Tumoticidal and antimicrobial compositions and methods

Abstract

The present invention relates to tumor-killing and antimicrobial compositions and methods. The present invention provides pharmaceutical compositions comprising tumor-killing and / or antimicrobial components isolated from the supernatant of the NK-92 cell culture medium, and methods of using the compositions to kill cancer cells.

Description

Technical Field

[0001] The present invention generally relates to pharmaceutical compositions having tumoricidal and / or antimicrobial properties, and methods of using said compositions to kill tumors and / or microorganisms. Background Art

[0002] Skin cancer is a leading type of cancer in humans and many domesticated animals. The incidence of skin cancer and melanoma is increasing due to a variety of factors, including unprotected exposure to ultraviolet rays, such as those found in the sun or tanning beds. If diagnosed in the early stages, these cancers can be easily treated by removing the cancer and surrounding tissue. However, as with any surgical procedure, there is still the possibility that abnormal cancer cells may remain at the site of removal, leading to cancer recurrence. This is particularly true in the case of melanoma, where failure to remove all tumor cells can lead to metastasis, which is accompanied by a higher incidence of cancer.

[0003] Aggressive measures are available to minimize the risk of recurrence, including conventional treatments and extensive testing by your doctor. Despite these options, melanoma tends to be very aggressive, with a reported recurrence rate of 2% to 65% within five years, depending on the stage of the cancer at the time of treatment. See, for example, www.aimatmelanoma.org / en / aim-for-answers / moving-on-after-treatment / follow-up-by-stage.html.

[0004] Animals are also susceptible to skin cancer, particularly melanoma, squamous cell carcinoma, and mast cell tumors. Skin cancer is especially common in animals that spend a lot of time in the sun.

[0005] Infection is a common problem worldwide. Many infections are caused by bacteria, fungi, and other microorganisms. Although current treatments for these infections rely heavily on antibiotics and antimicrobial drugs, an increasing number of bacterial infections are being found to be resistant to at least some antibiotics. According to the Centers for Disease Control (CDC), more than 2 million people in the United States are infected with antibiotic-resistant microorganisms each year, resulting in more than 23,000 deaths. See, for example, www.medicalnewstoday.com / articles / 266182.php. Skin and soft tissue infections are the third most common diagnosis in emergency care settings, and an estimated 7% to 10% of all hospitalizations have skin or soft tissue infections. Ki and Rotstein, Can. J. Infect. Dis. Med. Microbio1. 2008 March; 19(2): 173-184. Other common infections include systemic infections, respiratory infections, ear infections, gastrointestinal infections, and urinary tract infections. Infections are also common in domesticated animals and can be difficult to treat. Viral infections are also common, and few treatments are available for viral infections.

[0006] Thus, there remains a need for tumoricidal compositions that can be used to lyse cancer cells, particularly those associated with dermal and subcutaneous cancers.There also remains a need for new antimicrobial compositions that can be used to treat infections. Summary of the Invention

[0007] Certain cells of the immune system have cytotoxic activity against specific target cells. Natural killer (NK) cells, which typically account for about 10-15% of circulating lymphocytes, bind and kill target cells, including virus-infected cells and many malignant cells, non-specifically for antigens and without previous immune sensitization. Herberman et al., Science 214:24 (1981). Killing of target cells can occur by lysing cells. NK cells have been shown to be effective in both in vitro and in vivo treatment of patients with advanced cancer. However, endogenous NK cells (i.e., cells harvested from donors or patients) are still difficult to be effective in immunotherapy and applied in immunotherapy. NK cells are difficult to expand in vitro so that they maintain their tumor targeting, tumor-killing and virus-killing abilities in vivo, which is also a major obstacle to their clinical application in adoptive cellular immunotherapy. Melder et al., Cancer Research 48:3461-3469 (1988); Stephen et al., Leuk. Lymphoma 377-399 (1992); Rosenberg et al., New Engl. J. Med. 316:889-897 (1987). In addition, preparations of endogenous NK cells contain T cells and / or other immune effector cells that must be removed when NK cells are used to treat patients unrelated to the donor.

[0008] The NK-92 cell line is a unique cell line discovered to proliferate in the presence of interleukin-2 (IL-2). Gong et al., Leukemia 8: 652-658 (1994). Unlike NK cells, NK-92 cells are a cytolytic cancer cell line discovered in the blood of subjects with non-Hodgkin's lymphoma and then immortalized in vitro. This cell line has high cytolytic activity against a variety of cancers. The NK-92 cell line is a homogenous NK cell population, as this is associated with lytic activity. Phase I clinical trials have confirmed its safety profile, and anti-tumor responses have been observed in certain patients with advanced cancer.

[0009] Endogenous NK cells differ significantly from NK-92 cells, primarily due to their different origins: NK-92 cells are cancer-derived cell lines, whereas endogenous NK cells are obtained from a donor (or patient) and processed for infusion into the patient. Endogenous NK cell preparations are heterogeneous cell populations, while NK-92 cells are homogeneous clonal cell lines because they all exhibit lytic activity. NK-92 cells readily proliferate in culture while maintaining cytotoxicity, whereas endogenous NK cells cannot.

[0010] Cells (including NK cells) release multiple components into the culture medium in which they grow. Examples of such components include proteins, exosomes, and microvesicles. Exosomes are nanovesicles (up to 100 nanometers) released by normal cells and tumor cells. Microvesicles are similar to exosomes, but are larger in size (greater than 100 nanometers). Exosomes and microvesicles can be detected and separated from cell culture supernatants or from body fluids (such as blood).

[0011] Exosomes isolated from the cell culture supernatant of endogenous NK cells contain proteins, including CD56, perforin, FasL and Rab5B. Lugini et al. J Immunol. (2012) 189, 2833-2842. However, exosomes and other factors derived from endogenous NK cells are highly variable for the amount of exosomes recovered and the proteins contained therein. Ibid., p. 2839. Without being limited to theory, it is believed that this variability is because endogenous NK cells are obtained from individual donors and also because endogenous NK cells contain heterogeneous cell populations. In addition, differences in conditions (such as cell culture conditions, purification methods, starting cell populations and the ratio of cell types in culture) can all lead to changes in the composition and amount of exosomes that can be purified from endogenous NK cells. For example, certain cell populations in NK cell preparations can be selected considering the purification method and / or culture conditions used or selected for the purification method and / or culture conditions used.

[0012] The variability issues associated with endogenous NK cells do not apply to the NK-92 cell line. Because it is a cell line, large numbers of cells can be cultured and expanded over long periods of time. These cultures are homogenous cell populations that provide consistent, reproducible exosome and / or microvesicle preparations. The exosomes and / or microvesicles secreted by NK-92 cells contain proteins hypothesized to have tumoricidal properties, such as cytotoxic and / or cytolytic properties. Other components can be isolated from the culture medium containing NK-92 cells, including those with antimicrobial and immunomodulatory properties.

[0013] On the other hand, NK-92 cells are cancer cell lines. Some cancer cells have been shown to release exosomes containing factors (such as microRNA) that promote tumor growth in some cases. The exosomes and other factors released by many cancer cells are significantly different from those released by their non-malignant counterparts. For example, NK-92 cells release factors with antimicrobial properties, a feature not observed in endogenous NK cells. In one embodiment, the exosomes and / or microvesicles obtained from NK-92 cells are incubated in a suitable solution (such as PBS or isotonic saline) before use to extract these factors. This incubation period is envisioned to greatly reduce or eliminate those factors that promote tumor growth.

[0014] One aspect of the present invention provides a pharmaceutical composition comprising one or more components obtained from the supernatant of NK-92 cell culture medium. The components may be tumoricidal and / or antimicrobial. The components may also have immunomodulatory properties. These components are preferably exosomes and / or microvesicles separated from the supernatant. In one embodiment, the exosomes and / or microvesicles are cytotoxic. In one embodiment, the exosomes and / or microvesicles have the ability to lyse cancer cells. In one embodiment, the exosomes and / or microvesicles are antimicrobial. In one embodiment, the exosomes and / or microvesicles are antiviral. In one embodiment, the exosomes and / or microvesicles are antibacterial. In one embodiment, the exosomes and / or microvesicles are immunomodulatory.

[0015] In a preferred embodiment, the pharmaceutical composition does not contain living cells. In some embodiments, the pharmaceutical composition is an injectable composition. In some embodiments, it is envisioned that injection provides systemic immunomodulatory activity.

