RU2777993C2 - Particles pm21 for improvement of homing of nk-cells in bone marrow - Google Patents

Abstract

FIELD: cell biology; medicine.

SUBSTANCE: present invention relates to the field of cell biology and medicine, in particular to a method for the transfer of NK-cells to the bone marrow of a patient, and a method for the treatment of malignant neoplasm of the bone marrow. For the implementation of the specified methods, NK-cells are brought into contact with particles PM21 and/or feeding cells FC21 before the transfer of NK-cells to the body of the specified subject, and then, NK-cells are administered to the specified subject. In this case, the transfer of NK-cells to the bone marrow and, consequently, treatment is carried out due to that the specified contact of NK-cells with particles PM21 and/or feeding cells FC21 induces in NK-cells a cell mechanism inducing fucosylation of PSGL-1 on the surface of NK-cells.

EFFECT: present invention allows for the improvement of the efficiency of directed migration (homing) of NK-cells to bone marrow.

12 cl, 34 dwg, 2 ex

Description

This application claims priority in U.S. Provisional Application No. 62/464,747, filed Feb. 28, 2017, and incorporated herein by reference in its entirety.

I. STATE OF THE ART

Therapy using adoptive natural killer (NK) cells is a promising new type of intervention for oncological diseases, including malignant neoplasms of the bone marrow. Therefore, the efficiency of directed migration of the adopted adoptive NK cells is of great importance. There is a need for methods for efficient targeted migration of NK cells to the bone marrow.

II. BRIEF DESCRIPTION OF THE INVENTION

1. This application describes methods and compositions related to targeted migration of NK cells to the bone marrow, including bringing the NK cells into contact with PM21 particles and/or FC21 feeder cells. In one aspect, these methods may further include stimulation of NK cells with IL-2, IL-12, IL-18 and/or IL-18.

2. In addition, the present application describes methods for the treatment of malignant neoplasm of the bone marrow or congenital malignant neoplasm of the bone marrow and / or treatment of a viral infection (for example, a viral infection associated with the bone marrow, including a viral infection that is tropic in relation to the bone marrow or a viral infection that adversely affects the bone marrow) comprising bringing NK cells into contact with PM21 particles and/or FC21 nurse cells and adoptively transferring the NK cells into the subject's body. In some aspects, contact of the PM21 particles and/or FC21 nurse cells with NK cells may occur prior to transfer of the NK cells to the patient. In yet another aspect, the contact of PM21 particles and/or FC21 nurse cells with NK cells can occur in the patient's body.

III. BRIEF DESCRIPTION OF THE DRAWINGS

3. The accompanying drawings, incorporated herein and forming part of this specification, illustrate some embodiments and, together with the description, illustrate the compositions and methods described therein.

, черные) или РМ21 (

, синие) в количестве 200 мкг/мл в течение 27 дней, содержание клеток тестировали каждые 2-3 дня и продемонстрировали кратное увеличение количества NK-клеток (А) и процентного содержания клеток в суспензии (В). Частицы РМ21 (кратность 825±188, N=13, 4 донора) (синие) более эффективно стимулировали размножение NK-клеток по сравнению с частицами РМ15 (425±71, N=35, 9 доноров) (черные) на основании обобщенного анализа данных о размножении NK-клеток, полученных на 14 день (С). Мононуклеары периферической крови, выделенные от трех пациентов с ОМЛ на стадии ремиссии, культивировали в течение 14 дней с частицами РМ21 (200 мкг/мл), высевая из расчета 0,5×10⁶ NK-клеток/мл в SCGM с 10% FBS, 2 мМ Glutamax и 50 ед/мл ИЛ-2. Показана кратность увеличения количества NK-клеток по сравнению с первичными МПК (D) и содержания лимфоцитов (Е) (CD56⁺CD3^- NK-клетки (

, красный), CD56^-CD3⁺ Т-клетки (

, синий) и CD56 CD3⁺ NKT-клетки (

, черный)). МПК пациента F021 культивировали в течение 16 дней, как описано выше, и анализировали аутологичную цитотоксичность по отношению к ОМЛ-опухолям того же пациента (F). Размноженные РМ21-NK-клетки метили TFL4, совместно инкубировали (2 часа) при указанном соотношении Е:Т с клетками ОМЛ того же пациента на стадии активного заболевания и анализировали с помощью проточной цитометрии. Количество спонтанно погибших клеток-мишеней определяли с помощью контроля "Только мишень". Данные для каждой точки определяли в двух повторностях.4. Figure 1 shows that PM-21 particles efficiently and selectively promote the expansion of cytotoxic NK cells. Peripheral blood mononuclear cells (PBMCs) were isolated from a source of leukocytes and seeded at 0.1×10 ⁶ NK cells/ml in SCGM supplemented with 10% FBS, 2 mM Glutamax, and 50 U/ml IL-2. MICs were stimulated with PM15 particles (

, black) or PM21 (

, blue) at 200 µg/mL for 27 days, the cell content was tested every 2-3 days and showed a fold increase in the number of NK cells (A) and the percentage of cells in suspension (B). PM21 particles (825±188, N=13, 4 donors) (blue) stimulated NK cell proliferation more effectively than PM15 particles (425±71, N=35, 9 donors) (black) based on pooled data analysis about the expansion of NK cells obtained on day 14 (C). Peripheral blood mononuclear cells isolated from three AML patients in remission were cultured for 14 days with PM21 particles (200 μg/ml) seeding at 0.5×10 ⁶ NK cells/ml in SCGM with 10% FBS, 2 mM Glutamax and 50 U/ml IL-2. The multiplicity of the increase in the number of NK cells compared with the primary BMD (D) and the content of lymphocytes (E) (CD56 ⁺ CD3 ^- NK cells (

, red), CD56 ^- CD3 ⁺ T cells (

, blue) and CD56 CD3 ⁺ NKT cells (

, black)). Patient F021 PBMCs were cultured for 16 days as described above and analyzed for autologous cytotoxicity against AML tumors from the same patient (F). Expanded PM21-NK cells were labeled with TFL4, co-incubated (2 hours) at the indicated E:T ratio with AML cells from the same patient in active disease, and analyzed by flow cytometry. The number of spontaneously dead target cells was determined using the "Target Only" control. Data for each point was determined in duplicate.

, красный) и Т-клеток (

, черный) в виде доли от общих пСВ45⁺-клеток.5. Figure 2 shows that pre-activation of unselected PBMCs with PM21 particles induces NK cell expansion in vivo. NSG mice were injected ip with 2×10 ⁶ cells of non-activated PBMC (A and B) or PBMC previously activated ex vivo with PM21 particles and 100 U/ml IL-2 for 2 days (PM21-MPC) (C and D) . Mice in all groups received 1000 units of IL-2 ip three times a week. Groups of mice were also injected ip with 400 μg of PM21 particles twice a week (B and D). Peripheral blood was collected by sequential buccal bleeding and analyzed by flow cytometry for hCD45 ⁺ human lymphocytes twice a week starting on day 6. The number of NK-, T- and B-cells was determined based on staining for hCD3, hCD56, hCD19. The left graphs in each experimental group show the concentration of hNK cells per µl of RV. The right graphs show the percentage of hNK cells (

, red) and T cells (

, black) as a proportion of total PSV45 ⁺ cells.

6. Figure 3 shows that the proliferation assay confirms in vivo expansion of NK cells from PM21-MPC. MICs freshly thawed or pre-activated with PM21 particles and 100 U/ml IL-2 for two days (PM21-MPC) were labeled with Cell Trace (CT) Violet. 2×10 ⁶ non-activated MICs (A and B) or PM21-MICs (C and D) ip were administered to NSG mice. Mice in all groups received 1000 units of IL-2 ip three times a week. Two groups of mice were also injected ip with 400 μg of PM21 particles twice a week (B and D). Two mice in each group were euthanized on day 6 and peritoneal swabs were analyzed by flow cytometry for CT Violet hCD45 ⁺ , hCD3 ^- , hCD56 ⁺ NK cell fluorescence. CT Violet fluorescence histograms were analyzed by curve fitting using the Proliferation analysis package in the FlowLogic software.

, красный) и Т-клеток (

, черный) в виде доли от общих hCD45⁺-клеток. Анализ образцов РВ с 12 дня после в/б инъекции РМ21-МПК продемонстрировал увеличение количества hNK-клеток в РВ в зависимости от дозы частиц РМ21 in vivo (Е, слева), в то время как значимого увеличения количества общих CD3⁺Т-клеток в зависимости от дозы не наблюдали (Е, справа).7. Figure 4 shows that the use of PM21 in vivo can increase the number of NK cells in the peripheral blood. MICs were previously activated ex vivo with PM21 particles and 100 U/ml IL-2 for 2 days. PM21-MPC in an amount containing 0.2×10 ⁶ viable NK cells was injected ip into NSG mice. Mice in all groups received 1000 units of IL-2 ip three times a week. Mice were also injected ip with 0 (A), 400 (B), 800 (C), 1600 μg (D) of PM21 particles twice a week. Peripheral blood was analyzed by flow cytometry for hCD45 ⁺ -lymphocytes twice a week, starting on day 5, and the number of hNK-, hT- and hB cells was determined based on staining for hCD3, hCD56, hCD19. The left graphs in each experimental group show the concentration of hNK cells per µl of RV. The right graphs show the percentage of hNK cells (

, red) and T cells (

, black) as a proportion of total hCD45 ⁺ cells. Analysis of RV samples from day 12 after i.p. injection of PM21-MPC demonstrated an increase in the number of hNK cells in RV depending on the dose of PM21 particles in vivo (E, left), while a significant increase in the number of total CD3 ⁺ T cells in no dose dependence was observed (E, right).

