PM21 particles improve bone marrow homing of NK cells.

Abstract

Disclosed are compositions and methods for directing NK cells to the bone marrow through the use of PM21 particles. [Selection diagram] Figure 5

Description

[Technical Field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 464,747, filed February 28, 2017, which is incorporated herein by reference in whole. [Background technology]

[0002] I. Background Adoptive natural killer (NK) cell therapy is a promising new intervention for oncology, including for myeloid malignancies. Therefore, the trafficking efficiency of NK cells used as adoptive cells is of paramount importance. [Overview of the project] [Problems that the invention aims to solve]

[0003] What is needed is a method that can efficiently traffic NK cells into the bone marrow. [Means for solving the problem]

[0004] II. Overview 1. Disclosed are methods and compositions relating to trafficking NK cells into the bone marrow, comprising contacting NK cells with PM21 particles and / or FC21 feeder cells. In one embodiment, the method may further comprise stimulating the NK cells with IL-2, IL-12, IL-18 and / or IL-18.

[0005] 2. Also disclosed are methods for treating myeloma or malignant tumors of bone marrow origin, and / or methods for treating viral infections (such as bone marrow-associated viral infections, including bone marrow-tropic virus infections or viral infections that have adverse effects on the bone marrow). In some embodiments, the contact between PM21 particles and / or FC21 feeder cells and NK cells can be performed before the transfer of NK cells to the patient. In other embodiments, the contact between PM21 particles and / or FC21 feeder cells and NK cells can be performed within the patient's body.

[0006] III. Brief Explanation of the Figure 3. The accompanying drawings incorporated herein and forming part thereof illustrate several embodiments and, together with the description, illustrate the disclosed compositions and methods. [Brief explanation of the drawing]

[0007] [Figure 1]4. We demonstrate that PM21 particles efficiently and selectively amplify (expand) cytotoxic NK cells. Peripheral blood mononuclear cells (PBMCs) were isolated from a leukocyte source and seeded at 0.1 × 10⁶ NK cells / mL in SCGM supplemented with 10% FBS, 2 mM Glutamax, and 50 U / mL IL-2. PBMCs were stimulated for 27 days with 200 μg / mL PM15 (□, black) or PM21 (〇, blue) particles, and cell content was tested every 2-3 days to show the relative expansion factor of NK cell amplification (A) and the percentage of suspended cells (B). Based on a cumulative analysis of data at day 14 for NK cell amplification, PM21 particles (825 ± 188 times, N=13, 4 donors) (blue) were more efficient at amplifying NK cells compared to PM15 particles (425 ± 71 times, N=35, 9 donors) (black) (C). PBMCs isolated from three AML patients in remission were cultured with PM21 particles (200 μg / mL) for 14 days and seeded at 0.5 × 10⁶ NK cells / mL in SCGM containing 10% FBS, 2 mM Glutamax, and 50 U / mL IL-2. The NK cell amplification ratio from primary PBMCs (D) and lymphocyte content (E) (CD56+CD3-NK cells (●, red), CD56-CD3+T cells (■, blue), and CD56+CD3+NKT cells (▲, black)) is shown. PBMCs from patient F021 were cultured for 16 days as described above, and autologous cytotoxicity to AML tumors from the same patient was analyzed (F). Amplified PM21-NK cells were labeled with TFL4 and co-incubated (2 hours) with AML cells from the same patient in active disease at the E:T ratio shown, and analyzed by flow cytometry. The amount of naturally dying target cells was measured using a "target-only" control. Each data point was measured using a dual measurement method. [Figure 2]5. Pre-activation of non-selective PBMCs with PM21 particles induces in vivo NK cell amplification. NSG mice were intraperitoneally injected with either inactivated PBMCs (A and B) or PBMCs pre-activated ex vivo for 2 days with PM21 particles and 100 U / mL IL-2 (PM21-PBMCs) (C and D) in 2 × 10⁶ cells. All mice received 1000 U of IL-2 intraperitoneally three times a week. The mice also received 400 μg of PM21 particles intraperitoneally twice a week (B and D). Peripheral blood was collected by continuous buccal bleeding, and hCD45+ human lymphocytes were analyzed by flow cytometry twice a week, starting on day 6. The amounts of NK, T, and B cells were measured based on staining for hCD3, hCD56, and hCD19. The plot on the left for each experimental group shows the concentration of hNK cells per 1 μL of PB. The plot on the right shows the percentage of hNK cells (circles, red) and T cells (indentations, black) as a percentage of the total hCD45+ cells. [Figure 3] 6. Proliferation analysis demonstrates in vivo NK cell amplification from PM21-PBMCs. Freshly thawed PBMCs, or PBMCs pre-activated for 2 days with PM21 particles and 100 U / mL IL-2 (PM21-PBMCs), were labeled with Cell Trace (CT) Violet. 2 × 10⁶ inactivated PBMCs (A and B) or PM21-PBMCs (C and D) were intraperitoneally injected into NSG mice. All mice received 1000 U IL-2 intraperitoneally three times a week. Two mouse groups also received 400 μg of PM21 particles intraperitoneally twice a week (B and D). Two mice from each group were euthanized on day 6, and the peritoneal lavage fluid was analyzed by flow cytometry for CT Violet fluorescence of hCD45+, hCD3-, and hCD56+ NK cells. The histogram of CT Violet fluorescence was analyzed using curve fitting with the proliferation analysis suite in FlowLogic. [Figure 4]7. This study demonstrates that in vivo application of PM21 enables an increase in peripheral blood NK cells. PBMCs were pre-activated ex vivo for 2 days with PM21 particles and 100 U / mL IL-2. PM21-PBMCs containing 0.2 × 10⁶ viable NK cells were intraperitoneally injected into NSG mice. All mice received 1000 U of IL-2 intraperitoneally three times a week. Mice were also intraperitoneally injected with PM21 particles 0 (A), 400 (B), 800 (C), and 1,600 μg (D) twice a week. Peripheral blood was analyzed by flow cytometry twice a week, starting on day 5, for hCD45+ lymphocytes, and the amounts of hNK, hT, and hB cells were measured based on staining for hCD3, hCD56, and hCD19. The plot on the left for each experimental group shows the concentration of hNK cells per μL of PB. The plot on the right shows the percentage of hNK cells (circles, red) and T cells (indentations, black) as the proportion of total hCD45+ cells. Analysis of PB samples from 12 days after the initial intraperitoneal injection of PM21-PBMCs shows a dose-dependent increase in PB hNK cells in response to in vivo PM21 particle administration (E left), but no significant dose-dependent increase in total CD3+ T cells was observed (E right). [Figure 5]8. We demonstrate the in vivo distribution of in vivo amplified NK cells to important physiological sites, and that the in vivo distribution of said NK cells is increased by in vivo application of PM21 particles. As part of PM21-PBMC, pre-activated NK cells (0.2 × 10⁶ cells) were intraperitoneally injected into NSG mice for 2 days ex vivo with PM21 particles and 100 U / mL IL-2. All mice received 1000 U of IL-2 intraperitoneally three times a week. Mice were also intraperitoneally injected with 0 or 800 μg of PM21 particles twice a week. Mice were euthanized 16 days after the initial intraperitoneal injection of PM21-PBMC. On the day of euthanasia, bone marrow (thigh), spleen, lungs, brain, and liver were harvested, the organs were perfused, and the thigh was washed and cells were collected. Cells were stained with antibodies against hCD3, hCD45, hCD56, and hCD19 for flow cytometry analysis. Data from bone marrow, spleen, brain, lungs, and liver (from left to right) are shown along with the amount of hCD45+hCD56+hCD3-NK cells (plots above each organ) and the percentages of hCD45+hCD56+hCD3-NK cells (circles, red), hCD45+hCD3+ T cells (squares, blue), and other lymphocytes (triangles, black) of hCD45+, hCD56-hCD3- (plots below each organ). Each thick bar represents the mean. [Figure 6]9. Demonstrates consistency in in vivo NK cell amplification from different donor sources. The consistency of PM21 particles stimulated by in vivo NK cell amplification was tested using three different PBMCs obtained from healthy donors. PBMCs were pre-activated ex vivo for 2 days with PM21 particles and 100 U / mL IL-2 (PM21-PBMCs) and intraperitoneally injected into NSG mice. All mice received 1000 U of IL-2 intraperitoneally three times a week. Peripheral blood was analyzed for hCD45+ lymphocytes by flow cytometry twice a week, starting on day 5, and hNK, hT, and hB cell counts were measured based on staining for hCD3, hCD56, and hCD19. The concentration of hNK cells in the blood 12 days after intraperitoneal PBMC injection (A) and the amount of NK cells recovered in peritoneal lavage fluid 14 days after intraperitoneal PBMC injection (C) were similar among the different groups injected with different NK cell sources (PB p=0.84 and p=0.69). The corresponding cell content of hNK cells (circles, red), hT cells (squares, blue), and other hCD45+ cells (triangles, black) were also consistent among the groups injected with different PBMC sources in peripheral blood (B) and intraperitoneal (D). Each thick bar represents the mean. [Figure 7] HECA-452 staining of NK cells treated with soluble cytokines at stimulation days 10.0, 1, 7, and 10 is shown. [Figure 8] HECA-452 staining of NK cells cultured with K562 feeder cells following stimulation on days 11.10, 12, and 14 is shown. [Figure 9] This section shows a comparison of HECA-452 staining of NK cells stimulated under various conditions following the initial stimulation on December 10th. [Figure 10] 13. HECA-452 staining of NK cells cultured under various conditions following 10 days of stimulation is shown. [Figure 11] 14. HECA-452 staining of NK cells 3 days after rest following 10 days of stimulation. [Figure 12] 15. HECA-452 staining of PM21 particles before and after thawing, following 14-day amplification. [Figure 13] 16. This shows that STAT3 is involved in the IL-21-mediated modulation of FUT7 gene expression in human NK cells. (A) ChIP-seq was performed using an antibody against STAT3 in naive and expanded human NK cells stimulated with IL-21. Arrows indicate the direction of transcription. Scales are consistent for genes and islands; (B) RNA-seq reveals differential regulation of FUT7 gene expression in response to IL-21 stimulation; (C) IL-21 enhances STAT3 binding to the FUT7 gene in expanded NK cells; and (D) IL-21 upregulates FUT7 gene expression in expanded NK cells. MODE FOR CARRYING OUT THE INVENTION

[0008] IV. DETAILED DESCRIPTION 17. Before disclosing and describing the compounds, compositions, articles, devices, and / or methods of the present invention, it is to be understood that they are not limited to any particular synthetic method or particular recombinant biotechnological method unless otherwise specified, nor are they limited to particular reagents unless otherwise specified, and can of course vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0009] A. DEFINITIONS 18. As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a pharmaceutical carrier" includes a mixture of two or more such carriers, and the like.

