Use of plasma membrane particles, liposomes, and exosomes to assay the efficacy of immune cells

Abstract

This provides a method for determining the efficacy of immune cells. [Solution] This method includes the steps of contacting immune cells with an effective amount of cellular exosomes and detecting the amount of cytokines produced by the immune cells. A kit for assaying the efficacy of immune cells is also described. Efficacy assays are important to meet FDA requirements for novel biological agents such as immunotherapy cells. A method for using potent immune cells as immunotherapeutic treatment is described.

Description

Technical Field

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 805,359, filed on February 14, 2019 ( which is incorporated herein by reference in its entirety).

[0002] The present invention relates to immunotherapy, and more specifically, to testing the effector functions of immune cells. It does.

Background Art

[0003] Immunotherapy is the treatment of diseases by activating or suppressing the immune system. Cells inherent to the immune system can be used to improve immune functions and characteristics. In recent years, immunotherapy has become a major concern for researchers, clinicians, and pharmaceutical companies, especially in its promise to treat various forms of cancer. Immunomodulatory regimens often have fewer side effects than existing drugs, including a lower likelihood of developing resistance when treating microbial diseases. Conventional cancer treatments focus on killing or removing cancer cells using chemotherapy, surgery, and / or radiation. However, the field of therapeutic immune cells is growing rapidly and can be combined with conventional treatments or, in some cases, used instead of conventional treatments to treat, prevent, or delay the onset of cancer. Immune effector cells such as lymphocytes, macrophages, dendritic cells, natural killer cells (NK cells), and cytotoxic T lymphocytes (CTLs) naturally cooperate to defend the body against cancer by targeting abnormal antigens expressed on the surface of tumor cells. Recent developments in cancer treatment

[0004] The focus is on instructing the patient's immune system to attack and destroy the tumor. The strategy is being used, or research and testing are being conducted.

[0005] Adoptive cell transfer (ACT) is the transfer of cells to a patient for lung cancer, melanoma, and other conditions. It has been shown to be promising for this. The cells may be derived from the patient (autologous), and The cells may originate from a different individual (same species). Allogeneic therapy involves using cells from a different donor than the patient who received the cells. It contains isolated and proliferated cells, or adoptive cell transfer is used for subsequent transfusions. For example, self-extracted cells can be cultured and proliferated in vitro. Immunotherapy involves the use of natural killer cells and cytotoxic T lymphocytes derived from the subject's own peripheral blood. Extraction of epithelial cells and other related immune cells, proliferation of these cells in vitro, and then This involves reinjecting these cells into the target body.

[0006] In some therapies, cells (e.g., T cells) are in vitro before being returned to the same patient. They are genetically modified and proliferated in [location]. Chimeric antigen receptor T cell therapy (CAR-T) is targeted T cells are collected from the T cell receptor (TCR) gene, and then retrograde cells containing a copy of the T cell receptor (TCR) gene are used. This involves infecting T cells with a virus. The TCR gene is a tumor antigen (e.g., a chimeric antibody). The virus is specialized to recognize the protoreceptor (CAR). The virus uses the receptor to target the T cell. It is incorporated into the cell's genome. It causes the cell to proliferate nonspecifically and / or stimulates it. Then the cells The cells are reinjected to generate an immune response against the tumor cells.

[0007] The first CAR-T therapy was approved, and multiple for-profit companies became involved in multiple clinical trials. This field is expanding commercially, and the promising future of immunotherapy has been demonstrated. As the new clinical trials progress, the reliability of these therapeutic immune cells is increasing. The need for highly effective and reproducible efficacy tests has since increased. (Immune cell effectors) The industry's "gold standard" for testing functionality is the Kuromiu, developed in the 1960s. This is a radioactive release assay that involves the use and transformation of radioactive materials caused by target tumor cells. Despite concerns related to its dynamic nature, it is still in use. Available alternatives include Calsey. This is a tumor-based assay that uses different tumor targets to identify apoptotic bodies of tumor targets. It still exhibits a great deal of variability caused by the capture of calcein within the system.

[0008] To develop different methods for visually observing the effector functions of these immune cells Although other efforts have been made, these methods still use targeted tumor cells. Another surrogate method for checking the effector function of disease cells is to use these cells This involves checking the cytokines produced, and for that purpose, all conventional methods This uses target tumor cells to induce cytokine production from immune cells. The use of this method introduces biological variability into all of these tests due to the variability between tumor cell types. Add the following: Also, these assays require the hassle of introducing batch effects into these assays. Certain settings are required. Batch effects depend on the state of the target cells and the loading of the plate. Individual variability, plate condition, variability with various reagents, readout variability, etc. This is caused by eliminating all of these variability and obtaining reliable and reproducible results. An immunocyte potency assay capable of producing

[0009] is clearly required to evaluate the quality of immunocyte therapy products. A highly reliable and reproducible potency assay is needed. The approval process is strictly regulated, and pharmaceutical developers must submit a large amount of information about the drug to the regulatory authorities. This may include information regarding the potency of the drug product and the assay for determining this potency. As required by the FDA (21 CFR 610.10), the potency of a cell therapy product should be demonstrated by an appropriate test that shows the effector function of these therapeutic immunocytes, and this test will measure the production of relevant cytokines by these immunocytes.

Summary of the Invention

[0010] Details of one or more embodiments of the present invention are set forth in the accompanying drawings and the following description. Other features, objects, and advantages of the present invention will be apparent from the description and drawings, and from the claims.

[0011] In one aspect, a method for assaying the potency of immunocytes (e.g., T cells, macrophages, NK cells, NK T cells, CAR T cells, and / or CAR NK cells, etc.), comprising contacting the immunocytes with an effective amount of plasma membrane particles, liposomes (including artificial liposomes), or exosomes (including, but not limited to, engineered exosomes), and measuring one or more cytokines (e.g., IL-2, IL-6, IFN-γ, TNF-α, BAFF / TNFSF13B, CD163, CD30 / TNFR produced by the immunocytes. ​​SF8, Chitinase-like 1, gp130, IFN-α2, IL-6Rα, IL-8, IL -10, IL-11, IL-12(p40), IL-12(p70), IL-20, IL -22, IL-26, IL-29 / IFN-l1, IL-32, IL-34, IL-35 MMP-1, osteocalcin, OPN, pentraxin-3, TNF-R1, TNF -R2, TSLP, GM-CSF, MIP-1α, MIP-1β, RANTES, and The method includes detecting the amount of (or TWEAK / TNFSF12, etc.) This will be disclosed in detail. In one embodiment, the method controls the amount of cytokines produced in immunotherapy. This may further include comparing the cytokine efficacy level required for the use of immune cells. .

[0012] Furthermore, a method for assaying the efficacy of immune cells of any prior embodiment, cyto The amount of n is used in immunoassays (e.g., ELISA, intracellular cytokine staining, ELISp). ot, flow cytometry, Luminex xMAP(registered trademark), quantitative PCR (limited Detection is not confirmed, but can be performed using (including qRT-PCR and / or bead assays). The method for obtaining the output is disclosed herein.

[0013] In one embodiment, a method for assaying the efficacy of immune cells of any prior embodiment, Cells can be manipulated to absorb an effective amount of plasma membrane particles, liposomes, or exosomes (but not limited to these). (including the exosomes produced) and at least 5, 6, 7, 8, 9, 10, 15, 20, 2 5, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 , 95, 100, 105, 110, 115, 120, 150 minutes, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 30, 32, 36, 42, 48, 60 hours, 3, 4, 5, 6, 7, 8 , 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 2 2, 23, 24, 25, 26, 27, 28, 29, 30, 31, 45, 60, 61, 62 Methods for contacting for days, 3, 4, 5, or 6 months are disclosed herein.

