PM21 particles improve bone marrow homing of NK cells

Abstract

To provide compositions and methods for directing NK cells to the bone marrow through the use of PM21 particles.SOLUTION: Disclosed is a PM21 particle or FC21 feeder cell for use in a method for trafficking natural killer (NK) cells to the bone marrow, where the method for trafficking NK cells to the bone marrow comprises contacting NK cells with PM21 particles or FC21 feeder cells ex vivo or in vitro for six days to forty days before the transfer of the NK cell, where the contact elicits fucosylation of PSGL-1 on the surface of the NK cells.SELECTED DRAWING: Figure 5

Description

[Technical Field]

[0001] This application is incorporated by reference in its entirety. This application claims the benefit of U.S. Provisional Patent Application No. 62 / 464,747. [Background technology]

[0002] I. Background Adoptive natural killer (NK) cell therapy is a promising treatment for oncology, including myeloid malignancies. This is a novel intervention. Therefore, the efficiency of trafficking of adoptive NK cells is important. is very important. Summary of the Invention [Problem to be solved by the invention]

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

[0004] II. Overview 1. Disclosed is a method for treating NK cells with PM21 particles and / or FC21 feeder cells. and methods relating to trafficking NK cells to bone marrow, comprising contacting the In one embodiment, the method comprises inducing NK cells to induce IL-2, IL-12, IL-14, and IL-16. The method may further include stimulating with IL-18 and / or IL-18.

[0005] 2. Also disclosed is a method for treating NK cells with PM21 particles and / or FC21 feeders. and adoptively transferring the NK cells into the subject. Methods for treating malignancies or bone marrow derived malignancies, and / or viral infections (bone Bone marrow tropic viral infections or those with adverse effects on the bone marrow The present invention relates to a method for treating bone marrow-associated viral infections, including viral infections that affect the immune system. In some embodiments, PM21 particles and / or FC21 feeder cells and NK cells In another embodiment, the contact with PM can be performed prior to the transfer of the NK cells into the patient. Contact between 21 particles and / or FC21 feeder cells and NK cells occurs in the patient's body. It is possible to do so.

[0006] III. Brief description of the diagram 3. The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments. The figures, together with the description, exemplify the disclosed compositions and methods. [Brief explanation of the drawings]

[0007] [Figure 1]4. PM21 particles efficiently and selectively expand cytotoxic NK cells. Peripheral blood mononuclear cells (PBMCs) were isolated from a leukocyte source and seeded at 0.1 x 10 NK cells / mL in SCGM supplemented with 10% FBS, 2 mM Glutamax, and 50 U / mL IL-2. PBMCs were stimulated with 200 μg / mL PM15 (□, black) or PM21 (◯, blue) particles for 27 days, and cell content was tested every 2–3 days. The relative fold of NK cell expansion (A) and percentage of suspended cells (B) are shown. Based on cumulative analysis of NK cell expansion data through day 14, PM21 particles (825 ± 188 fold, N = 13, 4 donors) (blue) were more efficient at expanding NK cells than PM15 particles (425 ± 71 fold, 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 × 10 NK cells / mL in SCGM containing 10% FBS, 2 mM Glutamax, and 50 U / mL IL-2. The fold expansion of NK cells 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 previously described and analyzed for autologous cytotoxicity against AML tumors from the same patient (F). Expanded PM21-NK cells were labeled with TFL4 and co-incubated (2 h) with AML cells from the same patient during active disease at the indicated E:T ratio and analyzed by flow cytometry. The amount of apoptotic target cells was measured using a "target only" control. Each data point was measured in duplicate. [Figure 2]5. Preactivation of unselected PBMCs with PM21 particles induces in vivo NK cell expansion. NSG mice were intraperitoneally injected with 2 × 10 cells of either nonactivated PBMCs (A and B) or PBMCs preactivated ex vivo for 2 days with PM21 particles and 100 U / mL IL-2 (PM21-PBMCs) (C and D). All groups of mice received 1000 U of IL-2 intraperitoneally three times per week. Groups of mice were also intraperitoneally injected with 400 μg of PM21 particles twice per week (B and D). Peripheral blood was collected by serial cheek bleeds and analyzed by flow cytometry for hCD45+ human lymphocytes twice per week starting on day 6. NK, T, and B cell abundance was measured based on staining for hCD3, hCD56, and hCD19. For each experimental group, the left plot shows the concentration of hNK cells per μL of PB, and the right plot shows the percentage of hNK cells (circles, red) and T cells (vectors, black) as a percentage of total hCD45+ cells. [Figure 3] 6. Proliferation analysis demonstrates in vivo NK cell expansion from PM21-PBMCs. Freshly thawed PBMCs or PBMCs preactivated with PM21 particles and 100 U / mL IL-2 for 2 days (PM21-PBMCs) were labeled with Cell Trace (CT) Violet. 2 x 10 non-activated PBMCs (A and B) or PM21-PBMCs (C and D) were injected intraperitoneally into NSG mice. Mice in all groups received 1000 U IL-2 intraperitoneally three times per week. Two groups of mice also received 400 μg PM21 particles intraperitoneally twice per week (B and D). Two mice from each group were euthanized on day 6, and peritoneal lavage fluid was analyzed for CT Violet fluorescence of hCD45+, hCD3-, and hCD56+ NK cells by flow cytometry. Histograms of CT Violet fluorescence were analyzed through curve fitting using the proliferation analysis suite within FlowLogic. [Figure 4]7. In vivo application of PM21 increases NK cell numbers in peripheral blood. PBMCs were preactivated ex vivo with PM21 particles and 100 U / mL IL-2 for 2 days. PM21-PBMCs containing 0.2 × 10 viable NK cells were intraperitoneally injected into NSG mice. Mice in all groups received 1,000 U of IL-2 intraperitoneally three times per week. Mice were also intraperitoneally injected twice per week with 0 (A), 400 (B), 800 (C), or 1,600 μg (D) of PM21 particles. Peripheral blood was analyzed by flow cytometry for hCD45+ lymphocytes twice per week starting on day 5, and hNK, hT, and hB cell abundance was measured based on staining for hCD3, hCD56, and hCD19. The left plot for each experimental group indicates the concentration of hNK cells per μL of PB. The right plots show the percentages of hNK cells (circles, red) and T cells (black) as a percentage of total hCD45+ cells. Analysis of PB samples from day 12 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. In vivo expanded NK cells biodistribute to important physiological sites, and the biodistribution of these NK cells is shown to be increased by in vivo application of PM21 particles. NK cells (0.2 × 106 cells) preactivated ex vivo for 2 days with PM21 particles and 100 U / mL IL-2 as part of PM21-PBMC were intraperitoneally injected into NSG mice. Mice in all groups 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 sacrificed 16 days after the initial intraperitoneal injection of PM21-PBMC. On the day of euthanasia, bone marrow (femur), spleen, lung, brain, and liver were harvested, and the organs were perfused, and the femur was washed to recover cells. Cells were stained with antibodies against hCD3, hCD45, hCD56, and hCD19 for flow cytometry analysis. Data for bone marrow, spleen, brain, lung, and liver (left to right) are shown, along with the amount of hCD45+hCD56+hCD3- NK cells (top plots for each organ), and the percentages of hCD45+hCD56+hCD3- NK cells (circles, red), hCD45+hCD3+ T cells (squares, blue), and hCD45+hCD56-hCD3- other lymphocytes (triangles, black) (bottom plots for each organ). Each thick bar represents the mean. [Figure 6]9. Demonstration of consistent in vivo NK cell expansion from different donor sources. The consistency of PM21 particle-stimulated in vivo NK cell expansion was tested using three different PBMCs obtained from healthy donors. PBMCs were preactivated ex vivo with PM21 particles and 100 U / mL IL-2 for 2 days (PM21-PBMC) and injected intraperitoneally into NSG mice. Mice in all groups received 1000 U of IL-2 intraperitoneally three times a week. Peripheral blood was analyzed by flow cytometry for hCD45+ lymphocytes twice a week starting on day 5, and hNK, hT, and hB cell abundance was measured based on staining for hCD3, hCD56, and hCD19. The concentration of hNK cells in the blood on day 12 after intraperitoneal PBMC injection (A) and the amount of NK cells recovered in the peritoneal lavage fluid on day 14 after intraperitoneal PBMC injection (C) were similar among the different groups injected with different NK cell sources (p=0.84 for PB and p=0.69 for PB). The corresponding cell content of hNK cells (circles, red), hT cells (squares, blue), and other hCD45+ cells (triangles, black) was also consistent among the groups injected with different PBMC sources in peripheral blood (B) and intraperitoneally (D). Each thick bar represents the mean. [Figure 7] 10. HECA-452 staining of NK cells treated with soluble cytokines at 0, 1, 7, and 10 days of stimulation. [Figure 8] 11. HECA-452 staining of NK cells cultured with K562 feeder cells following 10, 12, and 14 days of stimulation. [Figure 9] 12. Comparison of HECA-452 staining of NK cells stimulated under various conditions following 10 days of stimulation. [Figure 10] 13. HECA-452 staining of NK cells cultured under various conditions following 10 days of stimulation. [Figure 11] 14. HECA-452 staining of NK cells after 3 days of rest following 10 days of stimulation. [Figure 12] 15. Shows pre-thaw and post-thaw HECA-452 staining following 14-day amplification of PM21 particles. [Figure 13] 16. We demonstrate that STAT3 is involved in IL-21-mediated modulation of FUT7 gene expression in human NK cells. (A) ChIP-seq was performed on IL-21-stimulated naive and expanded human NK cells using an antibody against STAT3. 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. DETAILED DESCRIPTION OF THE INVENTION

[0008] IV. Detailed Description 17. Disclose and describe the compounds, compositions, articles, devices, and / or methods of the present invention. Before using any of the above, it is important to understand that they are not derived from specific synthetic methods or specific recombinant bioengineering methods unless otherwise specified. It is not limited to, or unless otherwise specified, to, particular reagents, which of course may vary. It should be understood that the terminology used herein may be used to describe specific embodiments. It should also be understood that the information is for illustrative purposes only and is not intended to be limiting. .

[0009] A.Definition 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 mixtures of two or more such carriers, and the like. do.