[0010] 19. A range may be expressed herein as “about” one special value and / or “about” another special value. When such a range is expressed, another embodiment encompasses that one special value and / or another special value. Similarly, when a value is expressed as an approximation by the preceding use of “about,” it will be understood that the special value forms another embodiment. It will be further understood that each endpoint of a range is significant in relation to and independently of the other endpoints. It will also be understood that there may be multiple values ​​disclosed herein, each value disclosed as “about” a specific value in addition to that value itself. For example, if the value “10” is disclosed, “about 10” is also disclosed. If a value is disclosed as “less than or equal to” that value, it will be understood, as appropriately understood by those skilled in the art, that “greater than or equal to that value” and the possible range between the values ​​are also disclosed. For example, if the value “10” is disclosed, “less than or equal to 10” and “greater than or equal to 10” are also disclosed. Throughout this application, it will be understood that data will be provided in different formats, and that this data will be within the range of endpoints and starting points, and any combination of these data points. For example, if special data point "10" and special data point 15 are disclosed, it will be understood that greater than 10 and 15, greater than or equal to 10 and 15, less than or equal to 10 and 15, less than or equal to 10 and 15, and equal to 10 and 15 will be considered disclosed as well as between 10 and 15. It will also be understood that each unit between the two special units will also be disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 will also be disclosed.

[0011] 20. In this specification and the following claims, several terms will be used by reference as having the following meanings:

[0012] 21. "Optional" or "depending on the circumstances" means that the event or situation described thereafter may or may not occur, and that the description includes examples of when the event or situation occurs and examples of when it does not.

[0013] B. Method of using the composition 22. Adoptive natural killer (NK) cell therapy is a promising novel intervention for oncology, including for bone marrow malignancies and bone marrow-derived malignancies, and its veterinary applications. In another aspect, adoptive NK cells can be used therapeutically to treat bone marrow commensal viruses, such as parvovirus that induces aplastic anemia. Therefore, the efficiency of trafficking adoptively used NK cells is extremely important. In particular, how to induce the transplanted cells into bone marrow where they can be effectively treated is of paramount importance.

[0014] 23. One of the determinants of bone marrow homing is the ligand for selectin binding. In the case of L-selectin binding, a critical determinant is the fucosylation status of the sialyl Lewis x (sLex) carbohydrate chain attached to P-selectin glycoprotein ligand-1 (PSGL-1). In one embodiment, it is disclosed and intended herein that stimulation of NK cells with PM21 particles and / or FC21 feeder cells prepared from K562 cells (K562.mb21.41bbl) transformed to express manipulated membrane-bound forms of IL-21 and / or 41bbl induces efficient specific amplification of NK cells and induces total fucosylation of sLex.

[0015] 24. Accordingly, disclosed herein is a method relating to trafficking NK cells into the bone marrow, including contacting NK cells with PM21 particles and / or FC21 feeder cells, and adoptively transferring NK cells to a patient in need, and a method for treating myeloma or malignant tumors of bone marrow origin. In one embodiment, the method may further include stimulating NK cells with IL-2, IL-12, IL-15, IL-18, or IL-21 either ex vivo or in vivo (in the patient's body).

[0016] 25. In some embodiments, contact between PM21 particles and / or FC21 feeder cells and NK cells can be performed before the transfer of NK cells into the patient. In another embodiment, contact between PM21 particles and / or FC21 feeder cells and NK cells can be performed within the patient's body.

[0017] 26. In one embodiment, it is understood and intended herein that the efficacy of NK cell immunotherapy depends on the dose of NK cells administered to the patient or the dose of NK cells delivered after infusion through in vivo amplification. Currently available technologies are limited by their inability to achieve the level of NK cell amplification required to realize therapeutic effects in patients. The lack of more concise clinical amplification protocols is a major obstacle to the progress and widespread adoption of NK cell-based immunotherapy. Current ex vivo amplification protocols utilize combinations of high-dose cytokine and ligand activation expressed in leukemia-derived feeder / stimulating cell lines, posing considerable drawbacks to translating to the clinical environment found in most facilities and not being modifiable to direct in vivo amplification. The use of particle technologies, including exosomes, as described herein eliminates the requirement for stimulating cells, thus simplifying the methodology and enabling ex vivo amplification for adoptive therapy or in vivo application for selective in vivo amplification. Accordingly, in one embodiment, disclosed herein is a method for treating myelomas and myeloid-derived malignancies through adoptive transfer of NK cells, comprising contacting NK cells with one or more vesicles containing an NK cell effector agent, and / or a method for trafficking NK cells into the bone marrow. Also disclosed in one embodiment is a method for treating myelomas, myeloid-derived malignancies, and / or viral infections (including myeloid viruses), further comprising pre-activating or activating in vivo NK cells by contacting at least one type of NK cell with at least one or more stimulating cytokines. Accordingly, in one embodiment, diseases or syndromes of myeloid commensal viruses that induce aplastic anemia (e.g., parvovirus) can be treated using NK cells amplified ex vivo with PM21 particles and / or FC21 feeder cells, or NK cells directly stimulated in vivo with PM21 or FC21 feeder cells.

[0018] 27. The disclosed method completes the pre-activation or activation of NK cells by contacting at least NK cells with at least one or more stimulating cytokines (e.g., IL-2, IL-12, IL-15, IL-21 and / or IL-18). Accordingly, disclosed herein is a method for treating myelomas and malignancies derived from bone marrow through adoptive transfer of NK cells, and / or a method for trafficking NK cells into bone marrow, which includes pre-activating NK cells by contacting one or more NK cells with one or more stimulating cytokines selected from the group including IL-2, IL-12, IL-21, IL-15 and / or IL-18, or any combination thereof, such as contacting one or more NK cells with two or three stimulating cytokines. For example, specifically disclosed herein are methods in which the pre-activation or activation step comprises contact between NK cells and IL-2; IL-12; IL-15, IL-18, IL-12 and IL-15; IL-12 and IL-18; IL-15 and IL-18; or IL-12, IL-15, and IL-18. In one embodiment, a disclosed method for treating myelomas and myeloid-derived malignancies through adoptive transfer of NK cells, and / or trafficking NK cells into the bone marrow, may further comprise contacting NK cells with one or more cytokines selected from the group consisting of 4-1BBL, IL-2, IL-21, MICA / B, ULBP2, ICAM-1, 2B4, BCM1 / SLAMF2, CD155, CD112, CCR7, DAP12, Notch ligand, and / or DAP10, in soluble form or in the form of PM21 particles or FC21 feeder cells.

[0019] 28. It is understood and intended herein that the duration of pre-activation or activation in soluble form, or in the form of PM21 particles or FC21 feeder cells (i.e., the duration of contact between NK cells and stimulating cytokines (e.g., IL-2, IL-12, IL-15, IL-21 and / or IL-18)) may be any length of time required to achieve the desired pre-activation or activation of NK cells. For example, such contact may be as short as one minute or as long as seven days (e.g., culturing NK cells for seven days in the presence of IL-2, IL-12, IL-15, IL-21 and / or IL-18). In one embodiment, disclosed herein is a method for treating myelomas and myeloid-derived malignancies through adoptive transfer of NK cells, and / or a method for trafficking NK cells into the bone marrow, comprising pre-activating or activating NK cells by contacting one or more NK cells with IL-2, IL-12, IL-15, and / or IL-18 for 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, or 48 hours. It is understood and intended herein that the half-life of cytokines in culture may be shorter than the desired contact time. Therefore, disclosed herein are methods for contacting one or more types of NK cells with IL-2, IL-12, IL-15 and / or IL-18 at intervals of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours within a contact period (for example, at intervals of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours within a 24-hour contact period).

[0020] 29. Many hurdles associated with cytokine toxicity can be overcome through the use of plasma membrane (PM) particles, exosomes (EX), or feeder cells (FC) containing one or more NK cell effector agents (i.e., stimulating peptides, cytokines, and / or adhesion molecules) that come into contact with and activate and / or amplify NK cells. Examples of NK cell activators and stimulating peptides include, but are not limited to, 41BBL, IL-2, IL-12, IL-21, IL-18, MICA, LFA-1, 2B4, BCM / SLAMF2, CCR7, Notch ligand, and / or other homing-inducing signaling molecules. Examples of cytokines include, but are not limited to, IL-2, IL-12, IL-21, and IL-18. Examples of adhesion molecules include, but are not limited to, LFA-1, MICA, and BCM / SLAMF2. For example, plasma membrane (PM) particles, feeder cells (FC), or exosomes (EX) are prepared from feeder cells expressing membrane-bound IL-21 (FC21 cells, PM21 particles, and EX21 exosomes, respectively). FC21 cells, PM21 particles, and EX21 exosomes expressing membrane-bound IL-21 may further include, without limitation, one or more additional activators, stimulating peptides, cytokines, and / or adhesion molecules (e.g., PM21 particles, EX21 exosomes, or FC cells expressing 41BBL and membrane-bound interleukin-21), including 41BBL, IL-2, IL-12, IL-15, IL-18, MICA, LFA-1, 2B4, BCM / SLAMF2, and CCR7. Accordingly, in one embodiment, disclosed herein is a method for treating myelomas and myeloma-derived malignancies through adoptive transfer of NK cells, and / or a method for trafficking NK cells into the bone marrow, comprising contacting NK cells with at least one vesicle containing an NK cell effector agent, wherein the NK cell effector agent containing the vesicle is any combination of one or more of PM21 particles, EX21 particles, and / or FC feeder cells.For example, disclosed herein is a method for treating myelomas and myeloma-derived malignancies through adoptive transfer of NK cells, and / or a method for trafficking NK cells into the bone marrow, comprising, in addition to other steps, contacting NK cells with at least one vesicle containing an NK cell effector agent, wherein the NK cell effector agent containing the vesicle is comprised of PM21 particles; EX21 exosomes; FC21 feeder cells; PM21 particles and EX21 exosomes; PM21 particles and FC21 feeder cells; EX21 exosomes and FC21 feeder cells; or PM21 particles, EX21 exosomes, and FC21 feeder cells.