[0014] Furthermore, a method for assaying the efficacy of immune cells of any prior embodiment, wherein plasma membrane particles , liposomes, or exosomes (including, but not limited to, engineered exosomes) However, concentrations of 5 μg / mL to 1000 μg / mL (not limited to, but 50 μg / mL to 40 A method is disclosed herein that is provided at concentrations including 0 μg / mL.

[0015] In one embodiment, immune cells (e.g., T cells, macrophages, NK cells, NK T cells, A key for assaying the efficacy of CAR T cells and / or CAR NK cells, etc. A set containing an effective amount of plasma membrane particles and / or exosomes (but not limited to, manipulating) A container (for example, a microphone) containing the exosomes produced and a buffer suitable for immune cells. A kit including (such as a centrifuge tube) is disclosed herein. In some embodiments, The kit provides instructions on how to use it to stimulate cytokine production by immune cells. It may also include books.

[0016] Furthermore, a kit for assaying the efficacy of immune cells of any prior embodiment, membrane particles, liposomes, or exosomes (but not limited to, engineered exosomes) (including) concentrations of 5 μg / mL to 1000 μg / mL (not limited to, but 50 μg / mL) A kit is disclosed herein that is provided with concentrations ranging from L to 400 μg / mL.

[0017] In one embodiment, an immunotherapy method comprising a) immune cells of any prior embodiment (e.g., T Cells, macrophages, NK cells, NK T cells, CAR T cells, and / or CA The efficacy of R NK cells, etc., is assayed on multiple immune cells, and each immune cell a) Determining the efficacy of the cells, and b) detecting cytokines (e.g., IL-2, IL-6) , IFN-γ, TNF-α, BAFF / TNFSF13B, CD163, CD30 / TN FRSF8, Chitinase-like 1, gp130, IFN-α2, IL-6Rα, IL-8, IL-10, IL-11, IL-12(p40), IL-12(p70), IL-20, IL-22, IL-26, IL-29 / IFN-l1, IL-32, IL-34, IL- 35, MMP-1, Osteocalcin, OPN, Pentraxin-3, TNF-R1, T NF-R2, TSLP, GM-CSF, MIP-1α, MIP-1β, RANTES, Based on the amount of (and / or TWEAK / TNFSF12, etc.), at least one powerful The selection of immune cells, and c) a sufficient amount of potent immune cells for treatment, as immunotherapy drugs. An immunotherapy method is disclosed herein, which includes administering it to a subject in need. In one embodiment, the method involves assaying the efficacy of immune cells using allogeneic or autologous donors before assaying the efficacy of immune cells. This may further include extracting multiple immune cells.

[0018] Furthermore, any prior embodiment of an immunotherapy method, wherein a therapeutic amount of potent immune cells The immune system further includes proliferating at least one potent immune cell before delivering the immune agent. A therapeutic method is disclosed herein.

[0019] In one embodiment, an immunotherapy method of any prior embodiment, comprising a plurality of immune cells or a powerful This immunotherapy method further involves instructing immune cells to respond to specific antigens. This will be disclosed in the specification.

[0020] Furthermore, any prior embodiment of an immunotherapy method, comprising multiple immune cells or a strong immune Immunotherapy further includes genetically modifying cells to present chimeric antigen receptors. A method is disclosed herein.

[0021] In one embodiment, cancer and / or metastasis in a subject are treated, inhibited, reduced, or prevented. , and / or methods of mitigating a) one obtained from an allogeneic or autologous donor The immune cells above (e.g., T cells, macrophages, NK cells, NK T cells, CAR T cells) a) obtaining cells (and / or CAR NK cells, etc.) and b) immune cells in an effective quantity Plasma membrane particles, liposomes, or exosomes (but not limited to, engineered exosomes) c) bringing into contact with (including mu) and cytokines produced by immune cells (for example) , IL-2, IL-6, IFN-γ, TNF-α, BAFF / TNFSF13B, CD1 63, CD30 / TNFRSF8, Chitinase-like 1, gp130, IFN-α2, IL -6Rα, IL-8, IL-10, IL-11, IL-12(p40), IL-12(p 70), IL-20, IL-22, IL-26, IL-29 / IFN-l1, IL-32 IL-34, IL-35, MMP-1, osteocalcin, OPN, pentraxin - 3, TNF-R1, TNF-R2, TSLP, GM-CSF, MIP-1α, MIP-1 Detecting the amount of β, RANTES, and / or TWEAK / TNFSF12, etc. d) Based on the amount of cytokines detected, select at least one robust immune cell. This includes selecting and e) administering to the subject a therapeutically effective amount of potent immune cells, Methods are disclosed herein. In some embodiments, the methods involve self-donor or allogeneic donor This may further include extracting immune cells.

[0022] Furthermore, it treats, inhibits, reduces, and prevents any prior embodiment of cancer and / or metastasis. A method of reducing and / or alleviating at least one potent immune in a therapeutically effective amount This further includes growing at least one potent immune cell before delivering the disease cells. The method is disclosed herein.

[0023] Furthermore, the identity of at least one immune cell or cell population (e.g., differentiated Th1, Th 2, Th3, Th9, Th17, Effector Memory T (Tem) cells, Central Me Molly T (Tcm) cells, γδ T cells, or regulatory T (Treg) cells, resting NK cells The proliferated NK cells are identified based on the cytokine signature associated with their cell type. A method for doing so is disclosed herein. [Brief explanation of the drawing]

[0024] [Figure 1] This plot shows the correlation between exosome-induced NK cell cytokine release (derived from K562 cells) and PHA-induced NK cell cytokine release (at pg / million cells / hr). [Figure 2]This provides a plot showing the correlation between cytokine release from newly isolated NK cells (induced by K562 exosomes) and cytokine release from proliferating NK cells induced by exosomes (at pg / million cells / hr). [Figure 3] The total cytokine concentrations upon exposure to four different exosome concentrations (60, 100, 200, and 400 mg / mL) are shown. [Figure 4] This shows the dose-correlation between two different exosome concentrations. [Modes for carrying out the invention]

[0025] This invention involves bringing immune cells into contact with an effective amount of exosomes, and by the immune cells A method for determining the efficacy of immune cells, including detecting the amount of cytokines produced. Provided. This disclosure is given in connection with cancer immunotherapy, but the concepts disclosed herein are not limited to this disclosure. And these innovations can be applied to immunotherapy for other diseases and disorders. For example, autoimmune diseases, Used in immunotherapy for inflammatory diseases or disorders, viral diseases and / or bacterial infections. The immune cells used are also tested for efficacy using the assay disclosed herein. It is possible.

[0026] definition To clarify understanding and facilitate reference, the entire chapter of brief explanations and the rest of the specification are included. A list of terms used in various contexts is compiled here. Some terms are used throughout the field. These terms are commonly known through the body and are defined here for clarity, but some terms are originally... This is specific to the application and therefore needs to be defined in order to properly understand this application.

[0027] As used herein and in the claims, “one (a)” and “one (an)” are used in this specification and in the claims. The singular forms such as ")" and "the" should be used unless otherwise clearly indicated by the context. , including multiple referents. For example, the term "cell" can refer to multiple cells (a mixture of them). (including) and (including). When the plural form is used herein, it generally includes the singular form.