[0010] 19. Ranges are expressed herein as from "about" one particular value and / or from "about" one particular value. When expressing such a range, another embodiment may be is inclusive from that one special value and / or to another special value. Similarly, the value When values ​​are expressed as approximations, by use of the antecedent "about," the particular value represents another embodiment. It will be understood that each endpoint of a range is a It is further understood that the significance of the study is related to the clinical outcome and independent of other endpoints. There are multiple values ​​disclosed herein, and each value is expressly incorporated by reference in its entirety. It will also be understood that the value "10" may be disclosed as a specific value. , "about 10" is also disclosed. When a value is disclosed to be "less than or equal to" that value, one of skill in the art would understand that As will be understood by those skilled in the art, "greater than or equal to the value" and possible ranges between the values ​​are also disclosed. For example, if the value "10" is disclosed, it should be understood to also include "less than or equal to 10" and "10 or more" is also disclosed. Throughout this application, data is provided in different formats. and that this data will be used to determine endpoints and starting points, and It will also be understood that within any combination of data points. If a special data point "10" and a special data point "15" are disclosed, the and greater than 15, 10 and 15 or greater, less than 10 and 15, 10 and 15 or less, and equal to 10 and 15, are considered to be disclosed as well as between 10 and 15 It will be understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed. is shown.

[0011] 20. As used in this specification and the claims that follow, the following terms are defined to have the following meanings: There may be several terms referenced.

[0012] 21. "Optionally" or "optionally" means the event or circumstance described thereafter. may or may not occur, and the description is an example of when the event or circumstance occurs. This means that it includes examples where this does not happen.

[0013] B. Methods of Using the Composition 22. Adoptive natural killer (NK) cell therapy is a treatment for myeloid malignancies and bone marrow-derived malignancies. promising new interventions for oncology, including tumors and their veterinary applications. In another embodiment, adoptive NK cells are used to treat, for example, parvovirus-induced aplastic anemia. It can be used therapeutically to treat bone marrow-resident viruses, such as flu. The efficiency of NK cell trafficking is crucial. How to target the cells to the bone marrow where they can be effective as a treatment is of great importance.

[0014] 23. One of the determinants of bone marrow homing is the ligand for selectin binding. In the case of L-selectin binding, the critical determinant is the P-selectin glycoprotein ligand. Fucosylation of sialyl Lewis x (sLex) carbohydrate chains attached to PSGL-1 (PSGL-1) In one embodiment, the engineered membrane binding of IL-21 and / or 41bbl K562 cells transformed to express morphology (K562.mb21.41bbl) Stimulation of NK cells with PM21 particles and / or FC21 feeder cells prepared from Stimulation of NK cells induces efficient specific expansion and full fucosylation of sLex. is disclosed and contemplated herein.

[0015] 24. Thus, in one aspect, disclosed herein is a method for treating NK cells with PM21 particles. contacting the cells with NK cells and / or FC21 feeder cells; and methods for trafficking NK cells to the bone marrow, including adoptive transfer of NK cells to patients with leukemia. and methods for treating myeloid malignancies or malignancies of bone marrow origin. In this method, the NK cells are treated with IL-2, IL-12, IL-15, IL-18, IL-21. and further comprising stimulating the subject either ex vivo or in vivo (within the patient) with obtain.

[0016] 25. In some embodiments, PM21 particles and / or FC21 feeder cells The contact with the NK cells can occur prior to the transfer of the NK cells into the patient. Therefore, contact of NK cells with PM21 particles and / or FC21 feeder cells may contribute to the It can be done internally.

[0017] 26. In one embodiment, the efficacy of NK cell immunotherapy is evaluated by determining the NK cells or It is understood that the effect of NK cells on the immune system is dependent on the dose of NK cells delivered after infusion or through in vivo expansion. It is understood and contemplated herein that currently available technology is unable to achieve a therapeutic effect in patients. limited by the inability to achieve the levels of NK cell expansion required to The lack of simpler clinical expansion protocols has hindered the progress and success of NK cell-based immunotherapies. Current ex vivo amplification protocols are a major barrier to widespread adoption and widespread use of leukemia. Activation and activation of high doses of cytokines and ligands expressed in derived feeder / stimulator cell lines The combination of and is not amenable to direct in vivo amplification. The use of particle technologies such as ELISA eliminates the requirement for stimulator cells, thus simplifying the methodology. to allow ex vivo amplification for adoptive therapy or for selective in vivo amplification. Thus, in one aspect, disclosed herein is a method for treating NK cell contacting the NK cell with one or more vesicles containing an NK cell effector agent. Methods for treating myeloid and bone marrow-derived malignancies through adoptive transfer of K cells - Patent Application 20070122999 and / or a method of trafficking NK cells to the bone marrow. Also disclosed is a method for generating at least one NK cell using at least one or more stimulatory Preactivating or activating NK cells in vivo by contacting them with cytokines myeloid malignancies, myeloid derived malignancies, and / or viral infections, further comprising (including myelotropic viruses). Thus, in one embodiment, PM NK cells expanded ex vivo with FC21 particles and / or FC21 feeder cells NK cells stimulated in vivo directly with PM21 or FC21 feeder cells The cells can be used to treat bone marrow-resident viral diseases or syndromes that induce aplastic anemia (e.g., For example, parvovirus can be treated.

[0018] 27. The disclosed method comprises generating at least one or more stimulatory cytokines (e.g., IL-2, IL-12, IL-15, IL-21 and / or The pre-activation or activation of NK cells is accomplished by contacting the NK cells with IL-18. Thus, in one embodiment, disclosed herein is a method for treating one or more NK cells by: One or more NK cells are stimulated, for example, by contacting them with two or three stimulatory cytokines. , IL-2, IL-12, IL-21, IL-15 and / or IL-18, or one or more stimulatory cytokines selected from the group consisting of any combination thereof NK cell adoptive transfer, comprising preactivating the NK cells by contacting the NK cells with Methods for treating myeloid malignancies and bone marrow derived malignancies and / or bone marrow Specifically disclosed herein are methods for trafficking NK cells. IL-2; IL-12; IL-15 , IL-18, IL-12 and IL-15;IL-12 and IL-18;IL-15 and IL-18; or contact with IL-12, IL-15, and IL-18, In one embodiment, the disclosed method for treating myeloid malignancies and tumors through adoptive transfer of NK cells is and methods for treating bone marrow-derived malignancies, and / or trafficking NK cells to the bone marrow. The method involves inducing NK cells in a soluble form or in a form that is sensitive to PM21 particles or FC21 particles. Leader cell morphology, 4-1BBL, IL-2, IL-21, MICA / B, ULBP 2, ICAM-1, 2B4, BCM1 / SLAMF2, CD155, CD112, CCR 7, DAP12, Notch ligand and / or DAP10 The method may further comprise contacting the cells with one or more cytokines.

[0019] 28. Precipitation of soluble forms or forms of PM21 particles or FC21 feeder cells Pre-activation or duration of activation (i.e., NK cells and stimulatory cytokines (e.g., IL-1) -2, IL-12, IL-15, IL-21 and / or IL-18) The period of time is any time required to achieve the desired pre-activation or activation of NK cells. It is understood and contemplated herein that the contact may be for any length of time. For example, the contact may be for 1 minute or more. This can be as short as 10 days, or as long as 7 days (e.g., IL-2, IL-12, IL- 15, culturing NK cells in the presence of IL-21 and / or IL-18 for 7 days) In one embodiment, disclosed herein is a method for treating one or more NK cells with IL-2, IL-12, IL-15, and / or IL-18 and 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 Preactivate or activate NK cells by exposing them to IgG for 1, 36, or 48 hours. Treating myeloid malignancies and myeloid-derived malignancies through adoptive transfer of NK cells, including and / or methods of trafficking NK cells to bone marrow. It is understood that the half-life of the cytokine may be shorter than the desired contact time, and Thus, disclosed herein are methods for treating one or more NK cells with IL-1. -2, IL-12, IL-15 and / or IL-18, and 1, 2, 3, every 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours (e.g., 24-hour contact every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours within the It is a way to touch.

[0020] 29. One or more NK cells that are contacted to activate and / or expand NK cells effector agents (i.e., stimulatory peptides, cytokines, and / or adhesion molecules) The use of plasma membrane (PM) particles, exosomes (EX) or feeder cells (FC) containing Through this, many hurdles related to cytokine toxicity are overcome. Examples of agents and stimulatory peptides include 41BBL, IL-2, IL-12, IL-21 , IL-18, MICA, LFA-1, 2B4, BCM / SLAMF2, CCR7, No tch ligands and / or other homing-inducing signaling molecules, Examples of cytokines include, but are not limited to, IL-2, IL-12, IL-21, Examples of adhesion molecules include, but are not limited to, LF, IL-18, and IL-19. These include, but are not limited to, A-1, MICA, and BCM / SLAMF2. For example, plasma membrane particles (PM), feeder cells (FC), or exosomes (EX) are membrane-bound IL-21-expressing feeder cells (FC21 cells, PM21 particles, and and EX21 exosomes). FC21 cells expressing membrane-bound IL-21, PM21 particles and EX21 exosomes contain, but are not limited to, 41BBL, IL-2, IL- 12, IL-15, IL-18, MICA, LFA-1, 2B4, BCM / SLAMF2, One or more additional activators, including CCR7, stimulatory peptides, cytokines, and / or adhesion molecules (e.g., 41BBL and membrane-bound interleukin-21 The antibody may further comprise a PM21 particle, an EX21 exosome, or an FC cell expressing the antibody. Thus, in one aspect, disclosed herein are methods for treating NK cells as NK cell effectors. and contacting the NK cell fusion protein with at least one vesicle containing a targeting agent, the vesicle containing the vesicle. The vector agent is selected from PM21 particles, EX21 particles, and / or FC feeder cells. and the treatment of myeloid malignancies through adoptive transfer of NK cells, which may be one or more of any combination of: and methods for treating bone marrow derived malignancies and / or trafficking NK cells into bone marrow For example, disclosed herein are methods for, among other steps, inducing NK cell proliferation. contacting the cells with at least one vesicle comprising an NK cell effector agent; NK cell effector agents containing vesicles include PM21 particles; EX21 exosomes; and FC21 Feeder cells; PM21 particles and EX21 exosomes; PM21 particles and FC2 1 feeder cells; EX21 exosomes and FC21 feeder cells; or PM2 1. Adoptive Transfer of NK Cells Containing Particles, EX21 Exosomes, and FC21 Feeder Cells Methods and / or methods for treating myeloid malignancies and myeloid-derived malignancies through transplantation or a method of trafficking NK cells to the bone marrow.