[0016] Subpopulations of NK-92 cells are observed in culture and can be isolated. Subpopulations may differ in terms of expression of cell surface markers, protein expression, etc. In one aspect of the present invention, one or more subpopulations of NK-92 cells are isolated before obtaining one or more components from the supernatant of the NK-92 cell culture medium. The isolation of one or more defined subpopulations allows for the production of uniformly defined exosomes and / or microvesicles, particularly exosomes and / or microvesicles that exhibit low or no tumoricidal properties.

[0017] In some embodiments, the pharmaceutical compositions provided herein are administered to a part of the body after surgery to kill residual cancer cells and / or reduce the likelihood of cancer recurrence. In some embodiments, the pharmaceutical compositions are configured for topical use. In some embodiments, the pharmaceutical compositions are configured for subdermal application. In some embodiments, the pharmaceutical compositions comprise a phase-changing poloxamer.

[0018] In some embodiments, the pharmaceutical composition is in an injectable form comprising a phase change polymer, such that the composition is injected as a liquid and phase changes to a gel in vivo (eg, at body temperature), thereby providing a drug depot.

[0019] Another aspect of the present invention provides a pharmaceutical composition comprising an antimicrobial (including antibacterial, antifungal and / or antiviral) component obtained from the supernatant of NK-92 cell culture medium. In some embodiments, the antimicrobial component includes a tumoricidal component. In some embodiments, the antimicrobial component is an exosome and / or microvesicle separated from the supernatant. In a preferred embodiment, the antimicrobial component comprises microvesicles separated from the supernatant.

[0020] Another aspect of the present invention provides a method for lysing cancer cells, which comprises administering to a human or animal patient in need an anti-tumor or cytotoxic component separated from the supernatant of a growth medium of NK-92 cells. In one embodiment, the component is injected into a tumor (such as a solid tumor). In one embodiment, the component is injected into an area around or near a tumor. In one embodiment, the component is topically administered to a tumor (such as skin cancer). In one embodiment, the component is systemically administered.

[0021] In some embodiments, the cancer is a carcinoma, lymphoma, sarcoma, melanoma, astrocytoma, mesothelioma cell, ovarian cancer, colon cancer, pancreatic cancer, esophageal cancer, stomach cancer, lung cancer, urinary system cancer, bladder cancer, breast cancer, gastric cancer, leukemia, lung cancer, colon cancer, central nervous system cancer, ovarian cancer, cervical cancer, kidney cancer or prostate cancer. In a preferred embodiment, the cancer is skin cancer.

[0022] These and other aspects of the invention are set forth in detail below. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Shows the effect of NK-92 cells on the growth of Cryptococcus neoformans

[0024] Figure 2 Display and Figure 1 Same data as in , expressed as percentage of growth with Cryptococcus neoformans.

[0025] Figure 3A Photographs of Western blots analyzing protein components (lanes 4-7) contained in exosomes and / or microvesicles (EV / MV) isolated from NK-92 cells under various culture conditions. NK-92 cells (lane 3) serve as a positive control, while MCF-7 cells (lane 1) and exosomes (EV, lane 2) serve as positive controls for tubulin and / or Rab5B, and negative controls for cytolytic proteins.

[0026] Figure 3BPhotographs of Western blots analyzing the presence of markers for nuclear membrane (nucleoporins), mitochondria (antiproliferative proteins), exosomes (Rab5B), and exosome / microvesicle preparations in NK-92 cells.

[0027] Figure 4 Shown is the cytolytic activity of NK-92 exosome / microvesicle preparations on Jurkat cells in the propidium iodide assay. DETAILED DESCRIPTION

[0028] definition

[0029] Before disclosing and describing the articles and methods of the present invention, it should be understood that the following aspects are not limited to specific compositions, preparation methods or uses themselves, but may of course vary. It should also be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.

[0030] In this specification and the claims that follow, reference is made to a number of terms which shall be defined as having the following meanings:

[0031] It must be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, "a cytokine" includes a mixture of two or more cytokines, and so forth.

[0032] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0033] The term "comprising" means that compositions and methods include the recited elements, but do not exclude other elements. When used to define compositions and methods, "consisting essentially of" shall mean excluding other elements that are of any significance to the composition. For example, a composition on a substrate consisting of the elements defined herein does not exclude other elements that materially affect the basic and novel characteristics of the invention. "Consisting of" shall mean excluding more than trace amounts of other ingredients and the recited substantial method steps. Embodiments defined by each of these transitions are within the scope of the invention.

[0034] As used herein, a "patient" is any vertebrate organism, including but not limited to mammalian patients, such as humans, farm animals, domesticated pets, etc. In a preferred embodiment, the patient is a human.

[0035] When used herein before a numerical value, the term "about" means that the value can vary within a reasonable range, such as ±5%, ±1%, and ±0.2%.

[0036] The term "endogenous NK cells" as used herein refers to NK cells obtained from a donor (or patient), which is different from the NK-92 cell line. NK cells are immune system cells that kill target cells without specific antigenic stimulation and without the restriction of MHC classes. Endogenous NK cells are generally heterogeneous cell populations in which NK cells are enriched. Endogenous NK cells can be intended for autologous or allogeneic treatment of patients.

[0037] The term "NK-92 cells" includes wild-type NK-92 cells and modified NK-92 cells. NK-92 cells have been found to be more cytotoxic to tumor and infected cell types than NK cells.

[0038] The term "wild-type NK-92 cells" refers to an NK cell line, NK-92, that was originally obtained from a patient with non-Hodgkin's lymphoma and then immortalized in vitro. NK-92 cells can be obtained from the American Type Culture Collection (ATCC) under the accession number CRL-2407 and are described, for example, in U.S. Pat. No. 7,618,817, which is incorporated herein by reference in its entirety.

[0039] The term "modified NK-92 cells" refers to NK-92 cells that have been further processed to impart properties not possessed by the wild-type NK-92 cells from which they are derived. Such processing includes, for example, physical, chemical, and / or biological treatments. The processing imparts properties to the modified NK-92 cells that make them more advantageous for the desired purpose. Examples of modified NK-92 cells are described, for example, in U.S. Patents 7,618,817, 8,034,332, and 8,313,943, all of which are incorporated herein by reference in their entireties.

[0040] The term "extracellular vesicle" encompasses both exosomes and microvesicles, as well as any other vesicles secreted into the culture medium by cells (such as NK-92 cells). In general, exosomes are nanosomes with a diameter of less than about 100 nanometers (nm), and microvesicles have a diameter of about 100 nanometers or larger.

[0041] As used in describing the present invention, the terms "cancer," "tumor," and "malignant tumor" all refer to a proliferation of a tissue or organ. If the tissue is part of the lymphatic or immune system, the malignant cells may include non-solid tumors of circulating cells. Malignancies in other tissues or organs may produce solid tumors. In general, the methods of the present invention can be used to treat lymphocytes, circulating immune cells, and solid tumors.

[0042] The term "tumoricidal ingredient" refers to an ingredient that treats cancerous tumors and / or cancer cells. Treatment of tumors includes reducing or eliminating the tumor, killing cancer cells, and / or inhibiting the growth, proliferation, and / or metastasis of cancer cells. Preferably, tumor cells are killed, such as by cell lysis.

[0043] The term "antimicrobial ingredient" refers to an ingredient that treats or prevents microbial infection. Microorganisms include bacteria, fungi, molds, viruses, etc. Accordingly, antimicrobial ingredients also refer to antiviral ingredients, antibacterial ingredients, antifungal ingredients, etc.

[0044] As used to describe the present invention, the terms "cytotoxic" and "cytolytic" have the same meaning when used to describe the activity of effector cells such as NK cells. In general, cytotoxic activity is associated with killing target cells through a variety of biological, biochemical, and biophysical mechanisms. Cytolysis more specifically refers to an activity in which the effector cleaves the plasma membrane of the target cell, thereby destroying its physical integrity. This results in the killing of the target cell. Without wishing to be bound by theory, it is believed that the cytotoxic effect of NK cells is due to cell lysis.

[0045] As used herein, the terms "growth medium" and "culture medium" have the same meaning. Generally, the term refers to any culture medium or aqueous solution in which NK-92 cells can be grown or maintained and into which the NK-92 cells release exosomes, microvesicles, and / or other active ingredients. The culture medium can include growth medium (e.g., commercially available culture medium) such as X-VIVO 10 or RPMI. Alternatively, the culture medium can also include PBS or other aqueous solutions.