, красный), hCD45⁺hCD3⁺Т-клеток (

, синий) и hCD45⁺, hCD56^-hCD3^- других лимфоцитов (

, черный) (нижние графики для каждого органа). Толстая линия для каждого параметра представляет собой среднее значение.8. Figure 5 shows that NK cells expanded in vivo show a biological distribution in critical physiological areas, and the biological distribution of NK cells is increased by the use of PM21 particles in vivo. NK cells (0.2×10 ⁶ cells) as part of PM21-MPC, pre-activated ex vivo using PM21 particles and 100 U/ml IL-2 for 2 days, were administered ip to NSG mice. Mice in all groups received 1000 units of IL-2 ip three times a week. Mice were also injected ip with 0 or 800 μg of PM21 particles twice a week. Mice were sacrificed 16 days after the initial ip injection of PM21-MPC. On the day of euthanasia, bone marrow (from the femur), spleen, lungs, brain, and liver were collected, the organs were perfused, and the femur was washed to obtain cells. Cells were stained with antibodies against hCD3, hCD45, hCD56, hCD19 for flow cytometric analysis. Shown are data for bone marrow, spleen, brain, lung, and liver (from left to right) showing the number of hCD45 ⁺ hCD56 ⁺ hCD3 ^- NK cells (upper graphs for each organ) and the percentage of hCD45 ⁺ hCD56 ⁺ hCD3 ^- NK cells (

, red), hCD45 ⁺ hCD3 ⁺ T cells (

, blue) and hCD45 ⁺ , hCD56 ^- hCD3 ^- other lymphocytes (

, black) (lower plots for each organ). The thick line for each parameter represents the average value.

, красный), hT-клеток (

, синий) и других hCD45⁺-клеток (

, черный) также согласованно воспроизводилось в различных группах, получивших инъекции МПК из различных источников, в периферической крови (В) и в брюшной полости (D). Толстая линия для каждого параметра представляет собой среднее значение.9. Figure 6 shows that the expansion of NK cells from each organ is reproduced stably in vivo. The expansion consistency of NK cells stimulated with PM21 particles in vivo was tested using three different PBMCs obtained from healthy donors. MICs were pre-activated ex vivo with PM21 particles for 2 days and 100 U/ml IL-2 for 2 days (PM21-MIC) iv/b was administered to NSG mice. Mice in all groups received 1000 units of IL-2 ip three times a week. Peripheral blood was analyzed by flow cytometry for hCD45 ⁺ -lymphocytes twice a week, starting on day 5, and the number of hNK-, hT- and hB cells was determined based on staining for hCD3, hCD56, hCD19. The concentration of hNK cells in the blood 12 days after i.p. injection of MPC (A) and the number of NK cells collected by washing from the abdominal cavity 14 days after i.p. injection of MPC (C) were similar in different groups that received injections NK cells from various sources (p=0.84 for PB and p=0.69). The content of hNK cells (

, red), hT cells (

, blue) and other hCD45 ⁺ cells (

, black) was also consistently reproducible in different groups that received injections of PBMC from different sources, in peripheral blood (B) and in the abdominal cavity (D). The thick line for each parameter represents the average value.

10. Figure 7 shows HECA-452 staining of NK cells treated with soluble cytokines on days 0, 1, 7 and 10 of stimulation.

11. Figure 8 shows HECA-452 staining of NK cells cultured with K562 nurse cells after 10, 12 and 14 days of stimulation.

12. Figure 9 shows a comparison of HECA-452 staining of NK cells stimulated under different conditions after 10 days of stimulation.

13. Figure 10 shows HECA-452 staining of NK cells cultured under various conditions after 10 days of stimulation. NK cells were cultured for 10 days under various conditions as shown. The mAb HECA 452 conjugated with FITC was used as a probe for NK cells, which detected the fucosylated form of SLex. Culture cells were stained with HECA452-FITC and analyzed by flow cytometry with CD56+CD3- gated. (CSTX2 = CytoSen K562.mb21.41bbl; PC = feeder cells; PM21 = plasma membrane particles derived from CSTX2).

14. Figure 11 shows HECA-452 staining of NK cells three days of rest after 10 days of stimulation. NK cells were cultured for 10 days with CSTX2-PM21 particles (top) and then "left" in culture for 3 days after removal of PM21 particles (bottom). The mAb HECA452 conjugated with FITC was used as a probe for NK cells, which detected the fucosylated form of SLex. Culture cells were stained with HECA452-FITC and analyzed by flow cytometry with CD56+CD3- gated. (CSTX2 = CytoSen K562.mb21.41bbl; PC = feeder cells; PM21 = plasma membrane particles derived from CSTX2).

15. Figure 12 shows HECA-452 staining before and after freezing and thawing after 14 days of propagation with PM21 particles. NK cells were isolated from PBMCs (top), cultured for 10 days with CSTX2-PM21 particles (middle) and then frozen with cryopreservative and thawed (bottom). The mAb HECA452 conjugated with FITC was used as a probe for NK cells, which detected the fucosylated form of SLex. Culture cells were stained with HECA452-FITC and analyzed by flow cytometry with CD56+CD3- gated. (CSTX2 = CytoSen K562.mb21.41bbl; PC = feeder cells; PM21 = plasma membrane particles derived from CSTX2).

16. Figure 13 shows 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 anti-STAT3 antibodies. The arrows indicate the direction of transcription. The scale is the same for gene and islets; (B) RNA-seq revealed different 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 stimulates FUT7 gene expression in expanded NK cells.

IV. DETAILED DESCRIPTION OF THE INVENTION

17. Before disclosing and describing compounds, compositions, articles, devices and/or methods, it should be understood that they are not limited to specific synthetic methods or specific recombinant biotechnological methods, unless otherwise indicated, or to certain reagents, unless otherwise indicated, since such methods may, of course, vary. In addition, it should be understood that the terminology used herein is for the sole purpose of describing particular embodiments and is not intended to be limiting.

A. Definitions

18. In the present description and the appended claims, the singular forms include the plural unless the context clearly indicates otherwise. Thus, for example, reference to a "pharmaceutical carrier" includes mixtures of two or more such carriers, and the like.

19. As used herein, ranges may be expressed from "about" one specific value to "about" another specific value. If a range is expressed in this way, another implementation option includes values from one particular value and/or to another particular value. Similarly, if values are expressed as approximate values using the previous logical relation term "approximately", it should be understood that this particular value constitutes another implementation option. In addition, it should be understood that the end values of each of the ranges are meaningful both with respect to the other end value and independently of the other end value. In addition, it should be understood that a number of values are described herein, and that each value is also described herein as "approximately" that particular value, in addition to the value itself. For example, if the value "10" is described, then "about 10" is also described. In addition, it should be understood that when describing a value that is "less than or equal to" a value, the value that is "greater than or equal to the value" will also be described, as well as possible ranges between the values, as understood by a person skilled in the art. For example, if the value "10" is described as "less than or equal to 10", "greater than or equal to 10" is also described. In addition, it should be understood that in this application the data is presented in several different formats, and that these data represent end points and start points and ranges for any combination of data points. For example, if specific data point "10" and specific data point "15" are described, it should be understood that values greater than, greater than or equal to, less than, less than or equal to and equal to 10 and 15, and between 10 and 15 are also described. In addition, it should be understood that each unit between two specific units is also described. For example, if 10 and 15 are described, then 11, 12, 13, and 14 are also described.

20. A number of terms are referred to in this specification and the appended claims, which are defined using the following meanings:

21. "Optional" or "optional" means that the event or circumstance described after the term may or may not occur and that the description includes cases where the specified event or circumstance occurs and cases when it does not occur.

B. Methods of using the compositions

22. Adoptive therapy using donor natural killer (NK) cells is a promising new intervention for bone marrow and congenital bone marrow malignancies, including for veterinary use in such neoplasms. In yet another aspect, adoptive NK cells can be used therapeutically to treat resident bone marrow viruses, including, for example, parvoviruses that cause aplastic anemia. Therefore, the efficiency of targeted migration of adoptively used NK cells is of great importance. In particular, it is critical how to direct the transferred cells to the bone marrow, where they can be effectively used as a drug.

23. Bone marrow homing determinants include ligands for selectin binding. A key determinant for L-selectin binding is the fucosylation state of the sialyl-Lewis-x (sLex) carbohydrate chain attached to P-selectin glycoprotein ligand-1 (PSGL-1). In one aspect of the present invention, it is contemplated that stimulation of NK cells with PM21 particles and/or FC21 feeder cells derived from K562 cells transformed to express an engineered membrane-bound form of IL-21 and/or 41bbl (K562.mb21.41bbl) induces an effective specific expansion of NK cells and complete fucosylation of sLex.

24. Accordingly, in one aspect, the present invention provides methods for transferring NK cells to the bone marrow, and methods for treating bone marrow cancer or congenital bone marrow cancer, comprising contacting NK cells with PM21 particles and/or nourishing FC21 cells and adoptive transfer of NK cells to a patient in need thereof. In one aspect, these methods may further include stimulation of NK cells with IL-2, IL-12, IL-15, IL-18, IL-21 ex vivo or in vivo (in the patient).

25. In some aspects, contact of the PM21 particles and/or FC21 nurse cells with NK cells may occur prior to transfer of the NK cells to the patient. In yet another aspect, the contact of PM21 particles and/or FC21 nurse cells with NK cells can occur in the patient's body.

26. In one aspect of the present invention, it is contemplated that the efficacy of NK cell immunotherapy is dependent on the dose of NK cells administered to a patient or achieved after infusion via in vivo expansion. Currently available techniques are limited by the inability to achieve the level of NK cell expansion required to achieve a therapeutic effect in the patient. The lack of a simplified clinical multiplication protocol is a major obstacle to the progress and widespread adoption of NK cell immunotherapy. Current ex vivo propagation protocols use a combination of high doses of cytokines with activating ligands expressed on feeder/stimulator cell lines derived from leukemia cells, which is a significant disadvantage for clinical transfer in most centers; moreover, such cells cannot be directly propagated in vivo. The use of particle-based technology, including exosomes, as described herein eliminates the need for stimulator cells, thereby simplifying the methodology and enabling ex vivo propagation for adoptive therapy or in vivo selective propagation. Accordingly, in one aspect, the present invention provides methods for treating bone marrow malignancies and congenital bone marrow malignancies by adoptive NK cell transfer and/or methods for transferring NK cells to the bone marrow, comprising bringing the NK cells into contact with one or more vesicles. containing an NK cell effector agent. In addition, in one aspect, methods are described for treating bone marrow cancers, congenital bone marrow cancers, and/or viral infections (including viruses with bone marrow tropism), further comprising preactivating or activating NK cells in vivo by contacting at least one NK cell with at least one or more stimulatory cytokines. Thus, in one aspect, NK cells expanded ex vivo using PM21 particles and/or FC21 feeders, or using direct in vivo stimulation with PM21 or FC21 feeders, can be used to treat resident viral (e.g., parvovirus) conditions or bone marrow syndromes that cause aplastic anemia.