[0028] In this specification, the range is defined as "approximately" from one particular value and / or "approximately" from another particular value It can be expressed as, up to, etc. When such a range is expressed, another embodiment is that Includes a range from a specific value and / or up to another specific value. Similarly, values ​​are approximate values. When expressed as such, by using the antecedent "about", a specific value can be expressed in a different action form. It will be understood that a state is formed. Furthermore, each endpoint of the range, in relation to the other endpoints, It will be understood that both are important, independently of other endpoints. Numerical range by endpoint The enumeration includes all numbers within that range (for example, 1-5 includes 1, 1.5, 2 (including 2.75, 3, 3.80, 4, 5, etc.). Some values ​​disclosed herein Each value exists, and each value is disclosed herein as "approximately" the value itself, in addition to the value itself. It can also be understood as follows. For example, if the value "10" is disclosed, "approximately 10" is also disclosed. If a value is disclosed as "less than or equal to ~" so that it can be properly understood by the person, It is also understood that the possible range between values ​​"greater than or equal to " and values ​​" will be disclosed. For example, the value "10" If disclosed, then "10 or less" and "10 or more" will also be disclosed. Over the course of the journey, the data is provided in several different formats, and this data includes the endpoint and the start. It is also understood to represent a point, or a range of any combination of data points. For example. If a specific data point "10" and a specific data point 15 are disclosed, 1 In addition to the range between 0 and 15, the ranges are greater than 10, 10 or more, less than 10, 10 or less, and 10. Values ​​equal to, greater than, 15 or greater, less than, 15 or less, and equal to 15 are disclosed. It is understood that this is considered to be the case. Also, each unit between two specific units is also open. It is understood that they are shown. For example, if 10 and 15 are disclosed, then 11, 12, 1 Articles 3 and 14 are also disclosed.

[0029] As used herein, the term "includes" means that the compositions and methods listed are This is intended to mean that it includes elements but does not exclude other elements. Composition and When used to define a method, "to be essentially derived from" means a combination of This means excluding any other elements of essential importance. The composition, which is essentially composed of the elements defined herein, is obtained by isolation and purification methods from a small area. Contaminants and pharmaceutically acceptable carriers, such as phosphate-buffered saline and preservatives. This does not exclude. "~consists of" means that trace amounts of other components are required for administering the composition of the present invention. This means excluding anything beyond the basics, and the substantial method steps. The embodiments defined by each of these transitional terms are within the scope of the present invention.

[0030] "Increase" refers to any symptom, disease, composition, condition, or activity that results in a greater amount of symptoms, disease, composition, or activity. It can refer to a change. An increase is a statistically significant amount of any state, symptom, activity, or composition. This could be an increase in the individual values, median, or mean. Therefore, an increase is an increase in the median. As long as it is statistically significant, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 , 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, It could be an increase of 95 or even 100%.

[0031] "Reduction" means any less amount of symptoms, disease, composition, condition, or activity. It can refer to a change. A substance can also mean that the genetic output of the gene product containing that substance is If the output of the gene is less than that of a gene product that does not contain the substance, the genetic output of the gene is reduced. It is understood as reducing. Also, for example, a decrease means that the symptoms are less than previously observed. Such changes in the symptoms of the disorder may occur. A decrease in a statistically significant amount of the state, symptoms, or activity may occur. This could be a decrease in any individual value, median, or mean of the composition. Therefore, the decrease is As long as the decrease is statistically significant, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 5, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 It could be a decrease of 85%, 90%, 95%, or 100%.

[0032] "Inhibit," "the act of inhibiting," and "inhibition" all refer to activity, response, state, disease, and This means that other biological parameters will decrease. These include activity, response, state, This may include, but is not limited to, the complete elimination of the disease. This includes, for example, the complete elimination of the disease. This may also include a 10% reduction in activity, response, condition, or disease compared to natural or control levels. Therefore, the reduction is 10, 20, 30, 40 compared to natural or control levels. The reduction may be 50%, 60%, 70%, 80%, 90%, 100%, or any amount in between.

[0033] The word "reduce" or other forms of the word, such as "to reduce" or "reduce," means This means a decrease in the size or characteristic (e.g., tumor growth). This is typically due to several marks. It relates to the standard value or expected value; in other words, it is relative, but not to the standard value or relative. It is understood that referring to the backtrade value is not always necessary. For example, "lower tumor growth "To reduce" means to decrease the growth rate of a tumor compared to a standard or control. .

[0034] "To prevent" or other forms of the word, such as "to prevent" or "prevention," To halt a specific event or characteristic, or to stabilize the development or progression of a specific event or characteristic. To shorten or delay, or to minimize the possibility of a particular event or characteristic occurring. It means to keep it down. Prevention is typically more absolute than reduction, for example. No comparison with a control is required. When used herein, something can be reduced. However, there are cases where prevention is not possible, but where something can be prevented that can be reduced. There are also cases where something can be prevented but not reduced. In some cases, it may be possible to reduce something that is being prevented. When reduction or prevention is used It was understood that, unless specifically otherwise indicated, the use of other words would also be explicitly disclosed. stomach.

[0035] The term "effective in treatment" implies side effects (for example, side effects typically associated with alternative therapies). Activating agents (immunotherapy cells) that aim to reduce disease severity while avoiding ) It is intended to limit the number or amount of (etc.). One or more doses that are effective for treatment. It may be administered as such. Effective treatments include those that do not improve the disease outcome itself. If there are any, they are treatments that improve the quality of life for the individual.

[0036] "Effective amount" generally includes achieving the specific desired effect described in this application. For example, it can provide the desired local or systemic effect, such as effectively stimulating cytokine formation. It means the amount to be administered. For example, an effective amount is the amount that will produce a beneficial or desired clinical outcome. That is a sufficient amount.

[0037] The term "target" refers to any individual that is the target of administration or treatment. The target is the spine. It could be an animal, for example, a mammal. In one embodiment, the subject is a human, a non-human primate, a cattle, It could be a horse, pig, dog, or cat. The subject could also be a guinea pig, rat, or hamster. —It may be a rabbit, mouse, or mole. Therefore, the subject is human or animal. A patient can be a medical patient. The term "patient" refers to an individual under the treatment of a clinician, for example, a physician. To point.

[0038] The term "therapeutably acceptable carrier" generally refers to the preparation of safe and non-toxic compositions. It means a carrier or excipient useful for veterinary use and / or use in humans. The carrier is included. The intravenous delivery method includes (for example, osmotic pressure and p safe for intravenous use). Use a physiologically balanced and therapeutically acceptable carrier (at the H level). When used, the term "therapeutically acceptable carrier" refers to saline, Ringer's, and phosphate. Acid-buffered saline, water, dextrose aqueous solution, and emulsion (e.g., oil / water) Any of the standard carriers, such as water / oil emulsions, and various types of wetting agents. This includes any excipients, diluents. As used herein, the term "carrier" means any excipient, diluent. , used in fillers, salts, buffers, stabilizers, solubilizers, lipids, stabilizers, or therapeutic formulations This includes other materials well known in the relevant art. Therapeutically acceptable carriers also include immunosuppressants. Preservatives (including cryopreservatives) that maintain the viability and / or efficacy of disease cells. May include. As used herein and in the claims, “therapeutically permissible carrier” means 1 This includes both one such carrier and more than one such carrier.

