Method for the expansion of natural killer cells

Abstract

A method of expanding natural killer cells, comprising: providing a population of internal colloidal cells, wherein each internal colloidal cell comprises an internal colloidal volume and a fluid cell membrane comprising one or more membrane-bound proteins, each or collectively of which is capable of stimulating expansion of natural killer (NK) cells; and culturing a population of cells comprising NK cells capable of responding to the one or more membrane-bound proteins with the population of internal colloidal cells under conditions that allow expansion of the NK cells.

Description

[0001] CROSS-REFERENCE

[0002] This application claims priority to U.S. Provisional Application No. 62 / 984 / 060, filed March 2, 2020, the entire contents of which are incorporated herein by reference. BACKGROUND

[0003] Natural killer (NK) cells, which comprise 10-15% of peripheral blood lymphocytes, play an important role in immune surveillance, thanks to their innate ability to kill cancer and virus-infected cells without prior sensitization. See: Abel et al., Front. Immunol. 9, 1869 (2018); Cerwenka and Lanier, Nat. Rev. Immunol. 16, 112-123 (2016); Adams et al., J. Immunol. 197, 2963-2970 (2016); and Chiossone et al., Nat. Rev. Immunol. 18, 671-688 (2018). NK cells are identified by their surface expression of CD56 and the absence of the T cell marker CD3. A subset of NK cells express the FcyRIII protein, CD16, which enhances the cytotoxic function of NK cells through the mechanism of antibody-dependent cellular cytotoxicity (ADCC). See: Cerwenka and Lanier, Nat. Rev. Immunol. 16, 112-123 (2016); Adams et al., J. Immunol. 197, 2963-2970 (2016); and Freud et al., Immunity 47, 820-833 (2017).

[0004] NK cell function is primarily controlled by a family of cell surface activating and inhibitory receptors. Activating signals are transmitted through activating receptors such as NKG2D, which recognize ligands including the stress-induced protein MICA. Inhibitory receptors recognize molecules that are ubiquitously expressed on normal cells and frequently downregulated in cancer cells, such as MHC class I. By monitoring the balance between activating and inhibitory receptors, NK cells are able to recognize and kill stressed cells, such as infected cells or cancer cells. See: Cerwenka and Lanier, Nat. Rev. Immunol. 16, 112-123 (2016); Chiossone et al., Nat. Rev. Immunol. 18, 671-688 (2018); and Fujisaki et al., Cancer Res. 69, 4010-4017 (2009).

[0005] NK cells are involved in tumor immune surveillance based on many mouse models and human studies. Due to their potent anti-tumor activity, adoptive cell therapy using NK cells is an attractive anticancer treatment approach. See: Cerwenka and Lanier, Nat. Rev. Immunol. 16, 112-123 (2016); Fujisaki et al., Cancer Res. 69, 4010-4017 (2009); Cheung et al., Nat. Rev. Cancer 13, 397-411 (2013); and Brodeur et al., Nat. Rev. Cancer 3, 203-216 (2003).

[0006] Thus, there is a need to establish an amplification system to obtain a large number of high-potential NK cells for clinical applications. SUMMARY

[0007] The specifics of one or more embodiments are set forth in the accompanying drawings and in the following description. Other features, objects, and advantages of the embodiments will be apparent from the description and drawings, and from the claims.

[0008] In one aspect, a method of amplifying natural killer cells is described herein. The method comprises: providing an internal colloidal cell population, wherein each colloidal cell comprises an internal colloid and a fluid cell membrane containing one or more membrane-bound proteins, each or collectively of which is capable of stimulating amplification of natural killer (NK) cells; and culturing a cell population containing NK cells capable of responding to the one or more membrane-bound proteins with the internal colloidal cell population under conditions that allow amplification of the NK cells.

[0009] In some embodiments, the cell population is selected from the group consisting of peripheral blood mononuclear cells (PBMCs), enriched NK cells, iPSC-derived NK cells, embryonic stem cell-derived NK cells, tissue-resident NK cells, splenocytes, umbilical cord blood cells, and hematopoietic stem cell-derived NK cells.

[0010] In some embodiments, the one or more membrane-bound proteins are selected from the group consisting of 41BBL, IL-15, IL-21, B7-H6, BAT3, HLA-DP, HLA-E, HLA-C2, HLA-A, HLA-C, HLA-G, HLA-F, HLA-C, MICA / MICB, ULBP-1, ULBP-2, ULBP-3, ULBP-4, ULBP-5, ULBP-6, AICL, CD48, NTB-A, 2B4, CD2, CD58, CD11a, ICAM1, CRACC, OX40L, CD137L, Nectin-1, Nectin-2, Nectin-3, Nectin-4, necl-1, necl-2, necl-3, necl-4, necl-5, PCNA, AICL, IgG, CD27L, CD72, CEACAM-1, CEACAM-5, OCIL, N-cadherin, E-cadherin, R-cadherin, sialic acid, IL-1, IL-2, IL-4, IL-7, IL-9, IL-12, IL-18, IL-27, IL-33, IL-6, IL-11, CNTF, LIF, OSM, CT-1, CLC, IFN-a, INF-b, CCL-5, agonists of TLR-1, TLR-2, TLR-3, TLR-5, TLR-6, TLR-9, NOD-1, NOD-2, NOD-3, or NLRP3. For example, the one or more membrane-bound proteins can include 41BBL and IL-15.

[0011] In some embodiments, the culturing step is performed in the presence of IL-21 or IL-2.

[0012] In some embodiments, the ratio of the number of NK cells to the number of gel cells is 1:0.5-20 (e.g., 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:12, 1:15, or 1:20).

[0013] In some embodiments, the internalized gelated cell population is produced by steps comprising: providing a population of antigen presenting cells expressing one or more membrane-bound proteins; suspending the population of antigen presenting cells in phenol red-free DMEM containing a protease inhibitor cocktail to produce a first cell suspension; adding a gel solution to the first cell suspension to produce a second cell suspension, wherein the gel solution is capable of increasing membrane permeability of the antigen presenting cells and contains a photoreactive crosslinker and an optional photoinitiator; incubating the second cell suspension at room temperature for a sufficient time for the photoreactive crosslinker and the optional photoinitiator to enter the antigen presenting cells; centrifuging the second cell suspension to produce a cell pellet; resuspending the cell pellet in phenol red-free DMEM to produce a third cell suspension; applying light to the third cell suspension for a sufficient time for the photoreactive crosslinker to crosslink, thereby producing the internalized gelated cell population; and collecting and washing the internalized gelated cell population.

