40%, e.g., less than or equal to 30%, e.g., less than or equal to 20%, e.g., less than or equal to 10%, e.g., less than or equal to 5% CXCR3+ cells.

[0085] In some embodiments, the NK cell source, e.g., the cord blood unit, comprises less than or equal to 40%, e.g., less than or equal to 30%, e.g., less than or equal to 20%, e.g., less than or equal to 10%, e.g., less than or equal to 5% CD57+ cells.

[0086] In some embodiments, 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:el5115 (2010).

[0087] In some embodiments, NK cells in the NK cell source comprise the 158 V/V variant of CD16. 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).

[0088] In some embodiments, NK cells in the cell source comprises both the KIR B allele of the KIR receptor family and the 158 V/V variant of CD16.

[0089] In some embodiments, the NK cell source is CD3 {+) depleted. In some embodiments, the method comprises depleting the NK cell source of CD3(+) cells. In some embodiments, depleting the NK cell source of CD3(+) cells comprises contacting the NK cell source with a CD3 binding antibody or antigen binding fragment thereof. In some embodiments, the CD3 binding antibody or antigen binding fragment thereof is selected from the group consisting of OKT3, UCHT1, and HIT3a, and fragments thereof. In some embodiments, the CD3 binding antibody or antigen binding fragment thereof is OKT3 or an antigen binding fragment thereof. In some embodiments, the antibody or antigen binding fragment thereof is attached to a bead, e.g., a magnetic bead. In some embodiments, the depleting the composition of CD3(+) cells comprises contacting the composition with a CD3 targeting antibody or antigen binding fragment thereof attached to a bead and removing the bead-bound CD3(+) cells from the composition. The composition can be depleted of CD3 cells by immunomagnetic selection, for example, using a CliniMACS T cell depletion set ((LS Depletion set (162-01 ) Miltenyi Biotec).

B. Feeder Cells

[0090] Feeder cells can be used to stimulate the NK cells and help them to expand more quickly, e.g., by providing substrate, growth factors, and/or cytokines.

[0091] NK cells can be stimulated using various types of feeder cells, including, but not limited to peripheral blood mononuclear cells (PBMC), Epstein-Barr virus-transformed B- lymphoblastoid cells (e.g., EBV-LCL), myelogenous leukemia cells (e.g., K562), and CD4(+) T cells (e.g., HuT), and derivatives thereof.

[0092] In some embodiments, the feeder cells are inactivated, e.g., by γ-irradiation or mitomycin-c treatment.

[0093] Suitable feeder cells for use in the methods described herein are described, for example, in US 2020/0108096, which is hereby incorporated by reference in its entirety. [0094] In some embodiments, the feeder cell(s) are inactivated CD4(+) T ceil(s). In some embodiments, the inactivated CD4(+) T cell(s) are HuT-78 cells (ATCC® TIB-161TM) or variants or derivatives thereof. In some embodiments, the HuT-78 derivative is H9 (ATCC® HTB-176™).

[0095] In some embodiments, the inactivated CD4(+) T cell(s) express OX40L. In some embodiments, the inactivated CD4(+) T cell(s) are HuT-78 cells or variants or derivatives thereof that express OX40L (SEQ ID NO: 4) or a variant thereof.

[0096] In some embodiments, the feeder cells are HuT-78 cells 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 membrane bound TNFalpha (SEQ ID NO: 3) (“eHut-78 cells”), or variants thereof.

[0097] In some embodiments, the inactivated CD4(+) T cell(s) are HuT-78 (ATCC® TIB-161™) cell s or variants or derivatives thereof that express an ortholog of OX40L, or 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 an 4-1BBL ortholog or variant thereof, a membrane bound IL-21 ortholog or variant thereof, and membrane bound TNFalpha ortholog, or variant thereof.

[0098] In some embodiments, the feeder cells are HuT-78 cell(s) that express OX40L (SEQ ID NO: 4) and are engineered to express 4-1BBL (SEQ ID NO: 1), membrane bound IL- 21 (SEQ ID NO: 2), and membrane bound TNFalpha (SEQ ID NO: 3) (“eHut-78 cells”) or variants or derivatives thereof.

[0099] In some embodiments, the feeder cells are expanded, e.g., from a frozen stock, before culturing with NK cells, e.g., as described in Example 2.

C. Stimulating Factors

[0100] NK cells can also be stimulated using one or more stimulation factors other than feeder cells, e.g., signaling factors, in addition to or in place of feeder cells.

[0101] In some embodiments, the stimulating factor, e.g., signaling factor, is a component of the culture medium, as described herein. In some embodiments, the stimulating factor, e.g., signaling factor, is a supplement to the culture medium, as described herein.

[0102] In some embodiments, the stimulation factor(s) are cytokine(s). In some embodiments, the cytokine(s) are 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.

[0103] In some embodiments, the cytokine is IL-2. [0104] In some embodiments, the cytokines are a combination of IL-2 and IL-15.

[0105] In some embodiments, the cytokines are a combination of IL-2, IL-15, and IL-18.

[0106] In some embodiments, the cytokines are a combination of IL-2, IL-18, and IL-21 .

I). Culturing

[0107] The NK cells can be expanded and stimulated by co-culturing an \K cell source and feeder cells and/or other stimulation factors. Suitable NK. cell sources, feeder cells, and stimulation factors are described herein.

1. Culture Medium

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

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

[0110] Suitable basal culture media include, but are not limited to, DMEM, RPMI 1640, MEM, DMEM/F12, SCGM (CellGenix®, 20802-0500 or 20806-0500), LGM-3™ (Lonza, CC- 3211), TexMACS™(Miltenyi Biotec, 130-097-196), ALyS™ 505NK-AC (Cell Science and Technology Institute, Inc., 01600P02), ALyS™ 505NK-EX (Cell Science and Technology Institute, Inc., 01400P10), CTS™ AIM-V™ SFM (ThermoFisher Scientific, A3830801), CTS™ OpTmizer™ (ThermoFisher Scientific, A1048501, ABS-00I, StemXxVivoand combinations thereof.

[0111] The culture medium may comprise additional components, or be supplemented with additional components, such as growth factors, signaling factors, nutrients, antigen binders, and the like. Supplementation of the culture medium may occur by adding each of the additional component or components to the culture vessel either before, concurrently with, or after the medium is added to the culture vessel. The additional component or components may be added together or separately. When added separately, the additional components need not be added at the same time.

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

[0113] In some embodiments, the medium comprises and/or is supplemented with from or from about 0.5 % to or to about 10 % v/v plasma, e.g., human plasma. In some embodiments, the medium is supplemented with from or from about 0.5% to or to about 9%, from or from about 0.5% to or to about 8%, from or from about 0.5% to or to about 7%, from or from about 0.5% to or to about 6%, from or from about 0.5% to or to about 5%, from or from about 0.5% to or to about 4%, from or from about 0.5% to or to about 3%, from or from about 0.5% to or to about. 2%, from or from about 0.5% to or to about 1%, from or from about 1% to or to about 10%, from or from about 1% to or to about 9%, from or from about 1% to or to about 8%, from or from about 1 % to or to about 7%, from or from about 1 % to or to about 6%, from or from about 1% to or to about 5%, from or from about 1% to or to about 4%, from or from about 1% to or to about 3%, from or from about 1% to or to about 2%, from or from about 2% to or to about 10%, from or from about 2°% to or to about 9%, from or from about 2% to or to about 8%, from or from about 2% to or to about 7%, from or from about 2% to or to about 6%, from or from about 2% to or to about 5%, from or from about 2% to or to about 4%, from or from about 2% to or to about 3%, from or from about 3% to or to about 10%, from or from about 3% to or to about 9%, from or from about 3% to or to about 8%, from or from about 3% to or to about 7%, from or from about 3% to or to about 6%, from or from about 3% to or to about 5%, from or from about 3% to or to about 4%, from or from about 4% to or to about 10%, from or from about. 4% to or to about 9%, from or from about 4% to or to about 8%, from or from about 4% to or to about 7%, from or from about 4% to or to about. 6%, from or from about. 4% to or to about. 5%, from or from about 5% to or to about 10%, from or from about 5% to or to about 9%, from or from about 4% to or to about 8%, from or from about 5% to or to about 7%, from or from about 5% to or to about 6%, from or from about 6% to or to about 10%, from or from about 6% to or to about 9%, from or from about 6% to or to about 8%, from or from about 6% to or to about 7%, from or from about 7% to or to about 10%, from or from about 1% to or to about 9%, from or from about 7% to or to about 8%, from or from about 8% to or to about 10%, from or from about 8% to or to about 9%, or from or from about 9% to or to about 10% v/v plasma, e.g., human plasma. In some embodiments, the culture medium comprises and/or is supplemented with from 0.8% to1.2% 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.

[0114] In some embodiments, the culture medium comprises serum, e.g., human serum. In some embodiments, the culture medium is supplemented with serum, e.g., human serum. In some embodiments, the serum is inactivated, e.g., heat inactivated. In some embodiments, the serum is filtered, e.g., sterile-filtered. [0115] In some embodiments, the culture medium comprises glutamine. In some embodiments, the culture medium is supplemented with glutamine. In some embodiments, the culture medium comprises and/or is supplemented with from or from about 2.0 to or to about 6.0 mM glutamine. In some embodiments, the culture medium comprises and/or is supplemented with from or from about 2.0 to or to about 5,5, from or from about 2.0 to or to about 5.0, from or from about 2.0 to or to about 4.5, from or from about 2.0 to or to about 4.0, from or from about 2.0 to or to about 3.5, from or from about 2.0 to or to about 3.0, from or from about 2.0 to or to about 2.5, from or from about 2.5 to or to about 6.0, from or from about 2.5 to or to about 5.5, from or from about 2.5 to or to about 5.0, from or from about 2.5 to or to about 4.5, from or from about 2.5 to or to about 4.0, from or from about 2.5 to or to about 3.5, from or from about 2.5 to or to about 3.0, from or from about 3.0 to or to about 6.0, from or from about 3.0 to or to about 5.5, from or from about 3.0 to or to about 5.0, from or from about 3.0 to or to about 4.5, from or from about 3.0 to or to about 4.0, from or from about 3.0 to or to about 3.5, from or from about 3.5 to or to about 6.0, from or from about 3.5 to or to about 5.5, from or from about 3.5 to or to about 5.0, from or from about 3.5 to or to about 4.5, from or from about 3.5 to or to about 4.0, from or from about 4.0 to or to about. 6.0, from or from about 4.0 to or to about 5.5, from or from about 4.0 to or to about 5.0, from or from about 4.0 to or to about 4.5, from or from about 4.5 to or to about 6.0, from or from about 4.5 to or to about. 5.5, from or from about 4.5 to or to about. 5.0, from or from about 5.0 to or to about 6.0, from or from about 5.0 to or to about 5.5, or from or from about 5.5 to or to about 6.0 mM glutamine. In some embodiments, the culture medium comprises and/or is supplemented with from 3.2 mM glutamine to 4.8 mM glutamine. In some embodiments, the culture medium comprises and/or is supplemented with 4.0 mM glutamine. In some embodiments, the culture medium comprises and/or is supplemented with about 4.0 mM glutamine.

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

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

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

[0119] In some embodiments, the culture medium comprises and/or is supplemented with from 64 μ g/L to 96 μ g/L IL-2. In some embodiments, the culture medium comprises and/or is supplemented with 80 μ g/L IL-2 (approximately 1,333 lU/mL). In some embodiments, the culture medium comprises and/or is supplemented with about 80 μ g/L.

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

[0121] In some embodiments, the culture medium comprises and/or is supplemented with a combination of IL-2, IL-15, and IL-18.

[0122] In some embodiments, the culture medium comprises and/or is supplemented with a combination of IL-2, IL-18, and IL-21 , [0123] 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 from or from about 0.5 to or to about 3.5 g/'L glucose. In some embodiments, the culture medium comprises and/or is supplemented with from or from about 0.5 to or to about 3.0, from or from about 0.5 to or to about 2.5, from or from about 0.5 to or to about 2.0, from or from about 0.5 to or to about 1.5, from or from about 0.5 to or to about 1.0, from or from about 1.0 to or to about 3.0, from or from about 1.0 to or to about 2.5, from or from about 1.0 to or to about 2.0, from or from about 1.0 to or to about 1.5, from or from about 1.5 to or to about 3.0, from or from about 1.5 to or to about 2.5, from or from about 1.5 to or to about 2.0, from or from about 2.0 to or to about 3.0, from or from about 2.0 to or to about 2.5, or from or from about 2.5 to or to about 3.0 g/L glucose. In some embodiments, the culture medium comprises and/or is supplemented with from 1 .6 to 2.4 g/L glucose. In some embodiments, the culture medium comprises and/or is supplemented with 2.0 g/L glucose. In some embodiments, the culture medium comprises about 2.0 g/L glucose.

