Methods and compositions for natural killer cells

Abstract

Novel compositions and methods for stimulating and generating or expanding natural killer (NK) cells are provided. [Solution] A method for increasing the number of NK cells comprises contacting NK cells with at least one NK-stimulating exosome containing one or more stimulatory peptides present in the exosome membrane, the exosomes being extracellular products of exosome-secreting cells. Also provided is a method for treating cancer by administering an effective amount of a composition containing NK-stimulatory exosomes containing stimulatory peptides.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application is in accordance with the benefits of U.S. Provisional Application No. 62 / 069,057, filed on October 27, 2014. This assertion is incorporated herein by reference in its entirety.

[0002] (Field of Invention) This application generally relates to compositions and methods comprising natural killer (NK) cells. More specifically, this application relates to the in vivo, ex-analysis of endogenous natural killer (NK) cells. In vivo or in vitro stimulation and proliferation are associated with this endogenous natural killer NK cells attack and kill cancer cells, virus-infected cells, and certain immune cells. It is possible. [Background technology]

[0003] Hematopoietic stem cell transplantation (HSCT) from siblings with matching HLA genotypes is used for hematological malignancies. It also improved the long-term survival of patients with bone marrow failure syndromes. More than 10,000 people in the United States each year... For a person, a bone marrow transplant from an unrelated donor or umbilical cord blood unit is their only hope of a cure, life They suffer from diseases that threaten their health. However, more than 70% can be benefited from allogeneic stem cell transplantation. The patient does not have a matched sibling donor. These circumstances delay treatment and result in a partially mismatched donor. - This forces us to rely on suboptimal use, which ultimately leads to graft-versus-host disease. This can lead to increased rates of GVHD, transplant failure, and relapse, all of which affect the patient's survival rate. It dramatically reduces [the problem].

[0004] Further limitations include the duration of the transplant process and the high financial, emotional, and physical burdens involved. It is brought about by the burden and. Therefore, the application of HSCT from unrelated donors is appropriate. It is limited to younger, healthier patients who have adequate socioeconomic support and are able to withstand the process.

[0005] A further challenge is the high recurrence rate resulting from the inability to eradicate residual cancer cells. Although HSCT is considered curative, the cancer recurrence rate is alarming. Therefore, a novel, more targeted immune response that is more effective and preferably does not require a matched donor. Therapy is needed. Donor for the treatment of relapsed acute myeloid leukemia (AML) after HSCT. Lymphocyte infusion (DLI) was introduced in the 1990s. This approach involves the original donor... It consisted of lymphocyte administration to AML patients with relapsed disease. However, clinical benefits The benefits are limited and observed in only a small number of patients with less tumor burden, and T cell-mediated G VHD often worsened the outcome.

[0006] There is a great need for new and improved methodologies aimed at increasing the number of NK cells. It is being done.

[0007] Further advantages of the disclosed methods and compositions are partially described below. Some of these may be understood from the description or acquired through the practice of the disclosed methods and compositions. The advantages of the disclosed methods and compositions are as specifically noted in the attached "Claims". And will be realized and achieved using combinations of these. The general explanation above and the following Both "modes for carrying out the invention" are specific examples and are for illustrative purposes only, patent This is not intended to limit the scope of the claimed invention. [Overview of the project]

Means for Solving the Problem

[0008] This specification discloses an improved technique for enhancing the activity of natural killer (NK) cells. In certain embodiments, the methods disclosed herein result in an increase in the number of NK cells. In certain embodiments, the methods disclosed herein result in NK cells with improved activity. In certain embodiments, the methods disclosed herein result in an increase in the number of NK cells with improved activity.

[0009] This specification discloses a method for increasing the number of NK cells, which includes contacting at least one NK cell with at least one NK-stimulating exosome containing one or more stimulatory peptides present in the exosome membrane. The exosome can be an extracellular product of exosome-secreting cells generated in vitro. In some cases, the exosome is secreted from feeder cells. In some embodiments, one or more stimulatory peptides present in the exosome membrane can include 4-1BBL, IL-2, IL- 12, IL-18, IL-21, MICA / B, ULBP2, ICAM-1, 2B4, B CM1 / SLAMF2, CD155, CD112, CCR7, and / or other homing receptors, DAP12, DAP10, and / or other adapter proteins. In some cases, the exosome membrane does not contain IL-15. In some embodiments, one or more stimulatory peptides present in the exosome membrane include 4-1BBL and , IL-21. The stimulatory peptides can also include one or more membrane-inserting peptides. It may be bound. The membrane-inserted peptide may have affinity for the lipid bilayer, such as CD4 or Ig. It may contain a G segment. Alternatively, the membrane-inserted peptide may contain human Fc, GPI, It may contain a transmembrane T cell receptor or pHLIP. One or more types of membrane insertions. One or more irritating peptides bound to the peptide are encoded by recombinant DNA. It may contain composite proteins.

[0010] In some embodiments, exosomes are filtration during co-culture with NK cells. It is produced by co-culture cells. One drawback of current co-culture methods is the potential for contamination. Yes, there is. However, the disclosed method does not involve direct cellular indirectness between feeder cells and NK cells. Since contact is not necessary, cells have a membrane of a size that allows exosomes to pass through. Therefore, in this specification, we will not allow the passage of exosomes. A bioreactor containing feeder cells and NK cells separated by a membrane of a certain size. The bioreactor is also disclosed. Such a bioreactor comprises multiple compartments separated by a polymer porous membrane. Alternatively, hollow fiber type with a polymer porous membrane that allows exosomes to pass through but not cells. The design of such a bioreactor may be for cell activation, cell proliferation, or cell It can be incorporated as part of a larger device or system for processing.

[0011] The exosomes used herein have been modified to improve exosome expression. It may be produced by a cell line. In some cases, the cell line may be white, such as K562 cells. These are hematopoietic cell lines. In some cases, the cell line may be one such as 4-1BBL or IL-21. Alternatively, they are engineered to express two or more stimulating peptides. Therefore, several In one embodiment, the cell line includes K562-mb21-41BBL. In the application morphology, exosomes are produced from PBMCs. In some cases, the cell line is , Epstein-Barr virus-infected cell lines such as EBV-LCL cells, or cytomega These are cell lines infected with or co-infected with lovirus.

[0012] NK cells are stimulated by NK cells in vitro, in vivo, or ex vivo. They may be in contact with sosomes or exposed to NK-stimulated exosomes. NK cells are used in allogeneic transplantation, hapsigonine transplantation, or in vivo immunotherapy procedures. NK-stimulated exosomes may be brought into contact with the exosomes. In some embodiments, allografts, hemigrates, etc. The use of NK-stimulated exosomes in transplantation or in vivo immunotherapy is for grafts. It does not cause GVHD (graft-versus-host disease).

[0013] In some aspects, NK cells are found in populations of unselected peripheral blood mononuclear cells (PBMCs). It exists. In some embodiments, whole blood or PBMCs isolated from the subject are disclosed. The exosomes are brought into contact with the NK cells ex vivo, and NK cells are proliferated within PBMCs. In some embodiments, exosomes are induced pluripotent stem cells (IPSCs), PBMCs NK cells derived from umbilical cord blood, isolated NK cell precursors, or any combination thereof. Contact. Once in contact, whole blood, PBMCs, proliferated NK cells, isolated NK cells, or NK cell products, which have depleted other lymphoid cell types, can be transfused back into the subject. For sexualization or proliferation, by exosomes of NK cells or a population of NK cells containing NK cells Any of the stimuli are applied to a standard tissue culture plate or flask, or a closed-bag system. For example, the CliniMacs Prodigy system by Miltenyi, hollow Thread device (Quantum Cell Expansion S by TerumoBCT) This can be done using a ystem, G-Rex flask (Wilson Wolf), or other apparatus. It is possible.

[0014] This specification discloses a method for treating cells susceptible to NK-mediated lysis. This method involves administering an effective amount of a composition containing contacted NK cells to the cells. Includes. Contacted NK cells contain at least one NK cell present in the exosome membrane. Contact with at least one NK-stimulated exosome containing one or more stimulating peptides Exosomes may be produced by methods including contact, and exosomes secrete exosomes It is an extracellular product of cells. In some aspects, cells are susceptible to NK-mediated lysis. These cells may be infected with a virus. Cells that are susceptible to NK-mediated lysis are these However not limited to AML, breast cancer, bladder cancer, colorectal cancer, kidney cancer, lung cancer, prostate cancer, This includes cases where malignant cells, such as those associated with cancer, are present, including thyroid cancer and uterine cancer. be.

[0015] Methods to reduce the risk of recurrence after stem cell transplantation, and effective methods including contacted NK cells. A method for adjunctive therapy, comprising administering a certain amount of a composition, is disclosed, and contacting These NK cells include at least one NK cell that is present in the exosome membrane and one or two other types. Contacting at least one NK-stimulated exosome containing the above stimulating peptide Exosomes are produced by a method that includes the production of exosomes, and are extracellular products of exosome-secreting cells. .

[0016] A method for generating NK cell-stimulated exosomes has been disclosed, and this method involves exosomes This includes embedding one or more irritating peptides within the membrane. The irritating peptides are 4-1BBL, IL-2, IL-12, IL-18, IL-21, MICA / B, ULB P2, ICAM-1, 2B4, BCM1 / SLAMF2, CD155, CD112, CC R7 and / or other homing receptors, DAP12, DAP10, and / or other homing receptors It may also contain pteroprotein r. The stimulating peptide may be optionally a membrane-inserted peptide. It may be bound to CD4. The membrane insertion peptide has affinity for the lipid bilayer. It may contain IgG. Alternatively, the membrane-inserted peptide may contain human Fc, GPI, or transmembrane peptide. It may further contain type T cell receptors or pHLIP. One or more membrane insertion receptors One or more irritating peptides bound to the plutide are fusions encoded by recombinant DNA. It may contain proteins. In some embodiments, NK cell-stimulated exosomes. These may be derived from cell lines that have been manipulated to improve exosome expression, and this process is efficient. Cell lines include cell line K562-mb15-41BBL or cell line K562-mb21-41 BBL is one example, but it is not limited to these.

[0017] A composition containing an effective amount of NK-stimulated exosomes containing one or more stimulating peptides. A method for treating cancer, including the administration of a substance, is disclosed. NK-stimulated exosomes The use may include administering NK-stimulated exosomes to the subject. In this embodiment, the use of NK-stimulated exosomes ex viv The process involves contacting NK cells with o to obtain a group of NK cells that have been in contact, and the process of contacting NK cells with o This may include administering a group of cells to the subject.

[0018] A composition is disclosed that includes NK-stimulated exosomes containing one or more stimulating peptides. The irritating peptides are 4-1BBL, IL-2, IL-12, IL-18, and IL-2 1, MICA / B, ULBP2, ICAM-1, 2B4, BCM1 / SLAMF2, CD 155, CD112, CCR7, and / or other homing receptors, DAP12, DAP 10, and / or other adapter proteins may be included. The irritant peptide is as It may selectively bind to one or more membrane-inserted peptides. It may also contain a CD4 or IgG segment that has affinity for the lipid bilayer. Alternatively, the membrane insertion peptide could be human Fc, GPI, transmembrane T cell receptor, or pHLI. It may further contain P. One or more irritating peptides bound to the membrane-inserted peptide Even if the DNA contains a fusion protein encoded with recombinant or transgenic DNA Good. NK-stimulated exosomes are manipulated to improve or generate exosome expression. It may be derived from a cell line, and this cell line may include cell line K562-mb21-41BBL Examples include, but are not limited to, derivatives of, the composition. In some embodiments, the composition The composition may further contain a pharmaceutical carrier. An effective amount of the composition for enhancing NK cells is provided to the subject. A method for treating cancer is disclosed, which involves administering the composition to the exosome membrane. At least one NK-stimulated exosome containing one or more stimulating peptides The composition contains NK cells modified with [a specific compound]. An effective amount of the composition to enhance NK cells is administered to the subject. A method for treating cancer, which includes the action of having a composition present in the exosome membrane, is disclosed. Modified with at least one NK-stimulated exosome containing one or more stimulating peptides. It contains NK cells, and the stimulating peptides are 4-1BBL, IL-2, IL-12, IL -18, IL-21, MICA / B, ULBP2, ICAM-1, 2B4, BCM1 / S LAMF2, CD155, CD112, CCR7 and / or other homing receptors, DA Contains P12, DAP10, and / or other adapter proteins. Enhances NK cells. A method for treating cancer is disclosed, comprising administering an effective amount of a composition to a subject, The product contains at least one or more irritating peptides present in the exosome membrane. The composition contains NK cells modified with a single NK-stimulated exosome, and the composition is a membrane insertion peptide. It further includes one or more irritating peptides present in the exosome membrane. This involves administering an effective amount of a composition containing one NK-stimulated exosome to a cell population. A method for the proliferation of NK cells is disclosed. One or more stimuli present in the exosome membrane An effective amount of composition containing at least one NK-stimulated exosome containing a sex peptide is injected into cells. A method for promoting NK cell proliferation, including administration to a population, is disclosed, and the stimulating peptide is 4 -1BBL, IL-2, IL-12, IL-18, IL-21, MICA / B, ULBP 2, ICAM-1, 2B4, BCM1 / SLAMF2, CD155, CD112, CCR 7 and / or other homing receptors, DAP12, DAP10, and / or other adapters - Contains protein. Administer an effective amount of the composition to enhance NK cells to the subject. A method for proliferating NK cells, including the composition, is disclosed, and the composition is one of the components present in the exosome membrane. or modified with at least one NK-stimulated exosome containing two or more stimulating peptides The composition contains NK cells, and further comprises membrane-inserted peptides. A method for promoting NK cell proliferation is disclosed, which includes administering an effective amount of a composition to a subject. The product contains at least one or more irritating peptides present in the exosome membrane. The composition contains NK cells modified with one NK-stimulated exosome, and the composition contains membrane insertion peptides Further containing tides, membrane self-inserting peptides include human Fc, GP. I, transmembrane T cell receptor, or pHLIP. An effective amount for enhancing NK cells. A method for modulating the immune system is disclosed, comprising administering a composition to a subject, wherein the composition is At least one NK cell containing one or more irritant peptides present in the exosome membrane - Containing NK cells modified with stimulated exosomes, the composition further comprises membrane insertion peptides. .