[0021] 30. In some embodiments, PM21 particles, EX21 exosomes, or FC fillers Effector agents for T cell markers include membrane-inserted peptides (e.g., Fc, GPI, transmembrane T cell The stimulatory peptides include one or more stimulatory peptides coupled to a receptor (or pHLIP). The membrane-inserting peptide may be a molecule that promotes insertion into a membrane. It may contain a segment of CD4 or IgG that has affinity for lipid bilayers. Additionally, alternative membrane-inserting peptides include human Fc, GPI, transmembrane T cell receptors, or pH LIPs may also contain membrane self-inserting peptides. A membrane-inserting peptide is any peptide known to insert into a cell membrane. Depending on the use of the membrane self-inserting peptide conjugate, the specific membrane self-inserting peptide may Some peptides may be better choices than others. Those skilled in the art will appreciate which membrane self-inserting peptides are different. It will be appreciated that pHLIP membranes may be ideal for in vivo use. Self-inserting peptides may be suitable. pHLIP membrane self-inserting peptides can be synthesized under low pH conditions. Therefore, pHLIP conjugates only insert into membranes under normal physiological conditions. They do not insert into the cell membrane. However, when injected into the tumor environment, the tumor environment is transformed into a state similar to normal physiological conditions. pHLIP conjugates are more acidic than ATP, allowing them to insert into the plasma membrane of tumor cells. This insertion into the tumor environment allows for the activation of NK cells in the tumor area. The use of pHLIP therefore prevents unwanted insertion into random cell membranes.

[0022] 31. Membrane-inserting peptides can be coupled to one or more stimulatory peptides in a variety of ways. The peptides may be coupled to the stimulatory agent, and techniques for coupling peptides are well known in the art. The membrane-inserting peptide coupled to the activating peptide is called a membrane-inserting peptide conjugate. In some embodiments, the membrane-inserting peptide is coupled to The stimulatory peptide(s) may be a fusion protein encoded by recombinant DNA. Such fusion proteins may be produced in bacterial cells. In this embodiment, the fusion protein is loosely coupled to a liposome or cell membrane for immobilization. Conjugate or couple to lipophilic molecules such as hydrophobic peptides, GPI, or human Fc These fusion proteins may consist of one or more stimulatory peptides linked together. cDNA vectors for expression in bacterial (E. coli), insect, or mammalian cells The vector may be ligated into an expression plasmid that allows expression of the vector. Alternatively, the cDNA vector may be FLAG- or HIS-tagged. , Sambrook et al., Molecular Cloning: AL aboratory Manual.2nd ed. Cold Spring Harb or standard C as described in Laboratory Press (1989) Bacterial cells may also be transfected using the aCl transfection method. Alternatively, the cells may be cultured in LB medium, and the cells are harvested and lysed using a French press. The protein of interest can be isolated from the lysate by affinity chromatography. Palmitic acid-conjugated protein A and purified Fc The fusion proteins were mixed at 4°C in a 1:2 (w / w) ratio to obtain the desired fusion protein as described in the literature. The conjugate can then be injected directly into the tumor. or may be incorporated into liposomes.

[0023] 32. Types of coupling and methods for coupling are known to those skilled in the art As used herein, the term "couple" refers to the process by which a membrane self-inserting peptide is conjugated. A molecule that can be attached, linked, or otherwise connected to another molecule, such as a peptide or protein. For example, a membrane-inserting peptide coupled to a stimulatory peptide is a membrane-inserting peptide. It is a derivative protein in which the nucleotide is coupled to another protein via a disulfide bond. Coupling or conjugation can be performed by coupling a membrane self-inserting peptide to an NK cell antigen. This may mean that there is a chemical link between the effector agent and the agonist.

[0024] 33. In some embodiments, the one or more stimulatory peptides are membrane self-inserting peptides. for in situ self-assembly For example, 41-BBL and IL- 21 was coupled to the pHLIP peptide, which inserts independently into the cell membrane under acidic conditions. This may allow for the immobilization of stimulatory ligands to cells in the vicinity of the tumor. Peptides: 41BBL, IL-2, IL-12, IL-21, BCM / SLAMF2, CC R7 and / or other homing receptors may be produced within the bacterial cell, or In some cases, cDNA vectors for these proteins may be purchased from commercial sources. pTriEX allows expression in bacterial (E. coli), insect, or mammalian cells The cDNA vector may be ligated into an expression plasmid. The gene encoding the HIS-tag may be used for the expression of the bacterial cells. The cells can be transfected using the transfection method and cultured in LB medium. Cells can be harvested and lysed using a French press, and proteins of interest can be isolated by It may then be purified from the lysate by affinity chromatography.

[0025] 34. In some embodiments, pHLIP is 9-fluorenylmethyloxycarbonyl. The product may be prepared by solid phase peptide synthesis using hydroxyl chemistry, The pHLIP may then be purified by phase chromatography on a C18 column. by incubation with a cross-linking agent such as benzophenone-4-iodoacetamide. After multiple washes, the cells were then incubated for 1 hour at 4°C ..., at which time the cells were conjugated to a stimulatory human protein ligand. The conjugated pHLIP protein is resuspended in medium (e.g., saline) and The compounds may be injected intratumorally or intravenously. Based on the evidence, NK cells and IL-21 and IL-4 on the surface of such modified tumor cells. Interaction with stimulatory ligands such as 1-BBL stimulates NK cell expansion in situ. This type of stimulatory approach may be used in the treatment of ovarian cancer. can be used to treat solid tumors such as erythrocytes, where acidic pH conditions are used to grow tumor cells in situ. NK stimulatory ligands that intercalate into the cells were administered with low doses of IL-2 alone or in combination with NK cells. , can be injected into the peritoneal cavity of patients. 16) are essential for the efficacy of therapeutic antibody-based cancer treatment. Therefore, this approach should be used in combination with therapeutic antibodies. You can also be there.

[0026] 35. NK cell effector agents, including NK cells and vesicles (i.e., PM21 particles, EX21 The duration of contact with exosomes and / or FC feeder cells determines the number of memory NK cells. It is understood that the time period may be any length of time required to achieve the desired amplification of For example, the contact can be for as little as one minute or as long as 60 days. It may be (e.g., PM21 particles, EX21 exosomes, and / or FC feeders In one embodiment, the NK cells and the vesicles are cultured in the presence of NK cells for 7 days. The contact with the NK cell effector agent containing the NK cell effector agent can be for about 6 days to about 60 days, more preferably The contact can be for about 6 days to about 40 days. Cells were incubated with PM21 particles, EX21 exosomes, and / or FC feeder cells for 5 min. , 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 2 0, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 , 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 6 0, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or 72 Myeloid malignancies and bone marrow-derived NK cells are identified through adoptive transfer, including contact with the NK cells for 1 day. Methods for treating malignant tumors and / or trafficking NK cells to the bone marrow In some examples, the NK cells are combined with PM21 particles, EX21 exosomes, and and / or multiple contacts with FC feeder cells may be desirable and may be used. It is understood and contemplated herein that the NK cells may be immunized with, for example, PM21 particles, EX21 exons, 1, 2, 3, 4, 5, 6, 7, 8, 9, with FC feeder cells 10, 11, 12, 18, 24 hours, 2, 3, 4, 5, 6, 7, 8, 9, 0, 10, 11 , 1 dose every 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days Thus, in one aspect, disclosed herein is a method for treating a NK Cells were incubated once with PM21 particles, EX21 exosomes, and / or FC feeder cells. More contacts are made, and the contacts are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 , 18, 24 hours, 2, 3, 4, 5, 6, 7, 8, 9, 0, 10, 11, 12, 13, 1 NK cell cultures performed every 4, 15, 16, 17, 18, 19, 20, or 21 days Methods for treating myeloid malignancies and myeloid-derived malignancies through gene transfer; and / or This is a method of trafficking NK cells to the bone marrow.

[0027] 36. In one embodiment, the plasma membrane particle, feeder cell, or exosome is a NK The plasma membrane particles or the like disclosed herein may be purified from feeder cells that stimulate the cells. or for use in making exosomes, NK cell-stimulating feeder cells were irradiated autologous or allogeneic peripheral blood mononuclear cells (P BMC), or non-irradiated autologous peripheral blood mononuclear cells (PBMC) containing autologous or allogeneic peripheral blood mononuclear cells (PBMC). or PBMC, RPMI8866, HFWT, K562, SKOV3, or EB V-LCL, or non-irradiated transfected with membrane-bound IL-21 and 41BBL Irradiated autologous or PBMC, RPMI8866, HFWT, K562, SKOV3, In some embodiments, the NK cell fraction can be either NK cells or EBV-LCL cells. The leader cells were K562 cells transfected with membrane-bound IL-21 and 41BBL. It may be a cell.

[0028] 37. The disclosed compositions can be used to treat cancers, particularly those affecting or localizing in the bone marrow. Any disease that causes uncontrolled cell proliferation, such as malignant tumors, can be treated. A non-limiting list of types of cancer is as follows: lymphoma (Hodgkin's and non-Hodgkin's); Leukemia, carcinoma, carcinoma of solid tissue, squamous cell carcinoma, adenocarcinoma, sarcoma, glioma, hyperplasia High grade glioma, blastoma, neuroblastoma, plasmacytoma, cytocytoma, melanoma, adenocarcinoma tumors, hypoxic tumors, myeloma, AIDS-related lymphoma or sarcoma, metastatic cancer, or Or common cancer.

[0029] 38. A representative, non-limiting list of cancers that can be treated using the disclosed compositions is: , including: lymphoma, B-cell lymphoma, T-cell lymphoma, mycosis fungoides, Hojiki Cancer of the bladder, brain, nervous system, head and neck, squamous cell carcinoma of the head and neck, kidney kidney cancer, lung cancer, such as small cell lung cancer and non-small cell lung cancer g 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 nary cancer), esophageal carcinoma, head and neck carcinoma, colorectal cancer, hematopoietic cancer; testicular cancer; and rectal, prostate, or pancreatic cancer.