[0124] 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 from or from about 0. 1 to or to about 2.0 mM sodium pyruvate. In some embodiments, the culture medium comprises and/or i s supplemented with from or from about. 0.1 to or to about 1.8, from or from about 0.1 to or to about 1.6, from or from about 0.1 to or to about 1.4, from or from about 0.1 to or to about 1.2, from or from about 0.1 to or to about 1.0, from or from about 0.1 to or to about 0.8, from or from about 0.1 to or to about 0.6, from or from about 0.1 to or to about 0.4, from or from about 0. 1 to or to about 0.2, from or from about 0.2 to or to about 2.0, from or from about 0.2 to or to about 1.8, from or from about 0.2 to or to about 1.6, from or from about 0.2 to or to about 1.4, from or from about 0.2 to or to about 1.2, from or from about 0.2 to or to about 1.0, from or from about. 0.2 to or to about 0.8, from or from about 0.2 to or to about 0.6, from or from about 0.2 to or to about 0.4, from or from about 0.4 to or to about 2.0, from or from about. 0.4 to or to about 1.8, from or from about 0.4 to or to about 1.6, from or from about 0.4 to or to about 1.4, from or from about 0.4 to or to about 1.2, from or from about 0.4 to or to about 1.0, from or from about 0.4 to or to about 0.8, from or from about 0.4 to or to about 0.6, from or from about 0.6 to or to about 2.0, from or from about 0.6 to or to about 1 .8, from or from about 0.6 to or to about 1.6, from or from about 0.6 to or to about 1.4, from or from about 0.6 to or to about 1.2, from or from about 0.6 to or to about 1.0, from or form about 0.6 to or to about 0.8, from or from about 0.8 to or to about 2.0, from or from about 0.8 to or to about 1.8, from or from about 0.8 to or to about 1 .6, from or from about 0.8 to or to about 1 .4, from or from about 0.8 to or to about 1.4, from or from about 0.8 to or to about 1.2, from or from about 0.8 to or to about 1.0, from or from about 1.0 to or to about 2.0, from or from about 1.0 to or to about 1.8, from or from about 1.0 to or to about 1.6, from or from about 1.0 to or to about 1.4, from or from about 1.0 to or to about 1.2, from or from about 1.2 to or to about 2.0, from or from about 1.2 to or to about 1.8, from or from about 1.2 to or to about 1 .6, from or from about 1.2 to or to about 1 .4, from or from about 1.4 to or to about 2.0, from or from about 1.4 to or to about 1.8, from or from about 1.4 to or to about 1 .6, from or from about 1.6 to or to about 2.0, from or from about 1.6 to or to about 1.8, or from or from about 1.8 to or to about 2.0 mM sodium pyruvate. In some embodiments, the culture medium comprises from 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 pyuruvate.

[0125] In some embodiments, the culture medium comprises and/or is supplemented with sodium hydrogen carbonate. In some embodiments, the culture medium comprises and/or is supplemented with from or from about 0.5 to or to about 3.5 g/L sodium hydrogen carbonate. In some embodiments, the culture medium comprises and/or is supplemented with from or from about 0.5 to or to about 3.0, from or from about 0.5 to or to about 2.5, from or from about 0.5 to or to about 2.0, from or from about 0.5 to or to about 1.5, from or from about 0.5 to or to about 1.0, from or from about. 1.0 to or to about 3.0, from or from about 1.0 to or to about 2.5, from or from about 1.0 to or to about 2.0, from or from about 1.0 to or to about 1.5, from or from about 1.5 to or to about 3.0, from or from about 1.5 to or to about 2.5, from or from about 1.5 to or to about 2.0, from or from about 2.0 to or to about 3.0, from or from about. 2.0 to or to about 2.5, or from or from about 2.5 to or to about 3.0 g/L sodium hydrogen carbonate. In some embodiments, the culture medium comprises and/or is supplemented with from 1.6 to 2.4 g/L sodium hydrogen carbonate. In some embodiments, the culture medium comprises and/or is supplemented with 2,0 g/L sodium hydrogen carbonate. In some embodiments, the culture medium comprises about 2.0 g/L sodium hydrogen carbonate.

[0126] In some embodiments, the culture medium comprises and/or is supplemented with albumin, e.g., human albumin, e.g., a human albumin solution described herein. In some embodiments, the culture medium comprises and/or is supplemented with from or from about 0.5% to or to about 3.5% 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 from or from about 0.5% to or to about 3.0%, from or from about 0.5% to or to about. 2.5%, from or from about 0.5% to or to about 2.0%, from or from about 0.5% to or to about 1.5%, from or from about 0.5% to or to about. 1.0%, from or from about 1 .0% to or to about 3.0%, from or from about 1 .0% to or to about 2.5%, from or from about 1.0% to or to about 2.0%, from or from about 1.0% to or to about 1.5%, from or from about 1.5% to or to about 3.0%, from or from about 1.5% to or to about 2.5%, from or from about 1 .5% to or to about 2.0%, from or from about 2.0% to or to about 3.0%, from or from about 2.0% to or to about 2.5%, or from or from about. 2.5% to or to 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 from 1.6% to 2.4% 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 2.0% v/v of a 20% albumin solution, e.g., a 20% human albumin solution. In some embodiments, the culture medium comprises about 2.0% v/v of a 20% albumin solution, e.g., a 20% human albumin solution.

[0127] In some embodiments, the culture medium comprises and/or is supplemented with from or from about 2 to or to about 6 g/L albumin, e.g., human albumin. In some embodiments, the culture medium comprises and/or is supplemented with from or from about 2 to or to about 5.5, from or from about 2 to or to about 5.0, from or from about 2 to or to about 4.5, from or from about 2 to or to about 4, from or from about 2 to or to about 3.5, from or from about. 2 to or to about 3, from or from about 2 to or to about 2.5, from or from about 2.5 to or to about 6, from or from about 2.5 to or to about 5.5, from or from about 2.5 to or to about 5.5, from or from about 2.5 to or to about 5.0, from or from about 2.5 to or to about 4.5, from or from about 2.5 to or to about 4.0, from or from about 2.5 to or to about 3.5, from or from about 2.5 to or to about 3.0, from or from about 3 to or to about 6, from or from about 3 to or to about 5.5, from or from about 3 to or to about 5, from or from about 3 to or to about 4.5, from or from about 3 to or to about 4, from or from about 3 to or to about 3.5, from or from about 3.5 to or to about 6, from or from about 3.5 to or to about 5.5, from or from about 3.5 to or to about 5, from or from about 3.5 to or to about. 4.5, from or from about 3.5 to or to about. 4, from or from about 4 to or to about 6, from or from about 4 to or to about 5.5, from or from about 4 to or to about 5, from or from about. 4 to or to about 4.5, from or from about 4,5 to or to about 6, from or from about 4.5 to or to about 5.5, from or from about 4.5 to or to about 5, from or from about 5 to or to about 6, from or from about 5 to or to about 5.5, or from or from about 5.5 to or to about 6 g/L albumin, e.g., human albumin. In some embodiments, the culture medium comprises and/or is supplemented with from 3.2 to 4.8 g/L albumin, e.g., human albumin. In some embodiments, the culture medium comprises 4 g/L albumin, e.g., human albumin. In some embodiments, the culture medium comprises about 4 g/L albumin, e.g., human albumin [0128] In some embodiments, the culture medium is supplemented with Poloxamer 188. In some embodiments, the culture medium comprises and/or is supplemented with from or from about 0.1 to or to about 2.0 g/L Poloxamer 188. In some embodiments, the culture medium comprises and/or is supplemented with from or from about 0.1 to or to about 1.8, from or from about 0. 1 to or to about 1.6, from or from about 0. 1 to or to about 1.4, from or from about 0. 1 to or to about 1.2, from or from about 0.1 to or to about 1.0, from or from about 0.1 to or to about 0.8, from or from about 0.1 to or to about 0.6, from or from about 0.1 to or to about 0.4, from or from about 0. 1 to or to about 0.2, from or from about 0.2 to or to about 2.0, from or from about 0.2 to or to about 1.8, from or from about 0.2 to or to about 1.6, from or from about 0.2 to or to about 1.4, from or from about 0.2 to or to about 1.2, from or from about 0.2 to or to about 1 .0, from or from about 0.2 to or to about 0.8, from or from about 0.2 to or to about 0.6, from or from about 0.2 to or to about 0.4, from or from about 0.4 to or to about 2.0, from or from about 0.4 to or to about 1.8, from or from about 0.4 to or to about 1.6, from or from about 0.4 to or to about 1.4, from or from about. 0.4 to or to about 1.2, from or from about. 0.4 to or to about 1.0, from or from about 0.4 to or to about 0.8, from or from about 0.4 to or to about 0.6, from or from about 0.6 to or to about 2.0, from or from about. 0.6 to or to about 1.8, from or from about 0.6 to or to about 1.6, from or from about 0.6 to or to about 1.4, from or from about 0.6 to or to about 1.2, from or from about 0.6 to or to about 1.0, from or form about. 0.6 to or to about 0.8, from or from about 0.8 to or to about 2.0, from or from about 0.8 to or to about 1.8, from or from about 0.8 to or to about 1.6, from or from about 0.8 to or to about 1.4, from or from about 0.8 to or to about 1.4, from or from about 0.8 to or to about 1.2, from or from about 0.8 to or to about 1 .0, from or from about 1.0 to or to about 2.0, from or from about 1.0 to or to about 1.8, from or from about 1.0 to or to about 1 .6, from or from about 1.0 to or to about 1 .4, from or from about 1.0 to or to about 1.2, from or from about 1.2 to or to about 2.0, from or from about 1.2 to or to about 1.8, from or from about 1.2 to or to about. 1.6, from or from about 1.2 to or to about. 1.4, from or from about 1.4 to or to about 2.0, from or from about 1.4 to or to about 1.8, from or from about 1.4 to or to about 1.6, from or from about 1.6 to or to about. 2.0, from or from about 1.6 to or to about. 1.8, or from or from about 1.8 to or to about 2.0 g/L Poloxamer 188. In some embodiments, the culture medium comprises from 0.8 to 1.2 g/L Poloxamer 188. In some embodiments, the culture medium comprises 1 .0 g/L Poloxamer 188. In some embodiments, the culture medium comprises about 1.0 g/L Poloxamer 188.

[0129] In some embodiments, the culture medium comprises and/or is supplemented with one or more antibiotics.

[0130] A first exemplary culture medium is set forth in Table 1 ,

[0131] A second exemplary culture medium is set forth in Table 2.

2. CD3 Binding Antibodies

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

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

[0134] In some embodiments, the culture medium comprises and/or is supplemented with from or from about 5 ng/mL to or to about. 15 ng/mL OKT3. In some embodiments, the culture medium comprises and/or is supplemented with from or from about 5 to or to about 12.5, from or from about 5 to or to about 10, from or from about 5 to or to about 7.5, from or from about 7.5 to or to about 15, from or from about 7.5 to or to about 12.5, from or from about 7.5 to or to about 10, from or from about 10 to or to about 15, from or from about 10 to or to about 12,5, or from or from about 12.5 to or to about 15 ng/mL OKT3. In some embodiments, the culture medium comprises and/or is supplemented with 10 ng/mL OKT3. In some embodiments, the culture medium comprises and/or is supplemented with about 10 ng/mL OKT3.

3. Culture Vessels

[0135] In some embodiments, the culture vessel is selected from the group consisting of a flask, a bottle, a dish, a multiwall plate, a roller bottle, a bag, and a bioreactor.

[0136] In some embodiments, the culture vessel is treated to render 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, poy-L -lysine, fibronectin, extracellular matrix proteins, and combinations thereof.