27. In the described methods, pre-activation or activation of NK cells is carried out by bringing at least one NK cell into contact with at least one or more stimulatory cytokines (for example, IL-2, IL-12, IL-15, IL- 21 and/or IL-18). Thus, in one aspect, the present invention provides methods for treating bone marrow malignancies and congenital bone marrow malignancies by adoptive NK cell transfer and/or methods for transferring NK cells to the bone marrow, comprising pre-activating NK cells by bringing one or more NK cells in contact with one or more stimulatory cytokines selected from the group consisting of IL-2, IL-12, IL-21, IL-15 and/or IL-18, or any combination thereof, including bringing one or more NK- cells in contact with 2 or 3 stimulatory cytokines. For example, methods are specifically described herein in which the pre-activation or activation step comprises contacting NK cells with IL-2; IL-12; IL-15, IL-18, IL-12 and IL-15; IL-12 and IL-18; IL-15 and IL-18; or IL-12, IL-15 and IL-18. In one aspect, the described methods for treating bone marrow malignancies and congenital bone marrow malignancies by adoptive NK cell transfer and/or methods for transferring NK cells to the bone marrow may further comprise contacting the NK cell with one or more cytokines selected from the group consisting of 4-1BBL, IL-2, IL-21, MICA/B, ULBP2, ICAM-1, 2B4, BCM1/SLAMF2, CD155, CD112, CCR7, DAP12, Notch ligands and/or DAP10 in soluble form, or in the form of PM21 particles or FC21 feeding cells.

28. It should be understood, and is intended herein, that the duration of pre-activation or activation (i.e., the duration of contact between NK cells and stimulatory cytokines (e.g., IL-2, IL-12, IL-15, IL-21, and /or IL-18) in soluble or particulate form of PM21 or FC21 feeder cells can be any amount of time needed to achieve the desired pre-activation or NK cell activation. culturing NK cells in the presence of IL-2, IL-12, IL-15, IL-21, and/or IL-18 for 7 days.) In one aspect, the present invention provides methods for treating bone marrow cancers and congenital bone marrow cancers. brain through adoptive transfer of NK cells and/or methods of transferring NK cells to the bone marrow, including pre-activation or activation of NK cells by contacting one or more NK cells with IL-2, IL-12, IL-1 5 and/or IL-18 for 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 21, 22, 23, 24, 36 or 48 hours. It should be understood, and is intended herein, that the half-life of a cytokine in culture may be less than the desired contact time. Accordingly, methods are described herein in which one or more NK cells are brought into contact with IL-2, IL-12, IL-15 and/or IL-18 every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours during a contact period (for example, every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours during a 24-hour period contact).

29. Use of plasma membrane particles (PM), exosomes (EX), or nurse cells (FC) containing one or more of NK cell effector agents (i.e., stimulatory peptides, cytokines, and/or adhesion molecules) for contact and activation and/or expansion of NK cells eliminates many of the obstacles associated with cytokine toxicity. Examples of NK cell activating agents and stimulatory peptides include 41BBL, IL-2, IL-12, IL-21, IL-18, MICA, LFA-1, 2B4, BCM/SLAMF2, CCR7, Notch ligands, and/or others. signal molecules that induce homing, but are not limited to them. Examples of cytokines include, but are not limited to, IL-2, IL-12, IL-21, and IL-18. Examples of adhesion molecules include, but are not limited to, LFA-1, MICA, BCM/SLAMF2. For example, a plasma membrane particle (PM), nurse cells (FC), or exosomes (EX) are derived from nurse cells expressing membrane-bound IL-21 (FC21 cells, PM21 particles, and EX21 exosomes, respectively). FC21 cells expressing membrane-bound IL-21, PM21 particles, and EX21 exosomes may further contain one or more activating agents, stimulatory peptides, cytokines, and/or adhesion molecules, including 41BBL, IL-2, IL-12, IL-15, IL -18, MICA, LFA-1, 2B4, BCM/SLAMF2, CCR7 (eg, PM21 particle, EX21 exosome, or FC cell expressing 41BBL and membrane-bound interleukin-21), but not limited to. Accordingly, in one aspect, the present invention provides methods for treating bone marrow malignancies and congenital bone marrow malignancies by adoptive NK cell transfer and/or methods for transferring NK cells to the bone marrow, comprising bringing said cells into contact with at least one vesicle. containing the effector agent of NK cells; where the vesicle containing the NK cell effector agent is any combination of one or more PM21 particles, EX21 particles and/or FC21 feeding cells. For example, the present document describes methods of treating bone marrow cancers and congenital bone marrow cancers by adaptive transfer of NK cells and/or methods of transferring NK cells to the bone marrow, including, but not limited to, bringing said cells into contact with at least at least one vesicle containing the NK cell effector agent, wherein the vesicle containing the NK cell effector agent contains PM21 particles; exosomes EX21; feeding cells FC21; PM21 particles and EX21 exosomes; PM21 particles and FC21 feeder cells; EX21 exosomes and FC21 nurse cells; or PM21 particles, EX21 exosomes and FC21 nurse cells.

30. In some aspects, the effector agents of the PM21 particles, EX21 exosomes, or FC21 feeder cells comprise one or more stimulatory peptides associated with a membrane insertion peptide (eg, Fc, GPI, transmembrane T cell receptor, or pLIP). The membrane-embedding peptide may be a molecule that promotes incorporation into the membrane. Membrane embedding peptides may contain CD4 or IgG segments with affinity for the lipid bilayer. In addition, alternative membrane-integrating peptides may comprise a human Fc, GPI, a transmembrane T cell receptor, or pLIP. The membrane-embedding peptide may be any peptide known to be incorporated into the cell membrane. Depending on the application of the self-embedding peptide conjugate, some self-embedding peptides may be preferred over others. One skilled in the art will understand that a membrane self-integrating peptide is ideal under various circumstances. For example, the membrane self-integrating peptide pHLIP may be suitable for in vivo use. Self-integrating into the membrane, pHLIP peptides are incorporated into the membrane only at low pH. Therefore, pHLIP conjugates will not integrate into cell membranes under normal physiological conditions. However, when injected into the tumor environment, the pHLIP conjugate can be incorporated into the tumor cell membrane because the tumor environment is more acidic than normal physiological conditions. This incorporation into the tumor environment allows the activation of NK cells in the tumor region. Thus, the use of pLIP prevents unwanted incorporation into membranes of random cells.

31. Membrane-embedding peptides can be linked to one or more stimulatory peptides in a variety of ways, and methods of peptide binding are well known in the art. A membrane embedding peptide coupled to a stimulatory peptide may also be referred to as a membrane embedding peptide conjugate. In some aspects, the one or more stimulatory peptides associated with the membrane insertion peptide may comprise a fusion protein encoded by the recombinant DNA, and such fusion proteins may be produced in bacterial cells. In some embodiments, the fusion proteins may consist of one or more stimulatory peptides conjugated or linked to a lipophilic molecule, such as a hydrophobic peptide, GPI, or human Fc, for the purpose of attaching to liposomes or cell membranes. The cDNA vectors for these fusion proteins can be ligated into an expression plasmid that allows for expression in bacterial (E. coli), insect, or mammalian cells. In some embodiments, cDNA vectors may contain a FLAG or HIS marker. Bacterial cells can be transfected using standard CaCl transfection methods, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory Press (1989). In addition, bacterial cells can also be cultured in LB medium and harvested and lysed using a French press. Proteins of interest can be purified from the lysate using affinity chromatography. Fusion proteins containing palmitate-conjugated protein A and a purified Fc fragment can be conjugated as described in the literature by mixing in a ratio of 1:2 (w/w) at 4°C. The conjugates can then be injected directly into the tumor or incorporated into liposomes.

32. Types and methods of attachment are known to those skilled in the art. As used herein, the term "conjugated" refers to a peptide that is self-integrated into a membrane, conjugated, bound, or otherwise attached to another molecule, such as a peptide or protein. For example, membrane-integrating peptides coupled to stimulatory peptides can be fusion proteins in which a membrane-integrating peptide is attached to another protein via a disulfide bond. Attachment or conjugation may indicate the presence of a chemical bond between the membrane self-integrating peptide and the NK cell effector agent.

33. In some aspects, one or more stimulatory peptides can be attached to membrane self-integrating peptides or GPI anchor molecules for self-assembly in situ. For example, 41-BBL and IL-21 can be attached to the pHLIP peptide, which self-embeds into cell membranes under acidic conditions, which allows fixation of stimulatory ligands in cells near the tumor. Stimulating peptides 41BBL, IL-2, IL-12, IL-21, BCM/SLAMF2, CCR7 and/or other homing receptors can be produced in bacterial cells or purchased from commercially available sources, and cDNA vectors for these proteins can optionally be ligated into expression plasmid pTriEX, which allows expression in bacterial (E. coli), insect or mammalian cells. The cDNA vector may encode and express a FLAG or HIS marker. Bacterial cells can be transfected using standard CaCl transfection methods and cultured in LB medium. Cells can be harvested and lysed using a French press, and then proteins of interest can be purified from the lysate using affinity chromatography.

34. In some embodiments, pHLIP can be prepared by solid phase peptide synthesis using 9-fluorenylmethyloxycarbonyl based reactions and the product can be purified on a C18 column using reverse phase chromatography. The pHLIP can then be conjugated to human stimulatory protein ligands by incubation with a crosslinking agent such as benzophenone-4-iodoacetamide. After several washes, the pHLIP conjugated protein can be resuspended in a medium (eg, saline) and administered intratumorally or intravenously. Based on the data described in the previously published literature and presented in the experimental results, the interaction of NK cells with stimulatory ligands, for example, IL-21 and 41-BBL, on the surface of such modified tumor cells can stimulate the proliferation of NK cells in situ and trigger them. cytotoxic response to the tumor. This type of stimulatory approach can be applied to the treatment of solid tumors, such as ovarian cancer, in which NK-stimulating ligands, which are incorporated in situ into tumor cells at acidic pH values, can be administered intraperitoneally in patients with a low dose of IL-2 alone or together with NK cells. There is strong evidence that cytotoxic lymphocytes expressing high levels of FCγIIIR (CD16), such as NK cells, are critical to the effectiveness of cancer treatment with therapeutic antibodies. Thus, this approach can also be used in combination with therapeutic antibodies.