[0030] 39. The disclosed compositions can also be used to treat bone marrow-associated viral diseases. As used herein, a bone marrow-associated viral disease refers to a disease in which the bone marrow harbors the virus (i.e. , viral infections (including chronic, acute, latent, and persistent infections), or otherwise affecting the bone marrow. tropism), or bone marrow-inducing viruses that induce aplastic anemia, e.g., parvovirus Refers to a viral disease caused by a virus such as flu (in some cases, A disease or condition that adversely affects the bone marrow is a viral infection of the bone marrow. In one embodiment, disclosed herein is a method for treating NK cells with PM21 particles and / or F C21 feeder cells in a subject, and methods for treating viral infections (which adversely affect bone marrow) and for treating NK cells in response to viral infection. In one embodiment, the virus is a virus that is used to adoptively transfer a virus to a subject with aplastic anemia. may induce hematopoiesis and / or establish latent or chronic infection in the bone marrow The infection may be a myelotropic infection or a viral infection that causes the infection. For example, the virus may be a dengue virus. Hepatitis viruses (including but not limited to Hepatitis A virus, Hepatitis B virus, Hepatitis C virus) Hepatitis B, Hepatitis D, Hepatitis E, and Hepatitis G viruses), Epstein-Barr virus, Virus (also known as human herpesvirus 4), cytomegalovirus (also known as human herpesvirus 5), parvovirus (including but not limited to parvovirus B19, etc.), lymphocytic choriomeningitis virus (LCMV), human immunodeficiency virus HIV, and respiratory syncytial virus (RSV), among others. Not limited.

[0031] C. Composition 40. Disclosed are ingredients used to prepare the disclosed compositions, and These and other materials are the compositions themselves used in the methods disclosed herein. The materials disclosed herein include combinations, subsets, interactions, and groups of these materials. and the like, and various individual and collective combinations and permutations of these compounds are disclosed. Although specific references may not be explicitly disclosed, each is specifically contemplated and described herein. It will be understood that the feeder may be a special PM21 particle or FC21 feeder. Where cells are disclosed and discussed, multiple modifications may be made to multiple molecules, and are specifically contemplated. All combinations and variations of possible modifications are included unless specifically indicated to the contrary. Thus, a class of molecules A, B, and C is disclosed, and molecules D, E, and F classes, and examples of combined molecules A, B, C, and D are disclosed, each of which is Although not individually listed, each is contemplated individually and collectively, and in combination AE, AF, BD, BE, BF, CD, CE and CF are disclosing Likewise, any subset or combination of these also means Thus, for example, the subgroups AE, BF, and CE are expected to be disclosed. This concept applies, but is not limited to, to methods of making and using the disclosed compositions. This applies to all aspects of the present application, including steps in the If there are additional steps, each of these additional steps may be incorporated into the disclosed method. It will be understood that the present invention may be practiced in any specific embodiment or combination of embodiments.

[0032] 41. Disclosed myeloid malignancies and bone marrow-derived malignancies through adoptive transfer of NK cells The method for treating tumors and / or trafficking NK cells to bone marrow comprises: A species or species of cytokines (e.g., IL-12, IL-15 and / or IL-1 8) by, for example, adding PM21 particles, FC feeder cells, and / or EX21 exosomes. The disclosed methods are used in combination with vesicles containing NK cell effector agents such as leukocyte antigens. Providing the ingredients to be used in a package that allows a person to readily practice the disclosed method It is understood and contemplated herein that this would be advantageous.

[0033] Thus, in one aspect, disclosed herein is a method for treating a pulmonary arthritis with one or more cytokines. IL-2, IL-12, IL-15, and / or IL-18) and NK cells and one or more vesicles containing a vesicle effector agent. In one embodiment, the vesicles are used in the treatment of PM tumors with NK cells. EX21 particles, EX21 exosomes, and / or FC feeder cells. For example, the disclosed kits include IL-12 and PM21 particles; IL-15 and PM21 particles; IL-18 and PM21 particles; IL-12 and EX21 exosomes, IL-1 5 and EX21 exosomes; IL-18 and EX21 exosomes; IL-12 and and FC21 feeder cells; IL-15 and FC21 feeder cells; IL-18 and and FC21 feeder cells; IL-12, IL15, and PM21 particles; IL-12 , IL-18, and PM21 particles; IL-15, IL-18, and PM21 particles; I IL-12, IL-15, IL-18, and PM21 particles; IL-12, IL15, and and EX21 exosomes; IL-12, IL-18, and EX21 exosomes; IL -15, IL-18, and EX21 exosomes; IL-12, IL-15, IL-1 8, and EX21 exosomes; IL-12, IL15, and FC21 feeder cells cells; IL-12, IL-18, and FC21 feeder cells; IL-15, IL-18 , and FC21 feeder cells; IL-12, IL-15, IL-18, and FC2 1 feeder cells; IL-12, EX21 exosomes, and PM21 particles; IL-1 5, EX21 exosomes, and PM21 particles; IL-18, EX21 exosomes, and PM21 particles;IL-12, FC21 feeder cells, 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 Feeders cells, PM21 particles, and EX21 exosomes; IL-18, FC21 filters leader cells, PM21 particles, and EX21 exosomes; IL-12, IL15, EX 21 exosomes, and PM21 particles; IL-12, IL-18, EX21 exosomes IL-15, IL-18, EX21 exosomes, and PM 21 particles; IL-12, IL-15, IL-18, EX21 exosomes, and PM2 1 particle; IL-12, IL15, 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, IL15, EX21 exosomes, and FC21 feeder cells; IL-12, IL-18, EX21 exosomes endothelial cells, and FC21 feeder cells; IL-15, IL-18, EX21 exosomes IL-12, IL-15, IL-18, EX21 exosomes, and FC21 feeder cells; IL-12, EX21 exosomes, F C21 feeder cells, and PM21 particles; IL-15, EX21 exosomes, FC 21 feeder cells, and PM21 particles; IL-18, EX21 exosomes, FC2 1 feeder cells, and PM21 particles; IL-12, IL15, EX21 exosomes , FC21 feeder cells, and PM21 particles; IL-12, IL-18, EX21 axosomes, FC21 feeder cells, and PM21 particles; IL-15, IL-18, EX21 exosomes, FC21 feeder cells, and PM21 particles; or IL-1 2, IL-15, IL-18, EX21 exosomes, FC21 feeder cells, and May contain PM21 particles.

[0034] 42. NK cell effector agents contained in vesicles (e.g., PM21 particles, EX21 particles) lysosomes, and / or FC feeder cells) were treated with 4-1BBL, IL-2, IL-2 1, MICA / B, ULBP2, ICAM-1, 2B4, BCM1 / SLAMF2, CD 155, CD112, CCR7, DAP12, Notch ligand, and DAP10 It is understood and contemplated herein that the NK cell effector agent may be selected from the group consisting of: can be.

[0035] 43. The disclosed kit or device is capable of detecting IL-12, IL-15, and / or IL-16. It is understood and contemplated herein that the present invention may include cytokines in addition to IL-18. Thus, in one embodiment, 4-1BBL, IL-2, IL-12, IL-18, IL-21 , MICA / B, ULBP2, ICAM-1, 2B4, BCM1 / SLAMF2, CD1 55, and further including CD112, CCR7, DAP12, and DAP10 of NK cells. Methods of treating myeloid malignancies and myeloid-derived malignancies through adoptive transfer and / or or kits for methods of trafficking NK cells to bone marrow.

[0036] 44. In one embodiment, the disclosed kit or device comprises a non-selective collection of peripheral blood mononuclear cells. The present invention may be used in conjunction with NK cells obtained from a donor supply, such as NK cells obtained from a donor group. In some instances, myeloid malignancies and myeloid-derived malignancies are contemplated herein. Donor Supply for NK Cells Used in the Disclosed Kits for Treating Tumors The source can also be the recipient of NK cells. Thus, the NK cells are of an autologous source. In other examples, the donor source of the NK cells can be haploidentical. Or it may be an allogeneic donor source.

[0037] 45. This paper has identified some instances where it may be beneficial to provide NK cells in a kit or device. Further contemplated herein. Thus, in one aspect, disclosed herein is a method for producing a compound comprising: A kit for treating myeloid malignancies further comprising a K cell or NK cell line.

[0038] 1. Pharmaceutical Carriers / Pharmaceutical Product Delivery 46. ​​As previously described, the composition may also be administered in vivo in a pharmaceutically acceptable carrier. "Pharmaceutically acceptable" means that the compound is not biologically or otherwise undesirable. By "materials" is meant materials that do not induce any undesirable biological effects. or any of the other components of the pharmaceutical composition in which it is contained. The carrier may be administered to a subject together with the nucleic acid or vector. Naturally, the active ingredient(s) should be administered in a manner that minimizes any degradation of the active ingredient(s) and any adverse side effects in the subject. will be chosen to minimize

[0039] 47. The composition is administered topically, intranasally, or by inhalation, Orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, It may be administered dermally, ex vivo, topically, etc. As used herein, "topical intranasal administration" refers to administration of "Administer" refers to delivery of the composition to the nose and nasal passages through one or both nostrils, and includes spraying. delivery by a liquid or droplet mechanism or through aerosolization of the nucleic acid or vector Administration of the composition by inhalation may include nasal or intranasal administration via delivery by a spray or droplet mechanism. Delivery can be via the mouth or into any part of the respiratory system (e.g., lungs) via intubation. The exact amount of composition required will depend on the species, age, weight and the general condition, the severity of the allergic disorder being treated, the particular nucleic acid or vector used The exact dosage of each composition will vary from subject to subject, depending on the dosage, mode of administration, etc. It is not possible to specify an amount, but an appropriate amount can be determined by one skilled in the art given the teachings herein. can be determined using only routine experimentation.

[0040] 48. Parenteral administration of the composition, if used, is generally characterized by injection. The liquids may be in liquid solutions or suspensions, solid forms suitable for solution in liquid prior to injection, or in emulsion form. These may be prepared in conventional forms, either as an emulsion or as a more recent formulation for parenteral administration. A modified approach is to use delayed-release or sustained-release drugs so that a constant dose is maintained. See, for example, U.S. Patent No. 3,610, incorporated herein by reference. Please refer to No. 795.