[0021] 30. In some embodiments, the effector agents for PM21 particles, EX21 exosomes, or FC feeder cells include one or more stimulating peptides coupled to a membrane insertion peptide (e.g., Fc, GPI, transmembrane T cell receptor, or pHLIP). The membrane insertion peptide may be a molecule that facilitates insertion into the membrane. The membrane insertion peptide may contain a segment of CD4 or IgG having affinity for the lipid bilayer. In addition, alternative membrane insertion peptides may include human Fc, GPI, transmembrane T cell receptor, or pHLIP. The membrane self-inserting peptide may be any peptide known to insert into the cell membrane. Depending on the use of the membrane self-inserting peptide conjugate, certain membrane self-inserting peptides may be a better choice than others. Those skilled in the art will understand which membrane self-inserting peptides are ideal under different circumstances. For example, in in vivo use, the pHLIP membrane self-inserting peptide may be suitable. The pHLIP membrane self-inserting peptide inserts into the membrane only under low pH conditions. Therefore, pHLIP conjugates do not insert into cell membranes under normal physiological conditions. However, when injected into a tumor environment, the pHLIP conjugate can insert into the cell membrane of tumor cells because the tumor environment is more acidic than normal physiological conditions. This insertion into the tumor environment enables the activation of NK cells in the tumor area. Thus, the use of pHLIP prevents unwanted insertion into random cell membranes.

[0022] 31. Membrane insertion peptides may be coupled to one or more irritating peptides in various ways, and techniques for coupling peptides are well known in the art. Membrane insertion peptides coupled to irritating peptides may also be called membrane insertion peptide conjugates. In some embodiments, the one or more irritating peptides coupled to the membrane insertion peptide may include a fusion protein encoded by recombinant DNA, such fusion protein may be produced in bacterial cells. In certain embodiments, the fusion protein may consist of one or more irritating peptides conjugated or coupled to a hydrophobic peptide, GPI, or lipophilic molecule such as human Fc for immobilization in liposomes or cell membranes. cDNA vectors for these fusion proteins may be ligated into expression plasmids that enable expression in bacterial (E. coli), insect, or mammalian cells. In certain embodiments, the cDNA vector may be FLAG- or HIS-tagged. Bacterial cells may be transfected using a standard CaCl transfection method, such as that described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press (1989). Bacterial cells may also be cultured in LB medium, harvested, and lysed using a French press. The relevant proteins can be purified from the lysates by affinity chromatography. Palmitate conjugate protein A and the purified Fc fusion protein can be conjugated as described in the literature by mixing them in a 1:2 (w / w) ratio at 4°C. The conjugate may then be injected directly into the tumor or incorporated into liposomes.

[0023] 32. Types of coupling and methods for coupling are known to those skilled in the art. As used herein, “coupling” refers to the linking of a membrane self-insertion peptide to another molecule, such as a peptide or protein, by conjugate, linkage, or other means. For example, a membrane-insertion peptide coupled to a stimulant peptide may be an induced protein in which the membrane-insertion peptide is coupled to another protein via a disulfide bond. Coupling or conjugation may mean that a chemical link exists between the membrane self-insertion peptide and the NK cell effector agent.

[0024] 33. In some embodiments, one or more stimulating peptides may be coupled to a membrane self-insertion peptide or a GPI anchor for in situ self-assembly. For example, 41-BBL and IL-21 may be coupled to a pHLIP peptide for insertion into the cell membrane alone under acidic conditions, thereby enabling fixation of the stimulating ligand to cells near the tumor. Stimulating peptides: 41BBL, IL-2, IL-12, IL-21, BCM / SLAMF2, CCR7, and / or other homing receptors may be produced in bacterial cells or purchased from a commercial supplier, and cDNA vectors of these proteins may be ligated into a pTriEX expression plasmid to enable expression in bacterial (E. coli), insect, or mammalian cells, depending on the application. The cDNA vector may encode the expression of a FLAG- or HIS- tag. Bacterial cells can be transfected using a standard CaCl transfection method and may be cultured in LB medium. The cells can be harvested and lysed using a French press, and the relevant proteins may then be purified from the lysate by affinity chromatography.

[0025] 34. In some embodiments, pHLIP may be prepared by solid-phase peptide synthesis utilizing the chemistry of 9-fluorenylmethyloxycarbonyl, and the product may be purified by reverse-phase chromatography on a C18 column. pHLIP may then be conjugated to an irritating human protein ligand by incubation with a crosslinking agent such as benzophenone-4-iodoacetamide. After multiple washes, the conjugated pHLIP protein may be resuspended in a culture medium (e.g., physiological saline) and injected intratumorally or intravenously. Based on evidence from the aforementioned literature and experimental results, interaction between NK cells on the surface of such modified tumor cells and irritating ligands such as IL-21 and 41-BBL may stimulate NK cell amplification at its original site and induce a cytotoxic response to the tumor. This type of irritating approach can be used to treat solid tumors such as ovarian cancer, where NK stimulating ligands can be injected into the patient's peritoneal cavity, either with low doses of IL-2 alone or with NK cells, to be inserted into tumor cells at their original site under acidic pH conditions. There is strong evidence that cytotoxic lymphocytes expressing high levels of FCγ1IIR(CD16), such as NK cells, are essential for the effectiveness of cancer treatment using therapeutic antibodies. Therefore, this approach can also be used in combination with therapeutic antibodies.

[0026] 35. It is understood and intended herein that the contact period between NK cells and an NK cell effector agent containing vesicles (i.e., PM21 particles, EX21 exosomes, and / or FC feeder cells) may be any length of time required to achieve the desired amplification of memory NK cells. For example, such contact may be as short as one minute or as long as 60 days (e.g., culturing NK cells for 7 days in the presence of PM21 particles, EX21 exosomes, and / or FC feeder cells). In one embodiment, the contact between NK cells and an NK cell effector agent containing vesicles may be between about 6 days and about 60 days, more preferably between about 6 days and about 40 days. Also disclosed herein are NK cells with PM21 particles, EX21 exosomes, and / or FC feeder cells, and 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 4 A method for treating bone marrow malignancies and bone marrow-derived malignancies through adoptive transfer of NK cells, including contact for 4, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or 72 days, and / or a method for trafficking NK cells into the bone marrow. In some examples, it is understood and intended herein that multiple contacts between the NK cells and PM21 particles, EX21 exosomes, and / or FC feeder cells may be desirable and may be used. For example, the NK cells can be brought into contact with PM21 particles, EX21 exosomes, and / or FC feeder cells once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24 hours, or once every 2, 3, 4, 5, 6, 7, 8, 9, 0, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days.Accordingly, in one embodiment, disclosed herein is a method for treating myelomas and malignancies derived from bone marrow through adoptive transfer of NK cells, and / or a method for trafficking NK cells into bone marrow, wherein the NK cells are brought into contact with PM21 particles, EX21 exosomes, and / or FC feeder cells more than once, and such contact is performed every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24 hours, or every 2, 3, 4, 5, 6, 7, 8, 9, 0, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days.

[0027] 36. In one embodiment, the plasma membrane particles, feeder cells, or exosomes may be purified from NK cell-stimulating feeder cells. The NK cell-stimulated feeder cells for use in the claimed invention, for use in producing the plasma membrane particles or exosomes disclosed herein, may be any of the following: irradiated autologous or allogeneic peripheral blood mononuclear cells (PBMCs), or non-irradiated autologous or PBMCs containing autologous or allogeneic peripheral blood mononuclear cells (PBMCs), RPMI8866, HFWT, K562, SKOV3, or EBV-LCL cells, or non-irradiated autologous or PBMCs, RPMI8866, HFWT, K562, SKOV3, or EBV-LCL cells transfected with membrane-bound IL-21 and 41BBL. In some embodiments, the NK cell feeder cells may be K562 cells transfected with membrane-bound IL-21 and 41BBL.

[0028] 37. The disclosed compositions can be used to treat any disease causing unregulated cell proliferation, such as cancer, particularly malignant tumors affecting or localizing in the bone marrow. A non-exclusive enumeration of different types of cancer is as follows: lymphoma (Hodgkin and non-Hodgkin), leukemia, carcinoma, solid tissue carcinoma, squamous cell carcinoma, adenocarcinoma, sarcoma, glioma, high-grade glioma, blastoma, neuroblastoma, plasmacytoma, cell cytocytoma, melanoma, adenoma, hypoxic neoplasm, myeloma, AIDS-associated lymphoma or sarcoma, metastatic cancer, or common cancer.

[0029] 38. A representative and non-limiting list of cancers that can be treated with the disclosed compositions is as follows: lymphoma, B-cell lymphoma, T-cell lymphoma, mycosis fungoides, Hodgkin's disease, myeloid leukemia, bladder cancer, brain cancer, cancer of the nervous system, head and neck cancer, squamous cell carcinoma of the head and neck, kidney cancer, lung cancer such as small cell lung cancer and non-small cell lung cancer, neuroblastoma / glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, liver cancer, melanoma, squamous cell carcinoma of the mouth, throat, larynx and lung, colon cancer, cervical cancer, cervical carcinoma, breast cancer and epithelial cancer, renal cancer, genitourinary cancer, lung cancer, esophageal carcinoma, head and neck carcinoma, colorectal cancer, hematopoietic cancer; testicular cancer; colon and rectal cancer, prostate cancer, or pancreatic cancer.