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

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

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

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

[0141] In some embodiments, the bioreactor has a volume of from about 100 mL to about 1,000 L. In some embodiments, the bioreactor has a volume of from about 10 L to about 1,000 L. In some embodiments, the bioreactor has a volume of from about 100 L to about 900 L. In some embodiments, the bioreactor has a volume of from about 10 L to about 800 L. In some embodiments, the bioreactor has a volume of from about 10 L to about 700 L, about 10 L to about. 600 L, about 10 L to about 500 L, about 10 L to about 400 L, about 10 L to about. 300 L, about 10 L to about 200 L, about 10 L to about 100 L, about 10 L to about 90 L, about 10 L to about 80 L, about 10 L to about 70 L, about 10 L to about 60 L, about 10 L to about 50 L, about 10 L to about 40 L, about 10 L to about 30 L, about 10 L to about 20 L, about 20 L to about 1,000 L, about 20 L to about 900 L, about 20 L to about 800 L, about 20 L to about 700 L, about 20 L to about 600 L, about. 20 L to about. 500 L, about 20 L to about 400 L, about 20 L to about 300 L, about 20 L to about 200 L, about 20 L to about 100 L, about 20 L to about 90 L, about 20 L to about 80 L, about 20 L to about 70 L, about 20 L to about 60 L, about 20 L to about 50 L, about 20 L to about 40 L, about 20 L to about 30 L, about 30 L to about 1,000 L, about 30 L to about 900 L, about 30 L to about 800 L, about 30 L to about 700 L, about 30 L to about 600 L, about 30 L to about. 500 L, about 30 L to about 400 L, about 30 L to about 300 L, about. 30 L to about 200 L, about 30 L to about 100 L, about 30 L to about 90 L, about 30 L to about 80 L, about 30 L to about 70 L, about 30 L to about 60 L, about 30 L to about 50 L, about 30 L to about 40 L, about 40 L to about 1 ,000 L, about 40 L to about 900 L, about 40 L to about 800 L, about 40 L to about 700 L, about 40 L to about 600 L, about 40 L to about 500 L, about 40 L to about. 400 L, about 40 L to about 300 L, about 40 L to about 200 L, about 40 L to about 100 L, about 40 L to about 90 L, about 40 L to about 80 L, about 40 L to about 70 L, about 40 L to about 60 L, about 40 L to about 50 L, about 50 L to about 1 ,000 L, about 50 L to about 900 L, about 50 L to about 800 L, about 50 L to about 700 L, about 50 L to about 600 L, about 50 L to about 500 L, about 50 L to about 400 L, about 50 L to about 300 L, about 50 L to about. 200 L, about 50 L to about 100 L, about 50 L to about 90 L, about 50 L to about 80 L, about 50 L to about 70 L, about 50 L to about 60 L, about 60 L to about 1 ,000 L, about 60 L to about 900 L, about 60 L to about 800 L, about 60 L to about 700 L, about 60 L to about 600 L, about 60 L to about 500 L, about 60 L to about 400 L, about 60 L to about 300 L, about 60 L to about. 200 L, about 60 L to about 100L, about 60 L to about 90 L, about 60 L to about 80 L, about 60 L to about 70 L, about 70 L to about 1,000 L, about 70 L to about 900 L, about 70 L to about 800 L, about 70 L to about 700 L, about 70 L to about. 600 L, about 70 L to about 500 L, about 70 L to about 400 L, about 70 L to about 300 L, about 70 L to about 200 L, about 70 L to about 100 L, about. 70 L to about 90 L, about 70 L to about 80 L, about 80 L to about 1,000 L, about 80 L to about 900 L, about 80 L to about 800 L, about 80 L to about 700 L, about 80 L to about 600 L, about 80 L to about 500 L, about 80 L to about 400 L, about 80 L to about 300 L, about 80 L to about 200 L, about 80 L to about 100 L, about 80 L to about 90 L, about 90 L to about 1,000 L, about 90 L to about. 900 L, about 90 L to about 800 L, about 90 L to about 700 L, about. 90 L to about 600 L, about 90 L to about 500 L, about 90 L to about 400 L, about 90 L to about 300 L, about 90 L to about 200 L, about 90 L to about 100 L, about 100 L to about 1,000 L, about 100 L to about 900 L, about 100 L to about 800 L, about 100 L to about 700 L, about 100 L toa bout 600 L, about 100 L to about 500 L, about 100 L to about 400 L, about 100 L to about 300 L, about. 100 L to about 200 L, about 200 L to about 1,000 L, about 200 L to about 900 L, about 200 L to about 800 L, about 200 L to about 700 L, about 200 L to about 600 L, about 200 L to about 500 L, about 200 L to about 400 L, about 200 L to about 300 L, about 300 L to about 1,000 L, about 300 L to about 900 L, about 300 L to about 800 L, about 300 L to about 700 L, about 300 L to about 600 L, about 300 L to about 500 L, about 300 L to about 400 L, about 400 L to about 1,000 L, about 400 L to about 900 L, about 400 L to about 800 L, about 400 L to about 700 L, about 400 L to about 600 L, about 400 L to about 500 L, about 500 L to about 1,000 L, about 500 L to about 900 L, about. 500 L to about 800 L, about 500 L to about 700 L, about 500 L to about 600 L, about 600 L to about 1,000 L, about 600 L to about 900 L, about 600 L to about 800 L, about 600 L to about 700 L, about. 700 L to about 1,000 L, about. 700 L to about 900 L, about 700 L to about 800 L, about 800 L to about 1,000 L, about 800 L to about 900 L, or about 900 L to about 1,000 L. In some embodiments, the bioreactor has a volume of about 50 L.

[0142] In some embodiments, the bioreactor has a volume of from 100 mL to 1,000 L. In some embodiments, the bioreactor has a volume of from 10 L to 1,000 L. In some embodiments, the bioreactor has a volume of from 100 L to 900 L. In some embodiments, the bioreactor has a volume of from 10 L to 800 L. In some embodiments, the bioreactor has a volume of from 10 L to 700 L, 10 L to 600 L, 10 L to 500 L, 10 L to 400 L, 10 L to 300 L, 10 L to 200 L, 10 L to 100 L, 10 L to 90 L, 10 L to 80 L, 10 L to 70 L, 10 L to 60 L, 10 L to 50 L, 10 L to 40 L, 10 L to 30 L, 10 L to 20 L, 20 L to 1,000 L, 20 L to 900 L, 20 L to 800 L, 20 L to 700 L, 20 L to 600 L, 20 L to 500 L, 20 L to 400 L, 20 L to 300 L, 20 L to 200 L, 20 L to 100 L, 20 L to 90 L, 20 L to 80 L, 20 L to 70 L, 20 L to 60 L, 20 L to 50 L, 20 L to 40 L, 20 L to 30 L, 30 L to 1,000 L, 30 L to 900 L, 30 L to 800 L, 30 L to 700 L, 30 L to 600 L, 30 L to 500 L, 30 L to 400 L, 30 L to 300 L, 30 L to 200 L, 30 L to 100 L, 30 L to 90 L, 30 L to 80 L, 30 L to 70 L, 30 L to 60 L, 30 L to 50 L, 30 L to 40 L, 40 L to 1,000 L, 40 L to 900 L, 40 L to 800 L, 40 L to 700 L, 40 L to 600 L, 40 L to 500 L, 40 L to 400 L, 40 L to 300 L, 40 L to 200 L, 40 L to 100 L, 40 L to 90 L, 40 L to 80 L, 40 L to 70 L, 40 L to 60 L, 40 L to 50 L, 50 L to 1,000 L, 50 L to 900 L, 50 L to 800 L, 50 L to 700 L, 50 L to 600 L, 50 L to 500 L, 50 L to 400 L, 50 L to 300 L, 50 L to 200 L, 50 L to 100 L, 50 L to 90 L, 50 L to 80 L, 50 L to 70 L, 50 L to 60 L, 60 L to 1,000 L, 60 L to 900 L, 60 L. to 800 L, 60 L to 700 L, 60 L. to 600 L, 60 L to 500 L, 60 L. to 400 L, 60 L to 300 L, 60 L to 200 L, 60 L to 100L, 60 L to 90 L, 60 L to 80 L, 60 L to 70 L, 70 L to 1,000 L, 70 L to 900 L, 70 L to 800 L, 70 L to 700 L, 70 L to 600 L, 70 L to 500 L, 70 L to 400 L, 70 L to 300 L, 70 L to 200 L, 70 L to 100 L, 70 L to 90 L, 70 L to 80 L, 80 L to 1,000 L, 80 L to 900 L, 80 L to 800 L, 80 L to 700 L, 80 L to 600 L, 80 L to 500 L, 80 L to 400 L, 80 L to 300 L, 80 L to 200 L, 80 L to 100 L, 80 L to 90 L, 90 L to 1 ,000 L, 90 L to 900 L, 90 L to 800 L, 90 L to 700 L, 90 L to 600 L, 90 L to 500 L, 90 L to 400 L, 90 L to 300 L, 90 L to 200 L, 90 L to 100 L, 100 L to 1,000 L, 100 L to 900 L, 100 L to 800 L, 100 L to 700 L, 100 L to 600 L, 100 L to 500 L, 100 L to 400 L, 100 L to 300 L, 100 L to 200 L, 200 L to 1,000 L, 200 L to 900 L, 200 L to 800 L, 200 L to 700 L, 200 L to 600 L, 200 L to 500 L, 200 L to 400 L, 200 L to 300 L, 300 L to 1,000 L, 300 L to 900 L, 300 L to 800 L, 300 L to 700 L, 300 L to 600 L, 300 L to 500 L, 300 L to 400 L, 400 L to 1,000 L, 400 L to 900 L, 400 L to 800 L, 400 L to 700 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.

4. Cell Expansion and Stimulation

[0143] In some embodiments, the natural killer cell source, e.g., single unit of cord blood, is co-cultured with feeder cells to produce expanded and stimulated NK cells.

[0144] In some embodiments, the co-culture is carried out in a culture medium described herein, e.g., exemplary culture medium #1 (Table 1 ) or exemplary culture medium #2 (Table 2).

[0145] In some embodiments, the natural killer cell source, e.g., single unit of cord blood, comprises from or from about 1 x 10 ⁷ to or to about 1 x 10 ⁹ total nucleated cells prior to expansion. In some embodiments, the natural killer cell source, e.g., single unit of cord blood, comprises from or from about 1 x 10 ⁸ to or to about 1 .5 x 10 ⁸ total nucleated cells prior to expansion. In some embodiments, the natural killer cell source, e.g., single unit of cord blood, comprises 1 x 10 ⁸ total nucleated cells prior to expansion. In some embodiments, the natural killer cell source, e.g., single unit of cord blood, comprises about 1 x 10 ⁸ total nucleated cells prior to expansion. In some embodiments, the natural killer cell source, e.g., single unit of cord blood, comprises 1 x 10 ⁹ total nucleated cells prior to expansion. In some embodiments, the natural killer cell source, e.g., single unit of cord blood, comprises about 1 x 10 ⁹ total nucleated cells prior to expansion.

[0146] In some embodiments, cells from the co-culture of the natural killer cell source, e.g., single unit of cord blood and feeder cells are harvested and frozen, e.g., in a cryopreservation composition described herein. In some embodiments, the frozen cells from the co-culture are an infusion-ready drug product. In some embodiments, the frozen cells from the co-culture are used as a master cell bank (MCB) from which to produce an infusion-ready drug product, e.g., through one or more additional co-culturing steps, as described herein. Thus, for example, a natural killer cell source can be expanded and stimulated as described herein to produce expanded and stimulated NK cells suitable for use in an infusion-ready drug product without generating any intermediate products. A 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 drug product, or, alternatively, be used to produce another intermediate product, e.g., a second MCB. The second MCB can be used to produce expanded and stimulated NK cells suitable for an infusion-ready drug product, or alternatively, be used to produce another intermediate product, e.g., a third MCB, and so on.

[0147] In some embodiments, the ratio of feeder cells to cells of the natural killer cell source or MCB cells inoculated into the co-culture is from or from about 1 : 1 to or to about 4:1. In some embodiments, the ratio of feeder cells to cells of the natural killer cell source or MCB cell s i s from or from about 1 : 1 to or to about 3.5: 1, from or from about 1 : 1 to or to about 3:1, from or from about 1 : 1 to or to about 2.5: l, from or from about 1.1 to or to about 2: l, from or from about 1 : 1 to or to about 1.5: 1, from or from about 1.5: 1 to or to about 4: 1, from or from about 1.5: 1 to or to about 3.5: 1, from or from about 1.5: 1 to or to about 3: 1, from or from about

I.5:1 to or to about 2.5: 1, from or from about 1 ,5: 1 to or to about 2: 1, from or from about 2: 1 to or to about 4: 1, from or from about 2: 1 to or to about 3.5: 1, from or from about 2: 1 to or to about 3: 1 , from or from about 2 : 1 to or to about 2.5: 1 , from or from about 2.5: 1 to or to about. 4: 1 , from or from ab out 2.5: 1 to or to ab out 3.5:1, from or from ab out 2.5:1 to or to ab out 3: 1, from or from about 3: 1 to or to about 4: 1, from or from about 3: 1 to or to about 3.5: 1, or from or from about 3.5: 1 to or to 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.

[0148] In some embodiments, the co-culture is carried out in a disposable culture bag, e.g., a IL disposable culture bag. In some embodiments, the co-culture is carried out in a bioreactor, e.g., a 501. bioreactor. In some embodiments, culture medium is added to the co- culture after the initial inoculation.

[0149] 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, 24 or more days. In some embodiments, the co- culture is carried out for a maximum of 16 days.

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

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

[0152] In some embodiments, the co-culture is carried out at a dissolved oxygen (DO) level of 50% or more. [0153] In some embodiments, exemplary culture medium #1 (Table 1) is used to produce a MCB and exemplary culture medium #2 (Table 2) is used to produce cells suitable for an infusion-ready drug product.

[0154] In some embodiments, the co-culture of the natural killer cell source, e.g., single unit of cord blood, with feeder cells yields from or from about 50 x 10 ⁸ to or to about 50 x 10 ¹² cells, e.g., MCB cells or infusion-ready daig product cells. In some embodiments, the expansion yields from or from about. 50 x 10 ⁸ to or to about 25 x 10 ¹⁰ , from or from about 10 x 10 ⁸ to or to about 1 x 10 ¹⁰ , from or from about 50 x 10 ⁸ to or to about 75 x 10 ⁹ , from or from about 50 x 10 ⁸ to or to about 50 x 10 ⁹ , from or from about 50 x 10 ⁸ to or to about 25 x 10 ⁹ , from or from about 50 x 10 ⁸ to or to about 1 x 10 ⁹ , from or from about 50 x 10 ⁸ to or to about 75 x 10 ⁸ , from or from about 75 x 10 ⁸ to or to about 50 x 10 ¹⁰ , from or from about 75 x 10 ⁸ to or to about 25 x 10 ¹⁰ , from or from about 75 x 10 ⁸ to or to about 1 x 10 ¹⁰ , from or from about 75 x 10 ⁸ to or to about 75 x 10 ⁹ , from or from about 75 x 10 ⁸ to or to about 50 x 10 ⁹ , from or from about 75 x 10 ⁸ to or to about 25 x 10 ⁹ , from or from about 75 x 10 ⁸ to or to about. 1 x 10 ⁹ , from or from about 1 x 10 ⁹ to or to about 50 x 10 ¹⁰ , from or from about 1 x 10 ⁹ to or to about 25 x 10 ¹⁰ , from or from about 1 x 10 ⁹ to or to about. 1 x 10 ¹⁰ , from or from about 1 x 10 ⁹ to or to about 75 x 10 ⁹ , from or from about 1 x 10 ⁹ to or to about 50 x 10 ⁹ , from or from about 1 x 10 ⁹ to or to about 25 x 10 ⁹ , from or from about 25 x 10 ⁹ to or to about 50 x 10 ¹⁰ , from or from about 25 x 10 ⁹ to or to about 25 x 10 ¹⁰ , from or from about 25 x 10 ⁹ to or to about 1 x 10 ¹⁰ , from or from about 25 x 10 ⁹ to or to about 75 x 10 ⁹ , from or from about 25 x 10 ⁹ to or to about 50 x 10 ⁹ , from or from about 50 x 10 ⁹ to or to about 50 x 10 ¹⁰ , from or from about 50 x 10 ⁹ to or to about 25 x 10 ¹⁰ , from or from about 50 x 10 ⁹ to or to about 1 x 10 ¹⁰ , from or from about 50 x 10 ⁹ to or to about 75 x 10 ⁹ , from or from about 75 x 10 ⁹ to or to about 50 x 10 ^lu , from or from about 75 x 10 ⁹ to or to about 25 x ¹⁰¹⁰ , from or from about 75 x 10 ⁹ to or to about 1 x 10 ¹⁰ , from or from about 1 x 10 ¹⁰ to or to about 50 x 10 ¹⁰ , from or from about. 1 x 10 ¹⁹ to or to about 25 x 10 ¹⁰ , or from or from about 25 x 10 ¹⁰ to or to about 50 x 10 ¹⁰ cells, e.g., e.g., MCB cells or infusion-ready drug product cells.