35. It should be understood, and is intended herein, that the duration of contact between NK cells and the vesicle containing the NK cell effector agent (e.g., PM21 particles, EX21 exosomes, and/or FC21 nurse cells) may be any amount of time necessary for achieving the desired multiplication of NK-memory cells. For example, contact can be as short as 1 minute or 60 days (eg, culturing NK cells in the presence of PM21 particles, EX21 exosomes, and/or FC21 feeder cells for 7 days). In one aspect, the contact between the NK cells and the vesicle containing the NK cell effector agent can be from about 6 days to about 60 days, more preferably, said contact can be from about 6 days to about 40 days. In addition, described herein are methods for treating bone marrow cancers and congenital bone marrow malignancies by adoptive NK cell transfer and/or methods for transferring NK cells to the bone marrow, including bringing NK cells into contact with PM21 particles, EX21 exosomes. and/or FC21 nurse cells at 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 , 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 or 72 days. It should be understood, and is intended herein, that in some cases it may be desirable to repeatedly contact NK cells with PM21 particles, EX21 exosomes, and/or FC21 feeder cells. For example, NK cells can be contacted with PM21 particles, EX21 exosomes, and/or FC21 nurse cells every 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, 14, 15, 16, 17, 18, 19, 20 or 21 days. Accordingly, in one aspect, the present invention provides methods for treating bone marrow malignancies and congenital bone marrow malignancies by adoptive NK cell transfer and/or methods for transferring NK cells to bone marrow NK cells, comprising more than once bringing NK cells into contact with PM21 particles, exosomes and/or FC21 nurse cells, said contact occurring every 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, 14, 15, 16, 17, 18, 19, 20 or 21 days.

36. In one aspect, plasma membrane particles, nurse cells, or exosomes can be cleared of nurse cells that stimulate NK cells. Nurse cells that stimulate NK cells for use in the claimed invention, for use in the manufacture of plasma membrane particles or exosomes described herein, can be irradiated autologous or allogeneic peripheral blood mononuclear cells (PBMCs), or non-irradiated autologous cells or PBMCs, cells RPMI8866, HFWT, K562, SKOV3, or EBV-LCL, including autologous or allogeneic peripheral blood mononuclear cells (PBMCs) or unirradiated autologous cells or PBMCs, RPMI8866, HFWT, K562, SKOV3, or EBV-LCL cells transfected with membrane-bound IL-21 and 41BBL. In some aspects, the NK cell feeder cells can be K562 cells transfected with membrane-bound IL-21 and 41BBL.

37. The described compositions can be used to treat any disease in which uncontrolled cell proliferation occurs, such as cancer and, in particular, malignancies that affect the bone marrow or localized in the bone marrow. A non-limiting list of different types of cancer is as follows: lymphomas (Hodgkin's and non-Hodgkin's lymphomas), leukemias, carcinomas, dense tissue carcinomas, squamous cell carcinomas, adenocarcinomas, sarcomas, gliomas, low-grade gliomas, blastomas, neuroblasts, neuroblastomas, plasmacytomas, histiocytomas, melanomas, adenomas , hypoxic tumors, myelomas, lymphomas or sarcomas associated with AIDS, metastatic cancer or cancer in general.

38. A typical, but non-limiting, list of cancers for which the described compositions can be used is as follows: lymphoma, B-cell lymphoma, T-cell lymphoma, mycosis fungoides, Hodgkin's disease, myeloid leukemia, bladder cancer, brain cancer brain, nervous system cancer, head and neck cancer, head and neck squamous cell carcinoma, kidney cancer, lung cancer such as small cell lung cancer and non-small cell lung cancer, neuroblastoma/glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer , liver cancer, melanoma, squamous cell carcinomas of the mouth, throat, larynx and lung, colon cancer, cervical cancer, cervical carcinoma, breast and epithelial cancer, kidney cancer, genitourinary system cancer, lung cancer, esophageal carcinoma, carcinoma head and neck, colon cancer, malignant neoplasms of hematopoietic organs; testicular cancer; colon and rectal cancer, prostate cancer, or pancreatic cancer.

39. The described compositions can also be used to treat viral diseases associated with the bone marrow. As used herein, the term "bone marrow-associated viral disease" refers to a viral disease in which the bone marrow contains viruses (i.e., the virus infects (including chronic, acute, latent, and persistent infections) or otherwise has a tropism for bone the marrow) or the bone marrow is adversely affected by viruses, such as the viruses that cause aplastic anemia, such as parvovirus (in some cases, a disease or condition that adversely affects the bone marrow is a viral infection of the bone marrow). Thus, in one aspect of the present invention, methods are provided for treating a viral infection in a subject, wherein the viral infection is associated with bone marrow (e.g., has an undesirable effect on the bone marrow), comprising contacting NK cells with PM21 particles and/or FC21 nurse cells. and adoptive transfer of NK cells to a subject with a viral infection. In one aspect, the virus may cause aplastic anemia and/or be an infection with bone marrow tropism or a viral infection leading to a latent or chronic infection of the bone marrow. For example, the virus may include dengue virus, hepatitis virus (including but not limited to hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, and hepatitis G virus), Epstein-Barr virus (also known as human herpesvirus 4), cytomegalovirus (also known as human herpesvirus 5), parvovirus (including but not limited to parvovirus B19), lymphocytic choriomeningitis virus (LCMV), human immunodeficiency virus (HIV), and respiratory syncytial virus (RSV) , but is not limited to them.

C. Compositions

40. Describes the components that should be used to obtain the described compositions, as well as the compositions themselves, which should be used in the methods described herein. These and other materials are described in this document, and it is understood that when describing combinations, subsets, interactions, groups, etc. of these materials, although specific references to each individual and collective combination and permutation of these compounds may not be explicitly described, each of them is specifically considered and described in this document. For example, when describing and discussing a particular PM21 particle or FC21 host cell and the possibility of making a number of modifications to a number of molecules, each combination and permutation of these possible modifications is specifically considered, unless otherwise explicitly stated. So, if the classes of molecules A, B and C are described, as well as the classes of molecules D, E and F and an example of a combined molecule A-D, then even if each of the individually and collectively considered meaningful combinations A-E, A-F, B-D, B-E , B-F, C-D, C-E and C-F are not listed separately, they are considered described. Likewise, any subset of these combinations is described. So, for example, a subgroup of A-E, B-F and C-E can be considered described. This applies to all aspects of the present application, including but not limited to the steps of the methods for preparing and using the described compositions. Thus, if there are many additional steps that can be performed, it should be understood that each of these additional steps can be performed with any particular implementation or combination of implementations of the described methods.

41. Disclosed are methods of treating bone marrow cancer and congenital bone marrow cancer by adoptive NK cell transfer and/or methods of transferring NK cells to the bone marrow using one or more of cytokines (e.g., IL-12, IL-15, and /or IL-19) in combination with a vesicle containing an NK cell effector agent, eg PM21 particles, feeding FC21 cells and/or EX21 exosomes. It should be understood, and is intended herein, that it is advantageous to present the components used in the methods described in a package that would enable a person to perform the methods described.

Thus, in one aspect, the present invention provides kits for the treatment of bone marrow malignancies and congenital bone marrow malignancies using NK cells containing one or more cytokines (e.g., IL-2, IL-12, IL-15 and/or IL-18) and one or more of the vesicles containing the NK cell effector agent. In one aspect, said vesicle may be PM21 particles, EX21 exosomes, and/or FC21 feeder cells. For example, the kits described may contain IL-12 and PM21 particles; IL-15 and PM21 particles; IL-18 and PM21 particles; IL-12 and exosomes EX21, IL-15 and exosomes EX21; IL-18 and exosomes EX21; IL-12 and feeding cells FC21; IL-15 and feeding cells FC21; IL-18 and feeding cells FC21; IL-12, IL15 and PM21 particles; IL-12, IL-18 and PM21 particles; IL-15, IL-18 and PM21 particles; IL-12, IL-15, IL-18 and PM21 particles; IL-12, IL15 and exosomes EX21; IL-12, IL-18 and exosomes EX21; IL-15, IL-18 and exosomes EX21; IL-12, IL-15, IL-18 and exosomes EX21; IL-12, IL15 and feeding cells FC21; IL-12, IL-18 and feeding cells FC21; IL-15, IL-18 and feeding cells FC21; IL-12, IL-15, IL-18 and FC21 feeding cells; IL-12, EX21 exosomes and PM21 particles; IL-15, EX21 exosomes and PM21 particles; IL-18, EX21 exosomes and PM21 particles; IL-12 feeding FC21 cells and PM21 particles; IL-15 feeding FC21 cells and PM21 particles; IL-18 feeding FC21 cells and PM21 particles; IL-12 feeding FC21 cells and EX21 exosomes; IL-15 feeding FC21 cells and EX21 exosomes; IL-18 feeding FC21 cells and EX21 exosomes; IL-12 feeding FC21 cells, PM21 particles and EX21 exosomes; IL-15 feeding FC21 cells, PM21 particles and EX21 exosomes; IL-18 feeding FC21 cells, PM21 particles and EX21 exosomes; IL-12, IL15, EX21 exosomes and PM21 particles; IL-12, IL-18, EX21 exosomes and PM21 particles; IL-15, IL-18, EX21 exosomes and PM21 particles; IL-12, IL-15, IL-18, EX21 exosomes and PM21 particles; IL-12, IL15, feeding FC21 cells and PM21 particles; IL-12, IL-18, feeding FC21 cells and PM21 particles; IL-15, IL-18, feeding FC21 cells and PM21 particles; IL-12, IL-15, IL-18, feeding FC21 cells and PM21 particles; IL-12, IL15, EX21 exosomes and FC21 feeding cells; IL-12, IL-18, EX21 exosomes and FC21 feeding cells; IL-15, IL-18, EX21 exosomes and FC21 feeder cells; IL-12, IL-15, IL-18, EX21 exosomes and FC21 feeder cells; IL-12, EX21 exosomes, feeding FC21 cells and PM21 particles; IL-15, EX21 exosomes, feeding FC21 cells and PM21 particles; IL-18, EX21 exosomes, feeding FC21 cells and PM21 particles; IL-12, IL15, EX21 exosomes feeding FC21 cells and PM21 particles; IL-12, IL-18, EX21 exosomes feeding FC21 cells and PM21 particles; IL-15, IL-18, EX21 exosomes feeding FC21 cells and PM21 particles; or IL-12, IL-15, IL-18, EX21 exosomes feeding FC21 cells and PM21 particles.