[0019] Therefore, pharmaceutical compositions containing the disclosed exosomes within a pharmaceutically acceptable vehicle The material will also be disclosed. For example, exosomes are a suitable carrier chemical component. It can be prepared as an injectable preparation containing a ponent.

[0020] The disclosed NK-stimulated exosomes and / or contacted NK cells can be used alone or to treat This includes therapeutic antibodies, cancer vaccines, immune checkpoint inhibitors, and adoptive immunotherapy (ACT). However, these may be administered to the subject in combination with other cancer immunotherapies.

[0021] This specification describes how to bring NK cells into contact with at least one NK-stimulated exosome. A method for stimulating NK cells, including the NK-stimulated exosome, has been disclosed, and the NK-stimulated exosome contains siR NK-stimulating functional nucleic acids such as NA or miRNA are used to fill the device. For example, in some cases NK-stimulated functional nucleic acids are inhibitors of A2AR, P2YR, or combinations thereof (e.g.) For example, an antagonist, expression inhibitor, or silencer. The accompanying drawings incorporated herein and constituting part of this specification illustrate the disclosed method. And several embodiments of the composition are illustrated, along with the following description of the disclosed method and composition It functions to explain the principles of things. [Brief explanation of the drawing]

[0022] [Figure 1] This figure illustrates how exosomes isolated from cultures of K562-mb21-41BBL cells containing stimulating ligands (IL-21 and 41BBL) can be used to stimulate NK cells in allogeneic, hapsicotic, autologous, and direct in vivo settings for cancer therapy. [Figure 2A]This figure shows the characterization of exosomes isolated from cultures of K562-mb21-41bbl cells. Exosomes were characterized by nanoparticle tracking analysis using video microscopy with NanoSight NS300 (Malvern). Single-frame light scattering images (Figure 2A) and binned histograms of particle size distribution (Figure 2B) are shown. The presence of IL-21 was confirmed immunochemically by Western blotting analysis using anti-IL-21 antibody (Figure 2C). Analysis using anti-IL-21 antibody-conjugated gold nanoparticles (GNP) (Figure 2D) shows the presence of IL-21 in exosome samples, with a time-dependent increase in dynamic light scattering intensity observed with anti-IL-21 antibody-conjugated GNP compared to no increase with isotype control antibody-conjugated GNP. [Figure 2B] This figure shows the characterization of exosomes isolated from cultures of K562-mb21-41bbl cells. Exosomes were characterized by nanoparticle tracking analysis using video microscopy with NanoSight NS300 (Malvern). Single-frame light scattering images (Figure 2A) and binned histograms of particle size distribution (Figure 2B) are shown. The presence of IL-21 was confirmed immunochemically by Western blotting analysis using anti-IL-21 antibody (Figure 2C). Analysis using anti-IL-21 antibody-conjugated gold nanoparticles (GNP) (Figure 2D) shows the presence of IL-21 in exosome samples, with a time-dependent increase in dynamic light scattering intensity observed with anti-IL-21 antibody-conjugated GNP compared to no increase with isotype control antibody-conjugated GNP. [Figure 2C]This figure shows the characterization of exosomes isolated from cultures of K562-mb21-41bbl cells. Exosomes were characterized by nanoparticle tracking analysis using video microscopy with NanoSight NS300 (Malvern). Single-frame light scattering images (Figure 2A) and binned histograms of particle size distribution (Figure 2B) are shown. The presence of IL-21 was confirmed immunochemically by Western blotting analysis using anti-IL-21 antibody (Figure 2C). Analysis using anti-IL-21 antibody-conjugated gold nanoparticles (GNP) (Figure 2D) shows the presence of IL-21 in exosome samples, with a time-dependent increase in dynamic light scattering intensity observed with anti-IL-21 antibody-conjugated GNP compared to no increase with isotype control antibody-conjugated GNP. [Figure 2D] This figure shows the characterization of exosomes isolated from cultures of K562-mb21-41bbl cells. Exosomes were characterized by nanoparticle tracking analysis using video microscopy with NanoSight NS300 (Malvern). Single-frame light scattering images (Figure 2A) and binned histograms of particle size distribution (Figure 2B) are shown. The presence of IL-21 was confirmed immunochemically by Western blotting analysis using anti-IL-21 antibody (Figure 2C). Analysis using anti-IL-21 antibody-conjugated gold nanoparticles (GNP) (Figure 2D) shows the presence of IL-21 in exosome samples, with a time-dependent increase in dynamic light scattering intensity observed with anti-IL-21 antibody-conjugated GNP compared to no increase with isotype control antibody-conjugated GNP. [Figure 3A] This figure shows that exosomes isolated from K562-mb21-41bbl cell cultures stimulate specific proliferation of NK cells derived from unselected PBMCs. Unselected PBMCs were cultured with exosomes isolated from K562-mb21-41bbl cell cultures at a total protein level of 35 ng / mL. After an initial delay, NK cells proliferated exponentially, on average 270-fold, over 20 days (Figure 3A), and the relative abundance of total lymphocytes increased to 74% (Figure 3B). All cultures proliferated in two ways, and the markers represent the mean with error bars representing the standard deviation. [Figure 3B]This figure shows that exosomes isolated from K562-mb21-41bbl cell cultures stimulate specific proliferation of NK cells derived from unselected PBMCs. Unselected PBMCs were cultured with exosomes isolated from K562-mb21-41bbl cell cultures at a total protein level of 35 ng / mL. After an initial delay, NK cells proliferated exponentially, on average 270-fold, over 20 days (Figure 3A), and the relative abundance of total lymphocytes increased to 74% (Figure 3B). All cultures proliferated in two ways, and the markers represent the mean with error bars representing the standard deviation. [Figure 4] This figure shows that NK cells stimulated and proliferated with exosomes are cytotoxic to K562 cells. Unselected PBMCs were cultured with exosomes isolated from K562-mb21-41bbl cell cultures at a total protein level of 35 ng / mL and used to analyze cytotoxicity to K562 cells. The cytotoxicity of NK cells proliferated with exosomes (Ex21-NK cells●) was slightly lower compared to NK cells proliferated with feeder cells (FC21-NK cells▲) or NK cells proliferated with IL-21-bound plasma membrane particles (PM21-NK cells■). [Figure 5A]This figure shows that exosomes generated by K562-mb21-41bbl in culture as feeder cells possessing PBMCS are taken up by NK cells. K562-mb21-41bbl cells were externally labeled with AlexaFluor 647 (AF647), co-incubated with PBMCs, and then imaged with a 10x objective lens for 18 hours. Adhesion of the AF647 label was observed between a few minutes and approximately one hour later (Figure 5A). After several hours (Figure 5B), the formation of intracellular endosomes and polyendoplasmic reticulum was observed. Subsequently, cell-free exosomes were observed (Figures 5C and 5D). Samples were obtained from the live-imaged co-culture, stained with anti-CD3 and anti-CD56, and imaged by fluorescence confocal microscopy (Figure 5E). NK cells took up or subsequently bound to the AF647 label, while T cells did not preferentially take up or bind to it. A wider area than indicated was examined for statistical validity, and 10 slices along the Z-axis were imaged to identify intracellular and extracellular events. Arrows indicate intracellular and extracellular particles or exosomes within the culture. [Figure 5B] This figure shows that exosomes generated by K562-mb21-41bbl in culture as feeder cells possessing PBMCS are taken up by NK cells. K562-mb21-41bbl cells were externally labeled with AlexaFluor 647 (AF647), co-incubated with PBMCs, and then imaged with a 10x objective lens for 18 hours. Adhesion of the AF647 label was observed between a few minutes and approximately one hour later (Figure 5A). After several hours (Figure 5B), the formation of intracellular endosomes and polyendoplasmic reticulum was observed. Subsequently, cell-free exosomes were observed (Figures 5C and 5D). Samples were obtained from the live-imaged co-culture, stained with anti-CD3 and anti-CD56, and imaged by fluorescence confocal microscopy (Figure 5E). NK cells took up or subsequently bound to the AF647 label, while T cells did not preferentially take up or bind to it. A wider area than indicated was examined for statistical validity, and 10 slices along the Z-axis were imaged to identify intracellular and extracellular events. Arrows indicate intracellular and extracellular particles or exosomes within the culture. [Figure 5C]This figure shows that exosomes generated by K562-mb21-41bbl in culture as feeder cells possessing PBMCS are taken up by NK cells. K562-mb21-41bbl cells were externally labeled with AlexaFluor 647 (AF647), co-incubated with PBMCs, and then imaged with a 10x objective lens for 18 hours. Adhesion of the AF647 label was observed between a few minutes and approximately one hour later (Figure 5A). After several hours (Figure 5B), the formation of intracellular endosomes and polyendoplasmic reticulum was observed. Subsequently, cell-free exosomes were observed (Figures 5C and 5D). Samples were obtained from the live-imaged co-culture, stained with anti-CD3 and anti-CD56, and imaged by fluorescence confocal microscopy (Figure 5E). NK cells took up or subsequently bound to the AF647 label, while T cells did not preferentially take up or bind to it. A wider area than indicated was examined for statistical validity, and 10 slices along the Z-axis were imaged to identify intracellular and extracellular events. Arrows indicate intracellular and extracellular particles or exosomes within the culture. [Figure 5D] This figure shows that exosomes generated by K562-mb21-41bbl in culture as feeder cells possessing PBMCS are taken up by NK cells. K562-mb21-41bbl cells were externally labeled with AlexaFluor 647 (AF647), co-incubated with PBMCs, and then imaged with a 10x objective lens for 18 hours. Adhesion of the AF647 label was observed between a few minutes and approximately one hour later (Figure 5A). After several hours (Figure 5B), the formation of intracellular endosomes and polyendoplasmic reticulum was observed. Subsequently, cell-free exosomes were observed (Figures 5C and 5D). Samples were obtained from the live-imaged co-culture, stained with anti-CD3 and anti-CD56, and imaged by fluorescence confocal microscopy (Figure 5E). NK cells took up or subsequently bound to the AF647 label, while T cells did not preferentially take up or bind to it. A wider area than indicated was examined for statistical validity, and 10 slices along the Z-axis were imaged to identify intracellular and extracellular events. Arrows indicate intracellular and extracellular particles or exosomes within the culture. [Figure 5E]This figure shows that exosomes generated by K562-mb21-41bbl in culture as feeder cells possessing PBMCS are taken up by NK cells. K562-mb21-41bbl cells were externally labeled with AlexaFluor 647 (AF647), co-incubated with PBMCs, and then imaged with a 10x objective lens for 18 hours. Adhesion of the AF647 label was observed between a few minutes and approximately one hour later (Figure 5A). After several hours (Figure 5B), the formation of intracellular endosomes and polyendoplasmic reticulum was observed. Subsequently, cell-free exosomes were observed (Figures 5C and 5D). Samples were obtained from the live-imaged co-culture, stained with anti-CD3 and anti-CD56, and imaged by fluorescence confocal microscopy (Figure 5E). NK cells took up or subsequently bound to the AF647 label, while T cells did not preferentially take up or bind to it. A wider area than indicated was examined for statistical validity, and 10 slices along the Z-axis were imaged to identify intracellular and extracellular events. Arrows indicate intracellular and extracellular particles or exosomes within the culture. [Modes for carrying out the invention]

[0023] The disclosed methods and compositions are described in the following "Modes for Carrying Out the Invention" of specific embodiments and By referring to the "Examples" included therein, as well as the figures and their surrounding descriptions, This specification, including international application PCT / US2013 / 048678, can be easily understood. All references cited herein are incorporated in their entirety.

[0024] The disclosed methods and compositions do not, unless otherwise specified, refer to specific synthesis methods or specific analytical techniques. It should be understood that this may vary, as it is not limited to a specific reagent. The technical terms used herein are for the purpose of describing specific embodiments only. It should also be understood that this is not intended to be limiting.

[0025] The majority of graft-versus-tumor (GVT) effects mediated by donor lymphocyte transfusion are due to Nazi It may be caused by normal killer (NK) cells. NK cells isolated from donor blood. Infusion of this substance may produce beneficial GVT effects without causing GVHD. Preclinical and Clinical data demonstrated the effectiveness of NK cell infusion leading to complete remission without GVHD. However, NK cell infusion is used in combination with autologous transplantation, or as a standalone treatment, with a matched donor. For patients who do not have it, have experienced a relapse, or are not suitable for transplantation, innovative and It offers a potentially very effective alternative.

[0026] NK cell infusion is used for hematological malignancies (such as acute myeloid leukemia or multiple myeloma) and several other conditions. NK cell lysis is used in solid tumors (e.g., brain tumors, Ewing's sarcoma, and rhabdomyosarcoma). This is a treatment option for cancer patients who are susceptible to the effects (Harada, Saijo et al.) al.2002;Ruggeri,Capanni et al.2002;Mille r,Soignier et al.2005;Cho,Shook et al.20 10). An increase in the number of functional NK cells is also associated with lymphoma, colorectal cancer, lung cancer, and breast cancer, among others. It can significantly enhance the effectiveness of therapeutic antibodies used to treat several cancers, including cancer. (Hatjiharissi, Xu et al. 2007; Triulzi, Ver. Tuani et al.2010;Houot,Kohrt et al.2011; Tai, Horton et al. 2012). However, a typical antibody costs tens of thousands of dollars. These types of individualized treatments, involving whole-body regimens, are very expensive. Furthermore, existing patients The expected effectiveness of the law is due to the lack of involvement of immune cells in cancer patients who are immune-deficient. This is often not achieved (Dewan, Takada et al. 2009; Mame (Ssier, Sylvain et al. 2011).