[0039] The term "treatment" refers to curing, alleviating, or stabilizing a disease, pathological condition, or disorder. Or it refers to the medical management of a patient with the intention of prevention. This term is active treatment, in other words This includes treatment specifically aimed at improving a disease, pathological condition, or disorder, and causal treatment. In other words, this includes treatment aimed at eliminating the cause of the associated disease, pathological condition, or disorder. In addition, this term refers to palliative treatment, i.e., the treatment of a disease, pathological condition, or disorder. Rather, it refers to treatments designed to alleviate symptoms, preventive treatments, i.e., related diseases, illnesses. To minimize the onset of a condition or disorder, or to partially or completely inhibit it. Treatment directed towards, and supportive treatment for, related diseases, pathological conditions, and This includes treatments used to supplement other specific therapies aimed at improving the disorder.

[0040] "Administration" to a subject includes any route through which the drug is introduced or delivered to the subject. This includes oral, topical, intravenous, subcutaneous, transcutaneous, and transdermal (transcutaneous) administration. (Intramuscular), intra-articular, parenteral, intra-arterial, intradermal, intraventricular, intracranial, abdominal Intralesional, intranasal, rectal, vaginal, by inhalation, via transplant reservoir, parenteral (e.g., subcutaneous) , intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intramedullary, abdominal, liver, lesion, and It can be administered by any suitable route, including intracranial injection or infusion techniques. The term "concurrent administration" as used herein refers to simultaneous administration. tion, administration in combination, simul taneous administration, or administered s imultaneously) means that the compounds are administered at the same time point in time, or Essentially, this means they are administered immediately after each other. In the latter case, the two compounds are observed The results obtained are indistinguishable from the results obtained when administered at the same time point. It is administered within a few minutes. "Systemic administration" means, for example, by administering to the entry points of the circulatory or lymphatic system. Through this process, the drug is introduced into a wide area of ​​the target body (for example, more than 50% of the body). This refers to introducing or delivering a drug to a target via a delivery route. In contrast, "Local administration" means introducing or delivering the drug to the area of ​​the administration site or an area directly adjacent thereto. Drugs are introduced or delivered to the target via routes that do not involve systemic introduction of therapeutically significant doses. This refers to the act of being able to detect drugs administered locally in the immediate vicinity of the administration site. It is possible, but it is either undetectable or detectable in negligible amounts in the distal parts of the target body. It is possible. Administration includes self-administration and administration by another person.

[0041] "To treat," "the act of treating," "treatment," and their grammatical variations are as follows: When used, it refers to the intensity or frequency of one or more diseases or conditions, or the symptoms of a disease or condition. , or to partially or completely prevent, delay, or cure the underlying cause of a disease or condition. Healing, relief, easing, soothing, changing, treating, reducing, improving, stabilizing, sedating This includes the administration of a composition intended to reduce, or for the purpose of reducing, and / or reducing. The treatment according to the present invention may be applied preventively, antidiagnosticly, mitigatingly, or symptomatically. Treatment is provided before the onset of symptoms (for example, before obvious signs of cancer) or during the early stages of cancer (for example, in the early stages of cancer). It is administered to the subject at the time of signs and symptoms, or after the development of established cancer. Prophylactic administration is This can be done from one day (or several days) before the onset of symptoms of the disease or infection, up to several years prior.

[0042] Throughout this application, various publications are referenced. The disclosures of these publications are as follows: Therefore, in order to more fully explain the state of the art relating to this application, the following are incorporated herein by reference: The disclosed references are also discussed in the texts that refer to them, and the materials contained therein. These are incorporated herein by reference individually and specifically.

[0043] Immunoassay In one embodiment, the present invention provides a method for determining the efficacy of immune cells. This method involves immunotherapeutic cells Cells contain an effective amount of plasma membrane particles, liposomes, or exosomes (e.g., cancer cell exosomes). The steps involve contacting the exosome (or engineered exosome) and production by immune cells. The procedure includes the step of detecting the amount of cytokines being produced. For example, exosomes are cultured in a cell culture medium. By suspending immune cells in a culture medium, the immune cells can be exposed to plasma membrane particles or It can come into contact with exosomes (including, but not limited to, manipulated, exosomes). Cut.

[0044] In some embodiments, this method is used to control the amount of cytokines produced in immunotherapy. This includes a step of comparing the cytokine efficacy level required for the use of immune cells. Sei characterizes the product (i.e., immune cells), monitors lot-to-lot consistency, and the product It helps to ensure stability and therefore can affect the mechanism of action and function of the product. It is possible to detect differences that are therefore highly sensitive enough to have potential clinical significance. It should be a certain degree. This assay is a predictive biomarker for cell-mediated immunotherapy. Alternatively, it can be used as a pharmacodynamic assay. The efficacy assay is used to estimate the efficacy of the product. It is preferable that the physiological / pharmacological activity is as close as possible to the actual activity. The efficacy assay described in this document has the ability to measure the efficacy values ​​within the product specifications and their potential clinical significance. High sensitivity for detecting differences in product mechanism of action and presumed physiological / pharmacological factors. It provides a close relationship with activity. Preferably, the efficacy assay also satisfies the following secondary criteria. To achieve (the accuracy required to support the product specifications), use a sufficiently low assay and It offers good inter-assay variability, sufficient robustness, and suitability for high-throughput analysis. In some embodiments, this assay involves T cells, macrophages, NK cells, and NK cells. For quantifying the function of T cells, CAR T cells, and / or CAR NK cells Clinical assays (for diagnosing NK cell immunodeficiency and monitoring the effectiveness of immunosuppressants or immunoactivators) It is used as a biomarker for [specific purpose].

[0045] As described above, the disclosed method is provided for determining the efficacy of immune cells. A cell is any cell of the immune system that produces cytokines, as defined herein. These are cytokine-producing immune cells. Examples of cytokine-producing immune cells include: Lymphocytes, neutrophils, macrophages, and natural killer cells are examples. The sphere contains both B cells and T cells (including CD4 and CD8 T cells). One embodiment So, immune cells include tumor-infiltrating lymphocytes (TILs), T cells, and natural killer (NK) cells. Cells, NK T cells, chimeric antigen receptor (CAR) T cells, and / or CAR NK It may include. Immune cells can be obtained from cell cultures or from subjects (e.g., allogeneic donors). It can be obtained from (or from a self-donor, etc.)

[0046] In some embodiments, the immune cells are T cells. T cells are involved in cell-mediated immunity. They play a psychological role, and the presence of T cell receptors on the cell surface allows B cells and na They can be distinguished from other lymphocytes such as natural killer cells. An example of a T cell is: T helper cells (TH cells), cytotoxic T cells (TC cells), memory T cells, regulatory T cells or "suppressive" T cells, and natural killer T cells (NKT cells, NK cells) It is distinguished from other molecules and recognizes glycolipid antigens rather than peptides presented by MHC molecules. Different types of T cells differ from one another in their cytokine production patterns. It can be. T cells can be CD4 T cells or CD8 T cells. Furthermore, T cells This may include chimeric antigen receptor (CAR) T cells or tumor-infiltrating lymphocytes (TILs).

[0047] In some embodiments, the immune cells are NK cells. Natural killer cells are immune cells. NK cells are a type of cytotoxic lymphocyte associated with disease. NK cells rapidly respond to virus-infected cells. They provide a response and respond to transformed cells. Typically, immune cells respond to infected cells. Peptides from pathogens presented by major histocompatibility complex (MHC) molecules on the surface It detects this, triggering cytokine release, which leads to lysis or apoptosis. However, NK cells can determine whether pathogen-derived peptides are present on the MHC molecule. These cells remain unique because they possess the ability to recognize stressed cells. The initial idea was that cells do not require prior activation to kill their targets, and therefore, It was named "natural killer." NK cells are large granular lymphocytes (LGLs) and are found in the bone marrow. It is known that they differentiate, mature, and then enter the cycle. In some embodiments, NK The cells may be CAR NK cells.