[0030] 39. Disclosed compositions may also be used to treat bone marrow-related viral diseases. Bone marrow-related viral diseases as used herein refer to viral diseases in which the bone marrow harbors a virus (i.e., a viral infection (such as chronic, acute, latent, and persistent infection), or is bone marrow-tropic), or in which the bone marrow is adversely affected by a virus that induces aplastic anemia, such as parvovirus (in some cases, a disease or illness that adversely affects the bone marrow is a viral infection of the bone marrow). Disclosed herein, in one embodiment, is a method for treating a bone marrow-related (e.g., adversely affecting the bone marrow) viral infection in a subject, comprising contacting NK cells with PM21 particles and / or FC21 feeder cells, and a method for adoptively transferring NK cells to a subject having a viral infection. In one embodiment, the virus may induce aplastic anemia and / or be a myeloid-tropic or viral infection that establishes a latent or chronic infection in the bone marrow. Examples of such viruses include, but are not limited to, dengue virus, hepatitis viruses (non-specifically, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, and hepatitis G virus), Epstein-Barr virus (also known as human herpesvirus 4), cytomegalovirus (also known as human herpesvirus 5), parvovirus (non-specifically, parvovirus B19, etc.), lymphocytic choriomeningitis virus (LCMV), human immunodeficiency virus (HIV), and respiratory syncytial virus (RSV).

[0031] C. Composition 40. Disclosed are the components used to prepare the disclosed compositions, and the compositions themselves used in the methods disclosed herein. These and other materials are disclosed herein, and combinations, subsets, interactions, groups, etc., of these materials are disclosed, and specific references to each of the various individuals and collective combinations and permutations of these compounds are not necessarily explicitly disclosed, but it will be understood that each is specifically intended and described herein. For example, if special PM21 particles or FC21 feeder cells are disclosed and discussed, and multiple modifications can be applied to multiple molecules, then specifically intended are all possible combinations and permutations of modifications, unless specifically indicated otherwise. Thus, if classes of molecules A, B, and C are disclosed, and classes of molecules D, E, and F, as well as an example of a combined molecule AD are disclosed, then even if each is not individually enumerated, it means that each is individually and collectively intended, and combinations: AE, AF, BD, BE, BF, CD, CE, and CF are expected to be disclosed. Similarly, any subset or combination of these is also disclosed. Therefore, for example, the subgroups AE, BF, and CE are expected to be disclosed. This concept applies non-limitingly to all aspects of this application, such as steps in methods for preparing and using the disclosed compositions. Thus, where various additional steps can be implemented, it will be understood that each of these additional steps can be implemented in any specific embodiment or combination of embodiments of the disclosed method.

[0032] 41. The disclosed methods for treating myeloid malignancies and myeloid-derived malignancies through adoptive transfer of NK cells, and / or methods for trafficking NK cells into the bone marrow, utilize one or more cytokines (e.g., IL-12, IL-15, and / or IL-18) in combination with vesicles containing NK cell effector agents such as PM21 particles, FC feeder cells, and / or EX21 exosomes. It is understood and intended herein that it would be advantageous to provide the components used in the disclosed methods in a package that allows the disclosed methods to be immediately carried out by a person.

[0033] Accordingly, in one embodiment, disclosed herein is a kit for treating myelomas and myeloid-derived malignancies with NK cells, comprising one or more cytokines (e.g., IL-2, IL-12, IL-15, and / or IL-18) and one or more vesicles containing NK cell effector agents. In one embodiment, the vesicles may be PM21 particles, EX21 exosomes, and / or FC feeder cells. For example, the disclosed kit may include: IL-12 and PM21 particles; IL-15 and PM21 particles; IL-18 and PM21 particles; IL-12 and EX21 exosomes, IL-15 and EX21 exosomes; IL-18 and EX21 exosomes; IL-12 and FC21 feeder cells; IL-15 and FC21 feeder cells; IL-18 and FC21 feeder cells; IL-12, IL-15, and PM21 particles; IL-12, IL-18, and PM21 particles; IL-15, IL-18, and PM21 particles; IL-12, IL-15, IL-18, and PM21 particles; IL-12, IL-15, and EX21 exosomes; IL-12, IL-18, and EX21 exosomes; IL-15, IL-18, and EX21 exosomes; IL-12, IL-15, IL-18, and EX21 exosomes; IL-12, IL-15, and FC2 1 feeder cell; IL-12, IL-18, and FC21 feeder cell; IL-15, IL-18, and FC21 feeder cell; IL-12, IL-15, IL-18, and FC21 feeder cell; IL-12, EX21 exosome, and PM21 particle; IL-15, EX21 exosome, and PM21 particle; IL-18, EX21 exosome, and PM21 particle; IL-12, FC21 feeder cell, and PM21 particles; IL-15, FC21 feeder cells, and PM21 particles; IL-18, FC21 feeder cells, and PM21 particles; IL-12, FC21 feeder cells, and EX21 exosomes; IL-15, FC21 feeder cells, and EX21 exosomes; IL-18, FC21 feeder cells, and EX21 exosomes; IL-12, FC21 feeder cells, PM21 particles, and EX21 exosomes;IL-15, FC21 feeder cells, PM21 particles, and EX21 exosomes; IL-18, FC21 feeder cells, PM21 particles, and EX21 exosomes; IL-12, IL-15, EX21 exosomes, and PM21 particles; IL-12, IL-18, EX21 exosomes, and PM21 particles; IL-15, IL-18, EX21 exosomes, and PM21 particles; IL-12, IL-15, IL-18, EX21 exosomes, and PM21 particles; IL-12, IL-15, FC21 feeder cells, and PM21 particles; IL-12, IL-18, FC21 feeder cells, and PM21 particles; IL-15, IL-18, FC21 feeder cells, and PM21 particles; IL-12, IL-15, IL-18, FC21 feeder cells, and PM21 particles; IL-12, IL-15, EX21 exosomes, and FC21 feeder cells; IL-12, IL-18, EX21 exosomes Sosomes and FC21 feeder cells; IL-15, IL-18, EX21 exosomes and FC21 feeder cells; IL-12, IL-15, IL-18, EX21 exosomes and FC21 feeder cells; IL-12, EX21 exosomes, FC21 feeder cells and PM21 particles; IL-15, EX21 exosomes, FC21 feeder cells and PM21 particles; IL-18, EX21 exosomes, FC21 feeder Cells and PM21 particles; IL-12, IL-15, EX21 exosomes, FC21 feeder cells, and PM21 particles; IL-12, IL-18, EX21 exosomes, FC21 feeder cells, and PM21 particles; IL-15, IL-18, EX21 exosomes, FC21 feeder cells, and PM21 particles; or may include IL-12, IL-15, IL-18, EX21 exosomes, FC21 feeder cells, and PM21 particles.

[0034] 42. It is understood and intended herein that the NK cell effector agents contained in the vesicles (e.g., PM21 particles, EX21 exosomes, and / or FC feeder cells) may be selected from the group of NK cell effector agents consisting of 4-1BBL, IL-2, IL-21, MICA / B, ULBP2, ICAM-1, 2B4, BCM1 / SLAMF2, CD155, CD112, CCR7, DAP12, Notch ligand, and DAP10.

[0035] 43. It is understood and intended herein that the disclosed kit or apparatus may contain cytokines in addition to IL-12, IL-15, and / or IL-18. Accordingly, in one embodiment, there is a kit for a method of treating myelomas and myeloma-derived malignancies through adoptive transfer of NK cells, and / or a method of trafficking NK cells into the bone marrow, further comprising 4-1BBL, IL-2, IL-12, IL-18, IL-21, MICA / B, ULBP2, ICAM-1, 2B4, BCM1 / SLAMF2, CD155, CD112, CCR7, DAP12, and DAP10.

[0036] 44. In one embodiment, it is intended herein that the disclosed kit or apparatus may be used in conjunction with NK cells obtained from a donor supply, such as NK cells obtained from an unselected population of peripheral blood mononuclear cells. In some examples, the donor source for NK cells used in the disclosed kit for treating bone marrow malignancies and bone marrow-derived malignancies may also be recipients of NK cells. Thus, the NK cells may be from an autologous source. In other examples, the donor source for the NK cells may be a haplotype-matched or homogeneous donor source.

[0037] 45. It is further intended herein that there are instances in which it is beneficial to provide NK cells in a kit or device. Accordingly, in one embodiment, disclosed herein is a kit for treating myeloma, further comprising NK cells or an NK cell line.

[0038] 1. Delivery of pharmaceutical carriers / pharmaceutical products 46. ​​As previously stated, the composition may also be administered in vivo in a pharmaceutically acceptable carrier. "pharmaceutically acceptable" means a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject together with a nucleic acid or vector without inducing any undesirable biological effects or interacting in a harmful manner with any of the other components of the pharmaceutically acceptable composition contained herein. The carrier will, as is well known to those skilled in the art, be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject.

[0039] 47. The composition may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, percutaneously, extracorporeally, or topically, including by topical intranasal administration or inhalation. As used herein, “topical intranasal administration” means delivery of the composition to the nose and nasal cavity through one or both nostrils, and may include delivery by a spray or droplet mechanism, or through aerosolization of nucleic acids or vectors. Administration of the composition by inhalation may be through the nose or mouth via delivery by a spray or droplet mechanism. Delivery may be directly to any area of ​​the respiratory system (e.g., lungs) via intubation. The exact amount of composition required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the allergic disorder being treated, the individual nucleic acids or vectors used, and the mode of administration. Therefore, it is not possible to specify the exact amount of each composition. However, a suitable amount can be determined by a person skilled in the art using only routine experiments, subject to the teachings provided herein.

[0040] 48. Parenteral administration of the composition, when used, is generally characterized by injection. The injection solution may be prepared in conventional forms, either as a liquid solution or suspension, a solid form suitable for suspension in a liquid before injection, or an emulsion. More recent and modified approaches for parenteral administration include the use of delayed-release or sustained-release systems to maintain a constant dose. See, for example, U.S. Patent No. 3,610,795, incorporated herein by reference.