[0027] For it to be an effective cancer treatment method, it is necessary to proliferate NK cells to a level that reaches an effective therapeutic dose. It is desirable to achieve this. Several studies have shown that receptors expressed on the surface of stimulated cells (4- To activate 1BBL, cytokines (such as IL-15 or IL-21) and ri When stimulated, NK cells increase exponentially in in vitro culture. It was shown to preferentially proliferate in a mixture of peripheral blood mononuclear cells (PBMCs) (Imai, I wamoto et al.2005;Cho and Campana 2009;L ee,Verneris et al.2010;Somanchi,Senyukov et al. 2011).

[0028] Regarding the cytokines IL-15 and IL-21, cross-reactivity of membrane-bound interleukins The presentation is even more potent than these soluble cytokines, as in normal dendritic cells. This induces the proliferation of NK cells. Furthermore, under these stimulating conditions, low concentrations of soluble IL-12 Since only NK cells are required for survival, PBMCs do not require observable proliferation of T cells. Selective proliferation of NK cells becomes possible within the mixture. Soluble forms of IL-15 and IL-21 are also available. Tokines or high doses of IL-2 stimulate T cell proliferation more powerfully than NK cell proliferation. Previous research published by Campana and his collaborators has shown that membrane-bound IL-15 and Stimulation of NK cells in vitro culture using the K562 cell line containing 4-1BBL This is a powerful increase in NK cells that is not observed in K562 cells expressing either molecule alone. This demonstrated that it leads to reproduction (Imai, Iwamoto et al. 2005; Fuj (isaki, Kakuda et al. 2009). However, how many NK cells proliferate? Limited to cell division, the cells achieve aging, cease proliferation, and are consistent with the observation of telomere shortening. In follow-up studies, stimulation with membrane-bound IL-21 instead of membrane-bound IL-15 was ...stimulates the continuous proliferation of NK cells over countless generations, and cultures are regularly performed with fresh stimulated cells. It has been found that continuous proliferation of NK cells is possible as long as they are regularly replenished (Soma). nchi,Senyukov et al.2011;Denman,Senyukov et al. 2012). These methods are efficient for in vitro NK cell While enabling proliferation, the need for living feeder cells makes this methodology suitable for large-scale applications. This makes it difficult to transfer to clinical settings that do not have GMP facilities and capabilities. Furthermore, when infusing to patients... NK cells may stop dividing due to a lack of continuous stimulation from feeders. It's expensive. Furthermore, if the patient is reinfused, the NK cells cultured in vitro will not meet the intended purpose. Information regarding their ability to function in cages is still lacking (Miller 2009). IL-2 administration is the only FDA-approved method for in vivo proliferation of NK cells. IL-15 is currently being used as an alternative approach to IL-2 administration in Phase I clinical trials. Although it has been tested, based on preclinical results, when administered systematically, it still Significant toxicity is expected. Therefore, both methods are expected to have significant toxicity to the patient. It transports sex and further induces the proliferation of T cells, including regulatory T cells, leading to the short-lived persistence of NK cells. This leads to an average of less than 21 days.

[0029] In a successful pilot study, purified NK cells isolated from donor blood were infused. It has been shown to be safe and to lead to complete remission of AML without GVHD. To reach therapeutic levels, NK cells are administered high doses of IL-2 daily, which stimulates lymphatic drainage. The cells were proliferated in vivo in patients with lymphocyte depletion. However, the intensive treatment required for lymphocyte depletion The pretreatment regimen and the high dose of IL-2 used in this study did not result in significant toxicity or prolonged hospitalization. This, in many cases, resulted in low in vivo proliferation of NK cells. Furthermore, systemic administration of IL-2 promotes the proliferation of regulatory T cells, which suppress the number and function of NK cells. Because of this, the persistence and efficiency of NK cells in patients are limited. Alternative approaches are needed for the in vivo or ex vivo proliferation of the cells.

[0030] The efficacy of NK cell immunotherapy is determined by the transfusion of cells administered to the patient or by proliferation in vivo. The dose of NK cells reached after injection is dependent on the currently available technology and the therapeutic effect in patients. This is limited by the inability to achieve the necessary NK cell proliferation levels to accomplish the desired outcome. The lack of simpler clinical proliferation protocols is a factor in the advancement of NK cell-derived immunotherapy and broader applications. This is a major obstacle to widespread adoption. Current ex vivo proliferation protocols use high doses. A combination of cytokines and activating ligands expressed in leukemia-derived feeder / stimulating cell lines. The use of comorbidity is a significant disadvantage in transitioning to clinical settings at most centers. This results in a condition that makes it unsuitable for direct in vivo proliferation. The expression described herein The use of sosome-containing particle technology eliminates the need for stimulating cells, thus simplifying the methodology. It purifies the substance and enables direct and selective in vivo propagation.

[0031] Several groups have been exploring ways to proliferate NK cells ex vivo. However, most ex vivo methods currently under development involve high concentrations of various cytochemicals. In, the system relies primarily on the presence of IL-2 and co-culture systems of tumor cell lines and NK cells (C See Ho and Campana 2009; Suck and Koh 2010. ). The cells used to induce NK cell proliferation were irradiated with autologous or allogeneic PBM. C, RPMI8866, HFWT, K562, K562-mb15-41BBL(4-1 K562 and membrane-bound IL-15 transfected with BBL, K562-mb21 -41BBL and EBV-LCL are examples (Harada, Saijo et al.) al.2004;Imai, Iwamoto et al.2005;Berg, Lun dqvist et al.2009;Fujisaki,Kakuda et al. 2009; Siegler, Meyer-Monard et al. 2010). N.K. Cell proliferation occurs in some of these cell lines (30 to 10,000 times within 7 to 21 days). While potentially significant, the use of feeder cells currently costs millions of dollars. Due to the requirements of manufacturing practice (cGMP) facilities, most This presents a significant drawback in transitioning to a clinical setting (Cho and C See ampana 2009; Suck and Koh 2010). Furthermore, Fee Continuous culture of Dar cells is expensive and requires the support of dedicated personnel. The National Institute of Biomedical Research (NIH) was formerly known as Production Assistance for C In the form of Cellular Therapy (PACT), for the production of cells for cell therapy. It provided support in that regard. However, NK cells lose their activity during cryopreservation. It seems so (presentation at the PACT workshop). Therefore, the production site The storage and transport of proliferated NK cells to the transplant center is crucial for the successful application of therapy. This is a disorder. Further concern is these transformed cells and culture components (e.g., fetal bovine blood). Live feeder cells and / or genetic material released from (Qing) are transferred to the recipient patient. This is a possibility.

[0032] Miltenyi Biotech uses antibody-coated beads to activate NK cells. We introduced an in vitro proliferation kit that cross-links cell receptors. However, this method , requires the use of high concentrations of IL-2. While useful for laboratory applications, high concentrations are required for site use. NK cells cultured using NK cells undergo rapid apposition after infusion for cytokine withdrawal. Because it undergoes cis, this method cannot be transitioned to a clinical setting (Miller 2 009).

[0033] NK cell proliferation within PBMCs was promoted during the first 5 days with high concentrations of IL-2 and anti-CD3 antibodies. Stimulation by has been reported (Carlens, Gilljam et al. 2001) (Alici, Sutlu et al. 2008). Overall NK cell proliferation was 100 Although the ratio was close to 0, the majority of NK cells were actually NK-like T cells (Berg an (d Childs 2010). Therefore, all of these methods are transitioning to clinical application. In contrast, this presents significant difficulties, and there are no methods available for direct in vivo propagation. .

[0034] Methods to increase the number of NK cells At least one NK cell is stimulated by one or more irritants present in the exosome membrane. This involves contacting at least one NK-stimulated exosome containing ptide with an NK cell. A method for increasing the number of exosomes has been disclosed, and the exosome secretion cells It is an extracellular product of the cell.

[0035] Stimulating peptides Stimulant peptides suitable for use in the methods disclosed herein include NK cell activators ( That is, irritant ligands, cytokines, or adhesion molecules may be cited, but these may be Not limited to NK cell activators and stimulating peptides, 41BBL, IL-2, IL-12, IL-21, IL-18, MICA, LFA-1, 2B4, BCM / SLA Examples include, but are not limited to, MF2, CCR7, and / or other homing receptors. Examples of cytokines include IL-2, IL-12, IL-21, and IL-18. However, it is not limited to these. Examples of adhesive molecules include LFA-1, MICA, BCM / SLAMF2 is one example, but it is not limited to these. In one embodiment of the present invention, exo Somems are vehicles used to transport irritant peptides. It can be present in the exosome membrane. Irritating peptides are membrane-bound, while other therapeutic agents or Diagnostic agents can be transported into plasma membrane vesicles.

[0036] Membrane-inserted peptides bound to irritating peptides At least one NK cell is stimulated by one or more irritants present in the exosome membrane. This involves contacting at least one NK-stimulated exosome containing ptide with an NK cell. A method for increasing the number of exosomes has been disclosed, and the exosome secretion cells The extracellular product of the cell, one or more stimulating peptides, optionally one or It may be bound to two or more membrane-inserted peptides.

[0037] Membrane insertion peptides may also be molecules that promote insertion into the membrane. Membrane insertion peptides are lipids It may also contain a CD4 or IgG segment that has affinity for the bilayer. Alternative membrane insertion peptides include human Fc, GPI, transmembrane T cell receptor, or pHLIP. It may also contain. Membrane self-insertion peptides are any peptide known to insert into the cell membrane. It may also be a peptide. Depending on the use of membrane self-insertion peptide conjugates, a specific membrane Self-inserting peptides may be a better choice than others. Those skilled in the art will know which membrane self-inserting peptides You will understand what is ideal under different circumstances. For example, in vivo use For this purpose, pHLIP membrane self-insertion peptides may be suitable. Plitter is inserted into the membrane only under low pH conditions. Therefore, pHLIP conjugate Under normal physiological conditions, the substance is not inserted into the cell membrane. However, when infused into the tumor environment... In this case, the tumor environment is even more acidic than normal physiological conditions, therefore pHLIP conjugate The toxin can be inserted into the cell membrane of tumor cells. This insertion into the tumor environment allows for the tumor to function within the tumor region. This enables the activation of NK cells. Therefore, pHLIP is used to randomly select cells. To prevent unnecessary insertion into the membrane.

[0038] The membrane-inserted peptide may be bound to one or more irritating peptides in various ways. Furthermore, techniques for binding peptides are known in this field. The bound membrane-inserted peptide can also be called a membrane-inserted peptide conjugate. In this manner, one or more irritating peptides bound to the membrane-inserted peptide are recombinant It may also contain a fusion protein encoded by DNA, and such a fusion protein is It can be produced in bacterial cells. In certain embodiments, the fusion protein is liposome or cell To anchor to the vesicle membrane, it is attached to lipophilic molecules such as hydrophobic peptides, GPI, or human Fc. It may consist of a jugate or one or more conjugated irritant peptides (Hunt, Rath et al.1997;Kueng,Leb et al.2007;Pau lick, Forstner et al.2007; Paulick, Wise et al. al.2007;Reshetnyak,Segala et al.2007). child These fusion protein cDNA vectors can be used in bacterial (E. coli) cells, insect cells, or mammalian cells. It can be ligated to an expression plasmid that enables expression in animal cells. In certain embodiments, The cDNA vector may be FLAG-tagged or HIS-tagged. Bacterial cells, Sambrook et al.,Molecular Cloning: A Labo ratory Manual.2nd ed.Cold Spring Harbor Standard calcium chloride, as described in Laboratory Press (1989) They can be transfected using the Cium transfection method. Bacterial cells can also The cells may be cultured in LB medium, and the cells should be collected and lysed using a French press. This can be done. The target protein is purified from the solubilized solution by affinity chromatography. It is possible. Palmitic acid conjugate protein A and purified Fc fusion protein are used in the text. As described in the document, by mixing these at 4°C in a 1:2 (w / w) ratio, It can be denjugated (Kim & Peacock, Journal of Immunity) nological methods,1993 Jan.14;158(1):57~ 65 and Liu et al., Journal of Immunology, 200 See 7 Mar.1;178(5);3301~3306). Conjugate is The substance may then be directly infused into the tumor or incorporated into liposomes.

[0039] The types and methods of bonding are well known to those skilled in the art. When used herein, the term "bonding" is used in a more general sense. "Conjugate" means to conjugate, link, or combine with another molecular entity such as a peptide or protein. Otherwise, it refers to the membrane-inserted peptide that is linked to it. For example, a peptide that binds to an irritant peptide. The membrane-inserted peptide can be a fusion protein, and the membrane-inserted peptide has a disulfide bond. It binds to another protein via a membrane self-insertion peptide. The binding or conjugate is with the membrane self-insertion peptide. This could mean that there is a chemical bond between the NK cell effector agent and the cell.

[0040] In some embodiments, one or more irritating peptides are used in situ It can be bound to membrane self-insertion peptides or GPI anchors for self-assembly. For example, 4 1-BBL and IL-21 are pHLIP peptides that insert themselves into the cell membrane under acidic conditions. Because it may bind to the tumor, it allows the irritant ligand to be tethered to cells near the tumor. Sex peptide 41BBL, IL-2, IL-12, IL-21, BCM / SLAMF2, C CR7 and / or other homing receptors are produced in bacterial cells or available from commercially available sources. You can purchase these protein cDNA vectors from a supplier, and these proteins can be selectively used in bacteria. pTriEX expression platform enables expression in E. coli cells, insect cells, or mammalian cells. It can be linked to Sumi. The cDNA vector controls the expression of the FLAG tag or HIS tag. It is acceptable to do so. Bacterial cells can be transfected using a standard calcium chloride transfection method. It can be transfected and cultured in LB medium. The cells are French pre-coated. The target protein can be collected and lysed using a chloroform, and then the affinity chloroform can be used to collect and dissolve it. It can be purified from the solubilized solution by matrixing.