42. It should be understood, and is intended herein, that NK cell effector agents contained in vesicles (e.g., PM21 particles, EX21 exosomes, and/or FC21 feeder cells) can be selected from the NK cell effector agent group consisting of 4 -1BBL, IL-2, IL-21, MICA/B, ULBP2, ICAM-1, 2B4, BCM1/SLAMF2, CD155, CD112, CCR7, DAP12, Notch ligands and DAP10.

43. It is to be understood, and is intended herein, that the disclosed kits or devices may contain cytokines in addition to IL-12, IL-15 and/or IL-18. Accordingly, in one aspect, kits are provided for methods of treating bone marrow malignancies and congenital bone marrow malignancies via adoptive NK cell transfer and/or methods for transferring NK cells to the bone marrow, further comprising 4-1BBL, IL-2, IL- 12, IL-18, IL-21, MICA/B, ULBP2, ICAM-1, 2B4, BCM1/SLAMF2, CD155, CD112, CCR7, DAP12 and DAP10.

44. In one aspect of the present invention, it is contemplated that the disclosed kits or devices can be used with NK cells derived from a donor source, including NK cells derived from an unselected population of peripheral blood mononuclear cells. In some cases, the donor source of NK cells used in the described kits for the treatment of malignant neoplasms of the bone marrow and congenital malignant neoplasms of the bone marrow may also be a recipient for NK cells. Accordingly, NK cells may be derived from an autologous source. In other cases, the donor source of NK cells may be a haploidentical or allogeneic donor source.

45. In addition, this document suggests that there are instances where it is useful to present NK cells in a kit or device. Accordingly, in one aspect, the present invention provides kits for the treatment of bone marrow cancer further comprising NK cells or an NK cell line.

1. Pharmaceutical carriers/delivery of pharmaceutical products

46. As described above, the compositions can also be administered in vivo with a pharmaceutically acceptable carrier. By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i. said material can be administered to a subject along with the nucleic acid or vector without any undesirable biological effects or harmful interaction with any of the other components of the pharmaceutical composition in which it is contained. The carrier, of course, should be chosen so as to minimize degradation of the active ingredient and undesirable side effects in the subject, well known to the person skilled in the art.

47. The compositions can be administered orally, parenterally (eg, intravenously), intramuscularly, intraperitoneally, transdermally, extracorporeally, topically, and the like, including topical intranasal administration or administration in a form for inhalation. As used herein, the term "topical intranasal administration" means delivery of compositions to the nose and nasal passages through one or both nostrils, and may include delivery by nebulization or droplet delivery, or aerosolization of the nucleic acid or vector. Administration of compositions in inhalation form may be via the nose or mouth via delivery via a spray or drip mechanism. In addition, delivery can also be made directly to any area of the respiratory system (eg lungs) via intubation. The exact number of compositions required varies from subject to subject, depending on the species, age, weight, and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its route of administration, and the like. Thus, it is not possible to specify the exact amount of each composition. At the same time, an appropriate amount can be determined by a person skilled in the art using routine experimentation, taking into account the information provided herein.

48. When parenteral administration of the composition is used, it is usually characterized by injection. Formulations for injection may be prepared in conventional forms, as liquid solutions or suspensions, as solid forms suitable for dissolving the suspension in a liquid prior to injection, or as emulsions. A recently revised approach for parenteral administration includes the use of a slow or sustained release system to maintain a constant dose. See, for example, US Pat. No. 3,610,795, incorporated herein by reference.

49. Materials may be in solution, suspension (eg, may be incorporated into microparticles, liposomes, or cells). They can target specific cell types through antibodies, receptors, or receptor ligands. The following sources are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, K.D., Br. J. Cancer, 60:275- 281 (1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother., 35:421-425, (1992), Pietersz and McKenzie, Immunolog. Reviews, 129:57-80, (1992), and Roffler, et al., Biochem. Pharmacol, 42 :2062-2065, (1991)). This includes carrier media such as "stealth" carriers and liposomes conjugated to other antibodies (including lipid-mediated drug targeting of colon carcinoma), receptor-mediated targeting of DNA via cell-specific ligands, targeted targeting of lymphocyte-mediated tumor and highly specific therapeutic targeting of retroviruses on mouse glioma cells in vivo. The following sources are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al., Cancer Research, 49:6214-6220, (1989); and Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187, (1992)). As a rule, receptors, both constitutive and ligand-induced, are involved in endocytosis pathways. These receptors cluster in clathrin-coated pits, enter the cell through clathrin-coated vesicles, pass through an acidified endosome where the receptors are sorted, and then return to the cell surface, stored within the cell, or degraded in lysosomes. Internalization pathways are used for various functions such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, ligand dissociation and degradation, and regulation at the receptor level. Many receptors are used in more than one intracellular pathway, depending on cell type, receptor concentration, ligand type, ligand valency, and ligand concentration. The molecular and cellular mechanisms of receptor-mediated endocytosis have been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)).

a) Pharmaceutically acceptable carriers

50. Compositions containing antibodies may be used therapeutically in combination with a pharmaceutically acceptable carrier.

51. Such carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995. Generally, a suitable amount of a pharmaceutically acceptable salt is used in the formulation to render the formulation isotonic. Examples of a pharmaceutically acceptable carrier include, but are not limited to, saline solution, Ringer's solution, and dextrose solution. The pH of the solution is preferably from about 5 to about 8, more preferably from about 7 to about 10. Further carriers include sustained release formulations, for example, molded semi-permeable matrices of solid hydrophobic polymers, for example, films, liposomes, or microparticles. Those skilled in the art will appreciate that certain carriers may be more preferred depending on, for example, the route of administration and the concentration of the composition administered.

52. Pharmaceutical carriers are known to those skilled in the art. Typically, they are standard vehicles for administering drugs to humans, including solutions such as sterile water, saline, and physiological pH buffered solutions. The compositions can be administered intramuscularly or subcutaneously. Other compounds can be administered in accordance with standard procedures used by those skilled in the art.

53. Pharmaceutical compositions may contain carriers, thickeners, diluents, buffers, preservatives, surface active agents, and the like. in addition to the chosen molecule. Pharmaceutical compositions may also include one or more of the active ingredients, for example, antimicrobial agents, anti-inflammatory agents, anesthetics, and the like.

54. Pharmaceutical compositions can be administered in a variety of ways depending on the preference for topical or systemic application and the area being treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal), oral, inhalation, or parenteral, for example, by intravenous drip, subcutaneous, intraperitoneal, or intramuscular. Described antibodies can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, into the body cavity or transdermally.

55. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffer media. Carrier media for parenteral delivery include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution, or fixed oils. Intravenous vehicles include liquid and nutritional supplements, electrolyte supplements (eg Ringer's dextrose supplements), and the like. Preservatives and other additives may also be present, for example, antimicrobials, antioxidants, chelating agents, inert gases, and the like.

56. Topical formulations may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Use of standard pharmaceutical carriers, water, powder or fat bases, thickeners, etc. may be necessary or desirable.

57. Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous vehicles, capsules, sachets or tablets. It may be desirable to use thickeners, flavors, diluents, emulsifiers, dispersants or binders.

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

b) Therapeutic use

59. Effective dosages and regimens for administering compositions can be determined empirically and such determinations can be made by those skilled in the art. Dosage ranges for administering the compositions are ranges sufficient to obtain the desired effect influencing the symptoms of the disorder. The dosage should not be so high as to cause unwanted side effects such as unwanted cross-reactions, anaphylactic reactions, and the like. As a rule, the dosage depends on the age, condition, sex and severity of the disease in the patient, the route of administration and the inclusion of other drugs in the regimen; it can be determined by a person skilled in the art. In case of contraindications, the dosage can be adjusted by the attending physician. The dosage may vary and may be administered in one or more doses daily for one or more days. In the literature, guidance can be found on appropriate dosages for these classes of pharmaceutical products. For example, guidance on the selection of appropriate doses of antibodies can be found in the literature on the therapeutic use of antibodies, for example, Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, N.J., (1985) ch. 22 and pp. 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York (1977) pp. 365-389. A typical daily dose of a single antibody used may range from about 1 μg/kg to 100 mg/kg or more per day, depending on the factors mentioned above.

D. Examples

60. The following examples are presented to provide those skilled in the art with a complete description of the methods for preparing and evaluating the compounds, compositions, articles, devices and/or methods presented herein, are intended solely for use as examples and are not intended to limit the invention. Efforts have been made to ensure the accuracy of numerical values (eg quantities, temperatures, etc.), but the possibility of errors and deviations should be taken into account. Unless otherwise indicated, parts are parts by weight, temperatures are in °C or room temperature, and pressures are at or near one atmosphere.

1. Example 1: PM21 particles stimulate NK cell proliferation in vivo

61. Natural killer (NK) cells are a component of the innate immune system, identified as CD56 ⁺ CD3 ^- , and can naturally recognize and lyse cells damaged by viruses or that are malignant. NK cell therapy is a promising cancer treatment, and numerous clinical trials are currently underway for the treatment of various types of cancer (AML, lymphomas, breast, ovarian, neuroblastoma, non-small cell lung cancer). For effective anti-cancer therapy using NK cells, three general aspects must be considered: 1) a sufficiently large dose of NK cells must be delivered; 2) NK cells should have high cytotoxicity; and 3) NK cells must reach, possibly locate at the disease site, persist, and specifically target tumor cells.

62. For clinical efficacy in the setting of AML, Miller and colleagues recommended reaching a dose that would provide at least 100 NK cells per µl of peripheral blood (PB) two weeks after infusion. In some instances where NK cell adoptive therapy has been effective, more than 1000 NK cells per µl of PB have been observed. These observations highlight the importance of efficient ways to expand NK cells to deliver a sufficient dose for overall treatment efficacy.