[0041] 49. The material may be in solution, suspension (e.g., microparticles, liposomes, or cells). These are targeted to specific cells via antibodies, receptors, or receptor ligands. The following references are related to targeting specific proteins to tumor tissue: An example of the use of this technology is shown in Senter, et al., Bioconjugation te Chem., 2:447-451, (1991);Bagshawe, KD, Br. J. Cancer, 60:275-281, (1989);Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988);Se Bioconjugate Chem., 4:3-9, (199 3);Battelli, et al., Cancer Immunol. ther., 35:421-425, (1992);Pietersz and McKe nzie, Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al., Biochem. Pharmacol, 42: 2062-2065 (1991). Vehicles such as "stealth" and other antibody controls Conjugated liposomes (e.g., lipid-mediated drug targeting for colon carcinoma), cell-specific ligands, Receptor-mediated targeting of DNA through endothelial cells, lymphocyte-directed tumor targeting, and in vivo Highly specific therapeutic retroviral targeting of murine glioma cells in vivo. The following references are available: An example of the use of this technology to target specific proteins to tumor tissue is shown in Hughes et al. et al., Cancer Research,49:6214-6220,(1989) and Litzinger and Huang, Biochimica et Bi ophysica Acta, 1104:179-187,(1992)). Generally accepted The body engages in pathways of endocytosis, either constitutive or ligand-induced. These receptors cluster in clathrin-coated pits, and clathrin binds to them. They enter cells via coated vesicles, pass through acidified endosomes, and then The receptors are then selected and recycled to the cell surface, where they are stored intracellularly. The internalization pathway involves the uptake of nutrients, activation of proteins, and degradation in the lysosomes. removal of proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, ligand They perform a variety of functions, including dissociating and degrading receptors, and regulating receptor levels. The receptors vary depending on the cell type, receptor concentration, ligand type, ligand valency, and ligand concentration. Depending on the degree, more than one intracellular pathway is followed. and cellular mechanisms are being reconsidered (Brown and Greene, DNA and d Cell Biology 10:6, 399-409 (1991)).

[0042] a) Pharmaceutically Acceptable Carriers 50. The composition containing the antibody can be used therapeutically in combination with a pharmaceutically acceptable carrier. Cut.

[0043] 51. Suitable carriers and their formulations are described in Remington: The Science ce and Practice of Pharmacy (19th ed.) ed. A .R. Gennaro, Mack Publishing Company, East on, PA 1995. Typically, an appropriate amount of a pharmaceutically acceptable salt is used. are used in the formulation to make the formulation isotonic. Examples of pharmaceutically acceptable carriers include Examples include, but are not limited to, 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 solid hydrophobic carriers containing antibodies in which the matrix is ​​in the form of a shaped body. Sustained-release preparations such as semipermeable matrices of hydrophilic polymers, e.g., films, liposomes. or microparticles. Depending, for example, on the route of administration and the concentration of the administered composition, It will be apparent to those skilled in the art that certain carriers may be more preferable.

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

[0045] 53. Pharmaceutical compositions may contain, in addition to the selected molecule, carriers, thickeners, diluents, buffers, preservatives, The pharmaceutical composition may contain an antibacterial agent, an anti-inflammatory agent, an anesthetic agent, a surfactant, etc. It may also contain one or more active ingredients.

[0046] 54. The pharmaceutical composition may be administered in a variety of ways, depending on whether local or systemic treatment is desired and the area to be treated. Administration may be by several methods depending on the type of drug. Administration may be topical (ophthalmic, vaginal, rectal, intranasal) ), orally, by inhalation, or parenterally, e.g., by intravenous drip, subcutaneously, intraperitoneally, or intramuscularly. The disclosed antibodies can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, or intramuscularly. , intravenously, or transdermally.

[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, Cole, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate Aqueous carriers include water, alcoholic, saline, and buffered media. Parenteral vehicles include aqueous solutions, emulsions, or suspensions. sodium solution, Ringer's solution with dextrose, dextrose and sodium chloride, lactated Intravenous vehicles include fluids and nutrients, such as cereals containing glutamic acid, cereals containing glutamic acid, and cereals containing glutamic acid. Fluid replacement, electrolyte replacement (such as those based on glucose-added Ringer's solution), etc. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents, and inert gases. An agent may also be present.

[0048] 56. Formulations for topical administration include ointments, lotions, creams, gels, drops, and suppositories. Conventional pharmaceutical carriers, aqueous, powder or In some cases, an oily base, a thickener, or the like may be necessary or desirable.

[0049] 57. Compositions for oral administration include powders or granules, suspensions in water or non-aqueous media. Suspensions or solutions, capsules, sachets, or tablets. A solvent, emulsifier, dispersing aid or binder may be desirable.

[0050] 58. Some of the compositions potentially contain hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiochloric acid, Inorganic acids such as ammonium, sulfuric acid, and phosphoric acid, as well as formic acid, acetic acid, propionic acid, glycosaminoglycans, and Acids include lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid. By reaction with organic acids such as sodium hydroxide, ammonium hydroxide, Inorganic bases such as potassium, and mono-, di-, trialkyl and aryl amines, and substituted ethanolamines, They may be administered as an acceptable acid or base addition salt.

[0051] b) Therapeutic use 59. Effective dosages and regimens for administering the composition are determined empirically. Such determinations are within the skill of the art. The dosage ranges for this purpose are those large enough to produce the desired effect which affects the symptoms of the disorder. The dosage is selected so as not to induce adverse side effects such as unwanted cross-reactions and anaphylactic reactions. Generally, the dosage will vary depending on the age, condition, sex and disease of the patient. These will vary depending on the severity of the disease, the route of administration, or whether other drugs are included in the regimen, and will be understood by those skilled in the art. The dosage may be determined by any counterindications. The dosage can be varied and can be adjusted by the individual physician in the event of may be administered once or multiple times daily for several days. Guidance regarding appropriate dosages can be found in the literature. For example, suitable doses of antibodies can be Guidance for selection can be found in the literature on therapeutic uses of antibodies, e.g., Handbook k of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, NJ, (1985)ch. 22 and pp. 303-357;Smith et al., A antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York (19 77) pp. 365-389. Dosage ranges from about 1 μg / kg to 100 mg / kg of body weight per day, depending on the factors mentioned above. It may be in the range of up to or greater than that.

[0052] D. Working Example 60. The following examples illustrate the compounds, compositions, articles, devices, and / or compositions claimed herein. or a complete disclosure and description of how the methods were made and evaluated. They are set forth for the purpose of providing, are purely exemplary, and are not intended to limit the present disclosure. While every effort has been made to ensure accuracy (e.g., amounts, temperature, etc.), slight errors and bias may occur. Differences must be taken into account. Unless otherwise indicated, parts are parts by weight and temperatures are ° C. or is at ambient temperature and the pressure is at or near atmospheric.

[0053] 1. Example 1: PM21 particles stimulate NK cell expansion in vivo 61. Natural killer (NK) cells express CD56 + CD3 - Identified by It is a component of the innate immune system that naturally recognizes virally contaminated or malignant cells. Cell therapy with NK cells is promising for cancer treatment and has been shown to Clinical trials have been completed and the drug is effective in treating various cancers (AML, lymphoma, breast cancer, ovarian cancer, neuroblastoma, non-small cell lung cancer, etc.). Currently in progress for the treatment of 3-cell lung cancer. Three general aspects must be considered: 1) A sufficiently large dose of NK cells is delivered; 2) NK cells must be highly cytotoxic; and and 3) NK cells reach the disease site, possibly localize and persist, and destroy tumor cells. Must be specifically targeted.

[0054] 62 For clinical efficacy in the AML setting, Miller and colleagues To provide at least 100 NK cells per μL of peripheral blood (PB) at 2 weeks post-injection In some cases where treatment with adoptive NK cell therapy is effective, PB More than 1,000 NK cells were observed per μL. These observations suggest that the overall treatment efficacy This highlights the importance of proficient NK cell expansion methods for the delivery of adequate doses for therapeutic use.

[0055] 63. Currently, there are three different clinical uses for NK cell expansion for adoptive cell therapy: First, IL-1 in combination with host lymphodepletion / irradiation is used. In vivo amplification with cytokines such as IL-5 and IL-2 was observed in relatively small amounts of injected Second, cytokines, mainly NK cells, can stimulate the in vivo expansion of primary NK cells. Ex vivo methods using IL-2 and IL-15 can activate NK cells However, amplification is relatively low and variable. After infusion, activated NK cells undergo cytokine withdrawal, and N K cells undergo apoptosis. Third, feeder cells for ex vivo NK cell expansion The cell method utilizes co-culture with other stimulatory cells. The cell method involves Epstein-Barr virus-transduced LCLs or engineered tumor cells. K562 expressing IL-15 (mb15) and 4-1BB ligand (41BBL) Co-culture with CML cells (K562-mb15-41BBL) resulted in NK cells within approximately 2 weeks. However, NK cells expanded by this method do not age well. In addition, IL-15-activated NK cells express the ADAM17 protein. Loss of surface CD16 due to degradation activity. Rather, expression of mb21 instead of mb15 K562 cells, which are derived from NK cells, are able to promote NK cell proliferation while avoiding telomere shortening and the resulting NK cell senescence. NK cell expansion with K562-mb21-41BBL was highly efficient. It is highly efficient, with an average of 48,000-fold amplification and >85% enrichment, typically within 3 weeks. All of these approaches are being actively investigated in clinical trials.

[0056] 64. NK cell expansion methods have improved, but still have drawbacks and challenges. IL-2 High toxic doses of NK cells are required for survival of infused NK cells regardless of the expansion method, The persistence of NK cells was limited. The ex vivo method with feeder cells produced a large number of NK cells. However, long-term ex vivo culture of NK cells was effective in expanding the NK cell population to generate new NK cells. This leads to the problem of losing the ability to home to the affected area. The overall benefits of in vivo versus ex vivo expansion are debated. is a method that has the expansion capabilities of ex vivo feeder cell-based methods, but The method can be performed either in vitro or in vivo.

[0057] 65. Rapid and selective expansion of cytotoxic NK cells starting from PBMCs A novel PM21 particle-based method for width. Particles corresponding to closed plasma membrane vesicles were analyzed using K Prepared from the plasma membrane of 562-mb15-41BBL cells (PM15 particles) within 14 days 250-fold and 1,265-fold selective NK cell expansion after 17 days, which The amplification efficiency was comparable to that using cultured K562-mb15-41BBL feeder cells. PM15 particle-activated NK cells, similar to those expanded with feeder cells, were exocytically activated. It was highly cytotoxic against CML and AML cells in vivo. These particles offer many advantages over feeder cell methods. First, they It is prepared, tested, and can be stored for more than one year and is confined to a single GMP facility. It can be used as an "off-the-shelf reagent" without any need for clinical logistics of adoptive NK cell therapy. Second, PM particles are used instead of feeder cells to stimulate NK cells. When using tumor-derived feeder cells, irradiation of the feeder cells is necessary. and the steps required for safety measurements when testing for presence and growth in the final product. Third, tumor-derived feeder cells can be injected as adjuvant therapy. Although PM particles cannot be injected to stimulate the in vivo expansion of NK cells, The advantages offered by PM particle-based methods for NK cell expansion are significant. Enables clinical benefit.