[0041] In some embodiments, pHLIP is 9-fluorenylmethyloxycarbon The product may be prepared by solid-phase peptide synthesis using chemistry, and the product can be prepared by reverse-phase chromatography. It can be purified by Raffy using a C18 column. pHLIP then performs benzophenone- By incubating with crosslinking agents such as 4-iodoacetamide, stimulation It can be conjugated to sex human protein ligands. After several washes, the conjugated... The pHLIP protein is then resuspended in a culture medium (e.g., physiological saline) and administered to the tumor or stasis. It can be administered intravenously. (Previous literature: Imai, Iwamoto et al. 2005;L) iu,Breiter et al.2007;Fujisaki,Kakuda et al. al.2009;Somanchi,Senyukov et al.2011;De Based on evidence shown in nman, Senyukov et al. (2012) and experimental results. Therefore, on the surface of such modified tumor cells, NK cells and IL-21 and 4 Interaction with stimulating ligands such as 1-BBL promotes in situ proliferation of NK cells. Stimulation can trigger these cytotoxic responses against tumors. This type of irritant The approach involves inserting an NK-stimulating ligand into tumor cells in situ under acidic pH conditions. Low doses of IL-2 can be infused into the patient's intraperitoneal space, either alone or together with NK cells. In addition, it can be used to treat solid tumors such as ovarian cancer (Geller, Cooley et al.). al.2011). NK cells and other cells express high levels of FCγIII R(CD16). A strong finding suggests that cytotoxic lymphocytes are important for the effectiveness of cancer treatment with therapeutic antibodies. There is evidence (Kute, Savage et al. 2009; Reim, Dombro wski et al.2009;Mamessier,Sylvain et al. (2011). Therefore, this approach can also be used in combination with therapeutic antibodies. can.

[0042] functional nucleic acid This specification discloses methods for modifying NK cell function (e.g., NK cell activation). This method involves delivering functional nucleic acids that regulate NK cell function. The method involves bringing NK cells into contact with at least one NK-stimulated exosome. Functional nucleic acids including but not limited to siRNA, shRNA, or miRNA are N This may include delivery to K cells, where NK-stimulated exosomes are filled with functional nucleic acids. For example, in some cases, functional nucleic acids are A2AR, P2YR, or combinations thereof. The intention is to regulate the level of expression of the cross-compatibility.

[0043] Functional nucleic acids have specific functions, such as binding to target molecules or catalyzing specific reactions. These are nucleic acid molecules that possess functional properties. Functional nucleic acid molecules can be classified into the following categories. However, it is not limited to these. For example, functional nucleic acids include antisense molecules, and Examples include ptamers, ribozymes, triple-chain-forming molecules, RNAi, and external guide sequences. Functional nucleic acid molecules are factors that influence, inhibit, modify, and stimulate the specific activity of target molecules. Functional nucleic acid molecules can act as exciters, or they can act independently of any other molecule. This allows it to acquire de novo activity.

[0044] Functional nucleic acid molecules are any polymer such as DNA, RNA, polypeptides, or carbohydrate chains. It can interact with its offspring. Therefore, functional nucleic acids are mRNA or genome D It can interact with NA or with polypeptides. In some cases, functional nucleic acids are based on sequence homology between the target molecule and the functional nucleic acid molecule. It is designed to interact with nucleic acids. In other situations, functional nucleic acid molecules and target molecules. Specific recognition between the functional nucleic acid molecule and the target molecule is not based on sequence homology. However, it is rather based on the formation of a tertiary structure that enables specific recognition to occur.

[0045] Antisense molecules interact with target nucleic acid molecules via either standard or non-standard base pairing. It is designed to act. The interaction between an antisense molecule and a target molecule is, for example, RN Designed to promote the destruction of target molecules by AseH-mediated RNA-DNA hybrid degradation. It is measured. Alternatively, antisense molecules are normally performed on the target molecule, such as through transcription or replication. This is designed to interrupt the processing function. The antisense molecule is based on the sequence of the target molecule. It can be designed by finding the most reachable region of the target molecule. Numerous methods exist for optimizing efficiency. An example method involves DMS and DEPC. The experiments used are in vitro selection experiments and DNA modification studies. Antisense molecules. However, 10 -6 , 10 -8 , 10 -10 , 10 -12 The following dissociation constant (K d ) target molecule Bonding is preferable. Representative methods and techniques useful for the design and use of antisense molecules. Typical examples include U.S. Patent Nos. 5,135,917, 5,294,533, and 5 , No. 627,158, No. 5,641,754, No. 5,691,317, No. 5, No. 780,607, No. 5,786,138, No. 5,849,903, No. 5,8 No. 56,103, No. 5,919,772, No. 5,955,590, No. 5,99 No. 0,088, No. 5,994,320, No. 5,998,602, No. 6,005 ,095, 6,007,995, 6,013,522, 6,017, No. 898, No. 6,018,042, No. 6,025,198, No. 6,033,9 No. 10, No. 6,040,296, No. 6,046,004, No. 6,046,31 This can be found in No. 9 and No. 6,057,437.

[0046] An aptamer is a molecule that preferably interacts with a target molecule in a specific way. Typically an aptamer is a small nucleic acid having a length of 15 to 50 bases that folds into a defined secondary and tertiary structure, such as a stem-loop or G-quartet. Aptamers can bind small molecules such as ATP ( U.S. Patent No. 5,631,146) and theophylline (U.S. Patent No. 5,580,737 ), as well as large molecules such as reverse transcriptase (U.S. Patent No. 5,786,462) and thrombin ( U.S. Patent No. 5,543,293). Aptamers can bind to target molecules with a K from target molecules of less than 10 μM and bind very strongly. The aptamer -12 preferably binds to the target molecule with a K d of less than 10 nM, 10 -6 pM, 10 -8 fM, or 10 <� -10 aM. Aptamers can bind target molecules with a very high degree of specificity. For example, aptamers have been isolated with a difference in binding affinity between a target molecule and another molecule that differs only at a single position on the molecule by more than 10,000-fold (U.S. Patent No. 5,543,29 -12 3). It is preferred that the aptamer has a K d for the target molecule that is at least 10-fold, 100-fold, 1000-fold, 10,000-fold, or 100,000-fold lower than the K [[ID=3ģ]] d for background binding molecules. For example, when comparing polypeptides, it is preferred that the background molecules are different polypeptides. Representative examples of how to make and use aptamers to d bind various different target molecules are described in U.S. Patent No. 5,476,766 . Examples of how to make and use aptamers to bind various different target molecules are described in U.S. Patent No. 5,476,766 ​​​​​​, No. 5,503,978, No. 5,631,146, No. 5,731,424, 5,780,228, 5,792,613, 5,795,721, 5,792,613, 5,795,721, No. 5,846,713, No. 5,858,660, No. 5,861,254, No. No. 5,864,026, No. 5,869,641, No. 5,958,691, No. 6 ,001,988, 6,011,020, 6,013,443, 6, Nos. 020,130, 6,028,186, 6,030,776, and 6 It can be found in publications 051 and 698.

[0047] Ribozymes are nucleic acid components capable of catalyzing chemical reactions, either intramolecular or intermolecular. It is a child. Therefore, ribozymes are catalytic nucleic acids. Ribozymes react to intermolecular reactions. It is preferable to use a fertilizer. A nuclease based on ribozymes found in natural systems or Hammerhead-type ribozymes (US 5) that catalyze nucleic acid polymerase type reactions. ,334,711, 5,436,330, 5,616,466, 5, No. 633,133, No. 5,646,020, No. 5,652,094, No. 5,7 No. 12,384, No. 5,770,715, No. 5,856,463, No. 5,86 No. 1,288, No. 5,891,683, No. 5,891,684, No. 5,985 , No. 621, No. 5,989,908, No. 5,998,193, No. 5,998, International Patent Application No. 203, International Publication No. 9858 by Ludwig and Sproat No. 058, No. 9858057 by Ludwig and Sproat, and Lu (Patent No. 9718312 by dwig and Sproat), hairpin-type ribozyme (For example, U.S. Patent Nos. 5,631,115, 5,646,031, and 5,68) No. 3,902, No. 5,712,384, No. 5,856,188, No. 5,866 (Nos. 701, 5,869,339, and 6,022,962), and Tet Rahimenaribozyme (e.g., U.S. Patent No. 5,595,873 and No. 5,652) There are many different types of ribozymes, such as No. 107. Also, in natural systems... Ribozymes that have not yet been found but have been engineered to catalyze specific reactions de novo There are many such patents (for example, U.S. Patent No. 5,580,967, U.S. Patent No. 5,688,670, (Patents No. 5,807,718 and No. 5,910,408). Preferred ribozymes are It cleaves RNA or DNA substrates, and more preferably RNA substrates. Ribozymes are a classic example. In terms of type, it cleaves nucleic acid substrates through the recognition and binding of the target substrate, and subsequent cleavage. This recognition is often based primarily on the interaction of standard or non-standard base pairs. Because the recognition of the target substrate is based on the target substrate sequence, ribozymes are targeted specifically to nucleic acids. To make them particularly good candidates for cleavage. How to prepare and use ribozymes, various Typical examples of catalyzing different reactions include U.S. Patent No. 5,646,042, and Nos. 5,6. No. 93,535, No. 5,731,295, No. 5,811,300, No. 5,83 No. 7,855, No. 5,869,253, No. 5,877,021, No. 5,877 ,022, No. 5,972,699, No. 5,972,704, No. 5,989, This can be found in publications No. 906 and No. 6,017,756.

[0048] Triple-stranding functional nucleic acid molecules are molecules that can interact with either double-stranded or single-stranded nucleic acids. When a triple-chain molecule interacts with a target region, a structure called a triple chain is formed. In this structure, the complex depends on both Watson-Crick and Hoogsteen-type base pairing. The body consists of three strands of DNA. Triple-strand molecules are targeted by high affinity and high specificity. It is preferable because it can bind the target region. The triple chain forming molecule is 10 -6 , 10 -8 , 10 -10 , or 10 -12 K less than d It is preferable to bind the target molecule in this way. This document describes how to construct and use sea urchin triple-chain forming molecules to bind various different target molecules. Examples include U.S. Patent Nos. 5,176,996, 5,645,985, and 5,650. No. 316, No. 5,683,874, No. 5,693,773, No. 5,834,1 Nos. 85, 5,869,246, 5,874,566, and 5,962,4 It can be found in issue 26.

[0049] An external guide sequence (EGS) is a molecule that binds to the target nucleic acid molecule that forms the complex. This complex is recognized by RNase P, which cleaves the target molecule. EGS is selected RNAse P can be designed to specifically target RNA molecules within the cell. It is useful for processing transfer RNA (tRNA). Target RNA: The EGS complex is a native tRNA group By using EGS to guide the substance to mimic quality, bacterial RNAse P can be virtually any R NA sequences can be recruited to cleave. (International Publication No. 92 / 0 by Yale) Issue 3566, and Forster and Altman, Science 238:4 07-409 (1990).

[0050] Similarly, EGS / RNAseP-induced RNA cleavage in eukaryotes occurs within eukaryotic cells. It can be used to cleave the target of the beam. (Yuan et al., Proc .Natl.Acad.Sci.USA 89:8006~8010(1992);Ya International publication No. 93 / 22434 by le; same publication No. 95 / 24489 by Yale; Y uan and Altman, EMBO J 14:159~168 (1995), and and Carrara et al., Proc. Natl. Acad. Sci. (USA) 92:2627~2631(1995)). How to prepare and use EGS molecules, A typical example of facilitating the cleavage of various different target molecules is U.S. 5,168,0 No. 53, No. 5,624,824, No. 5,683,873, No. 5,728,52 This can be found in No. 1, No. 5,869,248, and No. 5,877,162. .

[0051] Gene expression can also be effectively controlled in a highly specific manner by RNA interference (RNAi). It can be silenced. This silencing is originally an appendage of double-stranded RNA (dsRNA). Observed in Canada (Fire, A., et al. (1998) Nature, 391:8 06~11;Napoli, C., et al. (1990) Plant Cell 2 :279~89;Hannon,GJ(2002)Nature,418:244~ 51). When dsRNA enters a cell, it is processed by the RNase III-like enzyme, Dicer. A length that includes two nucleotide overhangs at the 3' end is considered a double-stranded small interfering RNA ( siRNA is cleaved into 21-23 nucleotides (Elbashir, SM, et al.) al.(2001)Genes Dev.,15:188~200;Bernstei n,E.,et al.(2001)Nature,409:363~6;Hammon d,SM,et al.(2000)Nature,404:293~6). APT In the dependency process, siRNA guides the siRNA to the target RNA sequence, which is called RNAi induction. Multi-subunit tanning, commonly known as Silencing Complex (RISC) It becomes incorporated into the protein complex (Nykanen, A., et al. (20 01) Cell, 107:309~21). At some point, the siRNA double strand is unwound. The antisense chain remains bound to RISC, and the endonuclease and exonuclease It appears that in combination with ase, it directs the degradation of complementary mRNA sequences (Mar Tinez, J., et al. (2002) Cell, 110: 563~74). However Furthermore, the effects or use of iRNA or siRNA may not be due to any type of mechanism. Not limited.

[0052] Short interfering RNA (siRNA) induces sequence-specific post-transcriptional gene silencing, It is a double-stranded RNA that can reduce or even inhibit gene expression. siRNA has a region of sequence identity between both the siRNA and the target RNA, mRN It causes specific degradation of homologous RNA molecules such as A. For example, see International Publication No. 02 / 4432. When molecule 1 is base-paired with the 3' overhang end, it causes sequence-specific degradation of the target mRNA. Possible siRNAs are disclosed, and methods for producing these siRNAs are provided in the references. This is incorporated herein. Sequence-specific gene silencing is performed by an enzyme dicer. Using synthetic short double-stranded RNA that mimics the generated siRNA, mammalian cells are subjected to This can be achieved (Elbashir, SM, et al. (2001) Natur e,411:494 498)(Ui-Tei,K.,et al.(2000)FEB S Lett 479:79~82). siRNA is administered chemically or in vitro. Short, double-stranded hairpin-like RNA that can be synthesized or processed into siRNA within cells. (shRNA) may be the result. Synthetic siRNA is generally algorithmic and conventional. Designed using a DNA / RNA synthesizer. Suppliers include Ambion (Aust in, Texas), ChemGenes (Ashland, Massachusetts) s), Dharmacon (Lafayette, Colorado), Glen Re search(Sterling,Virginia), MWB Biotech(Es bersberg, Germany), Proligo (Boulder, Colorado) Examples include (do) and Qiagen (Vento, The Netherlands). siRNA is also Ambion's SILENCER® siRNA Co. It can be synthesized in vitro using a kit such as an instruction kit. .