[0014] In some embodiments, the gel solution is prepared such that the osmolality of the second cell suspension is between 320 mOsmol and 290 mOsmol, greater than 320 mOsmol, or less than 290 mOsmol.

[0015] In some embodiments, the gel solution contains dimethyl sulfoxide (DMSO) such that the second cell suspension contains between 0.1 and 5 wt% DMSO.

[0016] In some embodiments, the concentration of the photoreactive crosslinker in the second cell suspension is between 5 wt% and 50 wt%.

[0017] In some embodiments, the photoreactive crosslinker is poly(ethylene glycol)-diacrylate (PEG-DA), the photoinitiator is 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, and the light is blue light at 365 nm. For example, the 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone can range from 0.01 to 1 wt% in the gel solution, and the average molecular weight of the PEG-DA is between 200 Da and 5000 Da, ranging from 2 to 80 wt% in the gel solution.

[0018] In some embodiments, the gel solution is prepared by dissolving 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone in DMSO to produce a solution, and mixing the solution with PEG-DA having an average molecular weight of 700 Da.

[0019] In some embodiments, the concentration of the PEG-DA in the second cell suspension is between 10 wt% and 40 wt%.

[0020] In some embodiments, the population of antigen presenting cells are artificial antigen presenting cells. For example, the artificial antigen presenting cells can be or be engineered from K562 cells, PBMCs, EBV-transformed LCLs, 721.221 cells, 8866 cells, Jurkat cells, Jurkat / KL-1 cells, U937 cells, BJAB cells, NB4 cells, 293T cells, MCF7 cells, Jeg3 cells, Hela cells, A549 cells, 1106mel cells, or CEM cells.

[0021] In some embodiments, the method further comprises administering the expanded NK cells to a subject in need thereof, e.g., a subject having cancer, an infection, an autoimmune disease, an NK cell deficiency disorder, or a disorder with unwanted cells.

[0022] In one aspect, described herein is a method of treating a disease comprising administering expanded NK cells produced from the colloidal cells described herein to a subject in need thereof. In some embodiments, the disease is cancer, an infection, an autoimmune disease, or an NK cell deficiency disorder, such as typical NK deficiency and functional NK deficiency, or a disorder with unwanted cells.

[0023] In another aspect, described herein is a method of producing a population of internal colloidal cells. The method comprises: providing a population of precursor cells expressing one or more membrane-bound proteins; suspending the population of precursor cells in phenol red-free DMEM containing a protease inhibitor cocktail to produce a first cell suspension; adding a colloidal solution to the first cell suspension to produce a second cell suspension, wherein the colloidal solution is capable of increasing the membrane permeability of the precursor cells and contains a photoreactive crosslinking agent and an optional photoinitiator; incubating the second cell suspension at room temperature for a sufficient time for the photoreactive crosslinking agent and optional photoinitiator to enter the precursor cells; centrifuging the second cell suspension to produce a cell pellet; resuspending the cell pellet in phenol red-free DMEM to produce a third cell suspension; applying light to the third cell suspension for a sufficient time for the photoreactive crosslinking agent to crosslink, thereby producing the population of internal colloidal cells; and collecting and washing the population of internal colloidal cells; wherein each internal colloidal cell comprises a colloidal interior and a fluid cell membrane containing one or more membrane-bound proteins.

[0024] In some embodiments, the colloidal solution is prepared such that the osmolality of the second cell suspension is between 320 mOsmol and 290 mOsmol, greater than 320 mOsmol, or less than 290 mOsmol.

[0025] In some embodiments, the colloidal solution contains DMSO such that the second cell suspension contains 0.1 to 5 wt% of DMSO.

[0026] In some embodiments, the concentration of the photoreactive crosslinker in the second cell suspension is 5 wt% to 50 wt%.

[0027] In some embodiments, the photoreactive crosslinker is poly(ethylene glycol)- diacrylate (PEG-DA), the photoinitiator is 2-hydroxy-4'-(2-hydroxyethoxy)-2- methylpropiophenone, and the light is blue light at 365 nm. For example, the range of 2- hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone in the colloidal solution can be 0.01 to 1 wt%, and the average molecular weight of PEG-DA in the colloidal solution can be between 200 Da to 5000 Da, ranging from 2 to 80 wt%.

[0028] In some embodiments, the preparation of the colloidal solution is by dissolving 2- hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone in DMSO to produce a solution, and mixing the solution with PEG-DA having an average molecular weight of 700 Da.

[0029] In some embodiments, the concentration of PEG-DA in the second cell suspension is 10 wt% to 40 wt%.

[0030] In some embodiments, the population of precursor cells is an artificial antigen presenting cell. For example, the artificial antigen presenting cell can be or be engineered from K562 cells, PBMCs, EBV-transformed LCLs, 721.221 cells, 8866 cells, Jurkat cells, Jurkat / KL-1 cells, U937 cells, BJAB cells, NB4 cells, 293T cells, MCF7 cells, Jeg3 cells, Hela cells, A549 cells, 1106mel cells, or CEM cells.

[0031] In some embodiments, each or collectively the one or more membrane-bound proteins are capable of stimulating amplification of natural killer (NK) cells.

[0032] In some embodiments, the one or more membrane-bound proteins are selected from the group consisting of 41BBL, IL-15, IL-21, B7-H6, BAT3, HLA-DP, HLA-E, HLA-C2, HLA-A, HLA-C, HLA-G, HLA-F, HLA-C, MICA / MICB, ULBP-1, ULBP-2, ULBP-3, ULBP-4, ULBP-5, ULBP-6, AICL, CD48, NTB-A, 2B4, CD2, CD58, CD11a, ICAM1, CRACC, OX40L, CD137L, Nectin-1, Nectin-2, Nectin-3, Nectin-4, necl-1, necl-2, necl-3, necl-4, necl-5, PCNA, AICL, IgG, CD27L, CD72, CEACAM-1, CEACAM-5, OCIL, N-cadherin, E-cadherin, R-cadherin, sialic acid, IL-1, IL-2, IL-4, IL-7, IL-9, IL-12, IL-18, IL-27, IL-33, IL-6, IL-11, CNTF, LIF, OSM, CT-1, CLC, IFN-a, INF-b, CCL-5, agonists of TLR-1, TLR-2, TLR-3, TLR-5, TLR-6, TLR-9, NOD-1, NOD-2, NOD-3, or NLRP3.