[0046] As used herein, "treat," "treat," and "treat" are defined as the application of an agent to a disease, disorder, or condition to reduce or ameliorate the deleterious or other adverse effects or symptoms of such disease, disorder, or condition. As used herein, "treat" encompasses the treatment of a patient and includes: (a) reducing the risk of developing the condition in a patient determined to be susceptible to the condition but not yet diagnosed as having the condition; (b) preventing the onset of the condition; and / or (c) alleviating the condition, such as causing regression of the condition and / or relieving one or more symptoms of the condition. "Treatment" of a condition or patient or "treatment" of a condition or patient refers to taking steps to obtain beneficial or desired results, including clinical results such as alleviation of symptoms. For purposes of the present invention, beneficial or desired results include, but are not limited to: reducing the size of a tumor or reducing the metastatic potential of a tumor; killing tumor cells; or reducing the severity of infection by an infectious agent (microorganism), such as by alleviating one or more symptoms, shortening the duration of infection, etc.

[0047] Components isolated from NK-92 cell culture supernatant

[0048] NK-92 cells can be expanded, modified, and / or maintained in culture. Any acceptable culture conditions can be used. In one embodiment, NK-92 cells are cultured in enriched alpha minimum essential medium (MEM; Sigma Chemical Co., St. Louis, Mo.) supplemented with fetal bovine serum (e.g., 12.5%; Sigma Chemical Co., St. Louis, Mo.) and / or horse serum (e.g., 12.5%; Sigma Chemical Co., St. Louis, Mo.). In another embodiment, NK-92 cells are cultured in XVivo 10 medium supplemented with human serum, human plasma, or human serum albumin (e.g., 5%). In a preferred embodiment, serum, plasma, or serum albumin has been depleted of exosomes prior to culturing the NK-92 cells.

[0049] The culture medium is optionally supplemented with other nutrients, cytokines, and / or growth factors, such as interleukin-2 (IL-2), L-asparagine, L-glutamine, and / or L-serine. NK-92 cells can release components such as proteins (e.g., cytokines), microvesicles, and / or exosomes into the culture medium while in the culture medium. When the cells are separated from the culture medium, for example, by centrifugation, the components released from the cells are retained in the supernatant.

[0050] Another suitable culture medium includes X-VIVO 10 medium, 5% human serum AB, 36 μM L-asparagine, 450 μM L-glutamine, 324 μM L-serine and 500 IU of iL-2.

[0051] In some embodiments, the culture medium is a serum-free medium, PBS or other aqueous solution, such as Ringer's solution, glucose solution, Kank's solution or other aqueous physiologically balanced salt solution. Without being limited to theory, certain components of the growth medium (such as serum) may contain exosomes, microvesicles and / or other components unrelated to NK-92 cells. Therefore, it may be beneficial to maintain NK-92 cells in a serum-free medium or other aqueous solution for a period of time before separating exosomes, microvesicles or other components from the culture medium. Alternatively, NK-92 cells can be cultured in serum or serum substitutes that are depleted of exosomes or without exosomes.

[0052] In some embodiments, one or more stimulants are added to the culture medium. Without being limited to theory, it is believed that stimulation of NK-92 cells by such stimulants can result in a more consistent, stable, and / or reproducible release of components, including exosomes and / or microvesicles. The stimulant includes, for example, a cytokine or a drug stimulant. In one embodiment, the stimulant is iL-15. In one embodiment, the stimulant is interferon gamma.

[0053] In some embodiments, the one or more tumoricidal and / or antimicrobial components comprise exosomes isolated from the supernatant. In some embodiments, the one or more tumoricidal and / or antimicrobial components comprise microvesicles isolated from the supernatant. In some embodiments, the one or more tumoricidal and / or antimicrobial components comprise exosomes and microvesicles isolated from the supernatant.

[0054] Exosomes are nanovesicles secreted by cells with a diameter of up to about 100 nanometers. In some embodiments, the diameter of an exosome is about 30-100 nanometers. Microvesicles are non-cellular and have a diameter greater than about 100 nanometers and preferably less than about 1.5 microns.

[0055] Exosomes and / or microvesicles can be isolated from the culture medium by a variety of methods. One method is ultracentrifugation. Other methods include commercially available exosome isolation kits (such as the Total Exosomes isolation kit [Life Technologies], Exo-spin TM Exosome purification kit (Exo-spin TM Exosome Purification Kit)[Cell Guidance Systems], or Exosome Isolation Kit ( Exosome Isolation Kit) [101Bio]); commercially available instruments, such as Human CD63-specific purification system or Streptavidin purification system (available from Life Technologies); filtration; or differential centrifugation methods (such as those described in S. Rani et al., Methods Mol Biol., 784: 181-95 (2011)). The presence, size, and purity of exosomes and / or microvesicles can be determined by methods such as Western blotting, transmission electron microscopy, flow cytometry, atomic force microscopy, nanoparticle tracking analysis, micro-Raman spectroscopy, resistive pulse detection, and transmission electron microscopy.

[0056] In some embodiments, the NK-92 cells comprise wild-type NK-92 cells.

[0057] In some embodiments, NK-92 cells comprise modified NK-92 cells. NK-92 cells can be modified by methods known in the art, such as those described in U.S. Patent No. 7,618,817, which is incorporated herein by reference in its entirety. For example, NK-92 cells can be modified to express Fc receptors on the cell surface. The Fc receptors can be activated Fcγ receptors, CD16 (FcγRiii-A), FCγRi (CD64), FCγRii (CD32), FCγRIII, FcRn, Fca, and Fcε, etc. Fc receptors have any binding affinity for their ligands or ligand fragments, including low- and high-affinity forms. NK-92 cells can be further modified to express one or more related auxiliary signaling polypeptides, such as FcεRi-γ or TCR-ζ, cytokines, or fragments thereof.

[0058] In some embodiments, the NK-92 cells comprise NK-92 cells modified to express an Fc receptor. In some embodiments, the NK-92 cells comprise NK-92 cells modified to express FcγRiiI-A, FCγRi, FCγRiI, FCγRIii, FcRn, Fca, or Fcε, or a combination thereof. In some embodiments, the NK-92 cells comprise NK-92 cells modified to express one or more chimeric antigen receptors. In some embodiments, the NK-92 cells comprise modified NK-92 cells available from the American Type Culture Collection, accession numbers PTA-8836, PTA-6967, PTA-8837, or PTA-6672, or a combination thereof. In some embodiments, the NK-92 cells comprise NK-92-CD16, NK-92-CD16-γ, or NK-92-CD16-ζ, or a combination thereof.

[0059] In some embodiments, the NK-92 cells comprise NK-92 cells modified to express cytokines. In some embodiments, the NK-92 cells comprise NK-92 cells modified to express cytokines and / or cytokine receptors that promote cell growth. In some embodiments, the NK-92 cells comprise NK-92 cells modified to express IL-2 and / or IL-2 receptors. In some embodiments, the NK-92 cells comprise NK-92 cells modified to express IL-15, IL-18, or IL-21, or their receptors. In some embodiments, the NK-92 cells comprise modified NK-92 cells available from the American Type Culture Collection, with accession numbers CRL-2408 or CRL-2409, or a combination thereof. In some embodiments, the NK-92 cells comprise NK-92MI, NK-92CI, or a combination thereof.

[0060] Without being limited to theory, it is believed that the exosomes and / or microvesicles from modified NK-92 cells are different from those from wild-type NK-92 cells. For example, based on the modification of the cells, the exosomes and / or microvesicles from modified NK-92 cells may contain different receptors and / or other proteins (e.g., cell lytic enzymes). The exosomes and / or microvesicles from modified NK-92 cells may also contain different or relative amounts of some receptors and / or other proteins.

[0061] Pharmaceutical composition

[0062] In one aspect, a pharmaceutical composition for killing cancer cells in a warm-blooded animal is provided, the composition comprising a pharmaceutically acceptable carrier and one or more components isolated from the supernatant of NK-92 cell growth culture medium.

[0063] In some embodiments, the pharmaceutical composition is a liquid at room temperature and is a gel after application to a patient. In some embodiments, the pharmaceutically acceptable carrier comprises a poloxamer.

[0064] In another aspect, provided is a pharmaceutical composition comprising exosomes and / or microvesicles isolated from the supernatant of NK-92 cell growth culture medium and a sterile aqueous carrier.

[0065] In another aspect, a kit is provided comprising a first pharmaceutical composition and a second pharmaceutical composition, wherein the first pharmaceutical composition comprises a pharmaceutically acceptable carrier and exosomes and / or microvesicles isolated from the supernatant of NK-92 cell growth medium; and the second pharmaceutical composition comprises a gel, wherein the first pharmaceutical composition and the second pharmaceutical composition are topical formulations. In some embodiments, the second pharmaceutical composition comprises a poloxamer. In some embodiments, the second pharmaceutical composition comprises exosomes and / or microvesicles isolated from the supernatant of NK-92 cell growth medium. In a preferred embodiment, the first and / or second pharmaceutical composition comprises microvesicles.