[0041] 49. The material may be in solution or suspension (e.g., as microparticles, liposomes, or taken up by cells). These may be targeted to specific cell types via antibodies, receptors, or receptor ligands. The following references illustrate the use of this technique to target specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, KD, Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie, Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al., Biochem. Pharmacol, 42:2062-2065, (1991)). Vehicles such as "Stealth" and other antibody-conjugated liposomes (e.g., lipid-mediated drugs targeting colon cancer species), receptor-mediated targeting of DNA via cell-specific ligands, lymphocyte-directed tumor targeting, and highly specific therapeutic retroviral targeting of mouse glioma cells in vivo. The following references provide examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al., Cancer Research, 49:6214-6220, (1989); and Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187, (1992)). Receptors are generally involved in either constitutive or ligand-induced endocytosis pathways.These receptors aggregate in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through acidified endosomes where the receptors are sorted and subsequently recycled to the cell surface for intracellular storage, or degraded within lysosomes. The internalization pathway has various functions, including nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, ligand dissociation and degradation, and regulation of receptor levels. Many receptors follow one or more intracellular pathways depending on cell type, receptor concentration, ligand type, ligand valence, and ligand concentration. The molecular and cellular mechanisms of receptor-mediated endocytosis are being re-examined (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)).

[0042] a) Medically acceptable carriers 50. Compositions containing antibodies may be used therapeutically in combination with medically acceptable carriers.

[0043] 51. Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. AR Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, a suitable amount of pharmaceutically acceptable salt is used in the formulation to make it isotonic. Examples of pharmaceutically acceptable carriers include, but are not limited to, physiological saline, Ringer's solution, and dextrose solution. The pH of the solution is preferably about 5 to about 8, more preferably about 7 to about 7.5. Further carriers include sustained-release preparations such as semipermeable solid hydrophobic polymer matrices containing antibodies in the form of molded bodies, e.g., films, liposomes, or microparticles. It will be apparent to those skilled in the art that certain carriers may be more preferred depending, for example, the route of administration and the concentration of the composition being administered.

[0044] 52. Pharmaceutical carriers are known to those skilled in the art. Most of these will be standard carriers for administering drugs to humans, typically including solutions such as sterile water, physiological saline, and buffered solutions of physiological pH. The compositions may be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.

[0045] 53. In addition to the selected molecules, the pharmaceutical composition may also contain carriers, thickeners, diluents, buffers, preservatives, surfactants, etc. The pharmaceutical composition may also contain one or more active ingredients such as antibacterial agents, anti-inflammatory agents, and anesthetics.

[0046] 54. The pharmaceutical composition may be administered in multiple ways depending on whether topical or systemic treatment is preferred and the area to be treated. Administration may be external (ophthalmic, vaginal, transrectal, intranasal), oral, inhaled, or parenteral, such as intravenous drip, subcutaneous, intraperitoneal, or intramuscular injection. The disclosed antibody may be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, coelomatically, or percutaneously.

[0047] 55. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including physiological saline and buffer media. Parenteral vehicles include sodium chloride solution, glucose-added Ringer's solution, dextrose and sodium chloride, lactated Ringer's solution, or non-volatile oils. Intravenous vehicles include fluid and nutrient replacement fluids and electrolyte replacement fluids (such as those based on glucose-added Ringer's solution). Preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents, and inert gases, may also be present.

[0048] 56. Examples of formulations for external administration include ointments, lotions, creams, gels, intravenous infusions, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners, etc., may be essential or desirable.

[0049] 57. Compositions for oral administration include powders or granules, suspensions or solutions in water or a non-aqueous medium, capsules, sachets, or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing agents, or binders may be desirable.

[0050] 58. Some of the compositions may potentially be administered as pharmaceutically acceptable acids or base addition salts formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, as well as organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with inorganic bases such as sodium hydroxide, ammonium hydroxide, and potassium hydroxide, as well as organic bases such as mono-, di-, trialkyl and arylamines, and substituted ethanolamines.

[0051] b) Therapeutic use 59. Effective dosages and administration regimens for administering the composition may be determined empirically, and such determinations are within the scope of skill in the art. The dosage range for administering the composition is large enough to produce the desired effect affecting the symptoms of the disorder. The dosage should not be so large as to induce adverse side effects, such as unwanted cross-reactions or anaphylactic reactions. Generally, the dosage may vary depending on the patient's age, condition, sex, and the severity of the disease, the route of administration, or whether other drugs are included in the regimen, and may be determined by those skilled in the art. The dosage may be adjusted by each physician in the event of any counterindications. The dosage may vary and may be administered once or multiple times a day for a day or several days. Guidance on appropriate dosages for a given class of pharmaceutical products can be found in the literature. For example, guidance for selecting an appropriate antibody dose can be found in the literature on the therapeutic use of antibodies, such as Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, NJ, (1985) ch. 22 and pp. 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York (1977) pp. 365-389. A typical daily dose of an antibody used alone may range from approximately 1 μg / kg body weight to 100 mg / kg body weight per day, or more, depending on the factors mentioned above.

[0052] D. Examples 60. The following examples are provided to give a complete disclosure and description of how the compounds, compositions, articles, apparatus and / or methods claimed herein were prepared and evaluated, and are purely illustrative and not limiting to the disclosure. While efforts have been made to ensure accuracy with respect to numbers (e.g., quantity, temperature, etc.), some errors and deviations should be taken into consideration. Unless otherwise indicated, parts are parts by weight, temperature is °C or ambient temperature, and pressure is atmospheric pressure or approximately atmospheric pressure.

[0053] 1. Example 1: PM21 particles stimulate NK cell amplification in vivo. 61. Natural killer (NK) cells are CD56 + CD3 - NK cells are components of the innate immune system identified as being able to spontaneously recognize and lyse virus-contaminated or malignant cells. Cell therapy with NK cells is promising as a cancer treatment, and several clinical trials have been completed and are currently underway for the treatment of various cancers (AML, lymphoma, breast cancer, ovarian cancer, neuroblastoma, and non-small cell lung cancer). For effective anti-cancer treatment with NK cells, three general aspects must be considered: 1) a sufficiently large dose of NK cells must be delivered; 2) the NK cells must be highly cytotoxic; and 3) the NK cells must reach the disease site, possibly localize, remain, and specifically target tumor cells.

[0054] 62. For clinical efficacy in the AML setting, Miller and collaborators recommended reaching a dose that delivers at least 100 NK cells per μL of peripheral blood (PB) two weeks after fluid administration. In several cases where adoptive NK cell therapy was effective, more than 1,000 NK cells per μL of PB were observed. These observations highlight the importance of skilled NK cell amplification techniques for delivering doses sufficient for overall treatment efficacy.

[0055] 63. Currently, there are three main clinically used methods for NK cell amplification for adoptive cell therapy. Firstly, in vivo amplification with cytokines such as IL-15 and IL-2, combined with host lymphocyte depletion / irradiation, can stimulate in vivo amplification from relatively small amounts of injected donor NK cells. Secondly, ex vivo methods using cytokines, mainly IL-2 and IL-15, can activate NK cells, but amplification is relatively low and variable. Similarly, NK cells activated ex vivo by cytokines undergo cytokine withdrawal after infusion, and the NK cells undergo apoptosis. Thirdly, the feeder cell method for ex vivo NK cell amplification utilizes co-culture with other stimulating cells. Feeder cell methods for NK cell stimulation include Epstein-Barr virus-LCL or engineered tumor cells. Co-culturing K562CML cells expressing membrane-bound IL-15 (mb15) and 4-1BB ligand (41BBL) (K562-mb15-41BBL) with NK cells can amplify them several hundredfold within approximately two weeks, but the NK cells amplified by this method undergo senescence. In addition, IL-15-activated NK cells lose surface CD16 due to the proteolytic activity of ADAM17. Rather, K562 cells expressing mb21 instead of mb15 significantly improve NK cell amplification while avoiding telomere shortening and the resulting NK cell senescence. NK cell amplification with K562-mb21-41BBL is highly efficient, with an average amplification of 48,000 times and enrichment rates exceeding 85% typically achieved within three weeks. All of these methods are being actively investigated in clinical trials.

[0056] 64. NK cell amplification methods have improved, but still have drawbacks and challenges. High toxic doses of IL-2 are necessary for the survival of intravenously administered NK cells regardless of the amplification method, but the persistence of NK cells was limited. Ex vivo methods using feeder cells were effective for amplification to produce large quantities of NK cells, but a problem arose: long-term ex vivo culture of NK cells led to a loss of homing ability to disease sites such as bone marrow. Thus, a debate arose regarding the overall benefits of in vivo vs. ex vivo amplification. The optimal NK cell amplification procedure is one with proliferative capacity based on ex vivo feeder cells, but it can be performed either ex vivo or in vivo.

[0057] 65. A novel PM21 particle-based method for rapid and selective amplification of cytotoxic NK cells starting from PB mononuclear cells (PBMCs). Particles corresponding to closed plasma membrane vesicles were prepared from the plasma membrane of K562-mb15-41BBL cells (PM15 particles), enabling selective NK cell amplification of 250-fold within 14 days and 1,265-fold after 17 days, which is equivalent to the amplification efficiency using co-cultured K562-mb15-41BBL feeder cells. PM15 particle-activated NK cells, similar to those amplified in feeder cells, showed high cytotoxicity against CML and AML cells ex vivo. Importantly, these particles offer several advantages over the feeder cell method. Firstly, they can be pre-prepared, tested, and stored for more than a year, and can be used as "off-the-shelf reagents" without being tied to a single GMP facility, greatly simplifying the clinical logistics of adoptive NK cell therapy. Secondly, using PM particles instead of feeder cells to stimulate NK cells eliminates steps required for safety measurements when using tumor-derived feeder cells, such as radiation therapy of feeder cells, and when testing their presence and proliferation in the final product. Thirdly, tumor-derived feeder cells cannot be injected as adjuvant therapy, while PM particles can be injected to stimulate in vivo amplification of NK cells. The advantages offered by PM particle-based methods for NK cell amplification enable significant clinical benefits.

[0058] 66. At this stage of the study, the efficacy of PM particles prepared from K562-mb21-41BBL was tested for in vivo amplification of adoptively transferred NK cells, pre-activated in a relatively short and simple procedure that can be easily performed in a clinical setting. This method overcomes the shortcomings of previous studies by allowing intravenous infusion of adoptive NK cells, which enables only very small amounts of in vivo NK cell amplification and limited persistence. In this study, efficacy is demonstrated for PM21 particle-stimulated ex vivo and in vivo amplification of NK cells from non-selective PBMCs injected into the peritoneal cavity, which is intended to act as the intrinsic site for incubation and PM21 particle stimulation. This method is expected to be useful for in vivo amplification of therapeutically relevant amounts of NK cells, which means making NK cell-mediated immunotherapy more widely available to patients.