[0033] In yet another aspect, described herein is an internal colloidal cell population produced by the methods described herein, wherein each cell comprises an internal colloidal interior and a fluid cell membrane containing one or more membrane-bound proteins.

[0034] In one aspect, described herein is a composition comprising an internal colloidal cell population.

[0035] In another aspect, provided herein is a method of inducing an immune response in a subject comprising administering to the subject the composition. BRIEF DESCRIPTION OF DRAWINGS

[0036] FIG. 1 Schematic of an exemplary method for making a colloidal cell.

[0037] FIG. 2 A set of images showing live K562 cells and colloidal K562 cells. Live K562 cells (left) and colloidal K562 cells (right) were visually confirmed to have similar cell morphology prior to further experiments.

[0038] FIG. 3Figure 1 is a set of graphs showing that NK cells are amplified by novel feeder cell systems. PBMCs were co-cultured with irradiated K526-41BBL-mbl5 cells (GM) and gelified cells prepared from K526-41BBL-mbl5 cells (GC) in the presence or absence of IL-21 (100 ng / ml). Total cell number (A), NK cell number (B), and amplification fold (C) were determined at day 0, 7, and 14 (n=3). Error bars represent mean ± SD.

[0039] FIG. 4 Figure 2 is a set of graphs showing that NK cells exhibit increased cytotoxicity after amplification by GC. PBMCs were co-cultured with GM or GC in the presence or absence of IL-21, and in the presence (B) or absence (A) of anti-CD137 antibody. Cytotoxicity of amplified NK cells was evaluated by killing assay. E:T ratios were 1:1, 0.5:1, and 0.25:1 (n=2). Error bars represent mean ± SD.

[0040] FIG. 5 Figure 3 is a set of graphs showing that GC increase cytolytic activity of NK cells. (A) Total cell number (left), NK population (middle), and NK cell number (right) of GM and GC amplification systems were determined. (B) Amplification fold of GM-amplified NK cells and GC-amplified NK cells was determined after 7 days of amplification. (C) Cytolytic activity of NK cells was determined after 7 days of amplification. (D) GM and GC populations in the system were determined during NK amplification.

[0041] FIG. 6 Figure 4 is a set of graphs showing optimization of GC amplification system conditions. (A) NK cells were amplified by different GCs with different stiffness. Total NK cell number was determined after 7 days of amplification. (B) Cytolytic activity of NK cells amplified from GCs was determined after 7 days of amplification.

[0042] FIG. 7 Figure 5 is a set of graphs showing optimization of amplification conditions for NK cells enriched from PBMCs. PBMC-enriched NK cells were amplified using different ratios of cells (NK: feeder cells = 1:10, 1:5, 1:2, and 1:0.5) with 10 IU / mL IL-12 (A-C) or 100 IU / mL IL-2 (D-F). (A) (D) Total cell number (left), NK population (middle), and NK cell number (right) of amplified NK cells were evaluated at different cell ratios at day 0 and day 7. (B) (E) Amplification fold was determined after 7 days of amplification at different cell ratios. (C) (F) Cytolytic activity of amplified NK cells was determined after 7 days of amplification at different cell ratios. DETAILED DESCRIPTION

[0043] Results show that the intracellular hydrogel technology can maintain the integrity of the cell plasma membrane while achieving exceptional stability. See: Lin et al., Nat. Commun. 10, 1057 (2019). By using this technology to prepare artificial antigen presenting cells (APCs) as feeder cells for NK cells, it was observed that the scaled-up system can induce similar amounts of NK cell proliferation compared to standard feeder cell systems. Surprisingly, this scaled-up system not only increases the expression of NK activating receptors, but also enhances the cytotoxicity of NK cells against tumors.

[0044] Accordingly, described herein is a method of scaling up NK cells. The method comprises: providing a population of internal gel cells, each of which comprises a gel interior and a fluid cell membrane containing one or more membrane-bound proteins, each or collectively capable of stimulating the scaling up of NK cells; and culturing a population of cells containing NK cells capable of responding to the one or more membrane-bound proteins under conditions that allow the scaling up of the NK cells with the population of internal gel cells. In other words, the internal gel cells serve as feeder cells.

[0045] The population of NK cells can be selected from the group consisting of peripheral blood mononuclear cells (PBMCs), NK cells enriched from PBMCs or other sources, iPSC-derived NK cells, embryonic stem cell-derived NK cells, tissue-resident NK cells, splenocytes, umbilical cord blood cells, and hematopoietic stem cell-derived NK cells.

[0046] The one or more membrane-bound proteins can be selected from the group consisting of 41BBL, IL-15, IL-21, B7-H6, BAT3, HLA-DP, HLA-E, HLA-C2, HLA-A, HLA-C, HLA-G, HLA-F, HLA-C, MICA / MICB, ULBP-1, ULBP-2, ULBP-3, ULBP-4, ULBP-5, ULBP-6, AICL, CD48, NTB-A, 2B4, CD2, CD58, CD11a, ICAM1, CRACC, OX40L, CD137L, Nectin-1, Nectin-2, Nectin-3, Nectin-4, necl-1, necl-2, necl-3, necl-4, necl-5, PCNA, AICL, IgG, CD27L, CD72, CEACAM-1, CEACAM-5, OCIL, N-Cadherin, E-Cadherin, R-Cadherin, Sialic acid, IL-1, IL-2, IL-4, IL-7, IL-9, IL-12, IL-18, IL-27, IL-33, IL-6, IL-11, CNTF, LIF, OSM, CT-1, CLC, IFN-a, INF-b, CCL-5, an agonist of TLR-1, TLR-2, TLR-3, TLR-5, TLR-6, TLR-9, NOD-1, NOD-2, NOD-3, or NLRP3. For example, the one or more membrane-bound proteins can include 41BBL and IL-15.

[0047] The culturing step can be performed in the presence of IL-21 (e.g., 50 to 200 ng / ml) or IL-2 (e.g., 5 to 200 IU / ml) in a culture medium suitable for culturing and expanding NK cells.