63. There are currently three different clinically used NK cell expansion strategies for adoptive cell therapy. First, in vivo expansion using cytokines such as IL-15 and IL-2, in combination with lymphocyte depletion/host irradiation, can stimulate in vivo expansion with relatively few donated NK cells. Secondly, ex vivo methods using cytokines, mainly IL-2 and IL-15, can activate NK cells, although reproduction occurs at a relatively low and variable level. In addition, ex vivo cytokine-activated NK cells are not affected by cytokines after infusion and undergo apoptosis. Third, methods for expanding NK cells ex vivo using nurse cells use co-cultivation with other cells that have a stimulatory effect. Methods for stimulating NK cells using nurse cells include the use of Epstein-Barr virus-infected LCL cells or recombinant tumor cells. Co-cultivation with K562 CML cells expressing membrane-bound IL-15 (mb15) and ligand 4-1BB (41BBL) (K562-mb15-41BBL) can expand NK cells several hundredfold in about two weeks, but NK cells propagated using this method are subject to aging. In addition, IL-15 activated NK cells lose surface CD16 due to the proteolytic activity of ADAM17. K562 cells expressing mb21 instead of mb15 significantly improve NK cell proliferation by avoiding telomere shortening and consequent NK cell senescence. Expanding NK cells using K562-mb21-41BBL is very efficient and typically achieves an average of 48,000-fold expansion with >85% enrichment in three weeks. All of these methods are being actively studied in clinical trials.

64. Although methods for the expansion of NK cells are improving, there are still shortcomings and problems at present. Survival of injected NK cells requires a high toxic dose of IL-2 regardless of propagation mode, although NK cell resistance is limited. While ex vivo propagation methods using nurse cells are effective in propagating to produce large numbers of NK cells, concerns have been raised that long-term ex vivo culture of NK cells causes loss of homing ability in a diseased area, such as the bone marrow. Thus, there is a debate about the overall benefits of in vivo or ex vivo propagation. An optimal procedure for propagating NK cells would be a proliferative method, such as the ex vivo feeder cell method, which could be performed both ex vivo and in vivo.

65. A novel method for the rapid and selective propagation of cytotoxic NK cells, starting from PB mononuclear cells (MPCs), is based on PM21 particles. Particles corresponding to closed plasma membrane vesicles were obtained from the plasma membrane of K562-mb15-41BBL cells (PM15 particles); these particles provided 250-fold selective expansion of NK cells after 14 days and 1265-fold after 17 days, which is comparable to the expansion efficiency using K562-mb15-41BBL feeder cells in co-culture. NK cells activated with PM15 particles, similarly to NK cells expanded with feeder cells, had high cytotoxicity against CML and AML cells ex vivo. It is important to note that these particles have many advantages over feeder cell methods. First, they can be obtained in advance, tested and stored for more than a year and used as a “ready reagent” without being limited to one GMP-compliant site, which greatly simplifies the clinical logistics of adoptive NK cell therapy. Secondly, the use of PM particles instead of nurse cells to stimulate NK cells eliminates the steps necessary for safety when using tumor-derived nurse cells, such as irradiating the nurse cells and checking for their presence and proliferation in the finished product. Thirdly, tumor-derived nurse cells cannot be administered as adjuvant therapy, while PM particles can be administered to stimulate NK cell expansion in vivo. The advantages of the PM particle-based NK cell expansion method result in significant clinically beneficial properties.

66. In this study, we tested the efficacy of PM particles derived from K562-mb21-41BBL to expand in vivo adoptively transferred NK cells pre-activated in a relatively short and simple procedure that is easily implemented in the clinical setting. This method overcomes the shortcomings of previous studies with intravenous infusion of adoptive NK cells, which provided only minimal expansion and limited resistance of NK cells in vivo. This study demonstrates the expansion efficiency of NK cells stimulated with PM21 particles ex vivo and the expansion of NK cells in vivo from unselected PBMCs injected into the peritoneal cavity used as the site of in situ incubation and stimulation with PM21 particles. It is expected that this method will be suitable for expansion of NK cells in vivo in therapeutically relevant quantities and will increase the availability of NK cell-mediated immunotherapy for patients.

a) MATERIALS AND METHODS

(1) Human samples

67. Primary leukemic blasts were obtained during active disease from patients who signed informed consent approved by the ESU; these patients received a comparable amount of RV during remission. A source of leukocytes (One Blood, Orlando, FL, USA) or fresh blood from healthy volunteers who signed an ESA-approved informed consent were used as normal samples. MICs were isolated using Ficoll-Paque (GE Healthcare, Pittsburgh, PA, USA). All samples were anonymized and stored in a frozen viable state.

(2) Reagents and cell lines

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

(3) Acquisition and characterization of plasma membrane particles

69. PM particles were obtained from K562-mb21-41BBL cells. Cells were grown in RPMI-1640 medium supplemented with 5% fetal bovine serum. Cells were collected by centrifugation (1000 x g, 10 minutes), washed with DPBS containing 2 mm EDTA. Cells were resuspended in lysis buffer containing 50 mM HEPES, pH 7.4, 150 mM NaCl, 2 mM MgCb and AEBSF, aprotinin, leupeptin, and pepstatin A. Cells were disrupted by nitrogen cavitation at 300 psi. inch for 30 minutes at 4°C (Parr Instruments, Moline, IL, USA). The cell lysate was centrifuged (1000 x g, 10 minutes), then the supernatant was centrifuged (100,000 x g) to precipitate untreated cell membranes. The untreated membranes were then purified by sucrose gradient centrifugation and a fraction corresponding to closed plasma membrane vesicles was collected. All procedures were performed in compliance with asepsis rules, the sterility of the product was checked in culture. The production of PM particles was quantified by protein concentration in the BCA assay and reported in μg membrane protein/mL. The presence of IL-21 and 41BBL in the PM particles was confirmed by solid phase ELISA and Western blotting.

(4) Propagation of NK cells from PBMCs ex vivo

70. NK cells were expanded from PBMC using PM21 particles. Briefly, MICs were seeded at 0.1×10 ⁶ NK cells/ml in SCGM (CellGenix, Portsmouth, NH, USA) supplemented with 10% FBS, 2 mM Glutamax, 100 U/ml IL-2 (Peprotech, Rocky Hill, NJ, USA) and 200 μg/ml PM21 particles. The supplemented medium was changed routinely every 2-3 days after day 5.

(5) NK cell cytotoxicity assays in an autologous patient

71. Cytotoxicity assay of patient-derived NK cells against autologous AML tumor cells was analyzed using annexin V (BD Bioscience). NK cells expanded for 16 days (NK cell content >90%) were stained with TFL4 dye. Tumor target cells were co-cultured at a rate of 0.5×10 ⁶ CD34 ⁺ cells/ml with NK cells in E:T ratios of 1:1, 2:1, 5:1 and 10:1 for 2 hours at 37° C, in an atmosphere of 5% CO ₂ . The cells were then centrifuged and resuspended in Annexin V labeling buffer containing V-FITC annexes, anti-CD34-PE antibody and anti-CD56-PC7 antibody, and incubated for 15 minutes at 4°C. Labeled cells were diluted to a volume of 250 μl and analyzed by flow cytometry on an Accuri instrument (BD Bioscience).

(6) In vivo expansion of NK cells in NSG mice

72. MICs freshly thawed or previously activated for two days with 200 µg/ml PM21 and 100 U/ml IL-2 were washed twice and resuspended in RPMI medium without phenol red. 1×10 ⁵ NK cells in a cell suspension of total PBMCs were administered ip to NSG mice (NOD-SCIN IL-2R-gamma ^null ). In addition, particles of PM21 (numbers indicated in the designations to the figure, twice a week) and IL-2 (1000 units, three times a week) were administered i.p. and RV was collected by buccal bleeding or cardiac puncture. Organs were harvested at autopsy and perfused to obtain single cell suspensions for analysis.

b) RESULTS

(1) Propagation of NK cells obtained from healthy donors and leukemia patients, ex vivo and in vivo.

73. Because it has been reported that K562 cells engineered to express mbIL21 have better NK cell expansion efficiency without senescence, PM particles were obtained from K562-mb21-41BBL cells and designated as PM21 particles. PM21 particles were characterized by size and consistency of mbIL21 content (Figure S1) and tested for NK cell expansion.

74. PBMCs were cultured with PM21 particles (200 µg/ml) for 28 days. NK cells stimulated with PM21 particles were expanded, and by day 14, the content of NK cells in cultures of NK cells stimulated with PM21 particles reached >90% (figure 1AB). A pooled analysis of NK cell expansion at day 14±1 of culture showed that PM21 particles (mean 825-fold expansion, range 163-2216, n=13) were significantly (p=0.021) more efficient than PM15 particles (mean 424-fold expansion, range 290-570, n=30) (FIG. 1C). In addition, NK cells stimulated with PM21 particles proliferated exponentially over 28 days, reaching more than 100,000-fold expansion, in contrast to the expansion of NK cells with PM15 particles, which ceased at day 22 of culture due to aging. Thus, PM21 particles have improved NK cell expansion efficiency compared to PM15 particles, and expansion of NK cells with PM21 particles was comparable to the expansion described for K562-mb21-41BBL feeder cells from which PM21 particles were derived. . PM21 expanded NK cells also showed cytotoxicity against leukemic cell lines (FIG. S2).

75. The ability of PM21 particles to stimulate NK cell proliferation was further tested with MICs obtained from leukemia patients in remission. PM21 particles relatively efficiently induced NK cell expansion in all three patient-derived samples during 14 days of culture (113 ± 7-fold expansion for F021, 810 ± 81-fold expansion for M038, and 352 ± 86-fold expansion for M050, figure 1D). Reproduction was specific to NK cells, with the percentage of NK cells predominantly increasing relative to the total number of hCD45 ⁺ cells (figure 1E). For sample F021, cytotoxicity of expanded NK cells was tested under autologous conditions against tumor blasts obtained from the same patient during active disease (FIG. 1F). At a relatively low ratio of effectors and targets (E:T) 1:1, 78±3% of tumor cells underwent apoptosis. Thus, this method can be applied in the context of autologous transplantation.