[0058] 66. At this point in the study, PM particles prepared from K562-mb21-41BBL The efficacy of this product will be tested for in vivo expansion of adoptively transferred NK cells to demonstrate its feasibility in a clinical setting. The preactivation was carried out in a relatively short and simple procedure that can be easily carried out. Intravenous infusion of adoptive NK cells allows only in vivo NK cell expansion and limited persistence This overcomes the shortcomings of previous tests. The mice were injected into the peritoneal cavity, which was to serve as the primary site for stimulation with ethanol and PM21 particles. PM21 particle-stimulated ex vivo and in vivo expansion of NK cells from unselected PBMCs This method is expected to be useful for the in vivo expansion of therapeutically relevant amounts of NK cells. This represents a significant advancement in making NK cell-mediated immunotherapy more widely available to patients.

[0059] a) Materials and methods (1) Human samples 67. Primary leukemic blast cells were used in an IRB-approved, informed consent-signed activity. Leukocyte supply was obtained from patients during disease and equivalent PB was collected from these patients during remission. Source (One Blood, Orlando, FL) or IRB-approved information Fresh blood collected from healthy volunteers who signed informed consent was used as the healthy sample. PBMCs were cultured on Ficoll-Paque (GE Healthcare, Pennsylvania). All samples were re-identified and confirmed to be viable. It was stored frozen at .

[0060] (2) Reagents and cell lines 68. The K562 cell line was obtained from ATCC (Manassas, VA). Cytotoxicity Annexin-V FITC Kit and Enumeration F for Assay Low-Count beads are manufactured by Beckman Coulter, Miami, Florida. The following dye-conjugated antibodies were used for phenotyping: CD16- FITC, NKG2A-PE, NKp46-PE, CD3-APC (Beckman Co ulter);CD4-APC-Cy7, CD8-PE, CD56-BV421, CD9 4-APC (BD Biosciences); CD3-Alexa488, NKG2D -APC, CD62L-PE-Cy7, CD45-eFluor450, CD45-AP C(eBiosciences);CD56-PE, KIR2D-APC(Milteny i);NKG2C-PE NKp44-APC, TRAIL-PE(R&D System ms).

[0061] (3) Preparation and characterization of plasma membrane particles 69. PM particles were prepared from K562-mb21-41BBL cells. The cells were incubated at 5% Cells were grown in RPMI-1640 medium supplemented with fetal bovine serum. The cells were collected by centrifugation at 1000×g for 10 minutes and washed with DPBS containing 2 mM EDTA. The cells were cultured in 50 mM HEPES (pH 7.4), 150 mM NaCl, 2 mM Mg Contains Cl2 and AEBSF, aprotinin, leupeptin and pepstatin A The cells were resuspended in lysis buffer. The cells were lysed in a nitrogen cabinet at 300 psi and 4°C for 30 minutes. Destroyed by stationation (Parr Instruments, Moline, IL) The cell lysate was centrifuged (1,000 × g, 10 min), and the supernatant was then centrifuged. The crude membranes were pelleted by centrifugation at 100,000×g. The fraction corresponding to the closed plasma membrane vesicles was collected by further purification using the following procedure. Aseptic technique was utilized and the sterility of the product was tested by culture. Protein concentration was determined by BCA assay and expressed as μg / mL membrane protein. The presence of IL-21 and 41BBL on PM particles was assessed by ELISA and Western blot. This was confirmed by blot.

[0062] (4) Ex vivo NK cell expansion from PBMCs 70. NK cells from PBMCs were expanded using PM21 particles. , PBMCs, 10% FBS, 2mM Glutamax, 100 U / mL IL-2 ( Peprotech, Rocky Hill, NJ) and 200 μg / mL SCGM supplemented with PM21 particles (CellGenix, Portland, NH) 0.1 x 10 6 NK cells / mL. Supplemented medium was routinely After the fifth day, the solution was replaced every 2–3 days.

[0063] (5) Cytotoxicity assay of patient's autologous NK cells 71. Cytotoxicity assays of patient-derived NK cells against autologous AML tumor cells were performed using Ann The NK cells expanded for 16 days were assayed with exin V (BD Bioscience). The target tumor cells (NK cell content >90%) were stained with TFL4 dye. 0.5 × 10 with NK cells at 1:1, 2:1, 5:1, and 10:1 6 CD34 + Thin The cells were co-cultured at 1000 cells / mL for 2 hours at 37°C and 5% CO2. and Annexin V-FITC, anti-CD34-PE, and anti-CD56-PC7. The cells were resuspended in Annexin V labeling buffer containing 1000 kJ / ml and incubated at 4°C for 15 minutes. The labeled cells were diluted in 250 μL and loaded onto an Accuri instrument (BD Biosciences). e) and analyzed by flow cytometry.

[0064] (6) In vivo expansion of NK cells in NSG mice 72. Freshly thawed PBMCs or 200 μg / ml PM21 and 100 PBMCs preactivated with 10 U / mL IL-2 for 2 days were washed twice and then phenol-less 1 x 10 cells were resuspended in RPMI medium without phosphate. 5 NK cells, NSG (NOD-scid IL-2Rgamma null ) Mice were injected intraperitoneally. PM21 particles (amounts specified in the figure legend, twice a week) and IL-2 (1,000 U, 3 times a week) 1 time) were also injected intraperitoneally, and PB was collected by cheek bleeding or cardiac puncture. They were collected at necropsy and perfused to obtain single cell suspensions for analysis.

[0065] b) Result (1) Ex vivo and in vivo expansion of NK cells obtained from healthy donors and leukemia patients width 73. K562 cells engineered to express mbIL21 promote NK cell proliferation without senescence. PM particles were treated with K562-mb21-4 because it was reported to have good efficiency depending on the width. PM21 particles were prepared from 1BBL cells and designated as PM21 particles. PM21 particles were characterized by size distribution and m We characterized the consistency of bIL21 levels (fig. S1) and tested their ability to expand NK cells. Ta.

[0066] 74. PBMCs were cultured with PM21 particles (200 μg / ml) for 28 days. PM NK cells stimulated by PM21 particles expanded, and the number of NK cells increased. In culture c, the percentage reached over 90% by day 14 (Fig. 1A-B). Cumulative analysis of NK cell amplification on day 1 showed that PM21 particles (average 825-fold amplification, range 163- 2,216, n = 13) were PM15 particles (average 424 times, range 290-570, n = 30 ) was shown to be significantly (p=0.021) more effective than (Fig. 1C). Furthermore, NK cells stimulated with PM21 particles underwent exponential proliferation and senescence over a 28-day period. In contrast to the NK cell expansion with PM15 particles, which was stopped by day 22 of culture, The amplification reached over 1000 times. PM21 particles were therefore improved over PM15 particles. PM21 particle-induced NK cell expansion was observed in mice with a high level of NK cell proliferation. This was comparable to that reported for K562-mb21-41BBL feeder cells. PM21-amplified NK cells were also cytotoxic against leukemia cell lines (fig. S2).

[0067] 75. The NK cell-expanding ability of PM21 particles was examined in PBMCs from leukemia patients in remission. PM21 particles were cultured for 14 days in NK cells from all three patient samples. The cells were relatively efficiently induced to amplify (113±7-fold in F021 and 810±7-fold in M038). 81-fold in M050 and 352 ± 86-fold in M050 (Fig. 1D). The amplification was specific to NK cells. Total hCD45 + The percentage of NK cells relative to the number of cells was preferentially increased ( Figure 1 E) For sample F021, the cytotoxicity of expanded NK cells was assessed in the same patient during active disease. The results were compared with those of the control group, which showed relatively low efficacy against tumor blasts obtained from the control group (Fig. 1F). At an E:T ratio of 1:1, 78 ± 3% of tumor cells were apoptotic. This method can therefore be used in the autologous transplant setting.

[0068] 76. The unprecedented ability of PM particles as an injectable solution to promote in vivo amplification. PM2 We tested whether 1 particles stimulate in vivo NK cell expansion and whether ex vivo preactivation was required. To determine whether PM21 particles are essential for the treatment of NSG mice, we injected naive PBMCs or PM21 particles into NSG mice. 0.1 × 10 as part of pre-activated PBMCs (PM21-PBMCs) 6 NK cells in the peritoneal cavity Mice injected with non-activated PBMCs had low levels of human NK cells (hNK cells) in the PB. ) cells, and only hT cells were found to be present in total hCD45 cells for 15 days post-injection. + Percentage of cells In sharp contrast, the expression of PM21-PBMCs increased with increasing IL-1 expression (Figure 2A). The PB of the injected mice showed an increase in the amount of hNK cells, which peaked on day 12 after intraperitoneal injection. The amount of NK cells was found to be significantly increased by hCD45 + Cells were concentrated to 53±8% In the same experiment, the efficacy was tested for in vivo intraperitoneal application of PM21 particles. Mice injected with normal PBMCs were compared to control mice to promote better in vivo NK cell expansion. In the case of rhesus, additional in vivo PM21 particles did not stimulate hNK cell expansion. In vivo application of PM21 particles to mice engrafted with PM21-PBMCs had no effect. PM21-PBMC-transplanted mice did not receive PM21 particles in vivo. was higher compared to (Fig. 2D).

[0069] 77. To provide evidence that PM21 particles induce NK cell proliferation in vivo Analysis using in vivo expanded CTViolet-labeled hNK cells on day 6 after intraperitoneal inoculation Cells from mice injected with non-activated PBMCs were used to detect CTViolet fluorescent protein. It was shown that there was no or very little reduction in light, and there was no or very little reduction in NK cell division. The PM21-PBMC-injected mice showed almost no cytotoxicity (Figure 3A). hNK cells showed a significant decrease in CTViolet fluorescence intensity (Fig. 3C-D). From the fitting of the light intensity, it was found that the decrease in intensity corresponds to seven cell divisions in vivo within six days. It was shown to correlate with the major population. One extra division was observed in hNK cells obtained from mice that received 21 particles intraperitoneally. This further doubling with administration of in vivo PM21 particles is This correlates with the higher NK cell abundance observed in PB with particles.