[0066] In some embodiments, the first pharmaceutical composition comprises a liquid and the second pharmaceutical composition comprises a gel, a poloxamer, or a composition that is a liquid at room temperature and a gel at body temperature. First, the liquid is applied to the treatment area, and then the gel is applied to the liquid. Without being limited to theory, it is believed that the liquid formulation provides rapid treatment to the treatment area, while the gel maintains the liquid at the applied site. In some embodiments, the gel comprises exosomes and / or microvesicles separated from the supernatant of NK-92 cell growth culture medium and a sterile aqueous carrier. Without being limited to theory, it is believed that the gel provides a slower release of exosomes and / or microvesicles to the treatment area, thereby providing sustained release and treatment for the diseased area.

[0067] In some embodiments, the pharmaceutical composition further comprises one or more cytokines.

[0068] The tumoricidal and / or antimicrobial component can be used in combination with a cytokine (such as IFN-γ, TGF-β, IL-4, IL-10, IL-13, IL-2, etc.) to maintain the functional efficiency of the composition comprising the tumoricidal and / or antimicrobial component. The term "combined" means that the cytokine can be administered a short time before the application of the composition comprising this component, or it can be administered simultaneously with the composition comprising this component, or a short time after the application of the composition comprising the tumoricidal component. This cytokine can also be administered at two such times relative to the time of administration of the composition comprising the tumoricidal component or at all three such times. In some embodiments, the cytokine and the component are administered in a single composition.

[0069] In some embodiments, the one or more cytokines include at least interleukin-2.

[0070] The pharmaceutical composition can be in a variety of formulations suitable for oral, topical, transdermal, rectal, inhalant, or parenteral (intravenous, intramuscular, or intraperitoneal) administration. The pharmaceutical composition can also be in a formulation suitable for injection into a tumor or at or around the tumor site. In one embodiment, the pharmaceutical composition is injected or applied to the tumor site after surgery to remove all or most of the tumor.

[0071] As used herein, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any or all solvents, dispersants or vehicles, coatings, antimicrobial agents, isotonic / hypotonic / hypertonic agents, absorption modifiers, etc., which are suitable for pharmaceutical use and are compatible with the tumoricidal and / or antimicrobial ingredients. In addition, other or supplemental active ingredients may also be incorporated into the final composition.

[0072] The pharmaceutical compositions described herein are administered in a variety of ways, depending on whether the treatment is local or systemic, and the area to be treated. In one aspect, the composition can be administered by injection, wherein the composition is formulated into a liquid or gel. In other aspects, the composition can be formulated for internal application to the patient. In other aspects, the composition can be applied topically, including to the eye, vagina, rectum, nasal area, oral administration, or directly to the skin. For example, a topical composition comprising exosomes and / or microvesicles can be applied to any accessible tumor or infection, such as skin tumors or other tumors (e.g., Kaposi's sarcoma); viral infections (e.g., warts, genital warts, and herpes); or bacterial infections.

[0073] In some embodiments, the pharmaceutical composition is an injectable formulation.

[0074] The pharmaceutical composition can be administered parenterally, such as intravenously, intramuscularly, subcutaneously, or intraperitoneally. The composition can be injected systemically or locally into or near the cancer site. A single intravenous or intraperitoneal dose can be administered. Alternatively, a slow, prolonged infusion or multiple, shorter daily infusions can be used, generally lasting 1-8 days. Alternating daily dosing with administration every few days can also be used.

[0075] Sterile injectable compositions are prepared by incorporating the tumoricidal and / or antimicrobial components in appropriate amounts into a suitable carrier. Suitable carriers include aqueous carriers such as water and aqueous buffers (such as phosphate buffered saline (PBS), citrate buffer, etc.); water-soluble organic solvents (such as polyethylene glycol 300, polyethylene glycol 400, ethanol, propylene glycol, glycerol, N-methyl-2-pyrrolidone, dimethylacetamide and dimethyl sulfoxide); organic liquids / semisolids (beeswax, tocopherol, oleic acid, medium chain monoglycerides or diglycerides); nonionic surfactants (polyethoxylated castor oil (such as Cremophor EL, Cremophor RH 40, Cremophor RH 50, Cremophor RH 60, Cremophor RH 70, Cremophor RH 80, Cremophor RH 90, Cremophor RH 10 ... 60), polysorbate 20, polysorbate 80, poloxamer 188, poloxamer 407, tocopheryl polyethylene glycol 1000 succinate, polyethylene glycol (15)-hydroxystearate, sorbitan monooleate, oleoyl polyethylene glycol-6 glyceride, linoleoyl polyethylene glycol-6 glyceride, caprylocaproyl polyethylene glycol-8 glyceride, and PEG 300, 400 or 1750 mono- or di-fatty acid esters, etc.); lipids (such as castor oil, corn oil, cottonseed oil, olive oil, peanut oil, peppermint oil, safflower oil, sesame oil, soybean oil, hydrogenated vegetable oil, hydrogenated soybean oil, and medium-chain triglycerides of coconut oil and palm kernel oil); cyclodextrins (such as α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, hydroxypropyl-β-cyclodextrin and sulfobutyl ether-β-cyclodextrin) and phospholipids (such as lecithin, cephalin, phosphatidylserine, distearoylphosphatidylglycerol, 1-dimyristoylphosphatidylcholine, 1-dimyristoylphosphatidylglycerol, etc.) or mixtures thereof. In some embodiments, the aqueous carrier comprises hyaluronic acid, saline, Ringer's solution, glucose solution, Hank's solution and other aqueous physiologically balanced salt solutions. In some embodiments, the non-aqueous carrier includes fixed oils, vegetable oils (such as olive oil and sesame oil), triglycerides, propylene glycol, polyethylene glycol or injectable organic esters such as ethyl oleate. In some embodiments, the pharmaceutically acceptable carrier further comprises a viscosity enhancer such as carboxymethylcellulose or its salts, sorbitol or dextran; substances that enhance isotonicity and chemical stability such as phosphate buffers, bicarbonate buffers and Tris buffers; preservatives such as thimerosal, cresols, formalin and benzaldehyde.

[0076] Injectable compositions can be solutions or suspensions, but should be able to be easily passed through an injection device such as a hollow needle. Suitable viscosity can be achieved and maintained by appropriate selection of solvents or excipients. In some embodiments, the pharmaceutically acceptable carrier comprises a viscosity enhancer. In some embodiments, the viscosity of the composition is about 5 centipoise (cP) to about 1x10 at 25°C. 6 cP, or about 5 cP - about 1x10 5 cP, or about 5 cP - about 1x10 4 cP, or about 5 cP - about 1x10 3 cP, or about 6 cP to about 100 cP.

[0077] In some embodiments, the pharmaceutical composition is an injectable extended-release formulation. The tumoricidal and / or antimicrobial components in the extended-release formulation can be released from the composition into the body over an extended period of time, such as at least a few minutes, at least an hour, at least several hours, at least a day, at least several days, or several weeks, to provide a long-term and / or sustained therapeutic effect.

[0078] In some embodiments, the pharmaceutical composition comprises a localizing agent that allows for local retention of the composition upon delivery to or near a tumor site, optionally providing for prolonged and / or sustained release of the tumoricidal and / or antimicrobial components of the composition. Such agents include thixotropic agents, phase change agents such as hydrogels, bioerodible biocompatible polymers, and collagen gels, among others. These compositions are injectable or liquid under ambient conditions and, upon application, form a viscous or gel-like bioerodible or biocompatible mass that restricts transport of the tumoricidal and / or antimicrobial components away from the delivery site and allows diffusion of the tumoricidal and / or antimicrobial components from the composition.

[0079] The hydrogels useful in the compositions can be chemically and / or physically crosslinked hydrogels. In situ chemical crosslinking can be achieved, for example, by photoinitiated, redox-initiated, or Michael-type addition polymerization reactions, which preferably involve the formation of covalent bonds. Physically crosslinked hydrogels self-assemble under external stimuli and are independent of the formation of covalent bonds. Temperature, pH, ionic concentration, and hydrophobic interactions are some of the external stimuli that can be used for the self-assembly of such hydrogels and for the fixation of such hydrogels.

[0080] Exemplary polymers suitable for use in the composition include polylactide, polyglycolide, poly(caprolactone), polyanhydrides, polyamines, polyesteramides, polyorthoesters, polydioxanones, polyacetals, polyketals, polycarbonates, polyphosphates, polyorthocarbonates, polyphosphazenes, succinates, poly(hydroxysuccinic acid), poly(amino acids), polyvinylpyrrolidone, polyethylene glycol, polyhydroxycellulose, polyphosphates, polysaccharides, chitin, chitosan, hyaluronic acid, and copolymers such as poloxamers, terpolymers, and mixtures thereof.