[0048] In some embodiments, the NK cells and the internal colloidal cells can be co-cultured in the presence of IL-21 or IL-2 at a ratio of NK cells to colloidal cells of 1:0.5-20 (e.g., 1:1, 1:2, 1:3, 1:4, 1:5, 1:10, or 1:15).

[0049] Internal gelated cell populations can be produced by a step that includes transiently permeabilizing the lipid membranes of the precursor cell population to allow introduction of an inactive but activatable crosslinking agent into the cell. After the crosslinking agent has entered the permeabilized cell, the cell is returned to its non-permeabilized state, thereby sealing the crosslinking agent inside the cell. Any remaining extravesicular crosslinking agent is then removed, for example, by washing the cells. The internal crosslinking agent is subsequently activated to effect internal gelation of the cell without disturbing the membrane. The permeabilization step can be performed in the presence of the crosslinking agent. See also WO 2018 / 026644.

[0050] The resulting internal gelated cells retain their native appearance and are less susceptible to environmental stress. The membrane lipids and proteins retain their fluidity upon internal gelation. The internal gelated cells produced by this method have a fixed or gelated interior surrounded by a lipid membrane that is substantially identical to that of their precursor cells. Properties of the precursor cells, such as sensitivity to surfactants, membrane fluidity, membrane protein mobility, membrane permeability, membrane content, surface charge, and membrane biological function, can be preserved in the gelated cells.

[0051] Various techniques known in the art can be applied to induce transient membrane poration or permeabilization in the precursor cells. Such techniques include, but are not limited to, freeze-thaw treatment, osmotic shock, sonication, electroporation, laser-induced membrane poration, shear-induced membrane poration, and other mechanical means-based techniques. For example, sonication occurs when cavitation events occur in close proximity to the lipid membrane. The interaction between microbubbles and the membrane creates transient pores through acoustic microstreaming, bubble oscillation, shock waves, and microjet formation that puncture the lipid membrane. The skilled person is able to determine how to apply the technique to create temporary pores in the membrane without permanently damaging the membrane. Typically, the pores created close spontaneously after the transient membrane permeabilization technique is stopped.

[0052] Any crosslinking agent that is able to enter the permeabilized lipid membrane and be activated within the cell to form a gelated interior can be used to produce internal gelated cells. In some embodiments, the crosslinking agent is a monomer or polymer that can be activated to crosslink to form a gel. Thermo-responsive hydrogel crosslinking, photo-responsive hydrogel crosslinking, pH-sensitive hydrogel crosslinking, chemically-responsive hydrogel crosslinking, and sol-gel silica crosslinking are exemplary activatable crosslinking techniques.

[0053] Photopolymerization or photoreactive crosslinking can be used. Photopolymerization is a crosslinking of polymers in which the properties change upon exposure to light, typically in the ultraviolet or visible region of the electromagnetic spectrum, resulting in the solidification and hardening of the material. This process can be performed in the presence or absence of a photoinitiator. Examples of photoinitiators include, but are not limited to, cationic photoinitiators (e.g., onium salts, organometallic salts, and pyridinium salts) and free radical photoinitiators (e.g., diphenyl ketone, xanthone, quinone, benzoin ether, acetophenone, benzoyl oxime, and acyl phosphine). Examples of photoreactive crosslinking agents include, but are not limited to, epoxides, urethanes, polyethers, and polyesters of any molecular weight. Photoreactive crosslinking agents are typically functionalized with acrylate groups for crosslinking. For example, polyethylene glycol diacrylate (PEGDA) of molecular weight 700 can be used with (2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (I-2959) as a photoinitiator.

[0054] Thermosensitive polymers typically contain hydrophobic groups or groups that readily aggregate in chains at a critical temperature. The thermosensitive polymer can be introduced into the permeated cells at a particular temperature (i.e., a non-reactive temperature), followed by crosslinking by changing the temperature to the critical temperature. Examples of thermosensitive polymers suitable for use in the internal gelation methods described herein include, but are not limited to, polyacrylamide derivatives containing hydrophobic side groups, PEG-PLGA-PEG triblock copolymers, hydroxyethyl methacrylate-methyl methacrylate (HEMA-MMA), polyacrylonitrile-polyvinyl chloride (PAN-PVC), poly(N-isopropylacrylamide) (polyNIPAM), poly(N-vinylcaprolactam), cellulose derivatives, oxirane-propylene, and Matrigel.

[0055] In any of the methods described herein, the internal gel cells can be produced by providing a precursor cell population expressing one or more membrane-bound proteins. The precursor cell population is then suspended in phenol red-free DMEM containing a protease inhibitor cocktail to produce a first cell suspension. A gel solution containing a photoreactive crosslinking agent and optionally a photoinitiator is added to the first cell suspension to produce a second cell suspension. The gel solution containing the photoreactive crosslinking agent and optionally the photoinitiator is capable of increasing the membrane permeability of the precursor cells. The second cell suspension is incubated at room temperature for a sufficient period of time to allow the photoreactive crosslinking agent and the photoinitiator to enter the precursor cells. The second cell suspension is then centrifuged to produce a cell pellet, which is resuspended in phenol red-free DMEM to produce a third cell suspension. The third cell suspension is subjected to light for a sufficient period of time to allow the photoreactive crosslinking agent to crosslink, thereby producing an internal gel cell population. The internal gel cells are collected and washed. Each internal gel cell produced in this manner comprises a gel interior and a fluid cell membrane containing one or more membrane-bound proteins expressed by the precursor cell.

[0056] A colloidal solution can be prepared by dissolving a photoinitiator (e.g., I-2959) in dimethyl sulfoxide (DMSO) to create a solution and mixing the solution with a photoreactive crosslinker (e.g., PEG-DA). In some embodiments, the range of I-2959 in the colloidal solution can be 0.01 to 1 wt% and the average molecular weight of PEG-DA can be between 200 Da to 5000 Da, ranging from 2 to 80 wt%. For example, a colloidal solution can be first prepared by dissolving 20 μΐ^ of 750 mg / mL of I-2959 in DMSO, followed by mixing the resulting solution with 200 μΐ^ of PEG-DA.

[0057] In some cases, a colloidal solution is prepared and added to the first cell suspension such that the osmolarity of the second cell suspension is 290 mOsmol to 320 mOsmol, greater than 320 mOsmol, or less than 290 mOsmol.