[0059] a) Materials and methods (1) Human sample 67. Primary leukocytes were obtained from patients with active disease who signed IRB-approved informed consent, and equivalent PB cells were recovered from these patients in remission. Fresh blood from a leukocyte source (One Blood, Orlando, Florida) or from healthy volunteers who signed IRB-approved informed consent was used as the nominee sample. PBMCs were isolated using Ficoll-Paque (GE Healthcare, Pittsburgh, Pennsylvania). All samples were re-identified and cryopreserved in a viable state.

[0060] (2) Reagents and cell lines The 68.K562 cell line was obtained from ATCC (Manassas, Virginia). The Annexin-V FITC kit and Enumeration Flow-Count beads for the cytotoxicity assay were purchased from Beckman Coulter (Miami, Florida). The following dye-conjugated antibodies were used for phenotyping: CD16-FITC, NKG2A-PE, NKp46-PE, CD3-APC (Beckman Coulter); CD4-APC-Cy7, CD8-PE, CD56-BV421, CD94-APC (BD Biosciences); CD3-Alexa488, NKG2D-APC, CD62L-PE-Cy7, CD45-eFluor450, CD45-APC (eBiosciences); CD56-PE, KIR2D-APC (Miltenyi); NKG2C-PE, NKp44-APC, TRAIL-PE (R&D Systems).

[0061] (3) Preparation and characterization of plasma membrane particles 69. PM particles were prepared from K562-mb21-41BBL cells. Cells were grown in RPMI-1640 medium supplemented with 5% fetal bovine serum. Cells were harvested by centrifugation (1,000 × g, 10 mins) and washed with DPBS containing 2 mM EDTA. Cells were resuspended in a lysis buffer containing 50 mM HEPES (pH 7.4), 150 mM NaCl, 2 mM MgCl2, and AEBSF, aprotinin, leupeptin, and pepstatin A. Cells were disrupted by nitrogen cavitation at 300 psi and 4°C for 30 minutes (Parr Instruments, Maureen, Illinois). Cell lysates were centrifuged (1,000 × g, 10 mins), and the supernatant was then centrifuged (100,000 × g) to pellet the crude cell membranes. The crude membrane was further purified by sucrose gradient centrifugation, and the fraction corresponding to closed plasma membrane vesicles was recovered. All procedures were performed using sterile techniques, and the sterility of the product was tested by culture. The PM particle preparation was quantified by protein concentration using a BCA assay and expressed as membrane protein μg / mL. The presence of IL-21 and 41BBL on the PM particles was confirmed by ELISA and Western blotting.

[0062] (4) Ex vivo NK cell amplification from PBMCs 70. NK cells from PBMCs were amplified using PM21 particles. Briefly, PBMCs were amplified at 0.1 × 10¹⁶ in SCGM (CellGenix, Portsmouth, New Hampshire) supplemented with 10% FBS, 2 mM Glutamax, 100 U / mL IL-2 (Peprotech, Rocky Hill, New Jersey) and 200 μg / mL PM21 particles. 6 Cells were seeded with NK cells / mL. The supplemented culture medium was replaced daily, and every 2-3 days after day 5.

[0063] (5) Cytotoxicity assay of patient's autologous NK cells 71. The cytotoxicity assay of patient-derived NK cells against autologous AML tumor cells was performed using Annexin V (BD Bioscience). NK cells amplified for 16 days (NK cell content >90%) were stained with TFL4 dye. Target tumor cells were subjected to 0.5 × 10⁶ NK cell assays with E:T ratios of 1:1, 2:1, 5:1, and 10:1. 6 CD34 + Cells were co-cultured at 37°C and atmospheric pressure in 5% CO2 for 2 hours at a concentration of cells / mL. The cells were then centrifuged and resuspended in Annexin V-labeled buffer containing Annexin V-FITC, anti-CD34-PE, and anti-CD56-PC7, and incubated at 4°C for 15 minutes. Labeled cells were diluted in 250 μL and analyzed by flow cytometry using an Accuri instrument (BD Bioscience).

[0064] (6) In vivo amplification of NK cells in NSG mice 72. Freshly thawed PBMCs, or PBMCs pre-activated for 2 days with 200 μg / ml PM21 and 100 U / mL IL-2, were washed twice and resuspended in phenol red-free RPMI medium. 1 × 10⁶ cells were collected from the total PBMC cell suspension. 5 NK cells are treated with NSG (NOD-scid IL-2Rgamma null ) was administered intraperitoneally to mice. PM21 particles (amounts specified in the legend in the figure, twice a week) and IL-2 (1,000 U, three times a week) were also administered intraperitoneally, and PB was collected by bleeding from the cheek or by cardiac puncture. Organs were recovered by necropsy and perfused for analysis to obtain single-cell suspensions.

[0065] b) Results (1) Ex vivo and in vivo amplification of NK cells obtained from healthy donors and leukemia patients Since K562 cells engineered to express mbIL21 have been reported to exhibit good efficiency in senescence-free NK cell amplification, PM particles were prepared from K562-mb21-41BBL cells and designated as PM21 particles. The PM21 particles were characterized for the consistency of their size distribution and mbIL21 levels (Figure S1) and tested for their NK cell amplification capacity.

[0066] 74. PBMCs were cultured with PM21 particles (200 μg / ml) for 28 days. NK cells stimulated by PM21 particles amplified, and the NK cell count in PM21-stimulated NK cell culture c reached over 90% by day 14 (Figure 1AB). Cumulative analysis of NK cell amplification at 14±1 days of culture showed that PM21 particles (average 825-fold amplification, range 163-2,216, n=13) were significantly more effective (p=0.021) than PM15 particles (average 424-fold amplification, range 290-570, n=30) (Figure 1C). Furthermore, NK cells stimulated by PM21 particles amplified exponentially over the 28-day period, reaching an amplification of over 100,000 times, in contrast to NK cell amplification with PM15 particles, which stopped by day 22 of culture due to aging. Therefore, PM21 particles exhibited improved NK cell amplification proficiency compared to PM15 particles, and NK cell amplification with PM21 particles was comparable to that reported with K562-mb21-41BBL feeder cells in which PM21 particles were induced. PM21-amplified NK cells were also cytotoxic to leukemia cell lines (Figure S2).

[0067] 75. The NK cell amplification capacity of PM21 particles was further tested in PBMCs from leukemia patients in remission. PM21 particles relatively efficiently induced NK cell amplification in all samples from three patients over 14 days of culture (113±7x for F021, 810±81x for M038, and 352±86x for M050, Figure 1D). The amplification was specific to NK cells and total hCD45 +The percentage of NK cells among total cells increased preferentially (Figure 1E). In the case of sample F021, the cytotoxicity of the expanded NK cells was tested in an autologous setting against tumor blasts obtained from the same patient during active disease (Figure 1F). At a relatively low effector-to-target ratio (E:T) of 1:1, 78±3% of the tumor cells were apoptotic. Therefore, this method can be used in an autologous transplantation setting.

[0068] 76. The unprecedented capability of PM particles is to promote in vivo amplification when used as an injection. To test whether PM21 particles stimulate in vivo NK cell amplification and determine whether ex vivo pre-activation is required, NSG mice were injected with 0.1×10 as part of untreated PBMC or PM21 particle-preactivated PBMC (PM21-PBMC) 6 NK cells via intraperitoneal injection. Mice injected with non-activated PBMC had a small number of human NK (hNK) cells in the peripheral blood (PB), and only hT cells persisted for 15 days after injection in total hCD45 + increased as a percentage of cells (Figure 2AB). In striking contrast, it was found that the PB of mice injected with PM21-PBMC exhibited an increase in hNK cell levels peaking at day 12 after intraperitoneal injection (Figure 2CD). NK cell content accounted for hCD45 + cells were concentrated to 53±8%. In the same experiment, efficacy was tested for in vivo intraperitoneal application of PM21 particles to promote better in vivo NK cell amplification. In mice injected with normal PBMC, additional in vivo PM21 particles did not stimulate hNK cell amplification. However, in vivo application of PM21 particles to mice transplanted with PM21-PBMC had an effect, and the hNK cell level was higher compared to PM21-PBMC transplanted mice that did not receive in vivo PM21 particles (Figure 2D).

[0069] 77. To provide evidence that PM21 particles induce in vivo NK cell proliferation, analysis was performed using CTViolet-labeled hNK cells amplified in vivo 6 days after intraperitoneal inoculation. Cells from mice injected with inactivated PBMCs showed no or minimal decrease in CTViolet fluorescence, indicating no or minimal NK cell division (Figure 3AB). hNK cells from mice injected with PM21-PBMCs showed a significant attenuation of CTViolet fluorescence intensity (Figure 3CD). Fluorescence intensity fitting showed that this decrease in intensity correlated with a major population that underwent 7 cell divisions in vivo within 6 days. In the case of hNK cells obtained from mice injected with PM21 particles intraperitoneally, one extra division may be observed. This further doubling with in vivo PM21 particle administration correlates with the higher NK cell count observed in PB with in vivo PM21 particles.

[0070] 78. To further investigate whether in vivo PM21 particles enhance in vivo NK cell amplification, the dose-dependence of in vivo PM21 particles was tested (Figure 4). A dose-dependent increase in hNK cells in PB was observed at 0–800 μg of PM21 particles per injection (Figure 4E). At an 800 μg dose (corresponding to approximately 100 ng of mbIL21), 470 ± 40 hNK cells per μg of PB were observed 12 days after intraperitoneal injection of PM21-PBMC. This NK cell concentration in PB was five times higher than the concentration generally considered to be therapeutically effective in an AML environment. The dose-dependent effect on in vivo amplification was specific to hNK cells that did not show a significant increase in T cell volume (Figure 4E). At doses higher than 1,600 μg per injection, PB hNK cell levels decreased in a manner similar to that observed with ex vivo. Approximately 200-400 μg / mL was optimal for PM21 or PM15 particles, and higher doses resulted in attenuated NK cell amplification.