[0048] Therefore, in one embodiment, immune cells (e.g., T cells, macrophages, NK cells, N) Assaying the potency of KT cells, CAR T cells, and / or CAR NK cells. A method for delivering immune cells to an effective amount of plasma membrane particles, liposomes, or exosomes. Contact with (but not limited to) manipulated exosomes, and by immune cells A method comprising detecting the amount of one or more cytokines produced by the above is described herein. Disclosed. In one embodiment, the method is used to control the amount of cytokines produced in immunotherapy. This could further include comparing the cytokine efficacy levels required for the use of immune cells.

[0049] This assay involves stimulating immune cells with exosomes, followed by their production. The procedure includes the step of detecting the amount of cytokines. When used herein, "cytokine" is used. The term "Immuno" refers to cellular signaling, particularly immunomodulation, which can be produced by immune cells. This refers to small proteins (approximately 5-20 kDa) that are important for the body. Examples of cytokines include: Chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors It includes. The detected cytokines are produced by the immune cells being evaluated. It may contain known cytokines, or its detection may be due to its production by immune cells. This includes a broader range of cytokines, including cytokines whose effects are not yet known. It is possible.

[0050] In some embodiments, the detected cytokines are T cells or natural killer cells. It includes cytokines known to be produced by cells. In some embodiments, This includes cytokines that are known to be produced by T cells. T cells include Th1 and Th2 cells, and Th1 cells are interferon (I FN)-γ (IFN-γ), tumor necrosis factor (TNF)-α (TNF-α), and IL- Th2 cells primarily produce interleukin (IL)-2 (IL-2) and IL-4. It produces IL-5, IL-6, IL-9, IL-13, and IL-22. Examples of cytokines produced by natural killer cells include IL-1α, IL-1β, IL-2, IL-5, IL-8, IL-10, IL-13, IFN-γ, T NF-α, granulocyte-macrophage colony-stimulating factor (GM-CSF), leukemia arresting factor ( LIF), and chemokine macrophage inflammatory protein (MIP)-1α(MIP Examples include -1α), MIP-1β, and RANTES, which determine the efficacy of immune cells. Other cytokines useful for this purpose include, but are not limited to, B cell activators / tumor necrosis factors. Child (TNF) ligand superfamily member 13B (BAFF / TNFSF13B) ), Differentiation cluster (CD)163 (CD163), CD30 / TNFRSF8, Kichina -ase3-like1, gp130, IFN-α2, IL-6Rα, IL-11, IL-12(p4 0), IL-12(p70), IL-20, IL-26, IL-29 / IFN-l1, I L-32, IL-34, IL-35, Matrix Metalloproteinase-1 (MMP- 1) Osteocalcin, osteopontin (OPN), pentraxin-3, tumor necrosis Factor (TNF)-receptor 1 (TNF-R1), TNF-R2, thymic interstitial lymphopoietin (TSLP), or TNF-related weak apoptosis inducer (TWEAK) / TN F Super Family Member 12 (TWEAK / TNFSF12) is one example. However, in one embodiment, immune cells (e.g., T cells, macrophages, NK cells, NK T cells) Those who assay the potency of cells (such as CAR T cells and / or CAR NK cells). A law that allows immune cells to be exposed to an effective amount of plasma membrane particles, liposomes, or exosomes (limited Although not produced by direct contact with (manipulated exosomes), immune cells produce One or more cytokines are produced (e.g., IL-2, IL-6, IFN-γ, TNF-γ) α, BAFF / TNFSF13B, CD163, CD30 / TNFRSF8, chitinase 3-like 1, gp130, IFN-α2, IL-6Rα, IL-8, IL-10, IL-11 , IL-12(p40), IL-12(p70), IL-20, IL-22, IL-26 , IL-29 / IFN-l1, IL-32, IL-34, IL-35, MMP-1, male Theocalcin, OPN, Pentraxin-3, TNF-R1, TNF-R2, TSLP, GM-CSF, LIF, MIP-1α, MIP-1β, RANTES and / or T A method is disclosed herein that includes detecting the amount of WEAK / TNFSF12, etc. Immune cells (e.g., T cells, macrophages, NK cells, NK T cells, CAR cells) A method for assaying the efficacy of T cells and / or CAR NK cells, etc., Epidemic cells are subjected to an effective amount of plasma membrane particles, liposomes, or exosomes (but not limited to) Contact with the exosomes produced, and one or more produced by immune cells The cytokines listed above (for example, IL-2, IL-6, IFN-γ, TNF-α, BAFF / TNFSF13B, CD163, CD30 / TNFRSF8, Chitinase 3-type 1, gp1 30, IFN-α2, IL-6Rα, IL-8, IL-10, IL-11, IL-12( p40), IL-12(p70), IL-20, IL-22, IL-26, IL-29 / IFN-1, IL-32, IL-34, IL-35, MMP-1, osteocalcin, OPN, Pentraxin-3, TNF-R1, TNF-R2, TSLP, GM-CSF, MIP-1α, MIP-1β, RANTES, and / or TWEAK / TNFSF1 A method is disclosed herein that includes detecting an amount of (2, etc.). In one embodiment, this method The law stipulates that the amount of cytokines produced must be used in immunotherapy for the site where immune cells are needed. This may further include comparing with the caine potency level. In some embodiments, multiple The level of itokine is determined. In a further embodiment, cytokines interlo Selected from the group consisting of ikin-2, interleukin-6, and interferon-γ. It can be done.

[0051] This assay includes a step to detect the amount of cytokines produced by immune cells. Hmm. A wide variety of methods for detecting cytokines are known to those skilled in the art, and they can be detected. It can change depending on the cytokine. In some embodiments, multiple different cytokines To detect and / or quantify the presence of [unclear], one or more methods are used. Cytokines can be used, for example, in specific reagent kits or immunoassays. It can be detected by use. Cytokines are Miltenyi Biotec (trademark). Luminex and Thermo Fisher Scientific (trademarks) It can be detected using kits available from any commercial provider. A good example of a kit suitable for detecting cytokines is the Rapid Cytokine Inspector (CD4 / CD8) kit, or sites formed by either T cells or NK cells This MACSPlex cytokine T / NK kit can detect cytokines. Both are sold by Miltenyi Biotec (trademark).

[0052] In some embodiments, the amount of cytokine is detected using an immunoassay. There are many different forms and variations of immunoassays. This involves adding reagents and washing away or separating multiple samples at different time points in the assay. It may be performed in steps. The immunoassay involves heterogeneous immunoassays with multiple steps. A homogeneous immunoassay involves simply mixing reagents and samples and performing physical measurements. It is known that immunoassays often contain the analyte of concern. Using a calibrator, which is a solution containing the analyte, the concentration of the analyte is generally known. The assay response generated by the calibrator and the response to the actual sample are being compared. By comparing the responses of the samples, the presence or concentration of the analyte in the sample can be determined. This makes it possible to interpret the signal strength. Types of immunoassays include competitive homogeneous immunoassays. Non-competitive immunoassays, competitive heterogeneous immunoassays, one-site non-competitive immunoassays, and two-site non-competitive immunoassays. This includes site-non-competitive immunoassays. Immunoassays include enzyme-linked immunosorbent assays. (ELISA), Lateral Flow Immunoassay, Enzyme-Linked Immunoadsorption Spot (ELI) spot) assay, flow cytometry, intracellular cytokine staining, antibody array assay Sei and bead-based assays, magnetic immunoassays, radioimmunoassays, and This also includes quantitative PCR (but is not limited to qRT-PCR). In one embodiment, Sei includes Luminex xMAP (registered trademark).