76. The ability of PM particles to promote in vivo proliferation when used as an injectable formulation is unprecedented. To test the ability of PM21 particles to stimulate NK cell proliferation in vivo and determine the need for ex vivo pre-activation, NSG mice were injected ip with 0.1 x 10 ⁶ NK cells in untreated PBMC or PBM pre-activated with PM21 particles (PM21-MPC). In mice injected with non-activated PBMCs, there was a small number of human NK cells (hNK) in the RV, and only the number of hT cells increased as a percentage of the total number of hCD45 ⁺ cells within 15 days after injection (figure 2AB). In contrast, the RV of mice injected with PM21-MPC was found to contain an increased amount of hNK cells, which peaked 12 days after ip injection (FIG. 2CD). The content of NK cells was enriched to 53±8% of the number of hCD45 ⁺ cells. In the same experiment, we tested the effectiveness of i.p. administration of PM21 particles in vivo in relation to stimulation of proliferation of NK cells in vivo. In mice treated with conventional PBMC, additional PM21 particles did not stimulate hNK cell proliferation in vivo. However, administration of PM21 particles in vivo to mice transplanted with PM21-MPC had an effect when the number of hNK cells was higher compared to mice with PM21-MPC that did not receive PM21 particles in vivo (Figure 2D).

77. To confirm that PM21 particles induce NK cell proliferation in vivo, an assay was performed with hNK cells labeled with CTViolet and expanded in vivo 6 days after ip inoculation. Mice cells injected with non-activated PBMCs showed a slight to very slight decrease in CTViolet fluorescence, indicating no or very little division of NK cells (FIG. 3AB). hNK cells from mice injected with PM21-MPK showed a significant decrease in CTViolet fluorescence intensity (FIG. 3CD). Approximation of the fluorescence intensity showed that the decrease in intensity correlates with the main population, in which 7 cell divisions occur in vivo within 6 days. For hNK cells obtained from mice that received ip injections of PM21 particles, another division was observed. This additional doubling upon administration of PM21 particles in vivo correlates with the higher number of NK cells observed in RV with PM21 particles in vivo.

78. To further test the ability of PM21 particles in vivo to stimulate proliferation of NK cells in vivo, dose-response effects of PM21 particles in vivo were studied (FIG. 4). A dose-dependent increase in the number of hNK cells in RV was observed at doses ranging from 0 to 800 μg of PM21 particles per injection (Figure 4E). At a dose of 800 μg (corresponding to approximately 100 ng mbIL21), 470 ± 40 hNK cells per μl of RV were observed 12 days after ip injection of PM21-MPC. This concentration of NK cells in RV was 5 times the concentration that is generally considered therapeutically effective in the setting of AML. The dose dependent effect on in vivo proliferation was specific to hNK cells, with no significant increase in T cell numbers observed (Figure 4E). At a higher amount of 1600 μg per injection, the number of hNK cells in RV decreased similar to the effect observed ex vivo, where a dose of ~200-400 μg/ml was optimal for PM21 or PM15 particles, and higher numbers attenuated the expansion of NK cells.

79. The observation of a significant number of hNK cells in the RV indicates that hNK cells proliferating from PM21-MPC administered via ip injection can migrate from the peritoneal cavity to the RV. To ensure that adoptively transferred hNK cells can migrate to potential disease foci, hNK cells were quantified in various organs (Figure 5). Human NK cells were found in every organ examined, and all organs of mice treated with 800 μg of PM21 particles in vivo, except for the liver, had a significantly increased number of hNK cells (p<0.05). In addition, in the organs of mice treated with 800 μg of PM21 particles, the percentage of hNK cells was increased as a proportion of the total number of hCD45 ⁺ cells.

80. The mouse studies described herein have shown that a procedure combining a brief pre-activation with PM21 particles ex vivo and administration of PM21 particles in vivo induces a pronounced expansion of NK cells in vivo, potentially in a therapeutically relevant range. To demonstrate the consistency required for clinical application, the procedure was applied to sources of leukocytes from three different donors (not those used in other experiments) (figure 6). The average number of hNK cells in both the RV and the abdominal cavity was relatively the same in different sources of leukocytes. The percentage of hNK-, hT-cells and other hCD45 ⁺ cells was also constant for mice in the group treated with PM21-BMC from a specific source of leukocytes (n=3), as well as between sources of leukocytes L8 and L10.

(2) Phenotype of NK cells propagated by PM21 particles.

81. Antitumor cytolytic activity of NK cells was determined by the balance of stimuli from activating and inhibitory signals. In this paper, we performed a detailed comparative examination of NK cells stimulated with PM21 particles: 1) expanded ex vivo with PM21 for 12 days, 2) expanded in vivo and isolated from RV, and 3) expanded in vivo and isolated from abdominal washings. cavities (AW). These comparisons were performed in parallel using cells from a single donor under all conditions (Figure S3).

82. The presence of CD 16 (Feγ receptor) on NK cells is required for effective antibody-dependent cytotoxicity (ADCC). Almost all NK cells expanded in vivo showed CD 16 expression (97% and 87% in PB and AW, respectively). CD94 is a surface receptor that forms heterodimeric complexes with NKG2C or NKG2A. Approximately half of the NK cells expanded ex vivo expressed CD94. For NK cells expanded in vivo, cells from AW (64±9%) were characterized by higher expression than NK cells from PB (38±13%). Both receptors of the NKG2 family bind to CD94, with NKG2C being the activating receptor and NKG2A being the inhibitory receptor. Ex vivo expanded NK cells were characterized by relatively low expression of NKG2C, but expression on AW NK cells was higher (53±8%) and even higher than on PB NK cells (61±2%). The proportion of NK cells expressing NKG2A was higher in AW (82±8%) compared to PB (67±12%) and in cells expanded ex vivo (74%). NKG2D is another important activating receptor found on NK cells and its expression was found on 61±6% NK cells from AW, 26±3% from PB, and approximately 75% of ex vivo expanded NK cells. Expression of CD62L, which is known to correlate with bone marrow homing, was higher for NK cells in RV (63±10%) and lower for NK cells in AW (39±14), which is consistent with increased expression in mobilized cells. NKp44 and NKp46 are members of the natural cytotoxicity receptor family and play a role in NK cell mediated cytolysis. NKp46 was expressed on NK cells from both PB (76±9%) and AW (89±5%). NKp44 was relatively weakly expressed on these NK cells from all sources. On the other hand, NKp46 was well expressed in both RV (89±5) and AW (76±9). TRAIL is a ligand on NK cells that induces target apoptosis via the cell death receptor pathway. TRAIL was expressed on 36±6% of AW NK cells, 20±4% of PB, and 26% of ex vivo expanded cells. KIR2D is an immunoglobulin-like killer receptor (KIR) subtype 2D; a minority (approximately 1/3) of NK cells expanded in vivo or ex vivo expressed KIR2D. The proportion of CD8 and CD4 T cells was analyzed and it was found that CD8 T cells in in vivo samples were more numerous than CD4 T cells. In addition, the presence of NK-suppressive Treg cells was examined; their numbers in in vivo samples were very low (<0.1% of all CD3 ⁺ cells).

c) DISCUSSION

(1) PM21 particles facilitate the expansion of NK cells ex vivo and in vivo to a therapeutically significant amount.

83. Adoptive therapy using NK cells has great promise as a way to treat cancer in the initial treatment and maintain remission of various tumors. The requirement for the therapeutic use of NK cells is a method for rapid and selective expansion of NK cells that is safe, simple, and generally therapeutically effective. Several cytokine and nurse cell based methods are currently being clinically tested, and the methodology using the K562-mb21-41BBL cell line is one of the most efficient for expanding NK cells ex vivo. While feeder cell methods effectively provide a high initial dose and may allow multiple administrations, this may affect the ability of ex vivo expanded NK cells to homing in the bone marrow, which is important for the treatment of leukemia, and the stability of NK cells administered by in vivo infusion may not be optimal. The combined ex vivo and in vivo NK cell propagation method based on PM21 particles described herein can significantly improve the efficacy of NK cell adoptive therapy.

84. Importantly, PM21 particles can be used for in vivo stimulation to induce proliferation and persistence in vivo. The methodology developed herein used a short ex vivo pre-activation of 2 days followed by in vivo administration of PM21 particles. The use of PM21 particles in vivo causes increased proliferation of NK cells in vivo depending on the dose of PM21 particles used in vivo. With the current optimized procedure, an average 360-fold increase in the number of in vivo NK cells in RV was observed from days 5 to 12 after i.p. injection of PM21-MPK and, possibly, a higher multiplication rate in the abdominal cavity. In comparison, a recent study showed that after an IV infusion of 1-2×10 ⁶ NK cells, only 5-17 NK cells per µl of blood were observed on day 14 post-infusion. In contrast, in this study using PM21 particle stimulation, >400 NK cells/µL of blood were detected on day 12 after ip infusion of 2.0×10 ⁶ PM21-MIC (11%, i.e. 0.2× 10 ⁶ NK cells). In addition, an earlier study used 5 mcg (~50,000 units) per injection (three times a week) of either IL-2 or IL-15, while this study used a relatively low dose of IL-2 (1,000 units/injection three times in Week). In another study, 30 x 10 ⁶ NK cells, preferably expanded ex vivo with K562-mb15-41BBL nurse cells, were injected iv, followed by monitoring of injected human lymphocytes using an anti-CD45 antibody (rather than a combination of an anti-CD56 antibody and an anti-CD56 antibody). CD3). With this method, a high dose of IL-2 (25,000 units/day) is necessary to maintain the number of lymphocytes, and the concentration of NK cells was not determined, but rather implied. Compared to these previous methods, the amount of NK cell proliferation stimulated by PM21 particles in vivo is unprecedented and represents the unique ability of PM21 particles.

85. Here, delivery of PM21-MPC to NSG mice was performed by ip injection, similar to previous preclinical studies. Compared to these previous studies, the PM21 particle method is advantageous in several respects. First, the combined ex vivo pre-activation and in vivo stimulation with PM21 particles allows the use of much fewer unselected PBMCs compared to cytokine activation of isolated NK cells, requiring large numbers of lymphocytes to be harvested by apheresis followed by extensive laboratory processing to enrich NK cells. . Second, the PM21 particle method requires only a brief pre-activation of 2 days instead of 2 weeks of propagation in culture, which may provide better retention of physiologically relevant functionality. Thirdly, the current method can significantly enhance in vivo expansion compared to previous methods that do not provide expansion or maintenance of cell numbers in vivo without the use of a high dose of IL-2 associated with clinical toxicity. In intraperitoneal tumors, the advantages of the current described method can significantly enhance the overall antitumor effect. In the absence of intraperitoneal tumors, the peritoneal cavity is a suitable medium to retain PM21 particles in its volume and promote successful reproduction in vivo, which is clearly shown in the proliferation assay with CTViolet, and then NK cells can migrate in significant numbers to the RV and organs. When using PM21 particles in vivo, NK cells were not only observed in RVs, but were also found in organs, and were also more numerous. The number of NK cells in the bone marrow was comparable to that in a study using NK cells derived from cord blood CD34 ⁺ stem cells, indicating that these NK cells are capable of homing in the bone marrow.