[0081] In some embodiments, the positioning agent is a poloxamer. Poloxamer is a nonionic triblock copolymer consisting of a central hydrophobic polyoxypropylene (e.g., polypropylene oxide) chain and two hydrophilic polyoxyethylene (e.g., polyethylene oxide) chains flanking it. In one aspect, a poloxamer has the formula:

[0082] HO(C2H4O) b (C3H6O) a (C2H4O) b OH

[0083] wherein a is 10-100, 20-80, 25-70, 25-70, or 50-70; and b is 5-250, 10-225, 20-200, 50-200, 100-200, or 150-200. In another aspect, the molecular weight of the poloxamer is 2,000-15,000, 3,000-14,000, or 4,000-12,000. The poloxamer useful in the present invention is sold under the trade name Non-limiting examples of poloxamers useful in the present invention include, but are not limited to, P103, P105, P123, F127 and L 121. At suitable concentrations, such as 10%-30% w / w poloxamer, poloxamer solutions are liquid at room temperature and form gels in the body.

[0084] Suitable collagen comprises the alkali treatment of the insoluble collagen such as extracted from various animals, or by processing with enzyme, such as pepsin, trypsin, chymotrypsin, papain or pronase.Collagen can be obtained from the skin, bone, cartilage, tendon or organ of bird or mammal etc.Collagen can be flexible after processing (curing), and only needs a short time to be cross-linked, in other words, only needs a short time to gelation.Collagen solution also can be prepared by dissolving collagen in a non-toxic solvent, and the example of non-toxic solvent comprises water, physiological saline and buffer, such as borate buffer, or saline (such as sodium chloride, sodium bromide, potassium bromide) or the aqueous solution of protein, sugar or lipid etc.

[0085] Collagen can also form gel even in the presence of moisture, such as in blood or in tumor, and can show high viscosity for living tissue. The collagen solution used in the present invention can be made into various concentrations, neutralized and prepared for injection. In various embodiments, the collagen concentration in the composition can be 0.2mg / mL, 0.5mg / mL, 0.75mg / mL, 1mg / mL, 2mg / mL, 3mg / mL, 4mg / mL, 5mg / mL, 6mg / mL, 7mg / mL, 8mg / mL, 10mg / mL, 20mg / mL, 30mg / mL, 40mg / mL and 50mg / mL, or any scope between two of these numerical values. When injected into an organ, freezing collagen gel can be thermogel (thermogel) when reaching body temperature or about 37 ℃.

[0086] In some embodiments, the pharmaceutical composition provides for prolonged, sustained and / or continuous release of the tumoricidal and / or antimicrobial components of the composition.

[0087] Sterilization of the composition can be accomplished by known methods, such as filtration.

[0088] The final form should be stable under the conditions of manufacture and storage.In addition, the final pharmaceutical form should be protected from contamination and therefore should be able to inhibit the growth of microorganisms such as bacteria or fungi.

[0089] Prevention or inhibition of microbial growth can be achieved by adding one or more antimicrobial agents such as chlorobutanol, ascorbic acid, parabens, thimerosal, etc. It may also be preferred to include agents that alter osmotic pressure such as sugars and salts.

[0090] Pharmaceutical compositions can also be prepared as sterile powders. Sterile powders containing tumoricidal and / or antimicrobial components can be prepared by vacuum drying or freeze-drying of liquid compositions, such as compositions containing an aqueous carrier. Sterile powders can be reconstituted with an appropriate amount of an aqueous carrier, such as PBS, to provide injectable compositions for administration to a patient.

[0091] In some embodiments, the composition is formulated as a topical composition that is applied directly to the skin to treat skin cancer. In some embodiments, the composition is formulated as a topical composition that is applied directly to other palpable cancers, such as cervical cancer or oral cancer. In some embodiments, the topical composition is applied to virally infected cells, such as warts. In some embodiments, the warts are genital (sexually transmitted) warts. In some embodiments, the warts are common warts, flat warts, filiform / digital warts, inlay warts, periungual warts, or plantar warts.

[0092] Preparations for topical administration can include emulsions, creams, aqueous solutions, oils, ointments, pastes, gels, lotions, emulsions, foams, suspensions, and powders. In some embodiments, the pharmaceutical composition is a cream or lotion. In one aspect, the topical composition can include one or more surfactants and / or emulsifiers. In one embodiment, the emulsifier does not change the structure of the exosomes and / or microvesicles. In one embodiment, the emulsifier changes the structure of the exosomes and / or microvesicles. For example, the emulsifier can destroy the exosome and / or microvesicle structure, thereby releasing the active factors in the exosomes and / or microvesicles.

[0093] Surfactants (or surface-active substances) that may be present are anionic, nonionic, cationic and / or amphoteric surfactants. Typical examples of anionic surfactants include, but are not limited to, soaps, alkylbenzenesulfonates, alkanesulfonates, olefinsulfonates, alkylethersulfonates, glycerylethersulfonates, α-methylestersulfonates, sulfofatty acids, alkyl sulfates, fatty alcohol ether sulfates, glycerylether sulfates, fatty acid ether sulfates, hydroxy mixed ether sulfates, monoglyceride (ether) sulfates, fatty acid amide (ether) sulfates, mono- and dialkylsulfosuccinates, mono- and dialkylsulfosuccinamates, sulfotriglycerides, amide soaps, ether carboxylic acids and their salts, fatty acid isethionates, fatty acid sarcosinates, fatty acid taurides, N-acylamino acids (such as acyl lactylates), acyl tartrates, glutamic acids, acyl aspartic acids, alkyl oligoglucosides sulfates, protein fatty acid condensates (especially wheat-based plant products) and alkyl (ether) phosphates. Examples of nonionic surfactants include, but are not limited to, fatty alcohol polyglycol ethers, alkylphenol polyglycol ethers, fatty acid polyglycol esters, fatty acid amide polyglycol ethers, fatty amine polyglycol ethers, alkoxylated triglycerides, mixed ethers or mixed methylals, optionally partially oxidized alkyl (en) oligoglycosides or glucuronic acid derivatives, fatty acid N-alkyl glucamides, protein hydrolysates (particularly wheat-based plant products), polyol fatty acid esters, sugar esters, sorbitan esters, polysorbates, and amine oxides. Examples of amphoteric or zwitterionic surfactants include, but are not limited to, alkyl betaines, alkyl amido betaines, aminopropionates, aminoglycinates, imidazolinium betaines, and sulfobetaines.

[0094] In some embodiments, the surfactant can be a fatty alcohol polyglycol ether sulfate, a monoglyceride sulfate, a mono- and / or dialkyl sulfosuccinate, a fatty acid isethionate, a fatty acid sarcosinate, a fatty acid taurate, a fatty acid glutamate, an α-olefin sulfonate, an ether carboxylic acid, an alkyl oligoglycoside, a fatty acid glucamide, an alkyl amido betaine, an amphoteric acetal and / or a protein fatty acid condensate.

[0095] Examples of zwitterionic surfactants include betaines, such as N-alkyl-N,N-dimethylammonium glycinates, such as cocoalkyldimethylammonium glycinates, N-amidopropyl-N,N-dimethylammonium glycinates, such as cocoamidopropyl-N,N-dimethylammonium glycinates, and 2-alkyl-3-carboxymethyl-3-hydroxyethylimidazolines (in each case the alkyl or acyl group has 8 to 18 carbon atoms), and cocoamidoethylhydroxyethyl-carboxymethylglycinate.