[0155] In some embodiments, the expansion yields from or from about 60 to or to about 100 vials, each comprising from or from about 600 million to or to about 1 billion cells, e.g., MCB cells or infusion-ready drug product cells. In some embodiments, the expansion yields 80 or about 80 vials, each comprising or consisting of 800 million or about 800 million cells, e.g., MCB cells or infusion-ready drug product cells.

[0156] In some embodiments, the expansion yields from or from about a 100 to or to about a 500 fold increase in the number of cells, e.g., the number of MCB cells relative to the number of cells, e.g., NK cells, in the natural killer cell source. In some embodiments, the expansion yields from or from about a 100 to or to about a 500, from or from about a 100 to or to about a 400, from or from about a 100 to or to about a 300, from or from about a 100 to or to about a 200, from or from about a 200 to or to about a 450, from or from about a 200 to or to about a 400, from or from about a 100 to or to about a 350, from or from about a 200 to or to about a 300, from or from about a 200 to or to about a 250, from or from about a 250 to or to about a 500, from or from about a 250 to or to about a 450, from or from about a 200 to or to about a 400, from or from about a 250 to or to about a 350, from or from about a 250 to or to about a 300, from or from about a 300 to or to about a 500, from or from about a 300 to or to about a 450, from or from about a 300 to or to about a 400, from or from about a 300 to or to about a 350, from or from about a 350 to or to about a 500, from or from about a 350 to or to about a 450, from or from about a 350 to or to about a 400 fold increase in the number of cells, e.g., the number of MCB cells relative to the number of cells, e.g., NK cells, in the natural killer cell source.

[0157] In some embodiments, the co-culture of the MCB cells and feeder cells yields from or from about 500 million to or to about 1.5 billion cells, e.g., NK cells suitable for use in an MCB and/or in an infusion-ready drug product. In some embodiments, the co-culture of the MCB cells and feeder cells yields from or from about 500 million to or to about 1.5 billion, from or from about 500 million to or to about 1.25 billion, from or from about 500 million to or to about 1 billion, from or from about 500 million to or to about 750 million, from or from about 750 million to or to about 1.5 billion, from or from about 500 million to or to about 1.25 billion, from or from about 750 million to or to about 1 billion, from or from about 1 billion to or to about 1.5 billion, from or from about 1 billion to or to about 1.25 billion, or from or from about 1.25 billion to or to about 1.5 billion cells, e.g., NK cells suitable for use in an MCB and/or an infusion-ready drug product.

[01.58 ] In some embodiments, the co-culture of the MCB cells and feeder cells yields from or from about 50 to or to about 150 vials of cells, e.g., infusion-ready drug product cells, each comprising from or from about 750 million to or to about 1.25 billion cells, e.g., NK cells suitable for use in an MCB and/or an infusion-ready drug product. In some embodiments, the co-culture of the MCB cells and feeder cells yields 100 or about 100 vials, each comprising or consisting of 1 billion or about 1 billion cells, e.g., NK cells suitable for use in an MCB and/or an infusion-ready drug product.

[0159] In some embodiments, the expansion yields from or from about a 100 to or to about a 500 fold increase in the number of cells, e.g., the number of NK cells suitable for use in an MCB and/or an infusion-ready drug product relative to the number of starting MCB cells. In some embodiments, the expansion yields from or from about a 100 to or to about a 500, from or from about a 100 to or to about a 400, from or from about a 100 to or to about a 300, from or from about a 100 to or to about a 200, from or from about a 200 to or to about a 450, from or from about a 200 to or to about a 400, from or from about a 100 to or to about a 350, from or from about a 200 to or to about, a 300, from or from about a 200 to or to about a 250, from or from about a 250 to or to about a 500, from or from about a 250 to or to about a 450, from or from about a 200 to or to about a 400, from or from about a 250 to or to about a 350, from or from about a 250 to or to about a 300, from or from about a 300 to or to about a 500, from or from about a 300 to or to about a 450, from or from about a 300 to or to about a 400, from or from about a 300 to or to about a 350, from or from about a 350 to or to about a 500, from or from about a 350 to or to about a 450, from or from about a 350 to or to about a 400 fold increase in the number of cells, e.g., the number of NK cells suitable for use in an MCB and/or an infusion-ready drug product relative to the number of starting MCB cells.

[0160] In embodiments where the cells are engineered during expansion and stimulation, as described herein, not all of the expanded and stimulated cells will necessarily be engineered successfully, e.g., transduced successfully, e.g., transduced successfully 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 comprise sorting engineered cells, e.g., engineered cells described herein, away from non-engineered cells.

[0161] In some embodiments, the engineered cells, e.g., transduced cells, are sorted from the non-engineered cells, e.g., the non-transduced cells using a reagent specific to an antigen of the engineered cells, e.g., an antibody that targets an antigen of the engineered cells but not the non-engineered cells. In some embodiments, the antigen of the engineered cells is a component of a CAR, e.g., a CAR described herein.

[0162] Systems for antigen-based cell separation of cells are available commercially, e.g., the CiiniMACS® sorting system (Miltenyi Biotec).

[0163] In some embodiments, the engineered cells, e.g., transduced cells, are sorted from the non-engineered cells, e.g., the non-transduced cells using flow cytometry.

[0164] In some embodiments, the sorted engineered cells are used as an MCB. In some embodiments, the sorted engineered cells are used as a component in an infusion -ready drug product.

[0165] In some embodiments, the engineered cells, e.g., transduced cells, are sorted from the non-engineered cells, e.g., the non-transduced cells using a microfluidic cell sorting method. Microfluidic cell sorting methods are described, for example, in Dalili et. al., “A Review of Sorting, Separation and Isolation of Cells and Microbeads for Biomedical Applications: Microfluidic Approaches,” Analyst 144:87 (2019).

[0166] In some embodiments, from or from about 1% to or to about 99% of the expanded and stimulated cells are engineered successfully, e.g., transduced successfully, e.g., transduced successfully 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, from or from about 1 % to or to about 90%, from or from about. 1% to or to about 80%, from or from about 1% to or to about 70%, from or from about 1% to or to about 60%, from or from about 1% to or to about 50%, from or from about 1% to or to about 40%, from or from about 1% to or to about 30%, from or from about 1% to or to about 20%, from or from about 1% to or to about 10%, from or from about 1% to or to about 5%, from or from about 5% to or to about 99%, from or from about 5% to or to about 90%, from or from about 5% to or to about 80%, from or from about 5% to or to about 70%, from or from about 5% to or to about 60%, from or from about 5% to or to about 50%, from or from about 5% to or to about 40%, from or from about 5% to or to about 30%, from or from about 5% to or to about 20%, from or from about 5% to or to about 10%, from or from about 10% to or to about 99%, from or from about 10% to or to about 90%, from or from about 10% to or to about 80%, from or from about 10% to or to about 70%, from or from about 10% to or to about 60%, from or from about 10% to or to about 50%, from or from about 10% to or to about 40%, from or from about 10% to or to about 30%, from or from about 10% to or to about 20%, from or from about 20% to or to about 99%, from or from about 20% to or to about 90%, from or from about 20% to or to about 80%, from or from about 20% to or to about 70%, from or from about 20% to or to about 60%, from or from about 20% to or to about 50%, from or from about 20% to or to about 40%, from or from about. 20% to or to about 30%, from or from about 30% to or to about 99%, from or from about 30% to or to about 90%, from or from about 30% to or to about. 80%, from or from about 30% to or to about 70%, from or from about 30% to or to about 60%, from or from about 30% to or to about 50%, from or from about 30% to or to about 40%, from or from about 40% to or to about 99%, from or from about 40% to or to about 90%, from or from about 40% to or to about 80%, from or from about 40% to or to about 70%, from or from about 40% to or to about 70%, from or from about 40% to or to about 60%, from or from about 40% to or to about 50%, from or from about 50% to or to about 99%, from or from about 50% to or to about 90%, from or from about 50% to or to about 80%, from or from about 50% to or to about 70%, from or from about 50% to or to about. 60%, from or from about 60% to or to about 99%, from or from about 60% to or to about 90%, from or from about 60% to or to about 80%, from or from about 60% to or to about 70%, from or from about 70% to or to about 99%, from or from about 70% to or to about 90%, from or from about 70% to or to about 80%, from or from about 80% to or to about 99%, from or from about 80% to or to about 90%, or from or from about 90% to or to about 99% of the expanded and stimulated cells are engineered successfully, e.g., transduced successfully, e.g., transduced successfully with a vector comprising a heterologous protein, e.g., a heterologous protein comprising a CAR and/or IL-15 as described herein.

[0167] In some embodiments, frozen cells of a first, or second MCB are thawed and cultured. In some embodiments, a single vial of frozen cells of the first or second MCB e.g., a single vial comprising 800 or about 800 million cells, e.g., first or second MCB cells, are 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 either as a second or third MCB or in an infusion-ready drug product. In some embodiments, the cells from the co-culture of the first or second MCB are harvested and frozen.

[0168] In some embodiments, the cells from the co-culture of the natural killer cell source, a first MCB, or a second MCB are harvested, 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 cry opreservation composition, e.g., a cry opreservation composition described herein.

[0169] In some embodiments, the cells are mixed with a cryopreservation composition, e.g., as described herein, before freezing. In some embodiments, the cells are frozen in cryobags. In some embodiments, the cells are frozen in cryovials.

[0170] In some embodiments, the method further comprises isolating NK cells from the population of expanded and stimulated NK cells.

[0171] An exemplary process for expanding and stimulating NK cells is shown in FIG. 1 .

[0172] In some embodiments, the expanded and stimulated NK cells comprises at least 80%, e.g., at least 90%, at least 95%, at least 99%, or 100% CD56+CD3- cells.

[0173] In some embodiments, the expanded and stimulated NK 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+ cell s. [0174] In some embodiments, the expanded and stimulated NK 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.

[0175] In some embodiments, the expanded and stimulated NK 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.

[0176] In some embodiments, the expanded and stimulated NK 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.

[0177] In some embodiments, the expanded and stimulated NK 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.

[0178] In some embodiments, the expanded and stimulated NK 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% or 0% CD3+ cells.

[0179] In some embodiments, the expanded and stimulated NK 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% or 0% CD 14+ cells.

[0180] In some embodiments, the expanded and stimulated NK 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% or 0% CD 19+ cells.

[0181] In some embodiments, feeder cells do not persist in the expanded and stimulated NK cells.

II. MULTISPECIFIC ENGAGERS

[0182] As used herein, the term "multispecific engager" refers to an antibody construct which is “at least bispecific", i.e., it comprises at least a first binding domain and a second binding domain, wherein the first binding domain binds to one antigen or target (here: NK cell receptor, e.g. CD 16a), and the second binding domain binds to another antigen or target (here: the target cell surface antigen CD30). Accordingly, antibody constructs as defined in the context of the present disclosure comprise specificities for at least two different antigens or targets. For example, the first domain preferably binds to an extracellular epitope of an NK cell receptor of one or more of the species selected from human, Macaca species and rodent species. [0183] Multispecific antibody constructs include, for exampie, bispecific and trispecific antibody constructs, or constructs having more than three (e.g. four, five...) specificities. Examples of multispecific antibody constructs are provided, 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 a. (MAbs. 2019 Jul, 11 (5): 899-918).

[0184] In some embodiments, the multispecific engager is a bispecific antibody or antigen binding fragment thereof compri sing a first binding domain that specifically binds to CDI6 (FcγRIII) and a second binding domain that specifically binds to CD30.

[0185] In some embodiments, the multispecific engager is a bispecific engager. In some embodiments, the bispecific engager comprises a CD16 binding domain and a CD30 binding domain.

[0186] In some embodiments, the CD 16 binding domain 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, and 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.

[0187] In some embodiments, the CD 16 binding domain comprises a light chain variable (VL) region comprising or consisting of an amino acid sequence having or having at least 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 or having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 19.

[0188] 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.

[0189] In some embodiments, the CD30 binding domain comprises: 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, 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.

[0190] In some embodiments, the CD30 binding domain comprises a light chain variable (VL) region compri sing or consi sting of an amino acid sequence having or having at least 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 consi sting of an amino acid sequence having or having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 21.

[0191] 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 .