[0058] A colloidal solution can be prepared and added to the first cell suspension such that the second cell suspension contains 0.1 to 5 wt% of DMSO.

[0059] The stiffness of the colloidal cells can be varied by adjusting the concentration of the crosslinker in the second cell suspension. For example, a colloidal solution can be added to the first cell suspension such that the concentration of the photoreactive crosslinker in the second cell suspension is 5 wt% to 50 wt% (e.g., 5 wt%, 10 wt%, 15 wt%, 20 wt%, 25 wt%, 30 wt%, 35 wt%, and 40 wt%).

[0060] The expanded NK cells produced by the methods described herein can be administered to a subject in need thereof. The expanded NK cells can be derived from a population of cells (e.g., PBMCs) obtained from the subject or another donor subject. The expanded NK cells can be used to treat cancer, infection, autoimmune disease, or NK cell deficiency disorders, such as classic NK deficiency and functional NK deficiency, or to eradicate unwanted cells.

[0061] The internal colloidal cells described herein retain the antigen-presenting ability of the antigen-presenting cells, but lack proliferative activity. Thus, the colloidal cells retain their ability to modulate an immune response without the risk of tumorigenicity. Accordingly, the colloidal cells can be administered to a subject in need thereof to treat a disorder or to induce an immune response. In some cases, the colloidal cells are used as a vaccine.

[0062] The colloidal cells can be formulated into pharmaceutical compositions suitable for a variety of administration routes, such as intravenous, intra-articular, conjunctival, intracranial, intraperitoneal, intrapleural, intramuscular, intrathecal, or subcutaneous administration routes. They can contain a pharmaceutically acceptable carrier, such as a buffer or excipient, or an adjuvant.

[0063] The following specific examples are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

[0064] Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present disclosure to its fullest extent. All publications cited herein are hereby incorporated by reference in their entirety.

[0065] Example 1: Colloidal artificial antigen presenting cells support NK cell expansion in vitro.

[0066] Established genetically engineered artificial antigen presenting cells (aAPC) were intracellularly hydrocolloidized for NK amplification. K562 cells were lentivirally transduced to express 41BBL and membrane-bound IL15 (K562-41BBL-mb15 feeder cells). See: Fujisaki et al., Cancer Res. 69, 4010-4017 (2009).

[0067] Peripheral blood mononuclear cells (PBMCs) from healthy donors were co-cultured with irradiated K562-41BBL-mb15 feeder cells (GM) or hydrocolloidized K562-41BBL-mb15 feeder cells (GC) to selectively support NK cell amplification. Given the key role of IL-21 in NK cell maturation and proliferation, the amplification of NK cells after recursive stimulation with GC and GM in the presence or absence of IL-21 was compared. As shown in Figure FIG. 3 (A), CD3 - CD56 + NK cells were highly enriched (83.5% ± 10.10%, 89.2% ± 2.35% for GM, IL-21 + GM, and 69.0% ± 22.76%, 93.0% ± 4.55% for GC, IL-21 + GC). By day 7, NK cells were observed to expand, on average, 13.1-fold when co-cultured with GC, compared to 5.4-fold with GM. Surprisingly, the addition of IL-21 to GC co-cultures showed a significant increase in expansion (12.6-fold for IL-21 + GC), despite the lower number of NK cells when co-cultured with GC. By day 14, similar trends in NK amplification were observed for aAPC (66.7 ± 13.0, 75.8 ± 33.5 for GM, IL-21 + GM, and 39.7 ± 9.5, 75.3 ± 6.7 for GC, IL-21 + GC), indicating that hydrocolloidized aAPC retain the ability to support NK cell expansion in vitro and promote the selective enrichment of amplified NK cells. See Figure FIG. 3 (C).

[0068] Example 2: Colloidal aAPC-supported NK cell expansion leads to different manifestation of NK cell receptors.

[0069] Since GC and GM are derived from the same genetically engineered aAPC, it was thus expected that NK cells expanded with GC or GM would be activated through the same signaling pathways and should produce similar immune phenotypes. To address this issue, CD3 - CD56 + NK cells were evaluated for surface expression of major NK cell receptors before or after expansion by CyTOF. Unexpectedly, as shown in unsupervised hierarchical clustering analysis, the three different groups of unexpanded, GC-expanded and GM-expanded NK cells were clustered together (data not shown). Notably, although both groups exhibited higher levels of activating receptors than unexpanded NK cells, GC-expanded NK cells showed increased expression of activating receptors, including NKp30, CD137, CRACC and NKG2D, as well as perforin, compared to GM-expanded NK cells (data not shown). These results suggest that co-culture with GC can provide a sustained activation signal to NK cells and lead to higher expression of activating receptors and perforin.

[0070] Example 3: Colloidal aAPC-expanded NK cells show enhanced cytotoxicity against tumor cell lines.

[0071] With high expression of activating receptors and perforin, GC-expanded NK cells can have higher cytotoxicity against tumor targets. To address this issue, cytotoxicity of expanded NK cells was evaluated by killing assay. Indeed, it was found that GC-expanded NK cells had much greater specific killing effect on target tumor cell line K562 than GM-expanded NK cells, in the presence or absence of IL-21. See FIG. 4 (A). In addition, since CD137 is more highly expressed in GC-expanded NK cells, it is thus possible to further activate GC-expanded NK cells by applying anti-CD137 agonist antibodies. As shown in FIG. 4 (B), GC-expanded NK cells had further enhanced cytotoxicity against K562 target cells in the presence of anti-CD137 agonist antibodies compared to the GM group, suggesting the possibility of combining anti-CD137 agonist with GC-expanded NK cells as a therapeutic strategy.

[0072] Example 4: Colloidal aAPC have higher persistence and generate expanded NK cells with higher cytolytic activity

[0073] To further assess NK cell expansion by the gelotrophs, gelotrophs were generated from K562-41BBL-mbl5 cells. PBMCs were co-cultured with either GM or GC. Total cell number, NK population, and NK cell number were determined at day 0, day 4, and day 7. See FIG. 5 (A). After 7 days of expansion, NK cells were enriched and analyzed for their cytolytic activity. See FIG. 5 (C). Although the NK cell population was similar between the two groups at day 7, GM expanded more NK cells than the GC group. See FIG. 5 (A) and FIG. 5 (B). On the other hand, NK cells expanded by GC exhibited higher cytolytic activity than those expanded by GM, suggesting that GC has a higher potential to promote NK activity than GM. See FIG. 5 (C). GC was also observed to have higher persistence than GM. See FIG. 5 (D). Thus, GC is able to stimulate NK cells for a longer period of time and induce higher cytolytic activity in NK cells.