[0053] A method for determining the efficacy of immune cells is to use immune cells to deliver an effective amount of plasma membrane particles and / or other particles. This includes a step of contacting exosomes (e.g., engineered exosomes). Membrane (PM) particles are made from the plasma membrane of cells or are artificially created (i.e., These are liposomes (vesicles). PM particles contain a lipid bilayer or simply a lipid single layer. PM particles can be prepared in single-lamellar, multi-lamellar, or inverted forms. PM particles are referred to herein by reference in U.S. Patent No. 9,623,082 (the entire disclosure thereof is provided herein by reference). Prepare known plasma membrane preparation protocols or liposomes, such as those described in (used) Using the protocol described herein, Fc-bound feeder cells It can be prepared from. In certain embodiments, the PM particles disclosed herein are average diameter The diameter is in the range of approximately 170 to 300 nm.

[0054] Exosomes are cell-derived vesicles that are present in the fluids of many, perhaps all, eukaryotes. Exosomes are RNA, proteins, lipids, and other components that reflect the cell type from which they originate. It contains metabolites. The reported diameter of exosomes is 30-100 nm. Exosomes are released from cells either when the polyvesicle fuses with the plasma membrane, or directly from the plasma membrane. They are either released or released. In some embodiments, exosomes are released from cancer cells. Obtained from. In some embodiments, the exosomes are leukemia cell exosomes. This disclosure is given in the context of using exosomes to determine the efficacy of immune cells. However, other extracellular vesicles may also be used to determine the efficacy of immune cells. When used in detail, the term "extracellular vesicle" refers to a vesicle released from a cell by any mechanism. This includes, but is not limited to, all vesicles that are released. "Extracellular vesicles" are multivesicular or This includes exosomes released from the cell surface and microvesicles that leak out from the cell surface. ("Extracellular vesicles") These include vesicles produced by exocytosis or ectocytosis. "Extracellular vesicles" include exosomes released from the polysporoplasm and small vesicles released by reverse budding. Cell division, membrane division (multiple cells possible), multivesicular endosome, ectosome, microvesicle, microparticle, and Hybrid containing vesicles released by apoptotic organisms, as well as plasma membrane components. Extracellular vesicles contain proteins, nucleic acids, lipids, and other substances common to the cell from which they originate. It may contain other molecules.

[0055] In one embodiment, plasma membrane particles, or exosomes, are immune cells (e.g., NK cells, etc.) It can be purified from feeder cells that stimulate it. Plasma membrane particles disclosed herein are produced For use in the claimed invention for use in preparing or exosomes The feeder cells that stimulate immune cells are irradiated autologous or allogeneic peripheral blood mononuclear cells. (PBMC) or unirradiated autologous or allogeneic PBMC, RPMI8866, H FWT, 721.221, K562 cells, EBV-LCL, one or more membrane-bound IL-21 , membrane-bound IL-15, membrane-bound 4-1BBL, membrane-bound OX40L and / or membrane-bound TNF- T cells transfected with α (for example, transfected with membrane-bound IL-21) T cells, T cells transfected with membrane-bound 4-1BBL, membrane-bound IL-15 and T cells transfected with 4-1BBL, membrane-bound IL-21 and 4-1BBL T cells transfected with IL-21, NK cells transfected with membrane-bound IL-21 Cells (not limited to, but including PBMC, RPMI8866, NK-92, NK-92MI, NK) -YTS, NK, NKL, KIL, KIL C.2, NK3.3, NK-YS, HFWT NK cells (including K562 cells), transfected with membrane-bound 4-1BBL (limited) However, PBMC, RPMI8866, NK-92, NK-92MI, NK-YTS , NK, NKL, KIL, KIL C.2, NK3.3, NK-YS, HFWT, K56 NK cells (including 2 cells) transfected with membrane-bound IL-15 and 4-1BBL Cells (not limited to, but including PBMC, RPMI8866, NK-92, NK-92MI, NK) -YTS, NK, NKL, KIL, KIL C.2, NK3.3, NK-YS, HFWT (including K562 cells), or transfected with membrane-bound IL-21 and 4-1BBL The targeted NK cells (not limited to, but including PBMC, RPMI8866, NK-92, NK- 92MI, NK-YTS, NK, NKL, KIL, KIL C.2, NK3.3, NK- (Including YS, HFWT, and K562 cells), as well as cells that do not express other HLAs, or This could be either a low-HLA-expressing cell line or a primary tumor derived from the patient.

[0056] Plasma membrane particles and / or exosomes used in the methods of this disclosure are immune cells (e.g.) If additional effector agents are used to proliferate and / or activate NK cells, etc. It may be included in. Therefore, in one embodiment, a method for assaying the efficacy of immune cells, opening The feeder cells used to generate the exosomes or plasma membrane particles shown are The cell surface further contains at least one additional immune cell effector agent, and at least Another additional immune cell effector agent is cytokines, adhesion molecules, or immune cell activation agents. sexualizing agents (e.g. 4-1BBL, IL-2, IL-12, IL-15, IL-18, IL -21, MICA, LFA-1, 2B4, CCR7, OX40L, UBLP2, BCM1 / SLAMF2, NKG2D agonist, CD155, CD112, Jagged1, J  2, Delta-1, Pref-1, DNER, Jedi, SOM-11, U Ingress, CCN3, MAGP2, MAGP1, TSP2, YB-1, EGFL7, C CR7, DAP12, and DAP10, Notch ligand, NKp46 agonist, NKp44 agonist, NKp30 agonist, other NCR agonists, CD16 agonist A method is disclosed herein that is a strobe. In one embodiment, at least one additional immune Cell effector agents include IL-21, 4-1BBL, IL-15, IL-21 and 4- Includes 1BBL, IL-21 and IL-15, or IL-15 and 4-1BBL. Therefore, in one embodiment, it is produced by the feeder cells described above and disclosed herein Plasma membrane particles and exosomes used in methods for assaying the efficacy of immune cells are immune epidermal cell activators (e.g. 4-1BBL, IL-2, IL-12, IL-15, IL-1 8, IL-21, MICA, LFA-1, 2B4, CCR7, OX40L, UBLP2, BCM1 / SLAMF2, NKG2D agonist, CD155, CD112, Jagge d1, Jagged2, Delta-1, Pref-1, DNER, Jedi, SOM- 11. Wingless, CCN3, MAGP2, MAGP1, TSP2, YB-1, EGF L7, CCR7, DAP12, and DAP10, Notch ligand, NKp46 jaw Nist, NKp44 agonist, NKp30 agonist, other NCR agonists, CD1 This may include any combination of agonists in a membrane-bound configuration. For example, exosomes or Plasma film particles have IL-15, IL-21, and / or 4-1BB on their film. It may have L.

[0057] Immune cells are induced to produce cytokines, particles, or exosomes during this period. It is understood and intended herein that exposure to must be necessary. In one aspect, immuno A method for assaying the efficacy of cells, wherein immune cells are subjected to an effective amount of plasma membrane particles, liposyl M, or exosomes (including, but not limited to, engineered exosomes) and at least 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 , 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115 , 120, 150 minutes, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 30, 32, 36, 4 2, 48, 60 hours, 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 Contact for 29, 30, 31, 45, 60, 61, 62 days, 3, 4, 5, or 6 months A method for doing so is disclosed herein.