[0053] The generation of siRNA from a vector is mediated through the transcription of short hairpin RNA (shRNA). This is more commonly done. Kits for generating vectors containing shRNA are available. For example, Imgenex's GENESUPPRESSOR (trademark) Construct ion Kits and Invitrogen's BLOCK-IT® inducible RNAi Plasmids and lentiviral vectors. This specification discloses the flame Based on the sequence of the symptomatic mediator, any shRNA designed as described above is disclosed. It will be done.

[0054] NK cells At least one NK cell is stimulated by one or more irritants present in the exosome membrane. This involves contacting at least one NK-stimulated exosome containing ptide with an NK cell. A method for increasing the number of exosomes has been disclosed, and the exosome secretion cells NK cells are extracellular products of cells and exist within a population of unselected peripheral blood mononuclear cells (PBMCs). To exist.

[0055] Human NK cells are characterized by the expression of CD56 or CD16 and the absence of the T cell receptor (CD3). This is a subset of peripheral blood lymphocytes defined as (Ljunggren and Ma (lmberg 2007; Woan and Reddy 2007). NK cells are the main It senses and kills target cells that lack a histocompatibility complex (MHC) class I molecule. NK cells that activate receptors include, in particular, the innate cytotoxic receptor (NKp30). NKp44 and NKp46), and lectin-like receptors NKG2D and DNAM-1, These ligands are expressed in stress cells, transformed cells, or infected cells. However, it is not expressed in normal cells, and causes normal cells to show resistance to NK cell killing activity. Bottino,Castriconi et al.2005;Gasser,Ors (ulic et al. 2005; Lanier 2005). Activation of NK cells is Ig-like receptor (KIR), NKG2A / CD94, and leukocyte I It is negatively regulated via inhibitory receptors such as g-like receptor-1 (LIR-1). The involvement of sex receptors may be sufficient to prevent target lysis (Bryceson, Lju (nggren et al. 2009). Therefore, NK cells are involved in many stress-induced processes. Efficiently target cells that express ligands but express very little MHC class I ligands. To target.

[0056] NK cells destroy tumor cells, stress cells, and virus-infected cells in various different ways. To destroy efficiently. First, directly bind to the target cells, allow them to pass through these membranes, and then... By infusing a protein that cleaves and activates the apoptotic protein, It initiates programmed cell death (apoptosis) in the target cells. Also, the surface of NK cells Receptors for tumor necrosis factor (TNF)-related apoptosis-inducing ligands (TRAILs) These receptors activate internal signals for apoptosis-programmed cell death. It contains a protein ligand that can bind to and activate on target cells. When stimulated, NK cells These cells not only inhibit viruses and tumors, but also inhibit signaling invasion to other immune cells. Furthermore, NK cells also secrete cytokines such as INFγ and TNFα. Its anti-cancer activity has generated significant interest in NK cells in the medical field.

[0057] NK cells play a significant role in the immune system, and therefore the ability to increase the number of NK cells is important. This provides therapeutic opportunities that were impossible or ineffective with a small number of NK cells. .

[0058] Exosomes As disclosed herein, a method for increasing the number of NK cells is provided, at least 1 This involves contacting one NK cell with at least one NK-stimulated exosome. The exosomes used in this specification are those that contain one or more stimuli present in the exosome membrane. Containing sex peptides, these exosomes are extracellular products of exosome-secreting cells. In a certain embodiment, exosomes are used for improved formation or release of exosomes. It is generated by a cell line that is manipulated, and such cell line includes the cell line K562-mb15- Examples include, but are not limited to, 41BBL or the cell line K562-mb21-41BBL. Exosomes are a natural vehicle secreted by many different types of cells. It is found in various bodily fluids (Immune modulation of T- cell and NK(natural killer)cell activity es by TEXs(tumour-derived exosomes)White side TL,Biochem Soc Trans.2013 Feb 1;41( 1):245~51). Exosome secretion is carried out by a highly regulated process. The resulting particles are 30-100 nm in size. Exosomes are composed of lipids and other substances. They consist of proteins, and the protein identity found in specific exosomes is that they produce these proteins. It depends on the cells. The identity and composition of proteins found in specific exosomes How do exosomes signal, influence, and interact with other cells? To determine this. Exosomes are characterized by their ability to regulate immune cells and tumor cells. Therefore, it can be used to manipulate the biological activity of immune cells and tumor cells.

[0059] Smaller exosomes are smaller in size compared to other larger plasma membrane particles. This can increase the diffusion of exosomes due to their physiological barriers and in vivo distribution. It has a high probability. Furthermore, the smaller size of exosomes allows for intravenous injection of exosomes. This makes it possible to improve the proliferation and in vivo distribution of NK cells via the circulatory system.

[0060] In some cases, exosomes have a diameter of 30-100 nm.

[0061] Exosome use At least one NK cell is stimulated by one or more irritants present in the exosome membrane. This involves contacting at least one NK-stimulated exosome containing ptide with an NK cell. A method for increasing the number of exosomes has been disclosed, and the exosome secretion cells NK cells are extracellular products of cells, and NK cells are found in vitro, in vivo, or exv NK-stimulated exosomes are brought into contact with ivo. NK cells are used in allogeneic transplantation procedures and haploid transplantation procedures. NK-stimulated exosomes can be contacted in the following or in vivo immunotherapy procedures. In some aspects, NK cells in allogeneic transplantation, haploid transplantation, or in vivo immunotherapy. - The use of stimulated exosomes does not cause graft-versus-host disease (GVHD).

[0062] Treatment method A method for treating cells susceptible to NK-mediated lysis has been disclosed, and this method The procedure involves administering an effective amount of a composition containing contacted NK cells. The cell stimulates at least one NK cell with one or more stimuli present in the exosome membrane. A method comprising contacting at least one NK-stimulated exosome containing a sex peptide. Exosomes are produced by and are extracellular products of exosome-secreting cells. In that embodiment, cells susceptible to NK-mediated lysis are those infected with the virus. Cells susceptible to NK-mediated lysis include AML, breast cancer, bladder cancer, and colorectal cancer. This may include kidney cancer, lung cancer, prostate cancer, thyroid cancer, and uterine cancer.

[0063] Methods to reduce the risk of recurrence after stem cell transplantation, and effective methods including contacted NK cells. A method for providing an adjunctive therapy comprising administering a certain amount of a composition is disclosed. The NK cells that came into contact with the exosome membrane contained at least one NK cell, and one of the NK cells present in the exosome membrane. This involves contacting at least one NK-stimulated exosome containing two or more stimulating peptides. Exosomes are produced by a method that includes the following: exosomes are produced extracellularly by exosome-secreting cells. It is an object.

[0064] Proliferated NK cells, compositions, and / or methods used to increase the number of NK cells The law applies to cancer patients who are susceptible to NK cell-mediated lysis, and to patients who have undergone hematopoietic stem cell transplantation. It can be used as a therapeutic method for increasing residual tumor cells. The NK cell proliferation composition and method can be used to increase residual tumor cells. After added clearance and / or stem cell transplantation for relapse prevention, the amount of cytotoxic NK cells It can be used to increase NK cells. NK cell proliferation compositions and methods can also be used for viral infection patients. It can also be used in the treatment of patients.

[0065] The NK cell proliferation composition and method can be used as a therapeutic method after NK cell infusion. The effectiveness of NK cell therapy (i.e., patients who have achieved remission and / or are maintaining remission) To increase the number of cytotoxic NK cells and their persistence in vivo. Let it.

[0066] NK cells containing or not containing an NK cell proliferation composition are used in lymphoma, colorectal cancer, lung cancer, Treatment for various cancers, including but not limited to colon cancer, head and neck cancer, and breast cancer. Used in combination with antibodies, it responds to antibody drug therapy, achieves remission and / or maintains remission. This will likely increase the number of patients.

[0067] Methods for increasing NK cells are used in cancer treatment, viral infection treatment, NK cell research, and multiple sclerosis treatment. It is beneficial for immune surveillance and the treatment of graft-versus-host disease. Any NK cell-related disorder is NK cells can be treated or affected by proliferation. For example, by increasing activated T cells. Diseases such as multiple sclerosis, which are known to cause this condition, can be treated with the disclosed composition. However, this is because these compositions increase the number of NK cells that target and kill activated T cells. This is because it causes proliferation. Therefore, the disclosed composition reduces activated T cells. It can be used to achieve this.

[0068] Method for generating exosomes A method for generating NK cell-stimulated exosomes has been disclosed, and this method involves exosomes This includes embedding one or more irritating peptides within the membrane. The irritating peptides are 4-1BBL, IL-2, IL-12, IL-18, IL-21, MICA / B, ULB P2, ICAM-1, 2B4, BCM1 / SLAMF2, CD155, CD112, CC R7, and / or other homing receptors, DAP12, DAP10, and / or other adapter proteins may be included. The stimulatory peptide may optionally be bound to one or two or more membrane insertion peptides. The membrane insertion peptide may include CD4 or IgG having an affinity for the lipid bilayer. Also, alternative membrane insertion peptides may include human Fc, GPI, transmembrane T cell receptor, or pHLIP. One or more stimulatory peptides bound to one or more membrane insertion peptides may include a fusion protein encoded by recombinant DNA. In some embodiments, the NK cell-stimulatory exosome may be derived from a cell line engineered to improve exosome expression, e.g., the NK cell- stimulatory exosome may be derived from the cell line K562-mb21-41BBL ).

[0069] Exosomes are natural vehicles secreted by many different types of cells and are found in various body fluids (Whiteside 2013). Exosomes consist of lipids and proteins, and the identity of the proteins found in a particular exosome depends on the cells that produced them. Thus, a cell line expressing a stimulatory peptide and / or a stimulatory peptide bound to a membrane insertion peptide may produce exosomes having one or more stimulatory peptides embedded in the membrane of the exosome.

[0070] Exosomes can be prepared using any technique known in the art. For example, exosomes secreted by cells can be isolated from cell culture media by filtration (Figure 1 ). General protocols for preparing exosomes can be used.

[0071] Cancer treatment method and exosome composition A composition containing an effective amount of NK-stimulated exosomes containing one or more stimulating peptides. A method for treating cancer, including the administration of a substance, is disclosed. NK-stimulated exosomes The use of this method may include administering NK-stimulated exosomes to the subject (Figure 1). In some embodiments, the use of NK-stimulated exosomes in contact with a population of NK cells To obtain this, NK-stimulated exosomes are brought into contact with NK cells ex vivo, This may include administering a population of NK cells that have been in contact with the subject (Figure 1).

[0072] A composition is disclosed that includes NK-stimulated exosomes containing one or more stimulating peptides. One or more irritating peptides include 4-1BBL, IL-2, IL-12, and I. L-18, IL-21, MICA, 2B4, BCM1 / SLAMF2, CCR7, and / Or it may include other homing receptors. The stimulating peptide may be one or two types, optionally. The above membrane-inserted peptides can be bound to the membrane-inserted peptides. The membrane-inserted peptides have affinity for the lipid bilayer. It may contain a CD4 or IgG segment. Alternatively, the membrane-inserted peptide may contain human Fc It may also contain GPI, transmembrane T cell receptor, or pHLIP. One or more irritating peptides bound to the recombinant DNA-encoded fusion protein It may contain keratin. NK-stimulated exosomes are manipulated to improve exosome expression. It may be derived from the cell line K562-mb21-41. NK-stimulated exosomes are derived from the cell line K562-mb21-41. It may be derived from BBL. In some embodiments, the composition further comprises a pharmaceutical carrier. That's good too.

[0073] A composition containing NK-stimulated exosomes containing one or more stimulating peptides is used to treat cancer. The healing occurs through proliferation or increase in the number of NK cells in the presence of these compositions. To obtain: Increased NK cell proliferation allows more NK cells to target and kill tumor cells. To make this possible, tumor cells are reduced, ultimately leading to the treatment of cancer or prevention of recurrence.

[0074] Disclosed herein are NK-stimulated exosomes containing one or more stimulating peptides. The composition may provide a preventive effect. NK cells are known to provide immune surveillance. Therefore, administering a composition that promotes the proliferation of NK cells will result in more NK cells. Cells provide immune surveillance, enabling the targeting and elimination of precancerous cells before cancer develops. .

[0075] In some embodiments, the use of NK-stimulated exosomes is directly NK-stimulated exosomes were administered to subjects by direct injection to stimulate NK cells in vivo. This may include causing proliferation.

[0076] In some embodiments, the use of NK-stimulated exosomes in the disclosed composition Administration to cell populations in vitro or ex vivo, and subsequent processing of those cells. This may include administering cells to a subject, for example, a composition containing NK-stimulated exosomes. This involves administering it to NK cells from PBMCs isolated from donors by apheresis. The NK cells that have been brought into contact with the patient can be transfused to the patient using an allogeneic transplantation procedure or a haploid transplantation procedure. as shown in (Figure 1). The composition can also be administered to NK cells from PBMC isolated by apheresis from a patient, and the contacted NK cells can be infused into the patient (Figure 1). as shown in (Figure 1). The composition can also be administered to NK cells from PBMC isolated by apheresis from a patient, and the contacted NK cells can be infused into the patient (Figure 1).