[0070] 78. To further verify whether in vivo PM21 particles enhance in vivo NK cell expansion, To investigate the effect of PM21 particles on hNK cells in vivo, we examined their dose-dependence (Figure 4). A dose-dependent increase in was observed between 0 and 800 μg of PM21 particles per injection (Figure 4E At the 800 μg dose (corresponding to approximately 100 ng of mbIL21), 4 μg of PB 70±40 hNK cells were observed 12 days after intraperitoneal injection of PM21-PBMC. The NK cell concentration in the PB of patients with AML is generally considered to be therapeutically effective in the setting of AML. The dose-dependent effect on in vivo expansion was significant with increasing T cell abundance. The effect was specific to hNK cells that had not been injected (Figure 4E). At higher doses, the amount of PB hNK cells was reduced, similar to the effect observed ex vivo. However, approximately 200 to 400 μg / mL is optimal for PM21 particles or PM15 particles, and Higher doses attenuated NK cell expansion.

[0071] 79. The observation of significant amounts of hNK cells in PB suggests that intraperitoneally injected PM21-PBM These results demonstrate that hNK cells expanded in C can migrate from the peritoneal cavity to the PB. To verify that the isolated hNK cells could migrate to potential disease sites, we investigated the ability of hNK cells in various organs. NK cells were quantified (Figure 5). Human NK cells were found in each organ examined, with higher abundance hNK cells were found in the organs of mice treated in vivo with 800 μg of PM21 particles. , and all except the liver were significant (p<0.05). Organs from treated mice were enriched for total hCD45 + A higher percentage of cells The patients had a high number of hNK cells.

[0072] 80. From the mouse-based studies described herein, ex vivo A procedure combining a short preactivation at RT and in vivo administration of PM21 particles could potentially It has been shown to induce significant in vivo NK cell expansion within the therapeutically relevant range. To demonstrate the consistency required for laboratory use, this procedure was performed on leukocytes from three different donors. The donor was different from that used in the other experiments (Fig. 6). The mean amount of hNK cells in both purged fluids was relatively consistent between leukocyte sources. hNK, hT cells and other hCD45 + The percentage of cells also varies depending on the specific white blood cell donor. In mice in the group injected with PM21-PBMC from the donor (n=3), and leukocyte supply There was also excellent agreement between sources L8 and L10.

[0073] (2) Phenotype of NK cells amplified by P21 particles 81. Antitumor cytolytic activity of NK cells is stimulated by activating and inhibitory signals. Here, detailed comparative tests were carried out: 1) PM21 for 12 days amplified ex vivo, 2) amplified in vivo and isolated from PB, and 3) in vivo Performed on PM21 particle-stimulated NK cells expanded and isolated from peritoneal lavage fluid (AW) These comparisons were performed using cells from a single donor in all of these settings. , carried out in equilibrium (Fig. S3).

[0074] 82. The presence of the Fcγ receptor CD16 on NK cells is essential for effective antibody-dependent cell proliferation. Nearly all NK cells from in vivo expansion express CD16 The expression of CD94 is 97% and 87% in PB and AW, respectively. It is a surface receptor that forms a heterodimeric complex with C or NKG2A. Approximately half of the expanded NK cells have CD94 expression. In vivo expanded NK cells In this case, cells from AW (64±9%) were more effective than NK cells from PB (38±13%). Highly expressed. NKG2C as an activating receptor and NKG2 as an inhibitory receptor. Both NKG2 family receptors, including A and B, bind to CD94. The expanded NK cells had relatively low expression of NKG2C, whereas NK cells from AW was higher in NK cells from PB (53±8%) than in NK cells from PB (61±2%). The percentage of NK cells expressing KG2A was 67 ± 12% in PB and ex vivo expanded. The NKG2D gene was found on NK cells, but its expression was higher in AW (82±8%) than in WT (74%). Its expression was found in 61±6% of AW NK cells and 61±6% of P B cells were found in 26±3% of the NK cells and approximately 75% of the ex vivo expanded NK cells. The expression of CD62L, which is known to correlate with NK cell proliferation, was higher in PB NK cells ( 63±10%), lower in AW (39±14%), and higher on recruited cells. NKp44 and NKp46 are members of the natural cytotoxicity receptor family. NKp46 is a member of the PB( NKp44 was expressed in NK cells from both AW (76±9%) and AW (89±5%). was relatively poorly expressed on these NK cells from all sources. On the other hand, NKp46 was abundantly expressed in both PB (89±5) and AW (76±9). TRAIL induces target apoptosis in NK cells via the death receptor pathway. TRAIL was expressed in 36±6% of NK cells from AW and 20±4% of NK cells from PB. KIR2D was expressed in 26% of ex vivo expanded cells and 2% of the ex vivo expanded cells. KIR2D subtype, expressed in vivo or ex vivo NK cells are a minor component (approximately 1 / 3) of IR2D. The proportions were analyzed to determine whether CD8 T cells were more abundant than CD4 T cells from the in vivo samples. The presence of NK suppressor Treg cells was also investigated and found to be very rare (total CD3 + less than 0.1% of cells) were observed in in vivo samples.

[0075] c) Discussion (1) PM21 particles induce therapeutically relevant ex vivo and in vivo NK cell expansion. Promote 83. Adoptive NK cell therapy is a cancer treatment for the initial treatment and maintenance of remission of various tumors. The therapeutic use of NK cells has high expectations. The requirements are safety, simplicity, and overall therapeutic effectiveness. This is a promising method for rapid and selective NK cell expansion. A method based on leader cells is currently under clinical investigation, using K562-mb21-41BBL cells. The cell line methodology is the most effective for ex vivo NK cell expansion. Cellular methods are effective in providing a high initial dose and allow for repeated administration, but they are not suitable for leukemia. The ability of ex vivo expanded NK cells to home to the bone marrow is important for treatment. and the in vivo persistence of infused NK cells may be suboptimal. The ex vivo and in vivo PM21 particle-based NK cell expansion described herein The combination of methods may significantly enhance the efficacy of NK cell adoptive therapy.

[0076] 84. Importantly, PM21 particles are capable of in vivo amplification and persistence. The methodology developed herein can be used for short, two-day ex vivo stimulation. Preactivation and subsequent in vivo administration of PM21 particles were used. In vivo application of PM21 particles resulted in higher in vivo NK cell counts in a dose-dependent manner. Using this optimized procedure, an average of 360-fold expansion of PB NK cells was achieved in vivo. An increase was observed between days 5 and 12 after intraperitoneal injection of PM21-PBMC, possibly due to the In the peritoneal cavity, amplification would have been higher. For comparison, 1–2 × 10 6 NK cell stasis After intravenous infusion, only approximately 5-17 NK cells / μL of blood were observable 14 days after infusion. In contrast, this study using PM21 particle stimulation showed is 2.0 x 10 6 400 NK cells / μL on day 12 after intraperitoneal infusion of PM21-PBMC It was observed that the blood 6 NK cells). The study consisted of 5 μg (approximately 50, In this study, a relatively low dose of IL-2 (1,000 U / injection) was used. In a different study, K562-mb15-41BBL feeders were used. - 30 × 10 cells preferentially ex vivo amplified 6 NK cells were injected intravenously, and then Humans injected with anti-CD45 antibodies (but not the combination of anti-CD56 and anti-CD3) Lymphocytes were tracked using a high dose of intraperitoneally injected IL-2 (25,000 U / day) is required for lymphocyte persistence, NK cell concentrations were not determined, and In comparison with these previous methods, PM21 particle-stimulated immune responses were The magnitude of NK cell expansion is unprecedented and a unique capability of the PM21 particles.

[0077] 85 Here, the route of delivery of PM21-PBMCs to NSG mice was determined based on previous preliminary clinical trials. In comparison to these previous studies, the PM The 21 particle-based method is advantageous in several respects. First, ex vivo preactivation The combination of irradiated erythrocytes and in vivo stimulation with PM21 particles was shown to be effective in reducing the amount of phosphorus released by apheresis. Isolated NK cells require cell harvesting and subsequent extensive laboratory processing for NK cell enrichment. Allows the use of significantly lower amounts of unselected PBMC compared to cytokine activation of cells Second, the PM21 particle-based method uses a short incubation period instead of a two-week culture-based amplification. Requires only 2 days of preactivation, resulting in better retention of physiologically relevant functionality Third, the method of the present invention allows for significantly greater amplification than previous methods that do not allow amplification. without the use of high doses of IL-2, which have been associated with significant in vivo proliferation or clinical toxicity. In peritoneal tumors, the advantage of the methods described herein is that it provides total anti-tumor activity. As clearly shown by proliferation analysis in CTViolet, In the absence of membrane tumors, the peritoneal cavity confines PM21 particles within this volume, allowing for good in vivo amplification. By promoting the width, a comfortable environment can be provided, and then NK cells will significantly NK cells have not only been observed in the PB but also in the organs. It was also found in the bone marrow and was more abundant in the in vivo application of PM21 particles. The amount of NK cells was determined by CD34 + The amount was the same as that used in the test using NK cells generated from umbilical cord blood stem cells. etc., indicating that these NK cells are competent for bone marrow homing.

[0078] 86 from the phenotyping of NK cells expanded ex vivo or in vivo in parallel (Fig. S3), showing that the cells obtained were similar regardless of the approach. This was observed in most of the NK cells expanded in vivo, but not in the ex vivo environment. NKG2A - and NKG2C + Interesting differences were observed regarding the amplification of the subpopulations. KG2C + NK cell populations are involved in "memory-like" responses during viral reactivation. It has been observed that IL-12 production is dependent on monocytes. NKG2C in patients with CMV reactivation after stem cell transplantation for AML + NK thin The presence of HLA-E-induced inhibition was also associated with better outcome and lower relapse. Resistant NKG2A - The presence of a significant population of NK cells is crucial for the treatment of multiple myeloma patients. This may be important for the development of NK cells, as cells downregulate HLA class I but express HLA-E. Approaches aimed at downregulating NKG2A induce NK cell cytotoxicity. Ex vivo expanded cells have been proposed as a means to improve the viability and therefore therapeutic potential of most Mostly NKG2A + Therefore, the time required for ex vivo culture and subsequent in vivo amplification was shortened. This allows for the development of NK cells with greater phenotypic diversity and potentially better cytotoxicity to their targets. Additional benefits may be provided in the production of cells.