[0192] In some embodiments, the multispecific engager is a tetravalent homodimer. In some embodiments, the tetravalent homodimer comprises a first and second polypeptide monomer, each comprising or consisting of, from the N-terminus to the C-terminus: a CD16A heavy chain variable domain (VH_CD16A) - a first linker (L 1) - a CD30 light chain variable domain (VL_CD30) - a second linker (L2) - a CD30 heavy chain variable domain (VH_CD30) - a third linker (L3) - and a CD16 light chain variable domain (VL_CD16). In some embodiments, the first and second polypeptides dimerize from head to tail through non-covalent interactions of the domains in the Ig heavy (VH) and light (VL) variable chains.

[0193] In some embodiments, the multispecific engager is a tetravalent homodimer. In some embodiments, the tetravalent homodimer comprises a first and second polypeptide monomer, each comprising or consisting of, from the N-terminus to the C-terminus: a CD30 heavy chain variable domain (VH_CD30) - a first linker (L 1) - a CD16A light chain variable domain (VL_CD16A) - a second linker (L2) - a CD16A heavy chain variable domain (VH_CD16A) - a third linker (L3) - and a CD16 light chain variable domain (VL_CD30). In some embodiments, the first and second polypeptides dimerize from head to tail through non- covalent interactions of the domains in the Ig heavy (VH) and light (VL) variable chains.

[0194] In some embodiments, VH_CD 16 comprises a heavy chain complementarity determining region 1 (CDR.H1 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. In some embodiments, VH_CD16 comprises or consists of SEQ ID NO: 19. [0195] 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 (CDR.L2) 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,

[0196] In some embodiments, VH_CD30 comprises 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. In some embodiments, VH_CD30 comprises or consists of SEQ ID NO: 21.

[0197] In some embodiments, VL_CD30 comprises a light chain complementarity determining region 1 (CDR.L1) 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.

[0198] In some embodiments, L 1 comprises or consists of SEQ ID NO: 26.

[0199] In some embodiments, L2 comprises or consists of SEQ ID NO: 27.

[0200] In some embodiments, L3 comprises or consists of SEQ ID NO: 28.

[0201] In some embodiments, the first and second polymeric monomers each comprise or consists of SEQ ID NO: 18.

[0202] In some embodiments, the multispecific engager comprises a tag, e.g., a histidine tag, e.g., a hexa-histidine tag (SEQ ID NO: 25).

[0203] 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 hereby 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 hereby incorporated by reference in its entirety, Rothe et al., “A Phase 1 Study fo the Bispecific Anti-CD30/CD16A Antibody Construct AFM13 in Patients with Relapsed or Refractory Hodgkin Lymphoma,” Blood 125(26):4024-31 (2015), which is hereby incorporated by reference in its entirety.

[0204] As used herein, the term "NK cell receptor" defines proteins and protein complexes on the surface of NK cells. Thus, the term defines cell surface molecules, which are characteristic to NK cells, but are not necessary exclusively expressed on the surface of NK cells but also on other cells such as macrophages or T cells. Examples for NK cell receptors comprise, but are not limited to FcγRIII (CD 16a, CD 16b), NKp46 and NKG2D.

[0205] As used herein, "CD 16a" refers to the activating receptor CD16a, also known as FcγRIII A, expressed on the cell surface of NK cells. CDl oa is an activating receptor triggering the cytotoxic activity of NK cells. The affinity of antibodies for CD 16a directly correlates with their ability to trigger NK cell activation, thus higher affinity towards CD 16a reduces the antibody dose required for activation. The antigen-binding site of the antigen-binding protein binds to CDI6a, but not to CD16b. For example, an antigen-binding site comprising heavy (VH) and light (VL) chain variable domains binding to CD16a, but not binding to CD16B, may be provided by an antigen binding site which specifically binds to an epitope of CD 16a which comprises amino acid residues of the C-terminal sequence SFFPPGYQ (SEQ ID NO: 23) and/or residues G130 and/or Y141 of CD16a (SEQ ID NO: 24) which are not present in CD16b.

[0206] As used herein, "CD16b" refers to receptor CD16b, also known as FcγRIIIB, expressed on neutrophils and eosinophils. The receptor is glycosylphosphatidyl inositol (GPI) anchored and is understood to not trigger any kind of cytotoxic activity of CD 16b positives immune cells.

[0207] As used herein, the term "target cell surface antigen" refers to an antigenic structure expressed by a cell and which is present at the cell surface such that it is accessible for an antibody construct as described herein. In some cases, the "target cell surface antigen" to which the multi specific antibody constructs described herein binds to is CD30. CD30 also known as TNFRSF8, is a cell membrane protein of the tumor necrosis factor receptor family and tumor marker.

[0208] Given that the antibody constructs as defined in the context of the invention are (at least) bispecific, they do not occur naturally and they are markedly different from naturally occurring products. A "multispecific" antibody construct or immunoglobulin is hence an artificial hybrid antibody or immunoglobulin having at least two distinct binding sides with different specificities. Multispecific antibody constructs can be produced by a variety of methods including fusion of hybridomas or linking of Fab’ fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol . 79:315- 321 (1990).

[0209] The at least two binding domains and the variable domains (VH / VL) of the antibody construct of the present disclosure may or may not comprise peptide linkers (spacer or connector peptides). In some embodiments, the term "peptide linker" comprises an amino acid sequence by which the amino acid sequences of one (variable and/or binding) domain and another (variable and/or binding) domain of the antibody construct defined herein are linked with each other. The peptide linkers can also be used to fuse the third domain to the other domains or an Fc part of the antibody construct defined herein. Preferably, such peptide linker does not comprise any polymerization activity.

[0210] As used herein, the term "binding domain" characterizes a domain which (specifically) binds to/interacts with/recognizes a given target epitope or a given target side on the target molecules (antigens), e.g. a NK cell receptor antigen, e.g. CD 16, and the target cell surface antigen CD30, respectively. The structure and function of the first binding domain (recognizing e.g. CD 16), and preferably also the structure and/or function of the second binding domain (recognizing the target cell surface antigen), is/are based on the structure and/or function of an antibody, e.g. of a full-length or whole immunoglobulin molecule and/or is/are drawn from the variable heavy chain (VH) and/or variable light chain (VL) domains of an antibody or fragment thereof. Preferably the first binding domain is characterized by the presence of three light chain CDRs (i.e. CDR1, CDR2 and CDF3 of the VL region) and/or three heavy chain CDRs (i.e. CDR1, CDR2 and CDR3 of the VH region). The second binding domain preferably also comprises the minimum structural requirements of an antibody which allow for the 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). In some cases, the first, and/or second binding domain is produced by or obtainable by phage-display or library screening methods rather than by grafting CDR sequences from a pre-existing (monoclonal) antibody into a scaffold.

[0211] In some embodiments, binding domains are in the form of one or more polypeptides. Such polypeptides may include proteinaceous parts and non-proteinaceous parts (e.g. chemical linkers or chemical cross-linking agents such as glutaraldehyde). Proteins (including fragments thereof, preferably biologically active fragments, and peptides, usually having less than 30 amino acids) comprise two or more amino acids coupled to each other via a covalent peptide bond (resulting in a chain of amino acids).

[0212] As used herein, the term "polypeptide" describes a group of molecules, which usually consist of more than 30 amino acids. Polypeptides may further form multimers such as dimers, trimers and higher oligomers, i.e., consisting of more than one polypeptide molecule. Polypeptide molecules forming such dimers, trimers etc. may be identical or non-identical. The corresponding higher order structures of such multimers are, consequently, termed homo- or heterodimers, homo- or heterotrimers etc. An example for a heteromultimer is an antibody molecule, which, in its naturally occurring form, consists of two identical light polypeptide chains and two identical heavy polypeptide chains. The terms "peptide", “polypeptide" and "protein" also refer to naturally modified peptides/ polypeptides / proteins wherein the modification is affected e.g. by post-translational modifications like glycosylation, acetylation, phosphorylation and the like. A "peptide", "polypeptide" or "protein" when referred to herein may also be chemically modified such as pegylated. Such modifications are well known in the art and described herein below.

[0213] Preferably the binding domain which binds to the NK cell receptor antigen, e.g. CD 16 and/or the binding domain which binds to the target cell surface antigen CD30 is/are human, humanized or murine derived chimeric binding domains. Antibodies and antibody constructs comprising at least one human binding domain avoid some of the problems associated with antibodies or antibody constructs that possess non-human such as rodent (e.g. murine, rat, hamster or rabbit) variable and/or constant regions. The presence of such rodent derived proteins can lead to the rapid clearance of the antibodies or antibody constructs or can lead to the generation of an immune response against the antibody or antibody construct by a patient. In order to avoid the use of rodent derived antibodies or antibody constructs, human or fully human antibodies/ antibody constructs can be generated through the introduction of human antibody function into a rodent so that the rodent produces fully human antibodies.

[0214] In some embodiments, this antigen-binding site for CD16A does not bind to CD16B and binds to the known CD16A allotypes F158 and V158 with similar affinity. Two allelic single nucleotide polymorphisms have been identified in human CD16A altering the amino acid in position 158, which is important for interaction with the hinge region of IgG. The allelic frequencies of the homozygous 158 F/F and the heterozygous 158 V/F alleles are similar within the Caucasian population, ranging between 35 and 52% or 38 and 50%, respectively, whereas the homozygous 158 V/V allele is only found in 10-15% (Lopez-Escamez JA et al BMC Med Genet 2011; 12: 2). Activation of NK-cells by this anti-CD16A domain in all patients due to the similar affinity is therefore advantageous. Further CD16A antigen-binding sites comprising heavy and light variable chain domains that bind to CD 16A, but not to CD16B are described in WO 2006/125668.

[0215] In some embodiments, the heavy and light chain domains incorporate immunologically active homologues or variants of the CDR or framework sequences described herein. In some embodiments, a CDR variant sequence is modified to change non-critical residues or residues in non-critical regions. Amino acids that are not critical 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. III. PHARMACEUTICAL COMPOSITIONS

[0216] Provided herein are pharmaceutical compositions comprising the natural killer cells described herein.

[0217] Pharmaceutical compositions typically include a pharmaceutically acceptable carrier. As used herein the language "pharmaceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.

[0218] In some embodiments, the pharmaceutical composition comprises: a) natural killer cell(s) described herein; and b) a cryopreservation composition.

[0219] Suitable cryopreservation compositions are described herein.

[0220] In some embodiments, the pharmaceutical composition comprises: a) a cryopreservation composition described herein; and b) therapeutic cell(s).

[0221] In some embodiments, the therapeutic cell(s) are animal cell(s). In some embodiments, the therapeutic cell(s) are human cell(s).

[0222] In some embodiments, the therapeutic cell(s) are immune cell(s). In some embodiments, the immune cell(s) are selected from basophils, eosinophils, neutrophils, mast cells, monocytes, macrophages, neutrophils, dendritic cells, natural killer cells, B cells, T cells, and combinations thereof.

[0223] In some embodiments, the immune cell(s) are natural killer (NK) cells. In some embodiments, the natural killer cell(s) are expanded and stimulated by a method described herein.

[0224] In some embodiments, the pharmaceutical composition further comprises: c) a buffer solution. Suitable buffer solutions are described herein, e.g., as for cryopreservation compositions.

[0225] In some embodiments, the pharmaceutical composition comprises from or from about 1x10 ⁷ to or to about 1x10 ⁹ cells/mL. In some embodiments, the pharmaceutical composition comprises 1x10 ⁸ cells/mL. In some embodiments, the pharmaceutical composition comprises about 1x10 ⁸ cells/mL.

[0226] In some embodiments, the pharmaceutical composition further comprises a multispecific engager, e.g., a multispecific engager described herein.

[0227] Pharmaceutical compositions are typically formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. [0228] Methods of formulating suitable pharmaceutical compositions are known in the art, see, e.g., Remington: The Science and Practice of Pharmacy, 21st ed., 2005; and the books in the series Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY). For example, solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0229] Pharmaceutical compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. 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 should be fluid to the extent that easy syringability exists. 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 a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

[0230] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

IV. METHODS OF TREATMENT

[0231] The NK cells described herein find use for treating cancer or other proliferative disorders.

[0232] Thus, also provided herein are methods of treating a patient suffering from a disorder, e.g., a disorder associated with a cancer, e.g., a CD30+ cancer|, comprising administering the NK. cells, e.g., the NK cells described herein, and a CD30 targeting multispecific engager, e.g., a multispecific engager described herein.

[0233] Also provided herein are methods of preventing, reducing and/or inhibiting the recurrence, growth, proliferation, migration and/or metastasis of a cancer cell or population of cancer cells in a subject in need thereof, comprising administering the NK cells, e.g., the NK cells described herein, and a CD30 targeting multispecific engager, e.g., a multispecific engager described herein.

[0234] Also provided herein are methods of enhancing, improving, and/or increasing the response to an anticancer therapy in a subject in need thereof, comprising administering the NK cells, e.g., the NK cells described herein, and a CD30 targeting multi specific engager, e.g., a multispecific engager described herein..

[0235] Also provided herein are methods for inducing the immune system in a subject in need thereof comprising administering the NK cells, e.g., the NK cells described herein, and a CD30 targeting multispecific engager, e.g., a multispecific engager described herein.