[0074] Example 5: Modified gelotrophs and culture conditions improve NK cell expansion and cytolytic activity

[0075] To improve the ability of GC to expand NK cells, the properties of GC were modified to optimize efficiency.

[0076] Previous studies have shown that the stiffness of the stimulating surface can modulate NK cell activation. See: Mordechay et al., Mechanical Regulation of the Cytotoxic Activity of Natural Killer Cells (2020), biorxiv.org, doi:10.1101 / 2020.03.02.972984. The stiffness of GC was modulated to assess its effect on NK cell activation. GC with varying degrees of stiffness were tested, including 4%, 10%, 20%, and 40%. See FIG. 6 (A) and FIG. 6 (B). The data showed that the correlation between GC stiffness and NK expansion efficiency was bell-shaped, which is consistent with previous studies. A stiffness of 10% showed the greatest expansion efficiency. See FIG. 6 (A). On the other hand, there was no significant difference in cytolytic activity between different GC groups. See FIG. 6 (B).

[0077] Interactions between NK cells and other immune cells, such as T cells, can facilitate NK activation and proliferation. See: Malhotra and Shanker, NK cells: immune cross-talk and therapeutic implications 37 (2012); and Lee et al., Sci Rep 7, 11075 (2017). However, these interactions can affect the quality of NK cells from different expansion batches. To eliminate this difference, NK cells were enriched from PBMCs before expansion. Enriched NK cells were co-cultured with feeder cells at different ratios of NK cells to feeder cells (1:10, 1:5, 1:2, and 1:0.5) in the presence of 10 IU / mL or 100 IU / mL of human IL-2. See FIG. 7 . The data show that NK cells expanded with 100 IU / mL of IL-2 showed higher expansion efficiency than cells expanded with 10 IU / mL of IL-2. See FIG. 7 (A), FIG. 7 (B), FIG. 7 (D), and FIG. 7 (E). In addition, expanded NK cells showed higher cytolytic activity in the presence of 100 IU / mL of IL-2. See FIG. 7 (C), and FIG. 7 (F). According to these data, NK cells expanded with GC at a hardness of 10%, a ratio of NK cells to feeder cells of 1:5, and in the presence of 100 IU / mL of IL-2 showed good expansion efficiency and cytolytic activity. See FIG. 7 . In addition, there were significant differences in expansion efficiency and NK cytolytic activity between cells expanded in GM and cells expanded in GC at a ratio of 1:10 with 100 IU / mL of IL-2. See FIG. 7 (D) to FIG. 2 (F).

[0078] Example 6: Materials and Methods

[0079] Cell lines

[0080] K562-41BBL-mb15 cells were a gift from Dr. Chang at NTUH. All cells were cultured in RPMI 1640 medium (Gibco) supplemented with fetal bovine serum (Hyclone), penicillin (100 U / mL), and streptomycin (100 ug / mL).

[0081] Colloidal cells

[0082] First, 20 pL of 750 mg / mL 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure D-2959; Sigma-Aldrich) in dimethyl sulfoxide (DMSO) was dissolved and 200 pL of poly(ethylene glycol)-diacrylate (PEG-DA; Mn = 700 Da; Sigma-Aldrich) was mixed to prepare a gel buffer. 5 x 10 6 K562 cells or genetically modified K562 cells were collected and suspended in 1 mL of phenol red-free DMEM (Dulbecco's Modified Eagle Medium) containing 1 x protease inhibitor (CA21063-029; ThermoFisher Scientific). The gel buffer was added to the cell suspension at a volume ratio of 1:10, so that the PEG-DA concentration in the cell suspension was 10 wt%. After incubation at room temperature for 5 minutes, the cells were precipitated and resuspended in 500 pL of phenol red-free DMEM without gel buffer, and subjected to 365 nm blue light irradiation in a UV oven for 5 minutes. The resulting gelated cells (GC) were washed once with PBS and visually evaluated before further experiments. See FIG. 2 .

[0083] Expansion of NK cells from PBMC

[0084] Peripheral blood mononuclear cells (PBMC) were co-cultured with irradiated K562-41BBL-mb15 cells (GM) or GC in the presence or absence of IL-21 (100 ng / ml) or in the presence or absence of IL-2 (10 IU / ml or 100 IU / ml) in X-VIVO medium (Lonza) supplemented with 5% human serum (Gemini Bio). To evaluate the expansion efficiency, the NK population and cell number were evaluated after 7 days of expansion. In addition, the expanded NK cells were fixed with paraformaldehyde and the NK activation and inhibition markers were determined by CyTOF. NK activity was evaluated by cytotoxicity assay.

[0085] NK population and cell number determination

[0086] PBMC or expanded cells were stained with APC-anti-CD3 (Biolegend) and PE-anti-CD56 (Biolegend), respectively, and the proportion of NK population (CD3 - CD56 + ) was verified by flow cytometry. In addition, the total cell number was determined by a hemocytometer. The NK cell number was calculated as follows: total cell number x NK population proportion.

[0087] Cytotoxicity assay

[0088] Cytotoxic function of NK cells was assessed by measuring luminescence. Target cells K562-luc + -GFP + Stable expression of luciferase marker. NK cells were co-cultured with target cells at the indicated ratios in triplicate for 4 hours. Cells were lysed and luminescence was measured in 96-well white plates by the luciferase assay system (promega). The percentage of cell lysis was calculated as follows: (luminescence of target cells alone - luminescence of NK-target co-culture) / (luminescence of target cells alone - blank) x 100%

[0089] Single cell mass cytometry (CyTOF)

[0090] Samples were fixed in 1.5% paraformaldehyde for 10 minutes at room temperature, followed by two washes in PBS with 0.5% BSA. Fixed cell samples were incubated with metal-conjugated antibodies against surface markers for 1 hour, washed once in PBS with 0.5% BSA, permeabilized in ice-cold methanol for 10 minutes, washed twice in PBS with 0.5% BSA, and then incubated with metal-conjugated antibodies against intracellular molecules for 1 hour. After intracellular staining, cells were washed once in PBS with 0.5% BSA and then incubated with an iridium-containing DNA intercalator (Fluidigm) in PBS with 1.5% paraformaldehyde for 10 minutes at room temperature. After intercalation / fixation, cell samples were washed once in PBS with 0.5% BSA and twice in water, and then measured on a CyTOF mass cytometer (Fluidigm). Standardization of detector sensitivity was performed as previously described. After measurement and standardization, each file was first analyzed by gating out doublets, debris, and dead cells based on cell length, DNA content, and cisplatin staining. Heat maps, histograms, and ViSNE plots were generated with software tools provided at cytobank.org.