[0071] 79. Observation of a significant amount of hNK cells in PB suggests that hNK cells amplified in PM21-PBMCs injected intraperitoneally can migrate from the peritoneal cavity to PB. To verify that adoptively transferred hNK cells can migrate to potential disease sites, hNK cells were quantified in various organs (Figure 5). Human NK cells were found in each organ examined, with larger amounts of hNK cells found in the organs of mice treated in vivo with 800 μg of PM21 particles, and these were significant in all organs except the liver (p<0.05). Furthermore, organs from mice treated with 800 μg of PM21 particles showed a total hCD45 + They had a higher percentage of hNK cells as a proportion of their total cells.

[0072] 80. Mouse-based studies described herein have shown that a procedure combining short ex vivo pre-activation with PM21 particles and in vivo administration of PM21 particles induces significant in vivo NK cell amplification within a potentially therapeutically relevant range. To demonstrate the consistency required for clinical use, this procedure was applied to leukocyte sources from three different donors (different from those used in other experiments) (Figure 6). The mean amounts of hNK cells in both PB and peritoneal lavage fluid were relatively consistent among the leukocyte sources. hNK, hT cells and other hCD45 + The cell percentages also showed great agreement in mice injected with PM21-PBMC from specific leukocyte sources (n=3), and also between leukocyte sources L8 and L10.

[0073] (2) Phenotype of NK cells amplified with P21 particles 81. The antitumor cytolytic activity of NK cells is determined by the balance of stimulation from activating and inhibitory signals. Here, detailed comparative tests were performed on PM21 particle-stimulated NK cells that were 1) amplified ex vivo with PM21 for 12 days, 2) amplified in vivo and isolated from peritoneal lavage fluid (PB), and 3) amplified in vivo and isolated from peritoneal lavage fluid (AW). These comparisons were performed using cells from a single donor in all environments and were carried out in equilibrium (Figure S3).

[0074] 82. The presence of the Fcγ receptor CD16 on NK cells is necessary for effective antibody-dependent cytotoxicity (ADCC). Almost all NK cells from in vivo amplification show CD16 expression (97% from PB and 87% from AW, respectively). CD94 is a surface receptor that forms a heterodimer complex with NKG2C or NKG2A. Approximately half of ex vivo amplified NK cells have CD94 expression. In the case of in vivo amplified NK cells, cells from AW (64±9%) have higher expression than NK cells from PB (38±13%). Both receptors in the NKG2 family, including NKG2C as an activating receptor and NKG2A as an inhibitory receptor, bind to CD94. Ex vivo amplified NK cells had relatively low NKG2C expression, but NK cells from AW had higher expression (53±8%) and NK cells from PB had higher expression (61±2%). The proportion of NK cells expressing NKG2A was higher in AW (82±8%) than in PB (67±12%) and ex vivo amplified cells (74%). NKG2D is another important activating receptor found on NK cells, and its expression was found in 61±6% of AW NK cells, 26±3% from PB, and approximately 75% of ex vivo amplified NK cells. CD62L expression, known to correlate with bone marrow homing, was higher in NK cells in PB (63±10%) and lower in AW (39±14%), consistent with higher expression on recruited cells. NKp44 and NKp46 are members of the innate cytotoxic receptor family and play a role in NK cell-mediated cytolysis. NKp46 was expressed in NK cells from both PB (76±9%) and AW (89±5%). NKp44 was relatively underexpressed in these NK cells from all sources. On the other hand, NKp46 was adequately expressed in both PB (89±5) and AW (76±9). TRAIL is a ligand on NK cells that induces target apoptosis via the cell death receptor pathway. TRAIL was expressed in 36±6% of NK cells from AW, 20±4% from PB, and 26% of ex vivo amplified cells.KIR2D is a killer immunoglobulin-like receptor (KIR) 2D subtype and represents a minority component (approximately 1 / 3) of NK cells from in vivo or ex vivo expressed KIR2D. Analysis of CD8 and CD4 T cell ratios revealed that CD8 T cells were more abundant than CD4 T cells from in vivo samples. The presence of NK-suppressive Treg cells was also explored, but they were very small (total CD3). + Less than 0.1% of cells were observed in the in vivo sample.

[0075] c) Discussion (1) PM21 particles promote ex vivo and in vivo NK cell amplification up to therapeutically relevant levels. 83. Adoptive NK cell therapy holds great promise as a cancer treatment for initial treatment and remission maintenance of various tumors. The requirements for the therapeutic use of NK cells are a method for rapid and selective NK cell amplification that is safe, concise, and has overall therapeutic efficacy. Methods based on multiple cytokines and feeder cells are currently being clinically studied, and the methodology using the K562-mb21-41BBL cell line is among the most effective for ex vivo NK cell amplification. While the feeder cell method is effective in providing a high initial dose and allows for repeated administration, the ability of ex vivo amplified NK cells for homing to the bone marrow, which is important for leukemia treatment, may be affected, and the in vivo persistence of infusion-administered NK cells may not be optimal. The combination of ex vivo and in vivo PM21 particle-based NK cell amplification methods described herein may significantly enhance the efficacy of NK cell adoptive therapy.

[0076] 84. Importantly, PM21 particles can be used for in vivo stimulation to promote in vivo amplification and persistence. The methodology developed herein utilized a short 2-day ex vivo pre-activation followed by in vivo administration of PM21 particles. In vivo application of PM21 particles induces higher in vivo NK cell amplification in a dose-dependent manner to the in vivo-applied PM21 particles. Using this optimized procedure, an average 360-fold in vivo increase in PB NK cells was observed between days 5 and 12 after intraperitoneal injection of PM21-PBMCs, which would likely have been a higher amplification factor in the intraperitoneal setting. For comparison, 1–2 × 10⁻⁶ 6 Recent studies have shown that after intravenous NK cell infusion, only about 5-17 NK cells / μL of blood were observable 14 days after infusion. In contrast, this study using PM21 particle stimulation yielded 2.0 × 10⁶ 6 Twelve days after intraperitoneal fluid resuscitation of PM21-PBMCs, a concentration exceeding 400 NK cells / μL blood was observed (11%, i.e., 0.2 × 10⁻⁶). 6 NK cells). Similarly, the previous study used 5 μg (approximately 50,000 U) of either IL-2 or IL-15 per injection (three times a week), but this study used a relatively lower dose of IL-2 (1,000 U / injection, three times a week). In a different study, 30 × 10¹⁶ cells were preferentially ex vivo amplified in K562-mb15-41BBL feeder cells. 6 NK cells were intravenously injected, and the injected human lymphocytes were subsequently tracked using an anti-CD45 antibody (not a combination of anti-CD56 and anti-CD3). In this method, high doses of intraperitoneally injected IL-2 (25,000 U / day) were necessary for lymphocyte persistence, and NK cell concentrations were not determined, but rather suggested. Compared to these previous methods, the scale of in vivo NK cell amplification stimulated by PM21 particles is unprecedented and represents the unique capabilities of these PM21 particles.

[0077] 85. Here, the delivery route of PM21-PBMCs to NSG mice was by intraperitoneal injection, similar to previous preliminary clinical trials. Compared to these previous trials, the PM21 particle-based method is advantageous in several ways. First, the combination of ex vivo pre-activation and in vivo stimulation with PM21 particles allows for the use of significantly smaller amounts of non-selective PBMCs compared to cytokine activation of isolated NK cells, which requires large-scale lymphocyte harvesting by apheresis and subsequent extensive laboratory processing for NK cell enrichment. Second, the PM21 particle-based method requires only a short two-day pre-activation instead of amplification based on two weeks of culture, allowing for better retention of physiologically relevant functionality. Third, the method of the present invention allows for significantly greater in vivo proliferation or in vivo persistence without the use of high doses of IL-2 associated with clinical toxicity, compared to previous methods that do not allow amplification. In peritoneal tumors, the advantages of the method described herein can significantly enhance the overall antitumor effect. As clearly demonstrated by proliferation analysis in CTViolet, in the absence of peritoneal tumors, the peritoneal cavity can provide a favorable environment for PM21 particles by confining them to this volume and promoting good in vivo amplification, after which NK cells can migrate in significant quantities to the peritoneal bone marrow (PB) and organs. NK cells were observed not only in the PB but also in organs, and were more abundant with in vivo application of PM21 particles. The amount of NK cells measured in the bone marrow was CD34 + The amount of NK cells used is equivalent to that used in tests with NK cells derived from umbilical cord blood stem cells, demonstrating that these NK cells are suitable for bone marrow homing.

[0078] 86. Phenotyping of NK cells amplified in parallel ex vivo or in vivo (Figure S3) showed that the resulting cells were similar regardless of the approach. NKG2A was observed in most in vivo amplified NK cells but not in the ex vivo environment. - and NKG2C + Interesting differences were observed regarding the amplification of subpopulations. NKG2C +NK cell populations have been observed during viral reactivation in connection with a “memory-like” response, and it has recently been shown that IL-12 production is monocyte-dependent. NKG2C in patients with CMV reactivation after stem cell transplantation for AML. + The presence of NK cells was also associated with favorable outcomes and low relapse rates. Similarly, NKG2A cells were resistant to HLA-E-induced inhibition. - The presence of a significant population of NK cells can be important in the treatment of multiple myeloma patients, as these cells downregulate HLA class I but express HLA-E, thereby eliciting an NK cell response. Approaches aimed at downregulating NKG2A have been proposed as a means to improve the cytotoxicity, and thus the therapeutic potential, of NK cells. Ex vivo amplified cells are mostly NKG2A. + Therefore, shortening the time between ex vivo culture and subsequent in vivo amplification may provide additional benefits in generating NK cells with greater phenotypic diversity and potentially better cytotoxicity to targets.