[0077] Administration The disclosed composition can be administered in vitro or in vivo. In some embodiments, the method includes a combination of in vitro and in vivo administrations. The composition can be administered in vivo with a pharmaceutically acceptable carrier. As is well known to those skilled in the art, the term "pharmaceutically acceptable" includes substances that are not biologically or otherwise undesirable, i.e., the substance does not cause undesirable biological effects or interact in a harmful manner with any of the other components of the pharmaceutical composition in which it is contained, and can be administered to a subject together with exosomes or membrane self-inserting peptide conjugates. or interact in a harmful manner with any of the other components of the pharmaceutical composition in which it is contained, and can be administered to a subject together with exosomes or membrane self-inserting peptide conjugates. or interact in a harmful manner with any of the other components of the pharmaceutical composition in which it is contained, and can be administered to a subject together with exosomes or membrane self-inserting peptide conjugates. The carrier is of course selected to minimize degradation of the active ingredient and to minimize any harmful side effects in the subject. The carrier is of course selected to minimize degradation of the active ingredient and to minimize any harmful side effects in the subject. The carrier is of course selected to minimize degradation of the active ingredient and to minimize any harmful side effects in the subject.

[0078] The compositions disclosed herein can be administered, for example, orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, by intratumoral administration, transdermally, ex vivo, locally, which includes administration by local intranasal administration or inhalation. As used herein, "local intranasal administration" includes delivery of the composition to the nose and nostrils through one or both nostrils, including delivery by a nebulization mechanism or droplet mechanism, or delivery by aerosolization of protoplast membrane vesicles. As used herein, "local intranasal administration" includes delivery of the composition to the nose and nostrils through one or both nostrils, including delivery by a nebulization mechanism or droplet mechanism, or delivery by aerosolization of protoplast membrane vesicles. As used herein, "local intranasal administration" includes delivery of the composition to the nose and nostrils through one or both nostrils, including delivery by a nebulization mechanism or droplet mechanism, or delivery by aerosolization of protoplast membrane vesicles. Administration of the composition by inhalation can be by a nebulization or droplet mechanismDelivery may be done via the nose or mouth. Delivery may also be done via intubation into the respiratory system (for example). It can be performed directly on any area of ​​the lungs. The exact amount of the required composition depends on the subject's race. Age, weight and overall health, severity of the disease being treated, specific composition to be used, composition The appropriate dose will vary depending on the subject, as it depends on the method of administration, etc. The appropriate dose is as described in this specification. In light of this, it can be determined by those skilled in the art using only conventional experiments.

[0079] Pharmaceutical carriers The compositions described herein are used therapeutically in combination with pharmaceutically acceptable carriers. It is possible.

[0080] Suitable carriers and formulations thereof are, for example, Remington: The Science e and Practice of Pharmacy(19th ed.)ed.A .R. Gennaro, Mack Publishing Company, Easto This is described in n,Pa.1995. Typically, an appropriate amount of pharmaceutically acceptable salt is used in the formulation. This is used to impart isotonicity to the formulation. Examples of pharmaceutically acceptable carriers are not limited to these. Examples include physiological saline, Ringer's solution, and dextrose solution. The pH of the solution is Preferably, it is about 5 to about 8, more preferably about 7 to about 7.5. Furthermore, the carrier is antibody Includes sustained-release formulations such as a semipermeable matrix of a solid hydrophobic polymer containing a matrix The drug is in the form of a molded article such as a film, liposome, or microparticles. For example, the route of administration and It is obvious to those skilled in the art that certain carriers are more preferable depending on the concentration of the composition being administered. It is likely.

[0081] Pharmaceutical carriers are known to those skilled in the art. These are most typically sterile water, physiological saline, and standard carriers for drug administration to humans, including solutions such as buffers with physiological pH. The composition may be administered intramuscularly or subcutaneously. Other compounds may be used by those skilled in the art. It will be administered according to standard procedures.

[0082] The pharmaceutical composition contains, in addition to the selected molecule, a carrier, a thickener, a diluent, a buffer, a preservative, and an insecticide. It may contain surfactants, etc. Pharmaceutical compositions may also contain antibacterial agents, anti-inflammatory agents, anesthetics, etc. It may contain one or more active ingredients.

[0083] The pharmaceutical composition depends on whether local or systemic treatment is desired, and on the area to be treated. Therefore, it can be administered in numerous ways. Administration can be local (ophthalmic, transvaginal, transrectal, nasal). (Including intravenous administration), orally, by inhalation, or parenterally, for example, by intravenous infusion, subcutaneously, or intraperitoneally. The administration may be performed by intravenous, intraperitoneal, or intramuscular injection. It can be administered subcutaneously, intracavitarially, or percutaneously.

[0084] Parenteral formulations include sterile aqueous solutions or non-aqueous solutions, suspensions, and emulsions. Examples of liquids include propylene glycol, polyethylene glycol, and olive oil. The oil and the injectable organic ester such as ethyl oleate are used as the aqueous carrier. Contains water, alcoholic / aqueous solutions, emulsions, or suspensions, including a buffering medium. Parenteral biliary ointment The solution is sodium chloride solution, Ringer's dextrose, and dextrose. Trose and sodium chloride, lactated Ringer's solution, or non-volatile oils. This includes intravenous vehicles, fluids and nutritional supplements, electrolyte supplements (e.g., Ringer's benzoate). This includes (strawberry-based) and other preservatives and other additives, such as antimicrobial agents. It can exist as an antioxidant, a chelating agent, and an inert gas.

[0085] Preparations for topical administration include ointments, lotions, creams, gels, drops, suppositories, and sprays. Examples include liquids and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases. Thickening agents and other additives may be necessary or desirable.

[0086] Compositions for oral administration may include powders or granules, suspensions in water or a non-aqueous medium, or solvents. Examples include liquids, capsules, pouches, or tablets. Thickeners, flavorings, diluents, emulsifiers, Dispersing agents or binders may be desirable in some cases.

[0087] Some compositions include hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and Inorganic acids such as phosphoric acid, and formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid Reactions with organic acids such as oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid. By means of, or by inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, etc. and organic compounds such as mono, di, trialkyl, arylamines, and substituted ethanolamines. Potentially, as a pharmaceutically acceptable acid or base addition salt formed by the reaction with bases, It can be administered locally.

[0088] Combination therapy Treatment methods for cancer, viral infections, multiple sclerosis, and graft-versus-host disease are disclosed. These methods involve one of the disclosed compositions and a known treatment for the disease or disorder being treated. This includes administering a combination of the above to a subject. For example, a method for treating cancer is disclosed. This method involves NK-stimulated exosomes containing one or more stimulating peptides. An effective amount of the composition and, but not limited to, chemotherapy, immunotherapy, radiotherapy, or pain This includes administering it in combination with known cancer treatments such as pain therapy.

[0089] There are two different types of immunotherapy: passive immunotherapy uses components of the immune system, It does not necessarily initiate an immune response within the patient, but rather targets the cytotoxic activity against cancer cells. On the other hand, active immunotherapy actively stimulates an endogenous immune response. Passive plans The use of monoclonal antibodies (mAbs) produced by B cells that respond to specific antigens. This includes the development of hybridoma technology and the identification of tumor-specific antigens in the 1970s, and immunology. This enables the development of mAbs that can specifically target tumor cells for destruction by the system. It was made possible. To date, mAbs are the greatest success story in immunotherapy, and in 2012... The top three best-selling anticancer drugs were mAbs. Rituximab (Rituxan) Genentech is one such company, and it specializes in non-Hodgkin lymphoma (NHL). It binds to the CD20 protein, which is highly expressed on the surface of B-cell malignancies such as [specific example of a specific type of tumor]. Ximmab is used in combination with chemotherapy to treat NHL and chronic lymphocytic leukemia (CLL). It is approved by the FDA for use in [unspecified purpose]. Other important mAbs include trastuzumab ( Herceptin (Genentech) is a drug that targets HER2 expression. This revolutionized the treatment of HER2 (human epidermal growth factor receptor 2)-positive breast cancer.

[0090] In order to generate the optimal "killer" CD8 T cell response, in addition to T cell receptor activation, Simultaneous stimulation is also necessary, and these include OX40 (CD134) and 4-1BB (CD137) ) may be provided via linkage of tumor necrosis factor receptor family members, including OX4. Treatment with the activating (agonist) anti-OX40mAb is effective in promoting T cell differentiation and cytolysis. It is particularly interesting because it enhances function and increases anti-tumor immunity against various tumors.

[0091] In some embodiments, the disclosed vaccine is a chimeric antigen receptor (CAR), Adoptive immunotherapy (AC) involves targeting T cell receptors (TCRs) and tumor-infiltrating lymphocytes (TILs). It is used in combination with T).

[0092] The term "tumor-infiltrating lymphocytes" or "TIL" refers to white blood cells that have left the bloodstream and entered the tumor. The proliferation of lymphocytes, including tumor-infiltrating lymphocytes such as T cells, is considered in the relevant technical field. As is well known, this can be achieved by any of several methods. For example, T cells Lymphocytes and interleukin-2 (IL-2), IL-7, IL-15, IL -21, or in the presence of a combination thereof, rapid T cell receptor stimulation using nonspecific T cell receptor stimulation They can proliferate. Nonspecific T cell receptor stimulation can be used, for example, mouse monoclonal anti-CD3 The antibody may contain approximately 30 ng / mL of OKT3 (Ortho-McNeil(R) (Raritan, NJ) or Miltenyi Biotec (Bergisch Available from Gladbach, Germany. Alternatively, T cells are about 200 ~400 IlU / mL, for example, 300 IlU / mL of IL-2 or IL-15 (IL-2 is In vitro in the presence of T cell growth factors such as (preferred) peripheral blood mononuclear cells (PB MC) contains one or more antigens (such as epitopes, their antigenic portion, or cancer cells). These are, for example, approximately 0.3 μM MART-1:26~35 (27 L) or gp 100:209~217 (210M) Human leukocyte antigen A2 (HLA-A2) binding By stimulating with a vector such as a plutidot (which can be selectively expressed), They can proliferate rapidly. T cells induced in vitro are HLA-A2 express antigen-containing It is rapidly expressed by restimulating the indicator cells with the same antigen as the cancer cells that were pulsed. Alternatively, T cells are, for example, irradiated autologous lymphocytes or irradiated HLA-A2+ allogeneic lymphocytes. Pacocytes and IL-2 can be restimulated. The specific tumor reactivity of proliferated TILs is relevant to this field. It can be tested in any known way, for example, after co-culturing with tumor cells. This can be tested by measuring tokine release (e.g., interferon-gamma). In one embodiment, the self-ACT method cultures CD8+ T cells before rapid cell proliferation. This includes enriching TILs. After culturing TILs with IL-2, T cells are, for example, (for example) CliniMACS <plus>CD8 Microbead System (Miltenyi Using CD8 microbead isolation with Biotec, for CD4+ cells Deplete and enrich for CD8+ cells. In some embodiments, autologous T cells T cell growth factors that promote proliferation and activation are absorbed simultaneously with or following the absorption of autologous T cells. It is administered to mammals. T cell growth factors promote the proliferation and activation of the body's own T cells. It can be any suitable growth factor. Examples of suitable T cell growth factors include interleukin (IL)-2, IL-7, IL-15, IL-12, and IL-21 are examples, and these It can be used alone, or in combination with IL-2 and IL-7, IL-2 and IL-15, IL-7 and and IL-15, IL-2, IL-7 and IL-15, IL-12 and IL-7, IL-1 It can be used in various combinations such as IL-2 and IL-15, or IL-12 and IL-2. It is possible.

[0093] Many anticancer drugs are also available for use in combination with the methods and compositions of the present invention. The following is a non-comprehensive list of anti-cancer (antineoplastic) drugs that may be used in combination with radiation therapy. Asibicin, Acralubicin, Acodazole Hydrochloride, Acronin, Adzelesin, Aldes Leukin, Altoletamine, Ambomycin, Amethantrone Acetate, Aminoglutethimide Amsacrin, anastrozole, anthramycin, asparaginase, asperyl Azacitidine, Azetepa, Azotomycin, Batymast, Benzodepa, Bicalta Mido, Bisanthren hydrochloride, Bisnafid dimesylate, Bizeresin, Bleomycin sulfur Salts, Brequinal sodium, Bropyrimine, Busulfan, Cactinomycin, Callus Theron, chalasemide, carvethymer, carboplatin, carmustine, carbicin hydrochloride , Calzelsin, Sedefingol, Chlorambucil, Siloremycin, Cisplatin, Cladribine, cristol mesylate, cyclophosphamide, cytarabine, dacarb Dinc, dactinomycin, daunorubicin hydrochloride, decitabine, dexormaplatin, de Zaguanine, Dezaguanine Mesylate, Diadiquan, Docetaxel, Doxorubicin, Doxorubicin hydrochloride, droloxifen, droloxifen citrate, propionic acid Dromostanolon, Duazomycin, Edatrexate, Eflomitine hydrochloride, Elsa Mitrusin, Enloplatin, Empromate, Epipropidine, Epirubicin hydrochloride, Erbulozole, esorubicin hydrochloride, estramustine, estramus Sodium phosphate, etanidazole, etiodized oil I 131, etoposide, ri Etoposide acid, etopurine, fadrozol hydrochloride, fazarabine, fenretinide, Loxuridine, fludarabine phosphate, fluorouracil, flurocitabine ine), fosquidone, fostriesin sodium, gemcitabine, gemcitabine hydrochloride Gold (Au) 198, Hydroxyurea, Idarubicin hydrochloride, Ifosfamide, Ilmofo Syn, iproplatin, irinotecan hydrochloride, lanreotide acetate, letrozole, lyu - Prolide acetate, rialozol hydrochloride, lometrexol sodium, lomustine, ro Soxantrone hydrochloride, masopropyl alcohol, metansine, mechloretamine hydrochloride, megurine acetate Strol, melenegestrol acetate, melphalan, menogalyl, mercaptopurine, Methotrexate, methotrexate sodium, methoprine, metsuredepa, mitindomi Mitocalcin, Mitochromin, Mitogiline, Mitomarcin, Mitomycin, Mythos Per, Mitotane, Mitoxantrone hydrochloride, Mycophenolic acid, Nocodazole, Nogalama Icin, Ormaplatin, Oxythran, Paclitaxel, Pegasparagaze, Perio Mycin, pentamustine, pepromycin sulfate, perphosphamide, pipobromane, Piposulfan, Pyroxantrone hydrochloride, Plicamycin, Promethane, Porfima - Sodium, porphyromycin, prednimastine, procarbazine hydrochloride, pew Romycin, puromycin hydrochloride, pyrazofulin, ribopurine, logretimide, saph Saffingol, Safingol hydrochloride, Semustine, Simtrazen, Sparphosate Natri Um, sparsomycin, spirogermanium hydrochloride, spiromustine, spiroplatin Streptonigrin, streptozocin, strontium chloride Sr 89, slophenurethane Lu, thalisomycin, taxane, taxoid, tecogalan sodium, tegafur, tegafur, tero Xanthrone hydrochloride, temoporfin, teniposide, teroxylone, testolactone, Amiprin, thioguanine, thiotepa, thiazophrine, tirapazamine, topotecan hydrochloride Toremifene citrate, Trestron acetate, Trisilibine phosphate, Trimethrexate Trimethrexate glucuronide, triptorelin, tubrosol hydrochloride, uracil Mustard, uredepa, bapreotide, verteporfin, vinblastine sulfate, vin sulfate Cristine, vindesine, vindesine sulfate, vinepidine sulfate, vincinate sulfate, Vinoleulosine sulfate, vinorelbine tartrate, vinrosidine sulfate, vinzolidine sulfate, vo Rozole, zeniplatin, dinostatin, zolubicin hydrochloride.