[0236] The methods described herein include methods for the treatment of disorders associated with abnormal apoptotic or differentiative processes, e.g., cellular proliferative disorders or cellular differentiative disorders, e.g., cancer, including both solid tumors and hematopoietic cancers. Generally, the methods include administering a therapeutically effective amount of a treatment as described herein, to a subject, who is in need of, or who has been determined to be in need of, such treatment. In some embodiments, the methods include administering a therapeutically effective amount of a treatment comprising an NK cells, e.g., NK cells described herein, and a CD30 targeting multi specific engager, e.g., a multispecific engager described herein.. [0237] As used herein, the terms ‘‘treatment,” “treat,” and “treating” refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disorder associated with abnormal apoptotic or differentiative processes. For example, a treatment can result in a reduction in tumor size or growth rate. Administration of a therapeutically effective amount of a compound described herein for the treatment of a condition associated with abnormal apoptotic or differentiative processes wall result in a reduction in tumor size or decreased growth rate, a reduction in risk or frequency of reoccurrence, a delay in reoccurrence, a reduction in metastasis, increased survival, and/or decreased morbidity and mortality, among other things. In some embodiments, treatment may be administered after one or more symptoms have developed. In other embodiments, 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., in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example to prevent or delay their recurrence.

[0238] As used herein, the terms "inhibition”, as it relates to cancer and/or cancer cell proliferation, refer to the inhibition of the growth, division, maturation or viability of cancer cells, and/or causing the death of cancer cells, individually or in aggregate with other cancer cells, by cytotoxicity, nutrient depletion, or the induction of apoptosis.

[0239] As used herein, “delaying” development of a disease or disorder, or one or more symptoms thereof, means to defer, hinder, slow, retard, stabilize and/or postpone development of the disease, disorder, or symptom thereof. This delay can be of varying lengths of time, depending on the history of the disease and/or subject being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the subject does not develop the disease, disorder, or symptom thereof. For example, a method that “delays” development of cancer is a method that reduces the probability of disease development in a given time frame and/or reduces extent of the disease in a given time frame, when compared to not using the method. Such comparisons may be based on clinical studies, using a statistically significant number of subjects.

[0240] As used herein, “prevention” or “preventing” refers to a regimen that protects against the onset of the disease or disorder such that the clinical symptoms of the disease do not develop. Thus, “prevention” relates to administration of a therapy (e.g., administration of a therapeutic substance) to a subject before signs of the disease are detectable in the subject and/or before a certain stage of the disease (e.g., administration of a therapeutic substance to a subject with a cancer that has not yet metastasized). The subject may be an individual at risk of developing the disease or disorder, or at risk of disease progression, e.g., cancer metastasis. Such as an individual who has one or more risk factors known to be associated with development or onset of the disease or disorder. For example, an individual may have mutations associated with the development or progression of a cancer. Further, it is understood that prevention may not result in complete protection against onset of the disease or disorder. In some instances, prevention includes reducing the risk of developing the disease or disorder. The reduction of the risk may not result in complete elimination of the risk of developing the disease or disorder.

[0241] An “increased” or “enhanced” amount (e.g., with respect to antitumor 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 or more times (e.g., 100, 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 2.1, 2.2, 2.3, 2.4, etc.) an amount or level described herein. It may also include an increase of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 500%, or at least 1000% of an amount or level described herein.

[0242] A “decreased” or “reduced” or “lesser” amount (e.g., with respect to tumor size, cancer cell proliferation or growth) 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 or more times (e.g., 100, 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6,

1.7. 1.8, etc.) an amount or level described herein. It may also 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%, or at least 90%, at least 100%, at least 150%, at least 200%, at least 500%, or at least 1000% of an amount or level described herein.

A. Disorders

[0243] Examples of cellular proliferative and/or different! ative disorders include cancer, e.g., carcinoma, sarcoma, metastatic disorders or hematopoietic neoplastic disorders, e.g., leukemias. A metastatic tumor can arise from a multitude of primary tumor types, including but not limited to those of prostate, colon, lung, breast and liver origin.

[0244] As used herein, the terms “cancer”, “hyperproliferative” and “neoplastic” refer to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. Hyperproliferative and neoplastic disease states may be categorized as pathologic, i.e., characterizing or constituting a disease state, or may be categorized as non-pathologic, i.e., a deviation from normal but not associated with a disease state. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness, “Pathologic hyperproliferative” cells occur in disease states characterized by malignant tumor growth. Examples of non-pathologic hyperproliferative cells include proliferation of cells associated with wound repair.

[0245] The terms “cancer” or “neoplasms” include malignancies of the various organ systems, such as affecting lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon 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.

[0246] The term “carcinoma” is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary’ system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. In some embodiments, the disease is renal carcinoma or melanoma. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term also includes carcinosarcomas, e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues. An “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.

[0247] The term “sarcoma” is art recognized and refers to malignant tumors of mesenchymal derivation.

[0248] Additional 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, e.g., arising from myeloid, lymphoid or erythroid lineages, or precursor cells thereof. Preferably, the diseases arise from poorly differentiated acute leukemias, e.g., erythroblastic leukemia and acute megakaryoblastic leukemia. Additional exemplary myeloid disorders include, but are not limited to, acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CAIL) (reviewed in Vaickus, L. (1991) Crit Rev. in Oncol. /Hem otol. 11 :267-97); lymphoid malignancies include, but are not limited to acute lymphoblastic leukemia (ALL) which includes B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WAI). Additional forms of malignant lymphomas include, but are not limited to non-Hodgkin lymphoma and variants thereof, peripheral T cell lymphomas, adult T cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease and Reed-Sternberg disease.

[0249] In some embodiments, the cancer is selected from the group consisting of: acute lymphoblastic leukemia (ALL), acute myeloid leukemia (ANIL), adrenocortical carcinoma, Kaposi sarcoma, AIDS-related lymphoma, primary CNS lymphoma, anal cancer, appendix cancer, astrocytoma, typical teratoid/rhabdoid tumor, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain tumor, breast cancer, bronchial tumor, Burkitt lymphoma, carcinoid, cardiac tumors, medulloblastoma, germ cell tumor, primary CNS lymphoma, cervical cancer, cholangiocarcinoma, chordoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloproliferative neoplasms, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, ductal carcinoma in situ, embryonal tumors, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, Ewing sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, eye cancer (e.g., intraocular melanoma or retinoblastoma), fallopian tube cancer, fibrous histiocytoma of bone, osteosarcoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumors (GIST), germ cell tumors, gestational trophoblastic disease, hairy cell leukemia, head and neck cancer, heart tumor, hepatocellular cancer, histiocytosis, Hodgkin lymphomas, hypopharyngeal cancer, intraocular melanoma, islet cell tumors, pancreatic neuroendocrine tumors, kidney (renal cell) carcinoma, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer, lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, pleuropulmonary blastoma, and tracheobronchial tumor), lymphoma, male breast cancer, malignant fibrous histiocytoma of bone, melanoma, Merkel cell carcinoma, mesothelioma, metastatic cancer, metastatic squamous neck cancer, midline tract carcinoma, mouth cancer, multiple endocrine neoplasia syndromes, multiple myeloma/plasma cell neoplasms, mycosis fungoides, myelodysplastic syndromes, myelodysplastic/myeloproliferative neoplasms, myeloproliferative neoplasms, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, oral cancer, lip and oral cavity cancer, oropharyngeal cancer, osteosarcoma, malignant fibrous histiocytoma, ovarian cancer, pancreatic cancer, pancreatic neuroendocrine tumors, papillomatosis, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytomas, pituitary tumor, plasma cell neoplasm, multiple myeloma, pleuropulmonary blastoma, pregnancy and breast cancer, primary central nervous system lymphoma, primary peritoneal cancer, prostate cancer, rectal cancer, recurrent cancer, renal cell cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma (e.g., childhood rhabdomyosarcoma, childhood vascular tumors, Ewing sarcoma, Kaposi sarcoma, osteosarcoma, soft tissue sarcoma, uterine sarcoma), Sezary syndrome, skin cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer, stomach cancer, T-cell lymphomas, testicular cancer, throat cancer, nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer, thryomoma and thymic carcinomas, thyroid cancer, tracheobronchial tumors, transitional cell cancer of the renal pelvis and ureter, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vascular tumors, vulvar cancer, and Wilms tumor.

[0250] In some embodiments, the cancer is a solid tumor.

[0251] In some embodiments, the cancer is metastatic.

[0252] In some embodiments, the cancer is a CD30+ cancer.

[0253] In some embodiments, the CD30+ cancer is a lymphoma. In some embodiments, the lymphoma is selected from the group consisting of classic Hodgkin lymphoma (CHL), anaplastic large cell lymphoma (ALCL), grey zone lymphoma (GZL), Epstein-Barr virus- positive diffuse large B-cell lymphoma (EBV+ DLBCL), and combinations thereof.

B. Patients

[0254] In some embodiments, the methods of treatment provided herein may be used to treat a subject (e.g., human, monkey, dog, cat, mouse) who has been diagnosed with or is suspected of having a cellular proliferative and/or differentiative disorder, e.g., a cancer. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0255] As used herein, a subject refers to a mammal, including, for example, a human.

[0256] In some embodiments, the mammal is selected from the group consisting of an armadillo, an ass, a bat, a bear, a beaver, a cat, a chimpanzee, a cow, a coyote, a deer, a dog, a dolphin, an elephant, a fox, a panda, a gibbon, a giraffe, a goat, a gopher, a hedgehog, a hippopotamus, a horse, a humpback whale, a jaguar, a kangaroo, a koala, a leopard, a lion, a llama, a lynx, a mole, a monkey, a mouse, a narwhal, an orangutan, an orca, an otter, an ox, a pig, a polar bear, a porcupine, a puma, a rabbit, a raccoon, a rat, a rhinoceros, a sheep, a squirrel, a tiger, a walrus, a weasel, a wolf, a zebra, a goat, a horse, and combinations thereof.

[0257] In some embodiments, the mammal is a human.

[0258] The subject, e.g., the human subject, can be a child, e.g., from or from about 0 to or to about 14 years in age. The subject can be a youth, e.g., from or from about 15 to or to about 24 years in age. The subject can be an adult, e.g., from or from about 25 to or to about 64 years in age. The subject can be a senior, e.g, 65+ years in age. [0259] In some embodiments, the subject may be a human who exhibits one or more symptoms associated with a cellular proliferative and/or differentiative disorder, e.g., a cancer, e.g., a tumor. Any of the methods of treatment provided herein may be used to treat cancer at various stages. By way of example, the cancer stage includes but is not limited to early, advanced, locally advanced, remission, refractory, reoccurred after remission and progressive. In some embodiments, the subject is at an early stage of a cancer. In other embodiments, the subject is at an advanced stage of cancer. In various embodiments, the subject has a stage I, stage II, stage III or stage IV cancer. The methods of treatment described herein can promote reduction or retraction of a tumor, decrease or inhibit tumor growth or cancer cell proliferation, and/or induce, increase or promote tumor cell killing. I n some embodiments, the subject is in cancer remission. The methods of treatment described herein can prevent or delay metastasis or recurrence of cancer.

[0260] In some embodiments, the subject is at risk, or genetically or otherwise predisposed (e.g., risk factor), to developing a cellular proliferative and/or differentiative disorder, e.g., a cancer, that has or has not been diagnosed.

[0261] As used herein, an “at risk” individual is an individual who is at risk of developing a condition to be treated, e.g., a cellular proliferative and/or differentiative disorder, e.g., a cancer. Generally, an “at risk” subject may or may not have detectable disease, and may or may not have displayed detectable disease prior to the treatment methods described herein. “At risk” denotes that an individual has one or more so-called risk factors, which are measurable parameters that correlate with development of a disease or condition and are known in the art. For example, an at risk subject may have one or more risk factors, which are measurable parameters that correlate with development of cancer. A subject having one or more of these risk factors has a higher probability of developing cancer than an individual without these risk factor(s). In general, risk factors may include, for example, age, sex, race, diet, history of previous disease, presence of precursor disease, genetic (e.g., hereditary) considerations, and environmental exposure. In some embodiments, the subjects at risk for cancer include, for example, those having relatives who have experienced the disease, and those whose risk is determined by analysis of genetic or biochemical markers.

[0262] In addition, the subject may be undergoing one or more standard therapies, such as chemotherapy, radiotherapy, immunotherapy, surgery, or combination thereof Accordingly, one or more kinase inhibitors may be administered before, during, or after administration of chemotherapy, radiotherapy, immunotherapy, surgery or combination thereof. [0263] In certain embodiments, the subject may be a human who is (i) substantially refractor}' to at least one chemotherapy treatment, or (ii) is in relapse after treatment with chemotherapy, or both (i) and (ii). In some of embodiments, the subject is refractory to at least two, at least three, or at least four chemotherapy treatments (including standard or experimental chemotherapies).

[0264] In some embodiments, the patient is diagnosed with or has been diagnosed with CD30+ cancer,

[0265] In some embodiments, the patient is diagnosed with or has been diagnosed with a CD30+ cancer by immunohistochemical staining of a biopsy or surgical sample of the cancer. In some embodiments, the patient is diagnosed with or has been diagnosed with a CD30+ cancer by chromogenic in situ hybridization. In some embodiments, the patient is diagnosed with or has been diagnosed with a CD30+ cancer by fluorescent in situ hybridization of a biopsy or surgical sample of the cancer. In some embodiments, the patient is diagnosed with or has been diagnosed with a CD30+ cancer by genetic analysis.

[0266] In some embodiments, the patient is refractory to or has a recurrence after treatment with a CD30 inhibitor.

[0267] In some embodiments, the patient is refractory to or has a recurrence after treatment with a chemotherapy drug.