[0096] In some embodiments, the emulsifier can be a non-ionic surfactant selected from the following: addition products of 2-30 moles of ethylene oxide and / or 0-5 moles of propylene oxide to linear fatty alcohols containing 8-22 carbon atoms, to fatty acids containing 12-22 carbon atoms, to alkylphenols containing 8-15 carbon atoms in the alkyl group, or to alkylamines containing 8-22 carbon atoms in the alkyl group; alkyl and / or alkenyl oligoglycosides containing 8-22 carbon atoms in the alkyl (alkenyl) group and ethoxylated analogs thereof; addition products of 1-15 moles of ethylene oxide to castor oil and / or hydrogenated castor oil; addition products of 15-60 moles of ethylene oxide to castor oil and / or hydrogenated castor oil; partial esters of glycerol and / or sorbitan with unsaturated linear or saturated branched fatty acids (containing 12-22 carbon atoms) and / or hydroxy hydrocarbon acids (containing 3-18 carbon atoms); , and its adducts with 1-30 moles of ethylene oxide; partial esters of polyglycerol (average self-condensation degree of 2-8), trimethylolpropane, pentaerythritol, sugar alcohol (such as sorbitol), alkyl glucoside (such as methyl glucoside, butyl glucoside, lauryl glucoside) and polyglycoside (such as cellulose) with saturated or unsaturated linear or branched fatty acids (containing 12-22 carbon atoms) and / or hydroxy hydrocarbon acids (containing 3-18 carbon atoms), and its adducts with 1-30 moles of cyclic adducts of ethylene oxide; mixed esters of pentaerythritol, fatty acids, citric acid and fatty alcohols and / or mixed esters of fatty acids containing 6 to 22 carbon atoms, methyl glucose and polyols (preferably glycerol or polyglycerol), mono-, di- and tri-alkyl phosphates and mono-, di- and / or tri-PEG alkyl phosphates and their salts; wool wax alcohol; polysiloxane-polyalkyl-polyether copolymers and corresponding derivatives; and block copolymers, such as polyethylene glycol-30 dipolyhydroxystearate.

[0097] In some embodiments, the emulsifier is a polyalkylene glycol, such as polyethylene glycol or polypropylene glycol. In some embodiments, the emulsifier is a polyethylene glycol with a molecular weight of 100Da to 5,000Da, 200Da to 2,500Da, 300Da to 1,000Da, 400Da to 750Da, 550Da to 650Da, or about 600Da.

[0098] In some embodiments, the emulsifier is a poloxamer described herein.

[0099] In some embodiments, the emulsifier is composed of one or more fatty alcohols. In some embodiments, the fatty alcohol is a linear or branched C6 to C 3s Fatty alcohols. Examples of fatty alcohols include, but are not limited to, octyl alcohol (1-octanol), 2-ethylhexanol, nonanoic acid alcohol (1-nonanol), decanoic acid alcohol (1-decanol, decanol), undecyl alcohol (1-undecanol, undecanol, undecanol), lauryl alcohol (dodecanol, 1-dodecanol), tridecyl alcohol (1-tridecyl alcohol, tridecanol, isotridecanol), myristyl alcohol (1-tetradecanol), pentadecyl alcohol (1-pentadecanol, pentadecyl alcohol), cetyl alcohol (1-hexadecanol), palmitoleic acid alcohol (cis-9-hexadecene-1-ol), heptadecyl alcohol (1-n-heptadecanol, heptadecanol), stearyl alcohol (1-octadecanol), isostearyl alcohol (16-methylheptadecanol), elaidolyl alcohol (9E-octadecene-1-ol), oleyl alcohol (cis-9-octadecene-1-ol), linoleyl alcohol (9Z , 12Z-octadecadien-1-ol), elaidolinoleyl alcohol (9E, 12E-octadecadien-1-ol), linolenyl alcohol (9Z, 12Z, 15Z-octadecatrien-1-ol), elaidolinolenyl alcohol (9E, 12E, 15E-octadecatrien-1-ol), ricinoleyl alcohol (12-hydroxy-9-octadecen-1-ol), nonadecanol (1-nonadecanol), arachidyl alcohol (1-eicosyl alcohol), heneicosyl alcohol (1-heneicosyl alcohol), behenyl alcohol (1-docosyl alcohol), erucyl alcohol (cis-13-docosyl-1-ol), lignoceryl alcohol (1-tetracosyl alcohol), cetyl alcohol (1-hexacosyl alcohol), montanol, policosanol (1-octacosanol), myristyl alcohol, melissinol (1-triacontol), geddyl alcohol (1-tetratriacontol) or cetearyl alcohol.

[0100] In some embodiments, the carrier used to produce the topical composition is a mixture of polyethylene and one or more fatty alcohols. For example, the carrier comprises about 50% to about 99% by weight, about 75% to about 99% by weight, about 90% to about 99% by weight, or about 95% by weight of polyethylene glycol and about 1% to about 50% by weight, about 1% to about 25% by weight, about 1% to about 10% by weight, or about 5% by weight of the fatty alcohol. In some embodiments, the carrier is a mixture of polyethylene glycol and cetyl alcohol.

[0101] The topical composition can also include other additional components that are suitable for this composition. In some embodiments, the topical composition can include one or more of the following components: fat, wax, pearlescent wax, thickener, thickening agent, fat-enhancing agent, stabilizer, polymer, silicone compound, lecithin, phospholipid, bioactive ingredient, deodorant, antimicrobial agent, antiperspirant, leavening agent, insect repellent, solubilizer, cosolvent, preservative, fragrance oil and dye. Each example of these ingredients is disclosed in U.S. Patent No. 8,067,044, which is incorporated herein by reference.

[0102] Topical compositions containing the tumoricidal and / or antimicrobial ingredients described herein can be prepared by mixing the ingredients with a carrier for a sufficient time to uniformly disperse the particles throughout the carrier. In cases where the carrier contains two or more ingredients, the ingredients can be mixed with each other prior to adding the tumoricidal and / or antimicrobial ingredient. The amount of the tumoricidal and / or antimicrobial ingredient present in the topical composition can vary depending on the intended use. In some embodiments, the tumoricidal and / or antimicrobial ingredient comprises 0.5% to 20%, 1% to 10%, 2% to 5%, or approximately 3% by weight of the topical composition.

[0103] It will be appreciated that the amount of the tumoricidal and / or antimicrobial component in the composition will vary in a particular case depending on the specific tumoricidal and / or antimicrobial component used, the specific composition formulated, the mode of application, and the specific site and patient being treated. The dosage for a given subject can be determined using conventional considerations, such as by routine comparison of the differential activity of the composition with known therapies (e.g., according to an appropriate conventional pharmacological regimen). Physicians and formulators skilled in the art of determining dosages of pharmaceutical agents will readily determine dosages based on standard recommendations (Physician's Desk Reference, Barnhart Publishing (1999)).

[0104] Unit dose or multiple dose forms are contemplated, each with advantages in a particular clinical setting. A unit dose contains a predetermined amount of tumoricidal and / or antimicrobial component calculated to produce a desired effect in the case of treating cancer. Multiple dose forms can be very useful in situations where multiple single doses or partial doses are required to achieve a desired endpoint. Any of these administration forms can have instructions for use that are specified based on or directly depend on the unique characteristics of the specific tumoricidal and / or antimicrobial component, the specific therapeutic effect to be achieved, the disease to be treated, and the patient's specific condition.

[0105] The unit dose contains a therapeutically effective amount sufficient to treat the disease in the patient and can include about 0.001 mg to 100 mg of the tumoricidal, cytotoxic and / or antimicrobial component. The amount of the tumoricidal, cytotoxic and / or antimicrobial component can be 0.001% to 90% w / w of the composition, such as 0.001%, 0.01%, 0.1%, 1%, 10%, 50% or 90%, or any range therebetween. The disease treated can be any disease that can be treated by the exosomes of the present invention, such as cancer, bacterial infection, viral infection or fungal infection.

[0106] The pharmaceutical composition may further be in the form of an oral preparation, such as edible tablets, buccal tablets, capsules, caplets, elixirs, suspensions, syrups, lozenges, wafers, dragees, and the like.

[0107] The composition can be a sustained release formulation. The composition can be encapsulated in a hard or soft capsule, can be compressed into a tablet, or can be added to a beverage, food, or otherwise added to a diet. The percentage of the final composition and the preparation can certainly vary, and can conveniently be 1% to 90% of the weight of the final form (e.g., tablet). The amount of such therapeutically effective composition makes it possible to obtain a suitable dosage.

[0108] Suitable formulations for oral compositions may also include: binders such as gum tragacanth, gum arabic, corn starch, gelatin; sweeteners such as lactose or sucrose; disintegrants such as corn starch, alginic acid, etc.; lubricants such as magnesium stearate; or flavorings such as oil of peppermint or wintergreen. Various other materials may also be present as coatings or otherwise modify the physical form of the oral dosage unit. Oral dosage units may be coated with shellac, sugar, or both. Syrups or elixirs may contain tumoricidal and / or antimicrobial ingredients, sucrose as a sweetener, methylparaben and propylparaben as preservatives, dyes, and flavorings. Any materials used should be pharmaceutically acceptable and substantially non-toxic.

[0109] Further instructions for preparing pharmaceutical compositions can be found in Remington's Pharmaceutical Sciences, 19th edition (19 th Edition of Remington's Pharmaceutical Sciences, published by Mack Publishing Co., Easton, Pa. 18040. Relevant parts thereof are incorporated herein by reference.