[0091] GC persistence test

[0092] To monitor the persistence of GM and GC in the NK expansion system, a high content imaging system was used to monitor the change in GM and GC numbers. More specifically, PBMC and feeder cells (GM and GC) were labeled with CellTracker Deep Red (Thermo Fisher Scientific) and CFSE (Thermo Fisher Scientific), respectively. PBMC and feeder cells were co-cultured with 10 IU / ml human IL-2 in X-VIVO medium (Lonza) supplemented with 5% human serum (Gemini Bio) for 3 days. Images were acquired every 3 hours by ImageXpress Micro system (Molecular Devices, Sunnyvale, CA) with 20x objective, FITC and Cy7 filters set, 9 fields of view per well. Image data were analyzed by ImageJ to assess cell numbers.

[0093] GC stiffness

[0094] By adjusting the PEG-DA concentration (i.e., 4 wt%, 10 wt%, 20 wt%, and 40 wt%) in the cell suspension containing the colloidal buffer, GCs with different stiffness were generated, including 4%, 10%, 20%, and 40%. PBMC and GCs were co-cultured with 10 IU / ml human IL-2 in X-VIVO medium (Lonza) supplemented with 5% human serum (Gemini Bio). The medium was refreshed on day 3 and day 5. Total cell numbers and NK cell cytolytic function were assessed on day 7.

[0095] NK expansion test with enrichment

[0096] NK cells were enriched from PBMC by NK isolation kit (Miltenyi Biotec). Then, NK cells were co-cultured with different ratios of NK cells to feeder cells (GM or GC) with 10 or 100 IU / ml human IL-2 in X-VIVO medium (Lonza) supplemented with 5% human serum (Gemini Bio). The medium was refreshed on day 3 and day 5. NK cell numbers, populations, and NK cell cytolytic function were assessed on day 7.

[0097] Other embodiments

[0098] All features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature having the same, equivalent, or similar purpose. Unless otherwise stated, each feature disclosed is an example of a generic series of equivalent or similar features.

[0099] From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of the described embodiments, and without departing from the spirit and scope thereof, can make various changes and modifications of the embodiments to adapt the same to various uses and conditions. Thus, other embodiments are also within the scope of the following claims.

Claims

1. A method of in vitro expansion of natural killer cells, comprising: An internal colloidal cell population is provided, wherein each internal colloidal cell comprises a colloidal interior and a fluid cell membrane comprising one or more membrane-bound proteins, each or collectively of which is capable of stimulating amplification of natural killer (NK) cells; and culturing a population of cells containing NK cells capable of reacting to the one or more membrane-bound proteins with the population of internally colloidal cells under conditions that allow for expansion of the NK cells; the population of internally colloidal cells is produced by steps comprising: providing a population of antigen presenting cells expressing one or more membrane-bound proteins; suspending the population of antigen presenting cells in phenol red-free DMEM containing a protease inhibitor cocktail to produce a first cell suspension; adding a colloidal solution to the first cell suspension to produce a second cell suspension, wherein the colloidal solution is capable of increasing membrane permeability of the antigen presenting cells and contains a photoreactive crosslinking agent and optionally a photoinitiator; incubating the second cell suspension at room temperature for a sufficient period of time to allow the photoreactive crosslinking agent and the optional photoinitiator to enter the antigen presenting cells; centrifuging the second cell suspension to produce a cell pellet; resuspending the cell pellet in phenol red-free DMEM to produce a third cell suspension; exposing the third cell suspension to light for a sufficient period of time to allow the photoreactive crosslinking agent to crosslink, thereby producing the population of internally colloidal cells; and collecting and washing the population of internally colloidal cells; wherein the colloidal solution is prepared such that the osmolality of the second cell suspension is between 320 mOsmol and 290 mOsmol.

2. The method of claim 1, wherein the population of cells is selected from the group consisting of peripheral blood mononuclear cells (PBMCs), enriched NK cells, iPSC-derived NK cells, embryonic stem cell-derived NK cells, tissue-resident NK cells, splenocytes, umbilical cord blood cells, and hematopoietic stem cell-derived NK cells.

3. The method of claim 1 or 2, wherein the one or more membrane-bound proteins are selected from the group consisting of 41BBL, IL-15, IL-21, B7-H6, BAT3, HLA-DP, HLA-E, HLA-C2, HLA-A, HLA-C, HLA-G, HLA-F, HLA-C, MICA / MICB, ULBP-1, ULBP-2, ULBP-3, ULBP-4, ULBP-5, ULBP-6, AICL, CD48, NTB-A, 2B4, CD2, CD58, CD11a, ICAM1, CRACC, OX40L, CD137L, Nectin-1, Nectin-2, Nectin-3, Nectin-4, necl-1, necl-2, necl-3, necl-4, necl-5, PCNA, AICL, IgG, CD27L, CD72, CEACAM-1, CEACAM-5, OCIL, N-cadherin, E-cadherin, R-cadherin, IL-1, IL-2, IL-4, IL-7, IL-9, IL-12, IL-18, IL-27, IL-33, IL-6, IL-11, CNTF, LIF, OSM, CT-1, CLC, IFN-a, INF-b, CCL-5, and agonists of TLR-1, TLR-2, TLR-3, TLR-5, TLR-6, TLR-9, NOD-1, NOD-2, NOD-3, or NLRP3.

4. The method of claim 2, wherein the one or more membrane-bound proteins are 41BBL and IL-15.

5. The method of claim 1, wherein the step of culturing the cell population containing NK cells is performed in the presence of IL-21 or IL-2.

6. The method of claim 1, wherein the ratio of the number of NK cells to the number of internal colloidal cells is 1:0.5-20 in the step of culturing the cell population containing NK cells.