[0268] In some embodiments, the chemotherapy drug is selected from the group consisting of cisplatin, docetaxel, carboplatin, gemcitabine, cisplatin, pemetrexed, or combinations thereof

[0269] In some embodiments, the patient is refractory to or has a recurrence 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.

C. Lymphodepletion

[0270] In some embodiments, the patient is lymphodepleted before treatment.

[0271] Illustrative lymphodepleting chemotherapy regimens, along with correlative beneficial biomarkers, are described in WO 2016/191756 and WO 2019/079564, hereby incorporated by reference in their entirety. In certain embodiments, the lymphodepleting chemotherapy regimen comprises administering to the patient doses of cyclophosphamide (between 200 mg/m ² /day and 2000 mg/m ² /day) and doses of fludarabine (between 20 mg/m ² /day and 900 mg/m ² /day). [0272] In some embodiments, lymphodepletion comprises administration of or of about 250 to about 500 mg/m ² of cyclophosphamide, e.g., from or from about 250 to or to about 500, 250, 400, 500, about 250, about 400, or about 500 mg/m ² of cyclophosphamide.

[0273] In some embodiments, lymphodepletion comprises administration of or of about 20 mg/m ² /day to or to about 40 mg/m ² /day fludarabine, e.g., 30 or about 30 mg/m ² /day.

[0274] In some embodiments, lymphodepletion comprises administration of both cyclophosmamide and fludarabine.

[0275] In some embodiments, the patient is lymphodepleted by intravenous administration of cyclophosphamide (250 mg/m ² /day) and fludarabine (30 mg/m7day).

[0276] In some embodiments, the patient is lymphodepleted by intravenous administration of cyclophosphamide (500 mg/m ² /day) and fludarabine (30 mg/m ² /day).

[0277] In some embodiments, the lymphodepletion occurs no more than 5 days prior to the first dose of NK cells. In some embodiments, the lymphodepletion occurs no more than 7 days prior to the first dose of NK cells.

[0278] In some embodiments, lymphodepletion occurs daily for 3 consecutive days, starting 5 days before the first dose of NK cells (i.e., from Day -5 through Day -3).

[0279] In some embodiments, the lymphodepletion occurs on day -5, day -4 and day -3.

D. Administration

1. NK Cells

[0280] In some embodiments, the NK cells are administered as part of a pharmaceutical composition, e.g., a pharmaceutical composition described herein.

[0281] In some embodiments, the NK cells, e.g., the NK cells described herein are administered at or at about 1 x 10 ⁸ to or to about 8 x 10 ⁹ NK cells per dose. In some embodiments, the NK cells are administered at or at about 1 x 10 ⁸ , at or at about 1 x 10 ⁹ , at or at about 4 x 10 ⁹ , or at or at about 8 x 10 ⁹ NK cells per dose.

[0282] In some embodiments, the NK cells are administered weekly. In some embodiments, the NK cells are administered for or for about weeks. In some embodiments, the NK cells are administered weekly for or for about 8 weeks.

[0283] In some embodiments, the NK cells are cryopreserved in an infusion-ready media, e.g., a cryopreservation composition suitable for intravenous administration, e.g., as described herein. [0284] In some embodiments, the NK cells are cryopreserved in vials containing from or from about 1 x 10 ⁸ to or to about 8 x 10 ⁹ cells per vial. In some embodiments, the NK cells are cryopreserved in vials containing a single dose.

[0285] In some embodiments, the cells are thawed, e.g., in a 37°C water bath, prior to administration.

[0286] In some embodiments, the thawed vial(s) of NK cells are aseptically transferred to a single administration vessel, e.g., administration bag using, e.g., a vial adapter and a sterile syringe. The NK cells can be administered to the patient from the vessel through a Y-type blood/solution set filter as an IV infusion, by gravity.

[0287] In some embodiments, the NK cells are administered as soon as practical, preferably less than 90 minutes, e.g., less than 80, 70, 60, 50, 40, 30, 20, or 10 minutes after thawing. In some embodiments, the NK cells are administered within 30 minutes of thawing.

[0288] In some embodiments, the pharmaceutical composition is administered intravenously via syringe.

[0289] In some embodiments, 1 mL, 4 mL, or 10 mL of drug product is administered to the patient intravenously via syringe.

2. MiMspecific Engagers

[0290] In some embodiments, the NK cell(s) described herein, e.g., the pharmaceutical compositions comprising NK cell(s) described herein, are administered in combination with a multispecific engager, e.g., a multispecific engager described herein, e.g., an CD30-targeting multispecific engager. In some embodiments, a multispecific engager is administered together with the NK cells as part of a pharmaceutical composition. In some embodiments, a multispecific engager is administered separately from the NK cells, e.g., as part of a separate pharm aceuti cal composition .

[0291] In some embodiments, the multispecific engager is administered before the NK cells. In some embodiments, the multispecific engager is administered after the NK cells.

[0292] In some embodiments, the NK cells are administered the day after the multispecific engager is administered.

[0293] In some embodiments, the NK cells are administered at each administration, while the multispecific engager is administered at a subset of the administrations. For example, in some embodiments, the NK cells are administered once a week and the multispecific engager is administered once a month. [0294] In some embodiments, a dose of multi specific engager is given prior to the first dose of cells. In some embodiments, a debulking dose of the multispecific engager is given prior to the first dose of cells.

E. Dosing

[0295] An “effective amount” is an amount sufficient to effect beneficial or desired results. For example, a therapeutic amount is one that achieves the desired therapeutic effect. This amount can be the same or different from a prophylactically effective amount, which is an amount necessary to prevent onset of disease or disease symptoms. An effective amount can be administered in one or more administrations, applications or dosages. A therapeutically effective amount of a therapeutic compound (i.e., an effective dosage) depends on the therapeutic compounds selected. The compositions can be administered one from one or more times per day to one or more times per week; including once every other day. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the therapeutic compounds described herein can include a si ngle treatment or a series of treatments.

[0296] Dosage, toxicity and therapeutic efficacy of the therapeutic compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining 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 toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit high therapeutic indices are preferred. While 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 tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[0297] The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds may be within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

V. VARIANTS

[0298] In some embodiments, the molecule(s) or components thereof described herein, the fusion protein(s) or components thereof described herein, or the NK cell genotypes described herein, are at least 80%, e.g., at least 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of an exemplary sequence (e.g., as provided herein), e.g., have differences at up to 1%, 2%, 5%, 10%, 15%, or 20% of the residues of the exemplary sequence replaced, e.g., with conservative mutations, e.g., including or in addition to the mutations described herein. In preferred embodiments, the variant retains desired activity of the parent.

[0299] To determine the percent identity of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non- homologous sequences can be disregarded for comparison purposes). The length of a reference sequence aligned for compari son purposes is at least 80% of the length of the reference sequence, and in some embodiments is at least 90% or 100%. The nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein nucleic acid "identity” is equivalent to nucleic acid "homology"). The percent identity between the 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, which need to be introduced for optimal alignment of the two sequences.

[0300] Percent identity between a subject polypeptide or nucleic acid sequence (i.e. a query) and a second polypeptide or nucleic acid sequence (i.e. target) is determined in various ways that are within the skill in the art, for instance, using publicly available computer software such as Smith Waterman Alignment (Smith, T. F. and M. S. Waterman (1981) J Mol Biol 147: 195-7); "BestFit" (Smith and Waterman, Advances in Applied Mathematics, 482-489 (1981)) as incorporated into GeneMatcher PlusTM, Schwarz and Dayhof (1979) Atlas of Protein Sequence and Structure, Dayhof, M.O., Ed, pp 353-358; BLAST program (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 needed to achieve maximal alignment over the length of the sequences being compared. In general, for target proteins or nucleic acids, the length of compari son can be any length, up to and including full length of the target (e.g., 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%). For the purposes of the present disclosure, percent identity is relative to the full length of the query sequence.

[0301] For purposes of the present disclosure, the comparison of sequences 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 extend penalty of 4, and a frameshift gap penalty of 5.

[0302] 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.

VI. DEFINITIONS

[0303] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0304] Throughout this application, various embodiments may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. [0305] As used in the specification and claims, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a sample” includes a plurality of samples, including mixtures thereof.

[0306] The terms “determining,” “measuring,” “evaluating,” “assessing,” “assaying,” and “analyzing” are often used interchangeably herein to refer to forms of measurement. The terms include determining if an element is present or not (for example, detection). These terms can include quantitative, qualitative or quantitative and qualitative determinations. Assessing can be relative or absolute. “Detecting the presence of” can include determining the amount of something present in addition to determining whether it is present or absent depending on the context.

[0307] The terms “subject,” “individual,” or “patient” are often used interchangeably herein.

[0308] The term “in vivo” is used to describe an event that takes place in a subject’s body.

[0309] The term “ex vivo” is used to describe an event that takes place outside of a subject’s body. An ex vivo assay is not performed on a subject. Rather, it is performed upon a sample separate from a subject. An example of an ex vivo assay performed on a sample is an “in vitro” assay.

[0310] The term “in vitro” is used to describe an event that takes places contained in a container for holding laboratory reagent such that it is separated from the biological source from which the material is obtained. In vitro assays can encompass cell-based assays in which living or dead cells are employed. In vitro assays can also encompass a cell-free assay in which no intact cells are employed.

[0311] As used herein, the term “about” a number refers to that number plus or minus 10% of that number. The term “about” a range refers to that, range minus 10% of its lowest value and plus 10% of its greatest value.

[0312] 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.

[0313] As used herein, the term "cell culture medium" refers to a mixture for growth and proliferation of cells in vitro, which contains essential elements for growth and proliferation of cells such as sugars, amino acids, various nutrients, inorganic substances, etc.

[0314] A buffer solution, as used herein, is not. a cell culture medium. [0315] As used herein, the term “bioreactor” refers to a culture apparatus capable of continuously controlling a series of conditions that affect cell culture, such as dissolved oxygen concentration, dissolved carbon dioxide concentration, pH, and temperature.

[0316] The term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self- replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Some vectors are suitable for delivering the nucleic acid molecule(s) or polynucleotide(s) of the present application. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as expression vectors.

[0317] The term “operably linked” refers to two or more nucleic acid sequence or polypeptide elements that are usually physically linked and are in a functional relationship with each other. For instance, a promoter is operably linked to a coding sequence if the promoter is able to initiate or regulate the transcription or expression of a coding sequence, in which case, the coding sequence should be understood as being “under the control of" the promoter.

[0318] 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 include “engineered cells,” “transformants,” and “transformed cells,” which include the primary engineered (e.g., transformed) cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0319] As appropriate, the host cells can be stably or transiently transfected with a polynucleotide encoding a fusion protein, as described herein.

[0320] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

VII. EXAMPLES

[0321] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

Example 1: Off-the-Shelf NK Cell Therapy Platform

[0322] One example of a method by which NK cells were expanded and stimulated is shown FIG. 1. [0323] A single unit of FDA-licensed, frozen cord blood that has a high affinity variant of the receptor CD 16 (the 158 V/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 the KIR-B genotype (KIR B allele of the KIR receptor family, see, e.g., Hsu et al., “The Killer Cell Immunoglobulin-Like Receptor (K IR ) 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 :el 5115 (2010)) was selected as the source of NK cells.

[0324] The cord blood unit was thawed and the freezing medium was removed via centrifugation. The cell preparation was then depleted of T cells using the QuadroMACS Cell Selection System (Miltenyi) and CD3 (T cell) MicroBeads. A population of nucleated cells (TNC) were labelled with the MicroBeads and separated using the QuadroMACS device and buffer. Following depletion of T cells, the remaining cells, which were predominantly monocytes and NK cells, were washed and collected in antibiotic-free medium (CellgroSCGM). The cell preparation was then evaluated for total nucleated cell count, viability, and % CD3+ cells. As shown in FIG. 1 , the cord blood NK cells were CD3 depleted.

[0325] The CD3- cell preparation was inoculated into a gas permeable cell expansion bag containing growth medium. The cells were co-cultured with replication incompetent engineered HuT-78 (eHUT-78) feeder cells to enhance expansion for master cell bank (MCB) production. The CellgroSCGM growth media was initially supplemented with anti-CD3 antibody (OKT3), human plasma, glutamine, and IL-2.

[0326] As shown in FIG. 1, the NK cells are optionally engineered, e.g., to introduce CARs into the NK cells, e.g., with a lentaviral vector, during one of the co-culturing steps.

[0327] The cells were incubated as a static culture for 12-16 days at 37 ^C C in a 5% CO2 balanced air environment, with additional exchanges of media occurring every 2 to 4 days. After the culture has expanded more than 100-fold, the cultured cells were harvested and then suspended in freezing medium at 1.0 x 10 ⁸ cells/mL and filled into 5 mF. cryobags. In this example, 80 bags or vials at 10 ^s cells per bag or vial were produced during the co-culture. The cryobags were frozen using a controlled rate freezer and stored in vapor phase liquid nitrogen (LN2) tanks below -150°C. These cryopreserved NK cells derived from the FDA-licensed cord blood unit served as the master cell bank (MCB). [0328] To produce the drug product, a bag of frozen cells from the MCB was thawed and the freezing medium was removed. The thawed cells were inoculated into a disposable culture bag and co-cultured with feeder cells, e.g., eHUT78 feeder cells to produce the drug product. In this example, the cells are cultured in a 50 L bioreactor to produce thousands of lots of the drug product per unit of cord blood (e.g., 4,000-8,000 cryovials at 10 ⁹ cells/vial), which are mixed with a cryopreservation composition and frozen in a plurality of storage vessels such as cryovials. The drug product is an off-the-shelf infusion ready product that can be used for direct infusion. Each lot of the drug product can be used to infuse hundreds to thousands of patients (e.g., 100-1,000 patients, e.g. with a target dose of 4 x 10 ⁹ cells).