[0110] The treatment method of the present invention

[0111] The compositions described herein can be used to treat a variety of diseases such as cancer. Examples of diseases that the compositions can treat include malignancies of the immune system, lymphatic system, and hematopoietic system, tumors formed, and solid tumors. Non-limiting examples of cancers that the compositions can treat include mast cell leukemia, acute myeloid leukemia (AML), erythroleukemia, bone marrow disorders (such as myeloid leukemia, multiple myeloma, and erythroleukemia), germ cell tumors, lung cancer, small cell lung cancer, gastrointestinal stromal tumors, neuroblastoma, cervical tumors, ovarian cancer, brain cancer, breast cancer, ovarian cancer, endometrial cancer, kidney cancer, thyroid cancer, bladder cancer, colon cancer, pancreatic cancer, prostate cancer, skin cancer (such as melanoma), adenoma (such as colon villous adenoma) and sarcoma (such as osteosarcoma) etc.

[0112] In some embodiments, the pharmaceutical compositions described herein are used to treat skin cancer, such as melanoma, squamous cell carcinoma, basal cell carcinoma, or mast cell tumor.

[0113] In some embodiments, the pharmaceutical compositions described herein are used to treat other epidermal cell cancers, such as cervical cancer or oral cancer. In some embodiments, the pharmaceutical compositions described herein are used to treat virally infected cells, such as warts.

[0114] In some embodiments, the pharmaceutical compositions described herein are used to modulate the immune system. For example, exosomes and / or microvesicles can induce cytotoxic T cell responses (e.g., anti-tumor) and / or induce apoptosis (e.g., apoptosis of activated immune cells). Exosomes and / or microvesicles can also play a role in immune surveillance.

[0115] In some embodiments, the pharmaceutical compositions described herein can be used to treat infections. In one embodiment, the pharmaceutical compositions described herein can be used to treat infections caused by pathogenic viruses. Pathogenic viruses include, but are not limited to, human papillomavirus, human immunodeficiency virus, Epstein-Barr virus, cytomegalovirus, Ebola virus, Marburg virus, influenza virus, respiratory syncytial virus, poxvirus, varicella-zoster virus, and herpes virus. In one embodiment, the pharmaceutical compositions described herein can be used to treat bacterial infections. Infectious bacteria include, but are not limited to, Streptococcus, Staphylococcus, Cryptococcus neoformans, Chlamydia, Escherichia, Pseudomonas, Clostridium, and Candida, including antibiotic-resistant strains of any of the above bacteria. In one embodiment, the pharmaceutical compositions described herein can be used to treat infections caused by other microorganisms, including fungi and yeasts.

[0116] For mammals (including humans and domesticated animals), an effective amount can be administered, for example, in a topical composition, based on the body surface area to be covered (e.g., the area of infection). Suitable dosages range from about 0.001 mg to about 100 mg equivalent of the tumoricidal and / or antimicrobial component per square meter of body surface area, for example, about 0.005 mg / m2 to about 50 mg / m2. 2 The dose may be administered daily, such as once, twice, three times or more per day, or every two or several days, or weekly, etc. If a delayed release formulation is used, the frequency of administration may be reduced.

[0117] In one embodiment, the effective amount can be determined based on tumor volume. A suitable dosage range is about 1:100 to about 1:10,000 of the exosome / microvesicle preparation to tumor volume. In one embodiment, a suitable dosage range is about 1:100 to about 1:1,000 of the exosome / microvesicle preparation to tumor volume. In one embodiment, a suitable dosage range is about 1:1000 to about 1:1,000 of the exosome / microvesicle preparation to tumor volume.

[0118] In one embodiment, the effective amount can be determined based on the patient's weight. A suitable dosage range is about 1 μg to about 100 mg of exosome / microvesicle preparation per kilogram of body weight. In one embodiment, a suitable dosage range is about 1 μg to about 10 mg of exosome / microvesicle preparation per kilogram of body weight. In one embodiment, a suitable dosage range is about 1 μg to about 100 μg of exosome / microvesicle preparation per kilogram of body weight. In one embodiment, a suitable dosage range is about 10 μg to 10 mg of exosome / microvesicle preparation per kilogram of body weight. In one embodiment, a suitable dosage range is about 100 μg to about 10 mg of exosome / microvesicle preparation per kilogram of body weight.

[0119] The dosage and frequency of administration may depend on the type of formulation, the disease being treated, the amount of tumoricidal and / or antimicrobial component, and the age, sex, race, and other conditions of the patient.

[0120] Combination therapy

[0121] On the other hand, the compositions described herein can be used in combination with other cancer therapies, such as surgery, radiotherapy, chemotherapy (such as cisplatin, carboplatin, oxaliplatin, satraplatin and picoplatin, especially cisplatin and carboplatin; taxanes, such as paclitaxel and docetaxel; anthracyclines, such as daunorubicin, doxorubicin, epirubicin, idarubicin or valrubicin, etc.), cell-based therapies (such as NK-92 cell therapy), antibody therapy, etc. In some embodiments, the compositions can be used in combination with one or more cytokines described herein.

[0122] The following examples are used to illustrate the present invention, but are not intended to limit the present invention. All publications or references cited in this specification are hereby incorporated by reference into this document.

[0123] Example

[0124] Example 1: Antimicrobial Effects of NK-92 Cells

[0125] NK-92 cells were incubated with Cryptococcus neoformans in Myelocult medium in round-bottom culture plates for 24 hours. The effector cell: target cell (NK-92 cells: Cryptococcus neoformans) ratio was 100:1 (1x10 6 : 1x10 4 Cryptococcus neoformans (1x10 4 NK-92 cells / well) were incubated in Myelocult medium for 24 hours without NK-92 cells as a control. After 24 hours of incubation, the cultures were serially diluted and plated on Sabouraud agar plates. The plates were incubated at room temperature for 48 hours. The number of colonies was measured for each dilution.

[0126] The number of colonies per plate at each dilution was Figure 1 Indicated in the middle. White bars: NK-92 cells + Cryptococcus neoformans, black bars: Cryptococcus neoformans alone. Figure 2 It was shown in Figure 2 that NK-92 cells inhibited the growth of Cryptococcus neoformans by approximately 60%.

[0127] Similar results were observed when NK-92 cells were incubated with another fungus, Aspergillus species. NK-92 cells caused hyphal damage to the fungus in a dose- and time-dependent manner.

[0128] Example 2: Isolation of exosomes and / or microvesicles from NK-92 cell supernatant

[0129] Extracellular vesicles (exosomes and / or microvesicles) were isolated by ultracentrifugation. Cultured NK-92 cells were centrifuged at 300 x g for 10 minutes, and the cell pellet was discarded. The supernatant was centrifuged at 2000 x g for 20 minutes, and the pellet (cell debris) was discarded. The resulting supernatant was subjected to ultracentrifugation at 100,000 x g for 80 minutes. The resulting pellet was washed with phosphate-buffered saline and subjected to ultracentrifugation at 100,000 x g for 80 minutes. The washed pellet containing exosomes and microvesicles (EV / MV preparation) was retained for further study.

[0130] Example 3: Characterization of extracellular vesicles

[0131] NK-92 cells were cultured under various conditions, and extracellular vesicles were isolated from the culture medium as described in Example 2. Culture conditions are shown in Table 1. Cells were conditioned in exofree FBS for at least 24 to 46 hours before exosome harvesting. ExoFree FBS was depleted of exosomes to prevent contamination of NK-92 cell extracellular vesicle preparations.

[0132] Table 1. NK-92 growth conditions road Cell number Culture medium ExoFree FBS (%) IL-2 (IU / mL) 4 <![CDATA[4.0x10 7 ]]> αMEM(NK) 5 500 5 <![CDATA[4.8x10 7 ]]> RPMI 10 500 6 <![CDATA[4.0x10 7 ]]> <![CDATA[X-Vivo10 1 ]]> 5 500 7 <![CDATA[3.6x10 7 ]]> <![CDATA[X-Vivo10 1 ]]> 0 500

[0133] 1 Lonza Group Ltd.

[0134] EV / MV preparations were analyzed by standard Western blotting techniques for the presence of several proteins, such as Figure 3A and Figure 3B As shown in . The MCF-7 cell line was used as a positive control for exosome production and a negative control for cell lysate protein. NK-92 cell pellet (NK-92 cells) served as a positive control. MCF-7 cells were cultured in DMEM supplemented with 10% ExoFree fetal bovine serum (FBS) and 2 mM L-glutamine.