[0079] (2) Potential clinical utility of PM21 particles 87. The ability of PM21 particles to expand NK cells has implications for cancer treatment and potentially other PM21 particles are currently undergoing clinical trials, allowing for the widespread use of adoptive NK cell therapy for the treatment of these diseases. It can easily replace feeder cells currently used in the field, making it easier to transport and reducing risks. The use of tumor-derived feeder cells is prohibited or difficult to obtain approval for. In the case of regulatory control areas, the PM21 particles are readily available for ex vivo amplification and activation. The use of PM21 particles for ex vivo amplification in a homogeneous environment provides a time-saving solution. Cell depletion can be performed prior to ex vivo NK cell expansion. Current clinical trials of cultivated NK cells utilize T cell depletion before NK cell expansion to prevent GvH. Furthermore, in vivo administration of PM21 particles further eliminates allogeneic T cells that can induce D. The unprecedented ability to expand NK cells in vivo and potentially reduce T cell expansion GvHD can be alleviated by using peritoneal cancer and persistent ovarian epithelial cancer or desmoplastic small intestine. This NK cell expansion method is expected to be clinically applicable for the treatment of other peritoneal tumors, such as peritoneal tumors. Antitumor efficacy experiments for the elimination of peritoneal tumors are currently underway. The use of PM21-PBMCs and PM21 particles for this purpose is possible and incorporates T cell depletion. The methodology for this is being explored for application in homogeneous environments.

[0080] 88. Importantly, NK cells expanded by this method were transported from the peritoneal cavity to the peripheral blood and The intraperitoneal route of injection is used to treat pulmonary arterial disease and to treat multiple organs that are potential sites of cancer and various other cancers. Although unconventional for the treatment of liquid malignancies, delivery of NK cells by this intraperitoneal route has been shown to be beneficial for AM L results in PB concentrations of NK cells associated with treatment.

[0081] 89. The particle-based approach for NK cell-specific signaling is For further targeted stimulation of NK cells to enhance antitumor cytotoxicity and persistence, et al. a signaling molecule or a packaged delivery vehicle for a drug The PM21 particles may be a platform for innovative NK cell-specific immunotherapy currently under development. (Checkpoint inhibitors, CAR, bispecific engager (BiKE), Treg DT-fused IL-2 for depletion, and in vivo NK cell stimulation with PM21. It may have beneficial effects and high complementarity during gene amplification. The methods described herein allow for an unprecedented way to test such combination methods. Of course, there are murine models, but human NK cells that can exist for significant periods in vivo are There are no other ways to test the cells.

[0082] 90. In summary, this procedure with PM21 particles is typically performed using exogenous feeder cells. This allows preferential in vivo expansion of NK cells at a level previously only achieved by in vivo expansion. , the need for cell culture with feeder cells or high cytokine doses that are toxic Furthermore, PM21-PBMCs obtained by in vivo delivery of PM21 particles were not isolated from autologous PBMCs. be used to take advantage of the beneficial synergistic effects of other immune cells on NK cell function. This can be further combined with other strategies such as anti-KIR antibodies or BiKE to enhance NK cell proliferation. This method is therefore simple and suitable for clinical translation. While being modifiable, it meets the criteria for NK cell generation for potential therapeutic efficacy and is suitable for cancer or or other ailments.

[0083] 2. Example 2: Bone marrow trafficking of NK cells through stimulation of fucosylation by PM21 particles cking Transformed to express engineered membrane-bound forms of IL-21 and 41 bbl PM21 particles prepared from K562 cells (K562.mb21.41bbl) were Induce efficient specific expansion of NK cells. Co-culture with NK cells using feeder cells (FC) 21) PM21 particles or K562.mb21.41bbl provided The stimulation was monitored by HECA-452 mAb binding by flow cytometry. Induce full fucosylation of sLex.

[0084] 92. NK cells were expanded with PM21 or FC21 and stained with HECA452 mAb. The cells were stained and analyzed by flow cytometry. HECA452 inhibited the expression of PSGL-1, CD4 PM21 specifically recognizes the fucosylated forms of PM44 and other E-selectin ligands. NK cells stimulated with FC21 were compared with untreated NK cells, NK cells treated with soluble cytokines, and .... NK cells treated with mbIL21 alone (without 41bbl) or NK cells treated with mbIL21 alone (without 41bbl) Compared to feeder cells, they had a significantly higher MFI by flow cytometry analysis. This suggests that stimulation with PM21 particles and / or FC21 feeder cells may promote N cell proliferation in the bone marrow. Able to induce K cell trafficking and stimulation with PM21 or FC21 bone marrow trafficking of therapeutic NK cells generated by These findings strongly suggest that this approach may improve the treatment of malignancies of myeloid origin and bone marrow origin.

[0085] 93. Figure 7 shows HECA452 staining of NK cells following stimulation on days 0, 1, 7, and 10. The effect of soluble cytokines on the IL-1 expression was shown in Figure 8. The effect of particle or feeder cell stimulation was shown in Figure 8. NK cells expressing K562 cells or membrane-bound IL-21 and 4 K562 cells with 1BBL were stimulated for 10, 12, or 14 days (FC21 cells or PM21 particles) stained with HECA452. FC21 feeder derived from K562 NK cells stimulated with cells or PM21 particles were expressed in IL-21-free K562 cells. NK cells stimulated with NK cells showed significant activation compared to NK cells stimulated with NK cells. The effect of 10 days of culture using various conditions on NK cells is shown. NK cells were cultured for 10 days under various conditions. Cells from the cultures were probed with ITC-conjugated HECA452 mAb. Stained with CA452-FITC and analyzed by flow cytometry using gating on CD56+CD3-. analyzed by cytometry (CSTX2 = K562.mb21.4 from CytoSen). 1 bbl; FC = feeder cells; PM21 = plasma membrane particles prepared from CSTX2).

[0086] 94. To understand the effect of resting period on stimulated NK cells (Figure 11), NK cells were incubated with CSTX2-PM21 particles for 10 days (top), and then PM21 particles were removed. After incubation, NK cells were cultured for 3 days to become "rested" (bottom). The cells from the culture were probed with FITC-conjugated HECA452 mAb to detect the cytotoxicity of HIV-1. Cells were stained with HECA452-FITC and analyzed by gating on CD56+CD3-. The cells were analyzed by flow cytometry (CSTX2 = CytoSen's K562.m b21.41bbl; FC = feeder cells; PM21 = plasmodium prepared from CSTX2 membrane particles).

[0087] NK cells stimulated with 95.PM21 particles were stable and survived the freeze-thaw process, with no discernible effect on the cells (Figure 12). NK cells were isolated from PBMCs (top), then cultured with CSTX2-PM21 particles for 10 days (middle), and then cryopreserved and thawed (bottom). NK cells were probed with FITC-conjugated HECA452 mAb, which detects the fucosylated form of Slex. Cells from the cultures were stained with HECA452-FITC and analyzed by flow cytometry by gating on CD56+CD3- (CSTX2 = CytoSen's K562.mb21.41bbl; FC = feeder cells; PM21 = plasma membrane particles prepared from CSTX2). The present invention includes the following aspects. <1> A method of trafficking NK cells to bone marrow, comprising contacting NK cells with one or more of PM21 particles and FC21 feeder cells. <2> further comprising stimulating the NK cells with IL-2, IL-12 and / or IL-18. <1> The method described below. <3> said contacting of said PM21 particles and / or FC21 feeder cells with said NK cells occurs prior to transfer of said NK cells into a patient; <1> The method described below. <4> said contacting of said PM21 particles and / or FC21 feeder cells with said NK cells occurs subsequent to transfer of said NK cells into a patient; <1> The method described below. <5> Inducing an intracellular mechanism in NK cells to induce fucosylation of PSGL-1 on the surface of said NK cells; <1> , <3> or <4> A method according to any one of the preceding claims. <6> Induce the expression of FUT7 in NK cells in correlation with the fucosylation of PSGL-1 on the surface of the NK cells. <1> , <3> or <4> A method according to any one of the preceding claims. <7> A method for treating a myeloid malignancy or a myeloid-derived malignancy in a subject, comprising contacting NK cells with PM21 particles and / or FC21 feeder cells and adoptively transferring the NK cells into the subject. <8> A method for treating a bone marrow-associated viral infection in a subject, comprising contacting NK cells with one or more of PM21 particles and FC21 feeder cells, and adoptively transferring the NK cells into the subject. <9> The contacting of the NK cells with the PM21 particles and / or FC21 feeder cells occurs prior to the transfer of the NK cells into the patient. <7> or <8> The method described below. <10> the contacting of the NK cells with the PM21 particles and / or FC21 feeder cells occurs subsequent to the transfer of the NK cells into the patient; <7> or <8> The method described below.

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Claims

1. 1. A fucosylated natural killer (NK) cell for use in a method of treating a myeloid malignancy or myeloid-derived malignancy in a subject, comprising: The treating comprises: contacting NK cells with PM21 particles or FC21 feeder cells ex vivo or in vitro for 6 to 40 days to induce fucosylation of PSGL-1 on the surface of said NK cells, thereby obtaining fucosylated natural killer (NK) cells; and adoptively transferring said fucosylated NK cells into said subject; Including, the PM21 particles or FC21 feeder cells contain membrane-bound IL-21 and membrane-bound 41BBL; Fucosylated natural killer (NK) cells.

2. 1. A method for treating a bone marrow-associated viral infection in a subject, comprising administering to said subject a fucosylated natural killer (NK) cell. The treating comprises: contacting NK cells with PM21 particles or FC21 feeder cells ex vivo or in vitro for 6 to 40 days to induce fucosylation of PSGL-1 on the surface of said NK cells, thereby obtaining fucosylated natural killer (NK) cells; and adoptively transferring the fucosylated NK cells into the subject; the PM21 particles or FC21 feeder cells contain membrane-bound IL-21 and membrane-bound 41BBL; Fucosylated natural killer (NK) cells.

3. The fucosylated natural killer (NK) cells of claim 1 or 2, wherein said treating further comprises administering one or more of IL-2, IL-12 and / or IL-18 to said subject after transferring said fucosylated NK cells into said subject.

4. The fucosylated natural killer (NK) cells of claim 1 or 2, wherein the contacting induces expression of FUT7 in the NK cells in correlation with fucosylation of PSGL-1 on the surface of the NK cells.

5. The fucosylated natural killer (NK) cells of claim 1 or 2, wherein the induction of fucosylation of PSGL-1 on the surface of the NK cells further comprises stimulating the NK cells ex vivo or in vitro with IL-2, IL-12 and / or IL-18.

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