[0094] In some embodiments, cancer therapeutics and NK-stimulated exosomes are prepared in the same composition. It is possible. In some embodiments, cancer drugs and NK-stimulated exosomes may be used in different combinations. It can be prepared in the product.

[0095] The present invention includes NK-stimulated exosomes containing one or more stimulating peptides, and a cancer treatment drug. The compositions may be administered simultaneously or at different times. In some embodiments, one or NK-stimulated exosomes containing two or more stimulating peptides are used to treat the disease or disorder. Therapeutic drugs for knowledge 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 1 5, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 It is administered 29, 30, or 31 days prior or after. In some embodiments, one or NK-stimulated exosomes containing two or more stimulating peptides are used in treating diseases or disorders. One, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve months prior to a known drug or It will be administered later.

[0096] Device A device is disclosed that includes NK-stimulated exosomes containing one or more stimulating peptides. For example, the container used during apheresis contains one or more irritating peptides. It may contain NK-stimulated exosomes, which therefore pass through the container during apheresis. The cells are incubated with NK-stimulated exosomes or brought into contact with them. It can be positioned in a certain state, enabling stimulation of NK cells and ultimately proliferation of NK cells. .

[0097] kit The substances mentioned above and other substances are used to carry out or assist in carrying out the disclosed method. As a kit, it can be packaged together in any suitable combination. The components of the kit in the pack are designed and adapted to be used together in the disclosed manner. In short, it is useful.

[0098] The disclosed kit may also contain stimulating peptides. The kit is an NK stimulating exorcist. It may further contain ingredients for preparing the mixture.

[0099] definition When used in this specification and the attached "Claims", the singular form "a", "n" and "the" indicate multiple references unless otherwise explicitly indicated. It is important to note that this includes elephants. Therefore, for example, "exosome (an exoso The reference to "me)" includes multiple such exosomes and "the stimulant peptide (the stimulat) The reference to "(ory peptide)" refers to one or more irritating peptides and those well known to those skilled in the art. This is a reference to its equivalent.

[0100] "Exosomes" refer to membrane vesicles produced or secreted by living cells. This is achieved by treating synthetic liposomes derived from free lipid components or disrupted cell lipid membranes. It does not contain the plasma membrane vesicles that it has formed. This term is also used for microvesicles, epididymosomes (ep ididimosome), algosome, exosome-like vesicle, prominosome, dex, te This includes oxosomes, archeosomes, and oncosomes, but these are This applies only when the vesicles are produced or secreted by cells. In some embodiments, the vesicles are multi When the vesicle fuses with the plasma membrane, it is released from the cell. In some cases, the vesicle fuses with the plasma membrane. It is released directly from there.

[0101] "NK-stimulated exosomes" or "NK cell-stimulated exosomes" or "NK-stimulated exosomes" "Sosomes" are organisms that can stimulate the generation of NK cells or increase the number of NK cells. Hormone to target cells lysed by sosomes and / or NK cell cytotoxic activity This includes, but is not limited to, enhancing NK cell activity by enhancing targeting / targeting. This refers to exosomes that are capable of [something]. "NK-stimulated exosomes" or "NK cell-stimulated exosomes" "Sosomes" or "NK-stimulated exosomes" contain one or more stimulating peptides. obtain.

[0102] "Plasma membrane vesicles" are preparations of plasma membranes from cells or artificially created plasma membranes. "ku" refers to liposomes.

[0103] "Membrane-inserted peptides" are peptides that can be inserted into or anchored to the cell membrane.

[0104] "Stimulating peptides" are stimulating ligatures that bind to activating receptors present on the surface of NK cells. It refers to the peptide. Stimulating peptides also promote the proliferation, stimulation, activation, or interaction of NK cells. This refers to drugs that induce adhesion. Irritant peptides include cytokines, adhesion molecules, or NK cells. It may be a cell activator. "Adjustment" or "adjusting" when used herein means increasing It refers to addition or reduction. Adjustment leads to any difference compared to normal function. For example. Regulating the immune system refers to increasing or decreasing the number of immune cells.

[0105] "Optional" or "optionally" refers to the event, situation, or substance described thereafter. However, whether it exists or not, the explanation is whether an event, situation, or substance occurs or exists. This means including examples of cases where they exist, and examples of cases where they occur or do not exist. ru.

[0106] "To bring into contact" as used herein means that entities may influence each other. It means bringing one or more entities into close proximity. "To bring into contact" means physical It may or may not include contact. "to make contact," "made to make contact," "made to make contact" "To be exposed to and affected by these deformations through direct or indirect interaction" The effect further includes providing, and the effect includes, but is not limited to, intercellular or molecular interactions. Whether or not it is mediated by an interaction that does not occur, and whether or not it cooperates with it. Alternatively, it may or may not be the result of this.

[0107] As used herein, the term "subject" means a disclosed composition used, for example, in experiments, medical examinations. This refers to any organism that can be administered for the purpose of stopping and / or treating illness. Typical subjects include , animals (for example, non-human primates, and mammals such as humans, birds, cats, dogs, sheep, goats, cattle) , domestic animals or livestock such as horses and pigs, experimental animals such as mice, rats and guinea pigs Examples include animals (such as animals, rabbits, fish, reptiles, zoo animals, and wild animals). Typically, these are referred to as "subjects." This includes animals, including humans and mammals such as primates. The subject may also be a cell or a cell It can refer to stocks.

[0108] In this specification, the range is defined as "about" one specific value and / or "about" another specific value. It may also be expressed as a value. Furthermore, if such a range is expressed, it may not be particularly indicated in the context. Unless otherwise indicated, a range from one specific value to and / or another specific value is given particular consideration. It is thought that it will be disclosed. Similarly, when a value is expressed as an approximation, the preceding "approximately" is used. Depending on the application, certain values ​​may be considered to be disclosable unless otherwise indicated in the context. It will be understood that this forms a specific embodiment of the consideration. Unless otherwise indicated in the context. As far as is the case, each endpoint of the range has both with respect to the other endpoints and independently of the other endpoints. It will be further understood that this is the intention. Finally, each individual included within the explicitly disclosed scope All values ​​and partial range values ​​are also specifically intended and, unless otherwise indicated in the context, should not be disclosed. It should be understood that in certain cases, these embodiments Regardless of whether some or all of the above are expressly disclosed, the foregoing applies. .

[0109] Unless otherwise defined, all technical and scientific terms used herein are disclosed. The same meaning as generally understood by those skilled in the art in the field to which the described method and composition belong. It has any method and substance that is similar or equivalent to those described herein. However, the methods and compositions of the present invention can be used in the implementation or testing of these methods, but particularly useful methods, The apparatus and materials are as described. The papers cited herein and those cited are also referenced. Any materials mentioned herein are incorporated specifically by reference. However, the present invention does not grant prior rights to such disclosures through the effect of prior inventions. It should not be interpreted as an admission that it has not been done. Any references prior to the original It cannot be considered to constitute a technology. The author's argument is stated in the discussion of the references. The applicant reserves the right to challenge the accuracy and appropriateness of the cited documents. Numerous papers are referenced in the specification, but such references are... This does not constitute an admission that either of these constitutes part of the general knowledge in the relevant technical field. That is clear.

[0110] Throughout this specification and the claims, the word "comprise" and The variations of this word (for example, "comprising" and "comprises") are This means "includes, but is not limited to," for example, other additives, components, This is not intended to exclude integers or processes. In particular, one or more processes In a method that includes a step or operation, each step is limited to (a step consisting of, for example, "consisting of") It is specifically intended to include the listed items (unless the term is explicitly stated), and this is each The process excludes, for example, other additives, components, integers, or processes not listed in the process. This is not intended to be the case.

[0111] This may be used for the disclosed methods and compositions, and may be used in combination therewith. Substances, compositions, and chemicals that can be used to prepare these, or are products thereof. Compounds are disclosed. These and other substances are disclosed herein, and combinations of these substances When combinations, subsets, interactions, groups, etc. are disclosed, those of these compounds Specific references to each of the various individual and collective combinations and permutations are explicitly disclosed. While there may be cases where this is not the case, please understand that each is specifically intended and described herein. For example, membrane self-insertion peptide conjugates have been disclosed and discussed, and membrane self Numerous modifications made to numerous molecules, including self-insertion peptide conjugates, are discussed. If so, the combination and substitution of membrane self-insertion peptide conjugates with possible modifications is All of these are intended to be concrete unless otherwise instructed. Therefore, Classes A, B, and C of molecules are disclosed, and similarly, classes D, E, and F of molecules and If examples of combinations of molecules A-D are disclosed, then each one is further listed individually. If none exist, each is considered individually and collectively. Therefore, in this example, A~E, A Each of the combinations ~F, B~D, B~E, B~F, C~D, C~E, and C~F is Specifically intended, A, B, and C; D, E, and F; and exemplary pairs of A-D It should be considered that disclosure will be made from the disclosure of the combination. Similarly, these any sub-components Sets or combinations will also be specifically considered and disclosed. For example, A-E, B The subgroups ~F and C~E are specifically intended, and A, B, and C; D, E, and F; and This concept should be considered to be disclosed from the disclosure of exemplary combinations A to D. This applies to all aspects of this application and the process of preparing and using the disclosed compositions. This includes, but is not limited to, various additional steps that may be performed. Each of these additional steps is a part of any particular embodiment or embodiment of the disclosed method. It can be performed in combination, and each such combination is specifically intended. It is believed that this information will be disclosed. [Examples]

[0112] Example 1: Proliferation of NK cells by preparation of crude exosomes result As demonstrated herein, NK cell proliferation is K562-mb21-41BB These cells can be generated by using exosomes derived from cultured L-stimulated cells. It promotes very strong proliferation of NK cells and is derived from a peripheral blood mononuclear cell (PBMC) mixture before the start of culture. Since it has been reported that there is no need to isolate NK cells, K562-mb21-41BBL The culture was selected for the isolation of exosomes. Furthermore, the irritant ligand 41BBL and The presence of mbIL21 can be easily tracked by antibody staining, and these on feeder cells The expression of these molecules and their presence in isolated exosomes can be confirmed. .

[0113] This experiment showed that exosomes derived from cultured stimulated cells are converted into NK cells in the same way as stimulated cells. This was done to test whether it supported proliferation. PBMCs were treated with 50 U / mL IL-2. Crude exo When the concentration of sosomes was reduced over 24 days, the NK cell contents in the PBMC mixture The concentration of exosomes increased (Figure 4). The concentration of exosomes is the concentration of proteins embedded in the exosomes. Indicated by degree. Crude exosome preparations are cultured at higher exosome concentrations. It is highly likely that it contains certain culture-derived substances that inhibit the growth of the organism. However, crude exoskeletons The preparation of the exosomes was more effective when diluted. The dilution concentration of the exosomes used was ( At 50 μg / mL, an approximately 240-fold proliferation of NK cells was observed, and the proportion of NK cells was The number increased by more than 70%. Therefore, this experiment showed that NK cells do not contain feeder cells. Using exosomes embedded with irritating ligands, selective growth is performed within the PBMC mixture. To show that it is possible.

[0114] Materials and methods Cell lines K562-mb15-41BBL and K562-mb21-41BBL (K56 2-clone9.mbIL21) are respectively Dr. Dario Campana(S t. Jude Children's Research Hospital) and Dr. Obtained from Dean Lee (MD Anderson). K562 cell line used. The American Tissue Culture Collection (ATC Purchased from C). The preparation of NK cells to stimulate crude exosomes was carried out as follows: K562-mb21-41BBL cells were cultured in RPMI medium supplemented with 10% FBS. Then, the culture was scaled up to 1L. After scaling up, K562-mb21-41 BBL cultures were treated with 2 micromoles of monesin. The culture medium was 10N (1,000 × g). The cells were removed from the cell culture by centrifugation and then pelletized. The removed culture medium was The sample was filtered using a 0.45 μm filter, and then a 100 kDa MWCO membrane was used. The proteins were concentrated using a BCA assay. The apparent protein concentration bound to the protein was determined.