86. Phenotyping of NK cells propagated ex vivo or in vivo in parallel (Figure S3) showed that the resulting cells were similar regardless of approach. Interesting differences were observed in relation to the expansion of NKG2A ^- and NKG2C ⁺ subpopulations, which were mainly observed for NK cells expanded in vivo, but not ex vivo. Populations of NKG2C NK cells have been observed during viral reactivation associated with an immune response similar to that mediated by memory cells; recently shown that they depend on the production of IL-12 by monocytes. The presence of NKG2C ⁺ NK cells in patients with CMV reactivation after stem cell transplantation for the treatment of AML was also associated with a better outcome and a lower recurrence rate. In addition, the existence of a significant population of NKG2A ^- NK cells resistant to HLA-E-induced inhibition - may be important in the treatment of patients with multiple myeloma, where the cells suppress HLA class I but express HLA-E to elude the NK cell response. . Approaches aimed at suppressing NKG2A have been proposed as a means of improving the cytotoxicity of NK cells and hence their therapeutic potential. Since the cells expanded ex vivo were mainly NKG2A ⁺ , shortening the ex vivo culture time followed by in vivo expansion may provide an additional advantage in generating NK cells with increased phenotypic diversity and potentially high target cytotoxicity.

(2) Potential clinical value of PM21 particles.

87. The ability of PM21 particles to stimulate the proliferation of NK cells may lead to the expansion of adoption therapy using NK cells for the treatment of cancer and possibly other diseases. PM21 particles are easy to replace with nurse cells, which are currently used in clinical trials to simplify logistics and reduce risks. In regulatory areas where the use of tumor-derived nurse cells is prohibited or difficult due to regulatory difficulties, PM21 particles represent a turnkey solution for ex vivo expansion and activation. To use PM21 particles for ex vivo expansion under allogeneic conditions, T cell depletion can be induced prior to ex vivo expansion of NK cells. Current clinical studies of NK cells grown with K562-mbIL21 use T cell depletion prior to NK cell expansion to eliminate allogeneic T cells that can cause GVHD. Moreover, in vivo administration of PM21 particles can further promote in vivo NK cell proliferation, which is an unprecedented ability and possibly attenuates T cell proliferation, mitigating the risk of GVHD. This method of expanding NK cells can be clinically adapted for the treatment of peritoneal cancer and other abdominal tumors, such as persistent epithelial ovarian cancer or desmoplastic small round cell tumor. Currently, experiments are being carried out to evaluate antitumor efficacy in the elimination of a tumor in the abdominal cavity. PM21-MIC and PM21 particles can be used for autologous treatment, and methodologies for applying T cell depletion under allogeneic conditions are currently being studied.

88. It is important to note that NK cells propagated in this way are biologically distributed from the abdominal cavity to the peripheral blood and various organs that are potential foci of various other cancers. Although the ip route of injection is unconventional in the treatment of hematological malignancies, delivery of NK cells by this ip route leads to a concentration of NK cells in the RV, which is important for the treatment of AML.

89. A particle-based approach for regulating NK cell-specific signaling pathways could provide a platform to include other signaling molecules or even a carrier to deliver packaged agents to further target NK cell stimulation to enhance homing, antitumor cytotoxicity, and maintaining cell numbers. PM21 particles can complement to a high degree all innovative methods of NK cell-specific immunotherapy being developed (based on inhibitors of key components of the immune response, CAR, bispecific activators (BiKE), IL-2 attached to DT to deplete Treg, etc.). and beneficial effects exacerbated by in vivo expansion of NK cells by PM21 stimulation. Even as a preclinical agent, the current method may provide an unprecedented way to explore such combined methods. Of course, there are mouse-based models, but there are no other ways to study human NK cells that can be present in vivo for a significant period of time.

90. In summary, this PM21 particle procedure provides preferential expansion of NK cells in vivo at levels normally only achieved with nurse cell expansion ex vivo, but does not require cell culture with nurse cells or high doses of cytokines. that are toxic. In addition, PM21-MPC with in vivo delivery of PM21 particles can be used in autologous conditions to take advantage of the favorable synergistic effect of other immune cells on NK cell function and further combined with other strategies such as anti-KIR or BiKE antibodies to maximize cytotoxicity. NK cells. Thus, this method meets the criteria for obtaining NK cells with potential therapeutic efficacy, as well as being simple, more suitable for clinical adaptation, and may be effective in the treatment of cancer or other diseases.

2. Example 2 Transfer of NK cells to the bone marrow by stimulating fucosylation with PM21 particles

91. PM21 particles derived from K562 cells transformed to express the engineered membrane-bound form of IL-21 and 41bbl (K562.mb21.41bbl) effectively induce specific expansion of NK cells. Stimulation provided by PM21 or K562.mb21.41bbl particles used as feeder cells (FC21) in co-culture with NK cells induces complete sLex fucosylation as observed by HECA-452 mAb binding on flow cytometry.

92. NK cells were expanded with PM21 or FC21 and stained with mAb HECA452 and analyzed by flow cytometry. HECA452 specifically recognizes the fucosylated form of PSGL-1, CD44, and other E-selectin ligands. NK cells stimulated with PM21 or FC21 had a significantly higher MFI on flow cytometric analysis compared to untreated NK cells, NK cells treated with soluble cytokines, or NK cells treated with feeder cells containing only mbIL21 (but not 41bbl). This strongly suggests that stimulation with PM21 particles and/or FC21 nurse cells can stimulate transfer of NK cells to the bone marrow, and that transfer to the bone marrow of therapeutic NK cells derived from stimulation with PM21 or FC21 may be beneficial. for the bone marrow and improve the treatment of malignant neoplasms of the bone marrow.

93. Figure 7 shows the effect of soluble cytokines on HECA-452 staining of NK cells after 0, 1, 7 and 10 days of stimulation. The effect of particle or feeder cell stimulation is shown in Figure 8 where NK cells were stimulated with K562 cells or K562 cells with membrane-bound IL-21 and 41BBL (FC21 cells or PM21 particles) for 10, 12 or 14 days and stained with HECA452. NK cells stimulated with K562-derived FC21 feeder cells or PM21 particles showed marked activation compared to NK cells stimulated with K562 feeder cells without IL-21. Figures 9 and 10 show the effect of 10 days of NK cell culture under various conditions. NK cells were cultured for 10 days under various conditions as shown. The mAb HECA 452 conjugated with FITC was used as a probe for NK cells, which detected the fucosylated form of SLex. Culture cells were stained with HECA452-FITC and analyzed by flow cytometry with CD56+CD3- gated. (CSTX2 = CytoSen K562.mb21.41bbl; PC = feeder cells; PM21 = plasma membrane particles derived from CSTX2).

94. To view the effect of rest period on stimulated NK cells (Figure 11), NK cells were cultured for 10 days with CSTX2-PM21 particles (top) and then "left" in culture for 3 days after removal of PM21 particles (bottom). The mAb HECA 452 conjugated with FITC was used as a probe for NK cells, which detected the fucosylated form of SLex. Culture cells were stained with HECA452-FITC and analyzed by flow cytometry with CD56+CD3- gated. (CSTX2 = CytoSen K562.mb21.41bbl; PC = feeder cells; PM21 = plasma membrane particles derived from CSTX2).

95. NK cells stimulated with PM21 particles were stable and survived freezing/thawing with no discernible effect on the cells (Figure 12). NK cells were isolated from PBMCs (top), cultured for 10 days with CSTX2-PM21 particles (middle) and then frozen with cryopreservative and thawed (bottom). The mAb HECA 452 conjugated with FITC was used as a probe for NK cells, which detected the fucosylated form of SLex. Culture cells were stained with HECA452-FITC and analyzed by flow cytometry with CD56+CD3- gated. (CSTX2 = CytoSen K562.mb21.41bbl; PC = feeder cells; PM21 = plasma membrane particles derived from CSTX2).

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SUBSTITUTE SHEET

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Claims (12)

1. A method for transferring NK cells to the bone marrow of a subject, comprising bringing the NK cells into contact with one or more of the PM21 particles and/or FC21 feeder cells prior to transferring the NK cells to said subject and introducing the NK cells to said subject, wherein this method induces in NK cells a cellular mechanism that induces fucosylation of PSGL-1 on the surface of NK cells. 2. The method according to claim 1, characterized in that said method further comprises stimulating NK cells with IL-2, IL-12 and/or IL-18. 3. The method of claim 1, wherein said method further comprises contacting one or more of the PM21 particles and/or FC21 nurse cells with NK cells after transfer of the NK cells to the subject. 4. The method according to any one of paragraphs. 1-3, characterized in that this method induces FUT7 expression in NK cells, which correlates with PSGL-1 fucosylation on the surface of NK cells. 5. A method of treating bone marrow cancer in a subject, comprising bringing NK cells into contact with PM21 particles and/or FC21 feeder cells prior to transferring the NK cells to said subject's body and adoptively transferring NK cells to the subject's body, wherein said method induces in NK cells, a cellular mechanism that induces fucosylation of PSGL-1 on the surface of NK cells. 6. The method of claim 1 or 5, comprising contacting the NK cells with PM21 particles prior to transferring the NK cells to the subject. 7. The method of claim 6, further comprising administering the PM21 particles to the subject after transfer of the NK cells to the subject. 8. The method of claim 7, further comprising administering one or more of IL-2, IL-12, and/or IL-18 to the subject after transfer of the NK cells to the subject. 9. The method according to any one of paragraphs. 1-5, characterized in that the FC21 cells are K562 cells that express membrane-bound IL-21 and/or 4-1BB ligand. 10. The method according to any one of paragraphs. 1-8, characterized in that the PM21 particles are plasma membrane particles derived from K562 cells that express membrane-bound IL-21 and/or 4-1BB ligand. 11. The method according to any one of paragraphs. 1-8 and 10, characterized in that the PM21 particles are plasma membrane particles derived from K562 cells that express membrane-bound IL-21 and 4-1BB ligand. 12. The method according to claim 5, characterized in that said method induces in NK cells FUT7 expression correlated with PSGL-1 fucosylation on the surface of NK cells.

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