[0079] (2) Potential clinical utility of PM21 particles 87. The ability of PM21 particles for NK cell amplification enables the widespread use of adoptive NK cell therapy for cancer treatment and potentially other diseases. The PM21 particles readily replace feeder cells currently used in clinical trials, facilitating transport and reducing risk. In regulatory zones where the use of tumor-derived feeder cells is prohibited or difficult to obtain approval for, the PM21 particles provide an immediate solution for ex vivo amplification and activation. In the use of PM21 particles for ex vivo amplification in an allogeneic environment, T cell depletion can be performed prior to ex vivo NK cell amplification. Current clinical trials of NK cells grown with K562-mbIL21 utilize T cell depletion prior to NK cell amplification to eliminate allogeneic T cells that could induce GvHD. Furthermore, in vivo administration of PM21 particles may, as an unprecedented ability, amplify NK cells in vivo and possibly reduce T cell amplification to mitigate GvHD. For the treatment of peritoneal cancer and other peritoneal tumors such as persistent ovarian epithelial carcinoma or fibrous small cell tumor, this NK cell amplification method can be translated to clinical applications. Antitumor efficacy experiments for the elimination of peritoneal tumors are currently underway. The use of PM21-PBMCs and PM21 particles for autotherapy is possible, and methodologies for incorporating T cell depletion are being explored for application in a homogeneous environment.

[0080] 88. Importantly, NK cells amplified by this method are biodistributed from the peritoneal cavity to the peripheral blood and multiple organs, which are potential sites for various other cancers. Although the intraperitoneal route of injection is unconventional for the treatment of hematological malignancies, delivery of NK cells via this intraperitoneal route results in PB concentrations of NK cells relevant to AML treatment.

[0081] 89. The particle-based approach for NK cell-specific signaling may be a platform including other signaling molecules or vehicles for packaged delivery of drugs for further targeted stimulation of NK cells to enhance homing, antitumor cytotoxicity, and persistence. The PM21 particles may have high complementarity with all of the innovative NK cell-specific immunotherapies under development (checkpoint inhibitors, CARs, bispecific engagers (BiKE), DT-fused IL-2 for Treg depletion, etc.) and the beneficial effects added during in vivo amplification of NK cells with PM21 stimulation. In terms of preclinical utility as well, the methods described herein enable unprecedented methods for testing such combinations. Of course, there are mouse models, but there are no other methods to test human NK cells that can be present for a significant period in vivo.

[0082] 90. In summary, this procedure with PM21 particles enables preferential in vivo NK cell amplification at a level typically only achievable through ex vivo amplification with feeder cells, without the need for cell culture with feeder cells or toxic high cytokine doses. Furthermore, PM21-PBMCs, delivered in vivo with PM21 particles, can be used in an autologous environment and benefit from the advantageous synergistic effects of other immune cells on NK cell function, and can be combined with other strategies such as anti-KIR antibodies or BiKE to maximize the cytotoxicity of NK cells. Thus, this method is simpler and more modifiable for clinical conversion, yet meets the criteria for NK cell production for potential therapeutic efficacy and may have an impact on the treatment of cancer or other diseases.

[0083] 2. Example 2: Bone marrow trafficking of NK cells through fucosylation stimulation by PM21 particles PM21 particles prepared from K562 cells (K562.mb21.41bbl) transformed to express manipulated membrane-bound forms of 91.IL-21 and 41bbl induce efficient and specific amplification of NK cells. Stimulation provided by PM21 particles or K562.mb21.41bbl used as feeder cells (FC21) in co-culture with NK cells induces total fucosylation of sLex, as observed by HECA-452 mAb binding by flow cytometry.

[0084] 92. NK cells were amplified with PM21 or FC21, stained with HECA452 mAb, and analyzed by flow cytometry. HECA452 specifically recognizes fucosylated forms of PSGL-1, CD44, and other E-selectin ligands. NK cells stimulated with PM21 or FC21 had a significantly higher MFI by flow cytometry analysis compared to untreated NK cells, NK cells treated with soluble cytokines, or NK cell-treated feeder cells containing only mbIL21 (without 41bbl). This strongly suggests that stimulation with PM21 particles and / or FC21 feeder cells can induce NK cell trafficking into the bone marrow, and that bone marrow trafficking of therapeutic NK cells generated by PM21 or FC21 stimulation can improve bone marrow conditions and improve the treatment of bone marrow-derived malignancies.

[0085] 93. Figure 7 shows the effect of soluble cytokines on HECA452 staining of NK cells following stimulation at 0, 1, 7, and 10 days. The effect of particle or feeder cell stimulation is shown in Figure 8, where NK cells were stimulated with K562 cells, or K562 cells with membrane-bound IL-21 and 41BBL (FC21 cells or PM21 particles) for 10, 12, or 14 days and stained with HECA452. NK cells stimulated with K562-derived FC21 feeder cells or PM21 particles showed significantly greater activation compared to NK cells stimulated with K562 feeder cells without IL-21. Figures 9 and 10 show the effect of 10-day culture under various conditions on NK cells. NK cells were cultured for 10 days under the various conditions shown. NK cells were explored with FITC-conjugated HECA452 mAb to detect the fucosylated form of Slex. Cells from the culture were stained with HECA452-FITC and analyzed by flow cytometry with gating using CD56+CD3- (CSTX2 = CytoSen K562.mb21.41bbl; FC = feeder cells; PM21 = plasma membrane particles prepared from CSTX2).

[0086] 94. To understand the effect of the resting period on stimulated NK cells (Figure 11), NK cells were cultured with CSTX2-PM21 particles for 10 days (top), and then cultured for 3 days after removing the PM21 particles to induce a "resting" period (bottom). NK cells were examined with FITC-conjugated HECA452 mAb, which detects the fucosylated form of Slex. Cells from the culture were stained with HECA452-FITC and analyzed by flow cytometry with gating using CD56+CD3- (CSTX2 = CytoSen K562.mb21.41bbl; FC = feeder cells; PM21 = plasma membrane particles prepared from CSTX2).

[0087] NK cells stimulated with 95.PM21 particles were stable, survived the freeze-thaw process, and showed no recognizable effects on the cells (Figure 12). NK cells were isolated from PBMCs (top), cultured with CSTX2-PM21 particles for 10 days (middle), and then cryopreserved and thawed (bottom). NK cells were searched for with FITC-conjugated HECA452 mAb to detect the fucosylated form of Slex. Cells from the cultures were stained with HECA452-FITC and analyzed by flow cytometry with gating with CD56+CD3- (CSTX2 = CytoSen K562.mb21.41bbl; FC = feeder cells; PM21 = plasma membrane particles prepared from CSTX2). The present invention includes the following embodiments. <1> A method for trafficking NK cells into bone marrow, comprising contacting NK cells with one or more PM21 particles and FC21 feeder cells. <2> The further method includes stimulating the NK cells with IL-2, IL-12, and / or IL-18. <1> Methods used. <3> The contact between the PM21 particles and / or FC21 feeder cells and the NK cells takes place before the transfer of the NK cells to the patient. <1> Methods used. <4> The contact between the PM21 particles and / or FC21 feeder cells and the NK cells occurs following the transfer of the NK cells to the patient. <1> Methods used. <5> Inducing intracellular mechanisms within NK cells to induce fucosylation of PSGL-1 on the surface of the NK cells, <1> , <3> or <4> One of the methods described above. <6> The expression of FUT7 in NK cells is induced in correlation with the fucosylation of PSGL-1 on the surface of the aforementioned NK cells. <1> , <3> or <4> One of the methods described above. <7> A method for treating a bone marrow malignancy or bone marrow-derived malignancy in a subject, comprising contacting NK cells with PM21 particles and / or FC21 feeder cells, and adoptively transferring the NK cells to the subject. <8> A method for treating a bone marrow-related viral infection in a subject, comprising contacting NK cells with one or more PM21 particles and FC21 feeder cells, and adoptively transferring the NK cells to the subject. <9> The contact between the PM21 particles and / or FC21 feeder cells and the NK cells takes place before the transfer of the NK cells to the patient. <7> or <8> Methods used. <10> The contact between the PM21 particles and / or FC21 feeder cells and the NK cells occurs following the transfer of the NK cells to the patient. <7> or <8> Methods used.

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Claims

1. A composition comprising PM21 particles and / or FC21 feeder cells for inducing fucosylation of PSGL-1 on the surface of natural killer (NK) cells in order to traffic natural killer (NK) cells to the bone marrow of a target that requires it, The NK cells are contacted ex vivo or in vitro with PM21 particles and / or FC21 feeder cells for 6 to 40 days before being transferred to the target, and then rested for 3 days after the PM21 particles and / or FC21 feeder cells are removed. The aforementioned contact induces fucosylation of PSGL-1 on the surface of the NK cells, The PM21 particles and / or FC21 feeder cells contain membrane-bound IL-21 and membrane-bound 41BBL. composition.

2. The composition according to claim 1, further comprising the contact stimulating the NK cells with IL-2, IL-12, and / or IL-18.

3. The composition according to claim 1, wherein the contact correlates with the fucosylation of PSGL-1 on the surface of the NK cells, thereby inducing the expression of FUT7 in the NK cells.

4. A pharmaceutical composition comprising natural killer (NK) cells for treating myeloma or malignant tumors of myelo-derived origin in a subject, The above procedure involves contacting the NK cells with PM21 particles and / or FC21 feeder cells ex vivo or in vitro for 6 to 40 days before adoptive transfer of NK cells to the subject, and then, after removing the PM21 particles and / or FC21 feeder cells, allowing the subject to rest for 3 days. The aforementioned contact induces fucosylation of PSGL-1 on the surface of the NK cells, The PM21 particles and / or FC21 feeder cells contain membrane-bound IL-21 and membrane-bound 41BBL. Pharmaceutical composition.

5. A pharmaceutical composition comprising natural killer (NK) cells for treating bone marrow-related viral infections in a subject, The above procedure involves contacting the NK cells with PM21 particles and / or FC21 feeder cells ex vivo or in vitro for 6 to 40 days before adoptive transfer of NK cells to the subject, and then, after removing the PM21 particles and / or FC21 feeder cells, allowing the subject to rest for 3 days. The aforementioned contact induces fucosylation of PSGL-1 on the surface of the NK cells, The PM21 particles and / or FC21 feeder cells contain membrane-bound IL-21 and membrane-bound 41BBL. Pharmaceutical composition.

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