[0058] Furthermore, a method for assaying the efficacy of immune cells of any prior embodiment, wherein plasma membrane particles , liposomes, or exosomes (including, but not limited to, engineered exosomes) However, a method is disclosed herein that provides the solution at concentrations of 5 μg / mL to 1000 μg / mL. In one embodiment, the concentrations of particles or exosomes are 5, 10, 15, 20, 25, and 30. , 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 125, 130, 140, 150, 160, 170, 175, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, The concentration is 800, 850, 900, 950, or 1000 μg / mL. One embodiment So, the concentration of exosomes or particles is approximately 50 μg / mL to 100 μg / mL, 50 μ g / mL~200μg / mL, 50μg / mL~300μg / mL, 50μg / mL~5 The concentration is 00 μg / mL, or 100 μg / mL to 500 μg / mL. Preferably, The concentration of sosomes or particles is approximately 50 μg / mL to 400 μg / mL.

[0059] In some embodiments, immune cells are derived from unmodified cancer cells such as unmodified K562. They are stimulated using exosomes. However, in other embodiments, antigen-specific cells They are stimulated using exosomes from antigen-expressing cells. For example, antigen-expressing exosomes Using the patient's blood cells, antigen-specific therapeutic cells (e.g., CAR-T cells, CA) are used. R-NK cells are cells that release exosomes from antigen-expressing K562, or target cell engagers. - Can be stimulated by (bispecific engagers, BiTE, BiKE, TriNKET) .

[0060] In one embodiment, the same cytokines produced to determine the efficacy of immune cells are used It is understood that it is also possible to identify cells that produce cytokines, and this is intended to be done herein. It is done. Immune cells have a clear expression profile that is well known in the field of technology. Also, few Even without this, the identity of a single immune cell or cell population (e.g., differentiated Th1, Th2, Th3) Th9, Th17, effector memory T (Tem) cells, central memory T ( Tcm cells, γδT cells, or regulatory T(Treg) cells, resting NK cells, proliferating A method for determining NK cells based on cytokine signatures associated with their cell type. , disclosed herein. Therefore, immune cells (e.g., T cells, macrophages, N Identify K cells, NK T cells, CAR T cells, and / or CAR NK cells, etc. A method for delivering immune cells to an effective amount of plasma membrane particles, liposomes, or exosomes. Contact with (but not limited to) manipulated exosomes, and by immune cells This produces one or more cytokines (e.g., IL-2, IL-6, IFN-γ, T) NF-α, BAFF / TNFSF13B, CD163, CD30 / TNFRSF8, Kichi Nase 3-like 1, gp130, IFN-α2, IL-6Rα, IL-8, IL-10, IL -11, IL-12(p40), IL-12(p70), IL-20, IL-22, IL -26, IL-29 / IFN-l1, IL-32, IL-34, IL-35, MMP-1 Osteocalcin, OPN, Pentraxin-3, TNF-R1, TNF-R2, TS LP, GM-CSF, MIP-1α, MIP-1β, RANTES, and / or TW This includes detecting the amount of EAK / TNFSF12, etc., and determining the identity of immune cells through expression. Methods revealed herein are derived based on the cytokine profiles obtained. It can be done.

[0061] A kit for evaluating the efficacy of immune cells Another aspect of the present invention relates to immune cells (e.g., T cells, macrophages, NK cells, NK cells). To determine the efficacy of T cells, CAR T cells, and / or CAR NK cells, etc. A kit for which an effective amount of particles or exosomes (e.g., exosomes (limited) It is not suitable for immune cells (such as engineered exosomes or plasma membrane particles) The kit is provided, which includes a container containing a buffer. In some embodiments, the kit contains Xosomes are supplied at concentrations ranging from 5 μg / mL to 1000 μg / mL. In one embodiment, granules The concentrations of exosomes are 5, 10, 15, 20, 25, 30, 35, 40, and 45. , 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 1 20, 125, 130, 140, 150, 160, 170, 175, 180, 190, 2 00, 225, 250, 275, 300, 325, 350, 375, 400, 425, 4 50, 475, 500, 550, 600, 650, 700, 750, 800, 850, 9 The concentration is 00, 950, or 1000 μg / mL. In one embodiment, exosomes Alternatively, the particle concentration is approximately 50 μg / mL to 100 μg / mL, or 50 μg / mL to 200 μg / mL. g / mL, 50μg / mL~300μg / mL, 50μg / mL~500μg / mL, The concentration is 100 μg / mL to 500 μg / mL. Preferably, exosomes or particles. The concentration is approximately 50 μg / mL to 400 μg / mL. In some embodiments, the container This refers to a microcentrifuge tube (for example, an Eppendorf microcentrifuge tube). The kit also includes tools for obtaining samples from subjects, such as one or more immune cells. A syringe for obtaining the sample may be included. A suitable buffer solution is RPMI.

[0062] The kit also includes the necessary components (e.g., solid phase) for performing the immunoassay. This may also be the case, and in contrast, capture antibody and / or sandwich immunoassay format An antibody that functions as a detection antibody binds to it. The solid phase can be magnetic particles, beads, test tubes, or micro Titer plates, cuvettes, membranes, scaffold molecules, quartz crystals, films, filter paper, The material may be a disc or a chip. The kit also contains antibodies that function as detection antibodies. It may contain detectable labels that can be conjugated to or conjugate antibodies such as the following: The detectable labels are, for example, enzymes, oligonucleotides, nanoparticle chemiluminophores, The direct label may be a phosphor, a fluorescent quencher, a chemiluminescent quencher, or biotin. The test kit optionally includes any additional reagents necessary to detect the label. You can stay like that.

[0063] This kit evaluates the efficacy of immune cells by measuring cytokine production by immune cells. Instructions for using the kit to stimulate may be included in some embodiments. This kit explains how to use the amount of cytokines to determine the potency of cells. Further includes a booklet. The instructions included in the kit may be attached to the packaging material or attached separately. It can be included as an appendix. Instructions are typically written or printed, but This is not limited to such examples. It involves memorizing such instructions and communicating them to end users. Any medium that can be used is contemplated by this disclosure. Such medium is not limited to However, electronic storage media (e.g., magnetic disks, tapes, cartridges, chips), optical This includes media (e.g., CD-ROM). When used herein, "instructions" refers to the instruction manual. The terminology may include the address of an internet site that provides instructions.

[0064] Immunotherapy methods The method for determining the efficacy of immune cells is to perform the procedure before using immune cells as an immunotherapy agent. This is possible. For example, a method for determining the efficacy of one or more immune cells is as described above. It can be administered, and then at least one powerful immune cell (detected cytochia) Based on the amount of immunotherapy, a powerful immune cell in an effective amount for treatment can be selected. It can be delivered to the target as a drug. Therefore, in one embodiment, it is an immunotherapy method. a) Immune cells disclosed herein (e.g., T cells, macrophages, NK cells, N Assaying the potency of KT cells, CAR T cells, and / or CAR NK cells. The method is performed on multiple immune cells to determine the efficacy of each immune cell, and b) detection The cytokines involved (e.g., IL-2, IL-6, IFN-γ, TNF-α, BAFF) / TNFSF13B, CD163, CD30 / TNFRSF8, Chitinase 3-type 1, gp 130, IFN-α2, IL-6Rα, IL-8, IL-10, IL-11, IL-12 (p40), IL-12(p70), IL-20, IL-22, IL-26, IL-29 / IFN-1, IL-32, IL-34, IL-35, MMP-1, Osteocalcin OPN, Pentraxin-3, TNF-R1, TNF-R2, TSLP, GM-CSF , MIP-1α, MIP-1β, RANTES, and / or TWEAK / TNFSF Based on the amount (12, etc.), select at least one potent immune cell and c) treatment Administering a sufficient amount of potent immune cells as an immunotherapy drug to a target that needs it. An immunotherapy method, including the following, is disclosed herein. In one embodiment, the method involves immune cells Before assaying efficacy, multiple immune cells are extracted from an allogeneic or autologous donor. It may include these.