[0115] PBMCs isolated from blood by Ficol-Paque density gradient were 10% FBS They were grown in SCGM Cell Gro medium supplemented with 50 U / mL of IL-2, and exo The concentration of osomes was reduced. The cells were maintained at 37°C in a humidified atmosphere of 5% CO2. From day 5, the culture medium is replaced every other day by replacing half of the medium with fresh medium. The cells were replaced, and the exosomes removed by replacing the culture medium were replaced. The cells were measured every other day. Yes, I checked the contents of the culture.

[0116] Example 2: Characterization of exosomes NK cell-stimulated exosomes were isolated from cultured K562-mb21-41bbl cells. The cells were approximately 1 x 10 6 Culture to a cell / mL density, wash, and use serum-free RPMI. The cells were resuspended and treated with 2 μM monensin. The cells were then incubated at 10 N (1000 × g) for 10 minutes. The residue was removed by centrifugation, and then filtered through a 0.22 μm PES membrane. The culture medium was then ultrafiltered using a 100,000 kDa MWCO membrane. Therefore, it was concentrated.

[0117] Figures 2A to 2D show exoskeletons isolated from cultured K562-mb21-41bbl cells. The exosomes are characterized. The exosomes are resuspended in PBS and then scanned with NanoSight N Nanoparticle tracking analysis using video microscopy with S300 (Malvern) (NT Characterized by A). NTA is determined by analyzing light scattering intensity and diffusion kinetics. This determines the single-frame light scattering image (Figure 2A) and the binned histology of the grain size distribution. Gram (Figure 2B) is shown. The presence of IL-21 was detected using an anti-IL-21 antibody. This was confirmed immunochemically by stamp blot analysis (Figure 2C). Anti-IL-21 antibody-conjugated gold Analysis using nanoparticles (GNP) (Figure 2D) shows GNP conjugated to isotype control antibodies. Compared to the absence of an increase in P, the time course of dynamic light scattering intensity with anti-IL-21 antibody-conjugated GNP An increase is observed, indicating the presence of IL-21 in exosome samples.

[0118] Figures 3A and 3B show exoskeletons isolated from cultured K562-mb21-41bbl cells. This shows that the system stimulates the specific proliferation of NK cells derived from unselected PBMCs. Unselected PBMCs with a 100,000 NK cell / mL count were supplemented with 10% FBS to form SC cells. In GM medium, K562-mb21-41bbl cells with a total protein content of 35 ng / mL The cells were cultured with exosomes isolated from the cell culture medium. After an initial delay, the NK cells remained in the culture medium for 20 days. Over the course of the period, the number of lymphocytes increased exponentially by an average of 270 times (Figure 3A), and the relative abundance of total lymphocytes increased by 7 It increased to 4% (Figure 3B). The culture was renewed every other day in fresh medium containing exosomes. It was obscured. A normal patient that does not have mbIL-21 and 4-1BBL expressed by gene transfer. Exosomes isolated from cultured non-transformed K562 cells did not induce NK cell proliferation. All cultures grew in two ways, and the markers represented the mean with error bars representing the standard deviation. vinegar.

[0119] NK cells proliferated with exosomes were shown to be cytotoxic to K562 CML tumor cells. The cells were analyzed. K562 cells were pre-labeled with TFL4 dye. Target tumor cells were raised at 37°C. In a 5% CO2 atmosphere for 2 hours, the E:T ratio of NK cells was 0.5 × 10⁶. 6 The cells were then co-cultured with K562 cells / mL. Subsequently, the cells were treated with Annexin V-FITC. Centrifugation and resuspending in Annexin V-labeled buffer, then incubation at 4°C for 15 minutes. The labeled cells were diluted to 250 μL and tested using an Accuri instrument (BD Biosci). Analysis was performed by flow cytometry (ence). Figure 4 shows the results of exosome stimulation. This shows that proliferated NK cells are cytotoxic to K562 cells. Unselected PBM C was obtained from K562-mb21-41bbl cell cultures with a total protein concentration of 35 ng / mL. We cultured isolated exosomes and used them to analyze cytotoxicity against K562 cells. For comparison, NK cells were also compared to K562-mb21-41bbl feeder cells and NK cells (E) were proliferated using PM21 particles (200 μg / mL). The cytotoxicity of x21-NK cells (FC21-N) is due to NK cells (FC21-N) proliferating on feeder cells. K cells (▲) or NK cells (PM21-NK cells■) proliferated with IL-21-bound plasma membrane particles. It is slightly lower compared to ).

[0120] Exotherapy in co-culture of PBMCs and K562-mb21-41bbl feeder cells The presence of the PBMC was confirmed by video microscopy. Figures 5A to 5E show the PBMC. As feeder cells (100,000 NK cells / mL), and with a 10-fold excess of feeder cells As cells, they are generated in culture by K562-mb21-41bbl, and then live cells. Perkin Elmer Ultraview Micros with image formation steps The 10x objective lens of the copy system imaged the images over an 18-hour period. The sosome is shown. Observe the adhesion of the AF647 label over a period of several minutes to approximately one hour (Figure 5A). After several hours (Figure 5B), the formation of intracellular endosomes and polyendoplasmic reticulum is observed. Subsequently, Cellular exosomes are observed (Figures 5C and 5D). Live imaging of cells from co-culture. Samples were obtained, stained with anti-CD3 and anti-CD56, and imaged using fluorescence confocal microscopy. (Figure 5E). NK cells ingested or subsequently bound to the AF647 label, but T cells preferred Do not ingest or bind to it. A wider area than indicated may be tested for statistical validity. Ten slices along the Z-axis were then imaged, and intracellular and extracellular events were identified.

[0121] The disclosed methods and compositions may change, so the specific methodology and protocol described may not apply. It will be understood that this does not apply to reagents, etc. The terminology used herein is specific to the following: This is for illustrative purposes only and is not subject to the attached "Claims". It should also be understood that this is not intended to limit the scope of the present invention, which may be limited in some ways. ru.

[0122] Those skilled in the art will be able to recognize or confirm, by routine experimentation alone, many equivalents to specific embodiments of the methods and compositions described herein. Such equivalents shall be encompassed by the following claims. This application also relates to the following aspects. (1) A method for increasing the number of NK cells, A method comprising contacting at least one NK cell with at least one NK-stimulated exosome containing one or more stimulating peptides present in the exosome membrane, wherein the exosome is an extracellular product of an exosome-secreting cell. (2) The method according to (1) above, wherein the one or more irritating peptides are selected from the group consisting of 4-1BBL, IL-2, IL-12, IL-18, IL-21, MICA / B, ULBP2, ICAM-1, 2B4, BCM1 / SLAMF2, CD155, CD112, CCR7, DAP12, and DAP10. (3) The method according to (1) above, wherein one or more irritating peptides are bound to the membrane-inserted peptide. (4) The method according to (3) above, wherein the membrane-inserted peptide comprises a membrane-insertable fusion peptide having affinity for a lipid bilayer, and the fusion peptide comprises a segment of IG4, CD4, or a combination thereof. (5) The method according to (3) above, wherein the one or more irritating peptides bound to the membrane-inserted peptide are fusion proteins encoded by recombinant DNA. (6) The method according to (3) above, wherein the membrane insertion peptide comprises human Fc, GPI, transmembrane T cell receptor, or pHLIP. (7) The method according to (1) above, wherein the NK cells are present in a population of unselected peripheral blood mononuclear cells (PBMCs). (8) The method according to (1) above, wherein the exosomes are produced by a cell line that is manipulated to improve the expression of exosomes. (9) The method according to (1) above, wherein the NK cells are in contact with the NK-stimulated exosomes in vitro, in vivo, or ex vivo. (10) The method according to (9) above, wherein the NK cells are proliferated in vivo in the epidural space, intraperitoneal space, subcutaneously, or intravenously. (11) The method according to (9) above, wherein the NK cells come into contact with the NK-stimulated exosomes in an allogeneic transplantation procedure, a haploid transplantation procedure, or an in vivo immunotherapy procedure. (12) The method described in (11) above, wherein the use of NK-stimulated exosomes in allogeneic transplantation, haploid transplantation, or in vivo immunotherapy does not cause graft-versus-host disease (GVHD). (13) A method for treating cells susceptible to NK-mediated lysis, comprising administering an effective amount of a composition containing NK cells that have been contacted according to the method described in (1) above. (14) The cell susceptible to NK-mediated lysis is infected with the virus, as described in (13) above. (15) The method according to (13) above, wherein cells susceptible to NK-mediated lysis include AML, breast cancer, bladder cancer, colorectal cancer, kidney cancer, lung cancer, prostate cancer, thyroid cancer, and uterine cancer. (16) A method for reducing the risk of recurrence after stem cell transplantation, comprising administering an effective amount of a composition containing NK cells that have been contacted according to the method described in (1) above. (17) A method for generating NK cell-stimulated exosomes, comprising embedding one or more stimulating peptides within the membrane of an exosome. (18) The method according to (17) above, wherein the one or more irritating peptides are selected from the group consisting of 4-1BBL, IL-2, IL-12, IL-18, IL-21, MICA / B, ULBP2, ICAM-1, 2B4, BCM1 / SLAMF2, CD155, CD112, CCR7, DAP12, and DAP10. (19) The method according to (17) above, wherein one or more irritating peptides are bound to a membrane-inserted peptide. (20) The method according to (19) above, wherein the membrane-inserted peptide comprises CD4 or IgG having affinity for the lipid bilayer. (21) The method according to (19) above, wherein the one or more irritating peptides bound to the membrane-inserted peptide are fusion proteins encoded by recombinant DNA. (22) The method according to (19) above, wherein the membrane insertion peptide comprises human Fc, GPI, transmembrane T cell receptor, or pHLIP. (23) The method according to (17) above, wherein the NK cell-stimulated exosomes are derived from a cell line that has been manipulated to improve exosome expression. (24) A method for treating cancer, comprising administering an effective amount of a composition comprising NK-stimulated exosomes containing one or more stimulating peptides. (25) A method for treating cancer, comprising (a) ex vivo contacting NK cells with NK-stimulated exosomes containing one or more stimulating peptides to obtain a population of contacted NK cells, and (b) administering the population of contacted NK cells to a subject. (26) A composition comprising NK-stimulated exosomes containing 4-1BBL and IL-21 stimulating peptides. (27) The composition according to (26) above, further comprising one or more irritating peptides selected from the group consisting of 4-1BBL, IL-2, IL-12, IL-18, MICA / B, ULBP2, ICAM-1, 2B4, BCM1 / SLAMF2, CD155, CD112, CCR7, DAP12, and DAP10. (28) The composition according to (26) above, wherein one or more irritating peptides are bound to a membrane-inserted peptide. (29) The composition according to (28) above, wherein the membrane-inserted peptide comprises a segment of CD4, IgG, or a combination thereof that has affinity for the lipid bilayer. (30) The composition according to (28) above, wherein the one or more irritating peptides bound to the membrane-inserted peptide are fusion proteins encoded by recombinant DNA. (31) The composition according to (28) above, wherein the membrane insertion peptide comprises human Fc, GPI, transmembrane T cell receptor, or pHLIP. (32) The composition according to (26) above, wherein the NK-stimulated exosomes are derived from a cell line that has been manipulated to improve exosome expression. (33) The composition according to (26) above, further comprising a pharmaceutical carrier. (34) A method for modifying NK cell function, comprising contacting at least one NK cell with at least one NK-stimulated exosome, wherein the NK-stimulated exosome is filled with a functional nucleic acid that modifies NK cell function. (35) The method described in (34) above, wherein the functional nucleic acid includes microRNA (miRNA), RNAi, or a combination thereof. < / plus>

Claims

1. A method for stimulating the proliferation of natural killer (NK) cells, comprising contacting at least one NK cell with at least one NK cell-stimulated exosome ex vivo or in vitro, The NK cell-stimulated exosome delivers nucleic acids selected from antisense molecules, aptamers, ribozymes, triple-stranding molecules, RNAi, and external guide sequences. The at least one NK cell-stimulated exosome is filled with the nucleic acid, The aforementioned at least one NK cell-stimulated exosome contains two or more NK cell-stimulating peptides present in the exosome membrane. A method wherein 4-1BBL and IL-21 are two of the NK cell-stimulating peptides, and the NK cell-stimulated exosome is an extracellular product of an exosome-secreting cell line selected from the cell line K562-mb21-41BBL or a derivative of the cell line K562-mb21-41BBL.

2. The method according to claim 1, wherein the nucleic acid comprises siRNA, shRNA, microRNA (miRNA), or a combination thereof.

3. The method according to claim 1 or 2, wherein the nucleic acid acts as an effector, inhibitor, modifier, or stimulator of a specific activity possessed by at least one NK cell.

4. The method according to claim 3, wherein the nucleic acid is an inhibitor of A2AR, P2YR, or a combination thereof.

5. The method according to any one of claims 1 to 4, wherein the two or more NK cell-stimulating peptides further comprise peptides selected from the group consisting of IL-2, IL-12, IL-18, MICA / B, ULBP2, ICAM-1, 2B4, BCM1 / SLAMF2, CD155, CD112, CCR7, DAP12, and DAP10.

6. The method according to any one of claims 1 to 5, wherein the two or more NK cell-stimulating peptides are bound to one or more membrane-inserted peptides.

7. The method according to claim 6, wherein the one or more membrane-inserted peptides include a CD4 or IgG segment having affinity for the lipid bilayer.

8. The method according to claim 6, wherein the one or more membrane-inserted peptides include a peptide selected from human Fc, GPI, transmembrane T cell receptor, or pHLIP.

9. The method according to any one of claims 1 to 8, wherein the at least one NK cell is present in a population of unpurified peripheral blood mononuclear cells.

10. The method according to any one of claims 1 to 9, wherein the at least one NK cell-stimulated exosome is produced by an exosome-secreting cell line that has been engineered to improve exosome expression.

11. The method according to claim 10, wherein the exosome-secreting cell line is manipulated to improve the expression of exosomes containing membrane-bound IL-21 and / or membrane-bound 4-1BBL.

12. The method according to claim 10 or 11, wherein the exosome-secreting cell line includes the cell line K562-mb21-41BBL.

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