Example 2: Feeder Cell Expansion

[0329] As one example, suitable feeder cells, e.g., eHut-78 cells, were thawed from a frozen stock and expanded and cultured in a 125 mL flask in growth medium comprising RPMI1640 (Life Technologies) 89% v/v, inactivated fetal bovine serum (FBS) (Life Technologies) (10% v/v), and glutamine (hyclone) (2 mM) at or at about 37°C and at or at about

3-7% CO2 for or for about 18-24 days. The cells were split every 2-3 days into 125mL-2L flasks until the total volume reached 9.6L or more. The cells were harvested by centrifugation and gamma irradiated at 9,995-10,005 cGy. The harvested and irradiated cells were mixed with a cryopreservation medium (Cryostor CS10) in 2mL cryovials and frozen in a controlled rate freezer, with a decrease in temperature of about. 15°C every 5 minutes to a final temperature of or of about -90°C, after which they w'ere transferred to a liquid nitrogen tank or freezer to a final temperature of or of about -150°C.

[0330] After freezing, cell viability was greater than or equal to 70% of the original number of cells (here, at least 1.0 x 10 ⁸ viable cells/mL), and 85% or more of the cells expressed tmTNF-α , 85% or more of the cells expressed mb IL-21+, and 85% or more of the cells expressed

4- 1 BBL.

Example 3: NK Cell Expansion and Stimulation

[0331] As one example, suitable NK cells can be prepared as follows using HuT-78 cells transduced to express 4-1 BBL, membrane bound IL-21 and membrane bound TNF alpha (“ eHut- 78P cells”) as feeder cells. The feeder cells are suspended in 1% (v/v) CellGro medium at 2.5* 10 ⁶ cells/ml and are irradiated with 20,000 cGy in a gamma-ray irradiator. Seed cells (e.g., CD3-depleted PBMC or CD3-depleted cord blood cells) are grown on the feeder cells in CellGro medium containing human plasma, glutamine, IL-2, andOKT-3 in in static culture at 37° C. The cells are split every 2-4 days. The NK cells are harvested by centrifugation and cryopreserved. Thawed NK are administered to patients in infusion medium consisting of: Phosphate Buffered Saline (PBS lx, FujiFilm Irvine), albumin (human) (OctaPharma albumin solution. Dextran 40 in Dextrose (Hospira Dextran 40 in Dextrose Injection, USP) and dimethyl sulfoxide (DMSO) (Avantor DMSL solution)..

[0332] In some case, the seed cells are CD3-depleted cord blood cells. A cell fraction can be depleted of CD3 cells by immunomagnetic selection, for example, using a CliniMACS T cell depletion set ((LS Depletion set (162-01) Miltenyi Biotec).

[0333] Preferably, the cord blood seed cells are selected to express CD 16 having the V/V polymorphism at Fl 58 (Fc gamma RIIIa-158 V/V genotype) (Musolino et al. 2008 J Clin Oncol 26: 1789). Preferably, the cord blood seed cells are KIR-B haplotype.

Example 4: Preloading AB-101 NK cells with AFM13

[0334] As one example, 17.6 billion of AB-101 NK cells (cord-blood derived and expanded NK cells selected for V/V and KIR-B) (including 10% extra amount considering cell loss during centrifugation and formulation) were taken at harvest and transferred to the lab.

[0335] For preloading the AB-101 cells with AFM13 (homodimer of SEQ ID NO: 18), 4 billion cells were incubated in 40 mL culture media in the presence of 100 μg/mL of AFM13 for 30 minutes at ambient temperature, then each replicate was washed twice in 10 mL culture media, resuspended in 40 mL freeze media and cryopreserved in 1 mL aliquots. This preloading process results in AB-101 NK cells with AFM13 bound to (precomplexed with) CD16A molecules on the cells.

[0336] For the control AB-101 cells, 8 billion cells were resuspended in 80 mL Freezing medium, wherein the cells were frozen in 1 mL aliquots. The total number of cells required was 17.6 billion cells.

Example 5: Antibody dependent cell-mediated cytotoxicity (ADCC) of AFM13 and AB-101 NK cells

[0337] As one example, antibody-mediated target cell lysis by AB-101 NK cells in vitro was assessed by quantifying the release of calcein into cell culture supernatants from calcein- labeled Karpas-299 target cells after 4 hours. The assay was carried out with: 1) non- precomplexed (empty) AB- 101 cells that had previously been washed and cryopreserved alone (“non-preloaded” condition); 2) “non-preloaded” AB-101 cells as in condition 1 , but in combination with fresh (never frozen) AFM13 ("non-preloaded + fresh excess AFM13” condition), and 3) AB-101 cells preloaded with AFM13 (as described in Example 4), followed by removal of unbound AFM13 (i.e., washing) and subsequent cryopreservation. Prior to the assay, ciyopreserved AB-101 cells were swiftly thawed at 37°C and washed in PBS buffer supplemented with 2% FCS and 0.6% citrate-dextrose solution. The assay was carried out with AB-101 cells derived from two different cord units (MCB1 and MCB2), n=3 and n=4, respectively.

[0338] Target cells were labeled with 10 μM calcein AM for 30 min in RPMI 1640 medium without FCS at 37°C. After gentle washing, calcein-labeled cells were resuspended in complete RPMI medium (i.e., RPMI 1640 medium supplemented with 10% h.i. FCS, 2 mM L glutamine, 100 U/mL penicillin G sodium, 100 μg/mL streptomycin sulfate) at a density of 1x10 ⁵ /mL. 1x10 ⁴ target cells were then seeded in individual wells of a round-bottom 96-well microtiter plate and, if not mentioned otherwise, mixed with thawed empty or preloaded AB-101 NK cells at decreasing effector-to-target cell (E:T) ratios starting at 10: 1 followed by two-fold serial dilutions. If indicated, 10 μg/mL of AFM13 were added to individual wells of empty or preloaded AB- 101 NK cells to a total volume of 200 μL/well in duplicates.

[0339] Spontaneous calcein-release and maximal release were determined in quadruplicate on each plate. Spontaneous release was determined by incubation of target cells in the absence of effector NK cells and in the absence of AFMI3. Maximal release was achieved by adding Triton X 100 to a final concentration of 1% in the absence of effector cells and in the absence of antibodies. After centrifugation for 2 min at 200xg, microtiter plates were incubated for 4 h at 37°C in a humidified atmosphere with 5% CO2. Following incubation, 100 pL cell- free cell culture supernatant was harvested from each well after centrifugation for 5 min at 500xg and transferred to black flat-bottom 96-w'ell microtiter plates. Fluorescence counts of released calcein were measured at 520 nm using a multimode plate reader. Specific cell lysis was calculated according to the following formula: [fluorescence (sample) - fluorescence (spontaneous)] / [fluorescence (maximum) - fluorescence (spontaneous)] x 100% wherein “Fluorescence (spontaneous)” and “Fluorescence (maximum)” are defined as fluorescence in absence of effector cells and antibodies and fluorescence induced by the addition of Triton X100, respectively.

[0340] As shown in FIG. 2, FIG. 3, and FIG. 4, ADCC activity of AB-101 cells in combination with AFM13 exhibited an ADCC response as strong or stronger than AB-101 alone, both when AFM13 was added fresh and when it was pre-loaded prior to cry opreservation. Exampie 6: Retention of bound AFM13 after crvopreservation by AFM13- preloaded cryopreserved AB-101 NK cells

[0341] Retention of bound AFM13 after cry opreservation by AFM13- preloaded cryopreserved AB-101 NK cells (here referred to as occupancy) was assessed by flow cytometry . Preloading procedures and thawing after cry opreservation was performed as described in Example 5.

[0342] Thawed empty or AFM 13 -preloaded AB-101 NK cells were washed and resuspended in FACS buffer (i.e., PBS supplemented with 2% FCS and 0.1% sodium azide) and seeded in individual wells of a round-bottom 96-well microtiter plate at 2-4x 10 ⁵ cells. Cells were incubated with rat anti-AFM13 clone 7 antibody (5 μg/mL, Affimed GmbH) diluted in FACS buffer or FACS alone for 30 min in the dark at 4°C, followed by washing and subsequent incubation with goat anti-rat FITC (1/100, Dianova) for 30 min in the dark at 4°C. After final washing twice, cells were measured on a CytoFlex S flow cytometer (Beckman Coulter) followed by analysis by FlowJo software. Positive detection of AFM13 on AFM 13 -preloaded AB-101 cells was determined in comparison of staining with goat anti-rat FITC only as well as relative to empty AB-101 cells (FIG. 5 and FIG. 6). Filled histograms represent anti -AFM 13 + secondary antibody; open histograms represent secondary antibody only. The fluorescence intensity of bound AFM 13 on pre-complexed AB- 101 cells was as high as that of empty AB- 101 cells freshly incubated with AFM13, indicating maximal loading/saturation.

[0343] For evaluation of maximal loading/saturation, control empty and control preloaded AB-101 were first freshly incubated with AFM 13 (10 μg/mL) prior to the staining procedures described above.

[0344] Additionally, empty, AFM13- preloaded AB-101 NK cells were incubated with mouse anti-human CD16 BV421 (clone 3G8, 1/100, Biolegend), with mouse anti-human CD56 BV785 (clone 5.1H11, 1/100, Biolegend) and analyzed compared to corresponding concentration-matched mouse isotype controls (ah from Biolegend). As shown in FIG. 7 (left: MCB2, right: MCB1), CD 16 expression is detected on preloaded cryopreserved AB- 101 cells after thawing. Filled histograms represent CD16 staining; open histograms represent isotype control antibody staining only. The detection of uniform high AFM13 binding relative to uniform high CD16 expression suggests saturated binding of CD16 by AFM13.

Example 7: AFM 13 mediated NK fratricide detection

[0345] NK-NK cell lysis (i.e., NK fratricide), potentially mediated by cross-linking of two CD16 molecules or of CD30 and CD16 (by AFM 13 ) on two adjacent NK cells, was assessed in vitro by quantifying the release of caicein into cell culture supernatants from caicein- lab eled NK cell after 4 hour co-culture with autologous non-labelled NK cells. Preloading procedures and thawing after cryopreservation was performed as described in Example 5.

[0346] Target empty or preloaded AB-101 NK cells were labelled with caicein as described in Example 5. 5xl0 ⁴ target NK cells were then seeded in individual wells of a round- bottom 96-well microtiter plate and, if not mentioned otherwise, mixed with effector empty or preloaded AB-101 NK cells at 1 : 1 E:T ratio. If indicated, 10 μg/mL of AFM 13 were added to individual wells to a total volume of 200 μL/well in duplicates.

[0347] The following conditions were tested: a) Target calcein-labelled AFM13- preloaded AB-101 cells with effector AFM 13 -preloaded AB-101 cells, b) Target calcein-labelled empty AB-101 cells with effector empty AB-101 cells, and c) Target calcein-labelled empty AB- 101 cells with effector empty AB-101 cells in the presence of excess (no wash) AFM13. As shown in FIG. 8 (MCB2) and FIG. 9 (MCB1), AB-101 cells used in combination with AFM13 exhibited low levels of NK fratricide both if preloaded and if freshly added in excess (‘co- admin’), despite the significant expression of CD30 (and CD 16) on AB-101 cells. In contrast, in primary buffy coat-derived NK cells, AFM13 can result in dose-dependent NK. fratricide.

Example 8:NK cell activation in response to target cells by AFM13- preloaded cryopreserved AB-101 NK cells

[0348] To monitor NK cell activation, up-regulation of CD107a (marker for NK cell degranulation) and intracellular IFN-y expression were assessed in response to Karpas-299 target cell lines and AFM13. Preloading procedures and thawing after cry opreservation were performed as described in Example 5.

[0349] Empty (negative control) and preloaded AB-101 NK cells, and AB- 101 NK cells with 0.5 μg/mL added (fresh) AFM13 were co-cultured with and without tumor target cells at 1 : 1 cell ratio (each 5 * 10 ⁴ cells) in the presence of anti-CD107a--FITC (1/100 v/v, clone H4A3, Biolegend) and GolgiPlug (1/1000 v/v, BD Bioscience) in complete RPMI 1640 medium in round-bottom 96-well plates for 4 hours. The percentage of NK cell positive for extracellular CD 107a+ (FIG. 10 (MCB2) and FIG. 1 1 (MCB 1 )) or intracellular IFN γ+ NK cells (FIG. 12 (MCB2) and FIG. 13 (MCB 1)) was determined by flow cytometry. Flow cytometry staining was performed as described in Example 5. As shown by FIG. 10, FIG. 11, FIG. 12, and FIG. 13, AFM13 -preloaded cells and empty AB-101 cells supplemented with AFM13 exhibited degranulation and increased production of IFN-y, specifically in response to Karpas-299 target cells, while AFM 13 -preloaded AB-101 cells and empty AB-101 cells supplemented with AFM13 did not exhibit significant degranulation or IFN-γ expression in the absence of target cells.

Example 9:Viability of cryopreserved AFM13 preloaded AB-101

[0350] Viability analysis of cryopreserved AF Ms-preloaded AB-101 (vs cryopreserved non-loaded) was performed after thawing and after additional 24 hour culture. As shown in FIG. 14, viability of AB-101 cells was over 80% post-thawing, and over 60% 24 hours post-thawing.

SEQUENCES

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.