7. The method of claim 1, wherein the second cell suspension contains 0.1 to 5 wt% DMSO.

8. The method of claim 1, wherein the concentration of the photoreactive crosslinker in the second cell suspension is 5 wt% to 50 wt%.

9. The method of claim 1, wherein the photoreactive crosslinker is poly(ethylene glycol)-diacrylate (PEG-DA), the photoinitiator is 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, and the light is blue light at 365 nm.

10. The method of claim 9, wherein the 2-hydroxy-4'-(2-hydroxyethoxy)-2- methylpropiophenone ranges from 0.01 to 1 wt% and the average molecular weight of the PEG-DA is between 200 Da and 5000 Da, ranging from 2 to 80 wt% in the colloidal solution.

11. The method of claim 10, wherein the preparation of the colloidal solution is by dissolving 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone in dimethyl sulfoxide (DMSO) to produce a solution, and mixing the solution with PEG-DA having an average molecular weight of 700 Da.

12. The method of claim 9, wherein the concentration of PEG-DA in the second cell suspension is 10 wt% to 40 wt%.

13. The method of claim 1, wherein the population of antigen presenting cells is artificial antigen presenting cells.

14. The method of claim 13, wherein the artificial antigen presenting cells are or are engineered from K562 cells, PBMCs, EBV-transformed LCLs, 721.221 cells, 8866 cells, Jurkat cells, Jurkat / KL-l cells, U937 cells, BJAB cells, NB4 cells, 293T cells, MCF7 cells, Jeg3 cells, Hela cells, A549 cells, 1106mel cells, or CEM cells.

15. A method of producing a population of internal colloidal cells in vitro, comprising: providing a population of precursor cells expressing one or more membrane-bound proteins; suspending the population of precursor cells in phenol red-free DMEM containing a cocktail of protease inhibitors to produce a first cell suspension; adding a colloidal solution to the first cell suspension to produce a second cell suspension, wherein the colloidal solution is capable of increasing the membrane permeability of the precursor cells and contains a photoreactive crosslinker and a photoinitiator; incubating the second cell suspension at room temperature for a sufficient period of time to allow the photoreactive crosslinker and the photoinitiator to enter the precursor cells; centrifuging the second cell suspension to produce a cell pellet; resuspending the cell pellet in phenol red-free DMEM to produce a third cell suspension; exposing the third cell suspension to light for a sufficient period of time to allow the photoreactive crosslinker to crosslink, thereby producing the population of internal colloidal cells; and collecting and washing the population of internal colloidal cells; wherein each of the internal colloidal cells comprises an internal colloidal interior and a fluid cell membrane containing one or more membrane-bound proteins; wherein the colloidal solution is prepared such that the osmolality of the second cell suspension is 320 mOsmol to 290 mOsmol.

16. The method of claim 15, wherein the second cell suspension contains 0.1 to 5 wt% DMSO.

17. The method of claim 15, wherein the concentration of the photoreactive crosslinker in the second cell suspension is 5 wt% to 50 wt%.

18. The method of claim 15, wherein the photoreactive crosslinker is poly(ethylene glycol)-diacrylate (PEG-DA), the photoinitiator is 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, and the light is blue light at 365 nm. ​ 19. The method of claim 18, wherein in the colloidal solution, the 2-hydroxy-4’-2- (hydroxyethoxy)-2-methylpropiophenone ranges from 0.01 to 1 wt% and the PEG-DA has an average molecular weight between 200 Da and 5000 Da, ranging from 2 to 80 wt%.

20. The method of claim 19, wherein the colloidal solution is prepared by dissolving 2- hydroxy-4’-(2-hydroxyethoxy)-2-methylpropiophenone in dimethyl sulfoxide (DMSO) to produce a solution, and mixing the solution with PEG-DA having an average molecular weight of 700 Da.

21. The method of claim 18, wherein the concentration of PEG-DA in the second cell suspension is 10 wt% to 40 wt%.

22. The method of claim 15, wherein the precursor cell population is an artificial antigen presenting cell.

23. The method of claim 22, wherein the artificial antigen presenting cell is or is engineered from a K562 cell, a PBMC, an EBV-transformed LCL, a 721.221 cell, an 8866 cell, a Jurkat cell, a Jurkat / KL-l cell, a U937 cell, a BJAB cell, a NB4 cell, a 293T cell, a MCF7 cell, a Jeg3 cell, a Hela cell, an A549 cell, a 1106mel cell, or a CEM cell.

24. The method of claim 22, wherein each or collectively the one or more membrane-bound proteins are capable of stimulating amplification of natural killer (NK) cells.

25. The method of claim 24, wherein the one or more membrane-bound proteins are selected from the group consisting of 41BBL, IL-15, IL-21, B7-H6, BAT3, HLA-DP, HLA-E, HLA-C2, HLA-A, HLA-C, HLA-G, HLA-F, HLA-C, MICA / MICB, ULBP-1, ULBP-2, ULBP-3, ULBP-4, ULBP-5, ULBP-6, AICL, CD48, NTB-A, 2B4, CD2, CD58, CD11a, ICAM1, CRACC, OX40L, CD137L, Nectin-1, Nectin-2, Nectin-3, Nectin-4, necl-1, necl-2, necl-3, necl-4, necl-5, PCNA, AICL, IgG, CD27L, CD72, CEACAM-1, CEACAM-5, OCIL, N-cadherin, E-cadherin, R-cadherin, IL-1, IL-2, IL-4, IL-7, IL-9, IL-12, IL-18, IL-27, IL-33, IL-6, IL-11, CNTF, LIF, OSM, CT-1, CLC, IFN-a, INF-b, CCL-5, and agonists of TLR-1, TLR-2, TLR-3, TLR-5, TLR-6, TLR-9, NOD-1, NOD-2, NOD-3, or NLRP3.

26. An internal colloidal cell population, produced by the method of any one of claims 15-25, wherein each cell comprises an internal colloidal interior and a fluid cell membrane containing one or more membrane-bound proteins.

27. A composition comprising the internal colloidal cell population of claim 26.

28. Use of the natural killer cells produced by the method of amplifying natural killer cells in vitro of claim 1 for the manufacture of a medicament for treating chronic myeloid leukemia in humans.

29. Use of the composition of claim 27 in the method of amplifying natural killer cells in vitro.

Images