[0135] Extracellular vesicles from both NK-92 cells and MCF-7 cells are positive for Rab5B (exosome marker). NK-92EV / MV preparations (not MCF-7EV / MV preparations) are also positive for several apoptosis-inducing and / or cell lysis proteins, which are known to be involved in NK cell activity, including perforin, Fas ligand (FasL), granzyme B, and granulysin. However, the amount of each of these proteins seems to depend on NK-92 cell growth conditions, including culture medium (such as aMEM (NK), RPMI, X-VIVO) and serum concentration.

[0136] EV / MV preparations from NK-92 cells were analyzed for contamination with other organelles. EV / MVs isolated from NK-92 cells showed some contamination with nuclear material (apoptotic bodies) but were free of other contaminating organelles, as shown in FIG3b .

[0137] Example 4: Cytotoxicity of EV / MV preparations

[0138] The cytotoxicity of EV / MV preparations against Jurkat cells was tested using the culture conditions shown in Table 1. Jurkat cells (2x10 4) were incubated for 2 or 20 hours in 170 μL of culture medium (containing ExoFree FBS) and 5, 15, or 30 μL of EV / MV preparation (total incubation volume of 200 μL was achieved with PBS). Cytotoxicity was determined by propidium iodide (PI) assay, and data are expressed as the percentage of cells positive for Pi (indicating dead cells).

[0139] like Figure 4 As shown in , EV / MV preparations from NK-92 cells (but not MCF-7 cells) have cytotoxic activity against Jurkat cells in vitro. The culture medium composition alters the lytic potential of EV / MVs, and the presence of serum in the growth medium stimulates NK-92 cells to produce lytic EV / MVs. This application relates to the following implementation schemes: 1. A composition comprising exosomes and / or microvesicles isolated from NK-92 cells. 2. A composition as described in embodiment 1, wherein the composition comprises a sterile aqueous solution. 3. A composition as described in embodiment 2, wherein the sterile aqueous solution comprises one or more pharmaceutically acceptable excipients. 4. The composition as described in embodiment 2, wherein the sterile aqueous solution comprises sterile PBS or sterile saline solution. 5. The composition as described in embodiment 2, wherein the sterile aqueous composition comprises the culture supernatant of NK-92 cells. 6. The composition as described in embodiment 1, wherein the exosomes and / or microvesicles are isolated from a defined NK-92 cell subpopulation. 7. The composition as described in embodiment 1, wherein the NK-92 cells are treated with at least one stimulating agent before isolating the exosomes and / or microvesicles. 8. The composition as described in embodiment 7, wherein the at least one stimulator is IL-15 and / or interferon gamma. 9. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and one or more components isolated from NK-92 cells, wherein the one or more components are tumoricidal and / or antimicrobial. 10. The pharmaceutical composition as described in embodiment 9, wherein the composition comprises a sterile aqueous solution. 11. The pharmaceutical composition as described in embodiment 10, wherein the sterile aqueous solution comprises the culture supernatant of NK-92 cells. 12. The pharmaceutical composition of embodiment 9, wherein the one or more components comprise exosomes and / or microvesicles. 13. A pharmaceutical composition comprising the composition described in embodiment 1 and a sterile aqueous carrier. 14. The pharmaceutical composition of any one of embodiments 9-13, wherein the NK-92 cells comprise wild-type NK-92 cells. 15. The pharmaceutical composition of any one of embodiments 9-13, wherein the NK-92 cells comprise modified NK-92 cells. 16. The pharmaceutical composition of any one of embodiments 9-13, wherein the NK-92 cells comprise NK-92 cells modified to express an Fc receptor, a cytokine, a cytokine receptor, and / or a chimeric antigen receptor. 17. The pharmaceutical composition of any one of embodiments 9-16, wherein the pharmaceutical composition is an injectable preparation. 18. The pharmaceutical composition of embodiment 17, wherein the pharmaceutical composition is formulated for subdermal or subcutaneous injection. 19. The pharmaceutical composition according to embodiment 17, wherein the pharmaceutical composition is an injectable extended-release formulation. 20. The pharmaceutical composition of any one of embodiments 9-19, wherein the pharmaceutically acceptable carrier comprises a thickening agent. 21. The pharmaceutical composition of any one of embodiments 9-16, wherein the pharmaceutical composition is a topical preparation. 22. A pharmaceutical composition as described in embodiment 21, wherein the topical formulation is a liquid at room temperature and a gel when applied to the body. 23. A pharmaceutical composition as described in embodiment 22, wherein the topical formulation comprises a poloxamer. 24. The pharmaceutical composition as described in embodiment 21, wherein the pharmaceutical composition is a cream or a lotion. 25. The pharmaceutical composition of any one of embodiments 9-24, wherein the pharmaceutical composition further comprises one or more cytokines. 26. The pharmaceutical composition as described in embodiment 25, wherein the one or more cytokines include at least iL-2. 27. The pharmaceutical composition of any one of embodiments 9-26, wherein the NK-92 cells are treated with at least one stimulating agent prior to separation of the one or more components. 28. The pharmaceutical composition as described in embodiment 27, wherein the at least one stimulatory agent is IL-15 and / or interferon gamma. 29. A method of treating a tumor in a patient, comprising administering to the patient an effective amount of the pharmaceutical composition of any one of embodiments 9-28 to treat the tumor. 30. A method of treating an infection in a patient, said method comprising administering to said patient an effective amount of the pharmaceutical composition of any one of embodiments 9-28 to treat said infection. 31. The method of embodiment 30, wherein the infection is a bacterial infection, a viral infection, a fungal infection, or a yeast infection. 32. A method for inducing an immune response in a patient, the method comprising administering to the patient an effective amount of the pharmaceutical composition according to any one of embodiments 9 to 28 to induce an immune response. 33. A method for inducing an immunomodulatory response in a patient, the method comprising injecting an effective amount of the pharmaceutical composition according to any one of embodiments 9 to 20 into the patient to induce an immunomodulatory response. 34. A kit comprising a first pharmaceutical composition and a second pharmaceutical composition, wherein the first pharmaceutical composition comprises a pharmaceutically acceptable carrier and exosomes and / or microvesicles isolated from NK-92 cells, and the second pharmaceutical composition comprises a gel, wherein the first pharmaceutical composition and the second pharmaceutical composition are topical formulations. 35. The kit of embodiment 34, wherein the second pharmaceutical composition comprises a poloxamer. 36. The kit of embodiment 34, wherein the second pharmaceutical composition comprises exosomes and / or microvesicles isolated from the supernatant of growth medium of NK-92 cells.

Claims

1. Use of a composition comprising exosomes and microvesicles isolated from NK-92 cells in the preparation of a medicament for treating leukemia in a patient, wherein the NK-92 cells are cultured in an X-Vivo 10.

2. The method of claim 1, wherein the composition comprises a sterile aqueous solution.

3. The method of claim 2, wherein the sterile aqueous solution comprises one or more pharmaceutically acceptable excipients.

4. The method of claim 2, wherein the sterile aqueous solution comprises sterile PBS or sterile saline solution.

5. The use as claimed in claim 2, wherein the sterile aqueous composition comprises the culture supernatant of NK-92 cells.

6. The use as claimed in claim 1, wherein the exosomes and microvesicles are isolated from a defined subpopulation of NK-92 cells.

7. The use according to claim 1, wherein the NK-92 cells comprise wild-type NK-92 cells.

8. The use of claim 1, wherein the NK-92 cells comprise modified NK-92 cells.

9. The use as claimed in claim 1, wherein the NK-92 cells comprise NK-92 cells modified to express Fc receptors, cytokines, cytokine receptors and / or chimeric antigen receptors.

10. The use as claimed in claim 1, wherein the NK-92 cells are treated with at least one stimulating agent before isolating exosomes and microvesicles.

11. Use as claimed in claim 10, wherein the at least one stimulator is IL-15, interferon gamma or both.

12. The use according to claim 1, wherein the pharmaceutical composition is an injectable preparation.

13. The use of claim 12, wherein the pharmaceutical composition is formulated for subdermal or subcutaneous injection.

14. The use according to claim 13, wherein the pharmaceutical composition is an injectable delayed-release preparation.

15. The use according to claim 13, wherein the pharmaceutically acceptable carrier comprises a thickener.

16. The use according to claim 1, wherein the pharmaceutical composition is a topical preparation.

17. The use as claimed in claim 16, wherein the topical formulation is a liquid at room temperature and is a gel when applied to the body.

18. Use as claimed in claim 17, wherein the topical formulation comprises a poloxamer.

19. The use as claimed in claim 16, wherein the pharmaceutical composition is a cream or a lotion.

20. The use of claim 1, wherein the pharmaceutical composition further comprises one or more cytokines.

21. The pharmaceutical composition of claim 20, wherein the one or more cytokines include at least IL-2.

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