[0065] In some embodiments, the immune cells are immunotherapeutic immune cells. Cells are useful in treating diseases such as cancer. (Becker et al., Can) cer Immunol. Immunother 65,477-484(2016). The use of proliferated NK cells for cancer treatment is described. Rezvani et al. al., Front Immunol., 6, 578 (2015). Numerous cases during immunotherapy. Since it is useful to be able to administer immune cells, in some embodiments, The cells are proliferated immune cells. Proliferated immune cells grow many immune cells These are immune cells that grow ex vivo. In some embodiments, proliferated immune cells These are autologous cells that can be easily administered to a target without inducing an immune response. However, in some embodiments, the proliferated immune cells are alloimmune cells, and Their unique alloreactivity can be an advantage. In a further embodiment, the proliferated immune cells To help immune cells target diseased tissue, they may contain chimeric antigen receptors. The genes are manipulated. The preparation of proliferated immune cells involves activating and proliferating immune cells. Includes. Koepsell et al., Transfusion, 53(2):404 -10 (2013). Several cytokines (IL-2, IL-12, IL-15, I L-18, IL-21, type I IFN, and TGF-β activate immune cells ex vivo. It has been shown to be useful for fertilization and propagation. For example, in some embodiments Therefore, the NK cells being evaluated are those proliferated by IL-21. In one embodiment, before delivering a therapeutically effective amount of potent immune cells, at least one potent An immunotherapy method, further comprising the proliferation of immune cells, is disclosed herein.

[0066] Proliferation refers to the ex vivo proliferation of NK cells, where the population of NK cells increases. NK cells can be proliferated, for example, from peripheral blood mononuclear cells. However, NK Cells can also be proliferated from other types of cells, such as hematopoietic stem cells or progenitor cells. Early blood or stem cells are obtained from the placenta, umbilical cord blood, placental blood, peripheral blood, spleen, or liver, etc. It can be isolated from various different sources. Growth occurs in cell culture medium. Suitable The cell culture medium is known to those skilled in the art. The grown cells can be provided as a cell line. A cell line is a group of cells that can be maintained in a cell culture. Therefore, in one embodiment, Before delivering a therapeutically effective amount of potent immune cells, at least one potent immune cell An immunotherapy method is disclosed herein, further comprising proliferation. In some embodiments Before immune cells carry out the methods that determine the efficacy of immune cells, they use known methods. They are extracted from the target. Alternatively, immune cells can be procured from the growth of cell cultures. can.

[0067] In some forms, immune cells are instructed to respond to specific antigens. To respond either before or after the method for determining its effectiveness. It can be instructed to do so. In some embodiments, immune cells respond to a specific antigen. The genes are modified in such a way. The antigen may be, for example, a tumor-specific antigen. Several embodiments So, the immunotherapy method involves (either before or after determining the efficacy of immune cells) immune cells This includes genetically modifying the organism to present a chimeric antigen receptor.

[0068] As has been pointed out throughout, the method for determining the efficacy of immune cells is adoptive cell transfer therapy. It can be used as part of a treatment. Powerful immune cells are targeted using therapeutically acceptable carriers. It can be delivered to. Intravenous delivery has traditionally been used to deliver immunotherapy cells. However, other methods can also be considered (for example, to local areas of the body that require immunotherapy). (Direct transplant).

[0069] The effective amount for treatment is the amount of cytokines produced by immune cells in immunotherapy. This is determined by comparing it to the cytokine efficacy level required for the use of immune cells. This is possible. The effective amount for treatment depends on the number of immune cells administered, the target being treated, and the treatment being performed. It is understood and intended herein that this depends on the disease, disorder, and / or condition. If you are a medical professional, you should use the appropriate dose of immune cells that are likely to be effective in treating the patient. They probably know that.

[0070] A therapeutically effective amount of potent immune cells includes multiple potent immune cells. For example, a small number of After selecting at least one powerful immune cell, the selected cell is grown in vitro. It can produce multiple powerful immune cells.

[0071] The target of receiving powerful immune cells is any target that could benefit from immunotherapy (for example) , autoimmune diseases, inflammatory diseases or disorders, viral diseases, and / or bacterial infections The subject may be a person who has (or has) cancer. In some embodiments, the subject may be a cancer patient. In some embodiments, the subjects are individuals at high risk of developing cancer, and diagnosed with cancer. Individuals that have been treated for cancer, individuals being treated for cancer, or individuals recovering from cancer after surgery. It is possible. In some embodiments, robust immune cells prevent the development of cancer or metastasis. It may be delivered to the target as a preventative agent to inhibit, delay, or stabilize the process.

[0072] Methods of treating diseases The potent immune cells identified herein are, but are not limited to, autoimmune diseases and inflammatory diseases. Alternatively, adoptive immunotherapy may be used to treat disabilities, viral diseases, and / or bacterial infections. It is understood that it can be used in the treatment of any disease or disorder that may be treated. This specification intends to describe cancer and / or metastasis in the subject. In one embodiment, cancer and / or metastasis are described in the subject. A method for treating, inhibiting, reducing, preventing, and / or mitigating the same as or one or more immune cells obtained from an autologous donor (e.g., T cells, macrophages, N cells) Obtain K cells, NK T cells, CAR T cells, and / or CAR NK cells, etc. b) immune cells to an effective amount of plasma membrane particles, liposomes, or exosomes (operation) c) contact with (including exosomes that have been processed) and cytoplasm produced by immune cells Tocaines (e.g., IL-2, IL-6, IFN-γ, TNF-α, BAFF / TNFS F13B, CD163, CD30 / TNFRSF8, Chitinase 3-type 1, gp130, I FN-α2, IL-6Rα, IL-8, IL-10, IL-11, IL-12(p40) , IL-12(p70), IL-20, IL-22, IL-26, IL-29 / IFN- l1, IL-32, IL-34, IL-35, MMP-1, osteocalcin, OPN, pentraxin-3, TNF-R1, TNF-R2, TSLP, GM-CSF, MIP- 1α, MIP-1β, RANTES, and / or TWEAK / TNFSF12, etc.) detecting the amount of, and d) based on the amount of the detected cytokine, selecting at least one potent immune cell, and e) administering to the subject a therapeutically effective amount of the potent immune cell are disclosed herein. In some embodiments, the method may further comprise extracting immune cells from the self or an allogeneic donor.

[0073] It is useful that a large number of immune cells can be administered during immunotherapy, and in some embodiments, it is understood that the immune cells are expanded immune cells, which are contemplated herein and are. Expanded immune cells are immune cells that grow ex vivo to grow a large number of immune cells. Thus, a method for treating, inhibiting, reducing, preventing, and / or alleviating autoimmune diseases, inflammatory diseases or disorders, viral diseases, bacterial infections, cancer and / or metastases, comprising further expanding at least one potent immune cell before delivering a therapeutically effective amount of at least one potent immune cell, is disclosed herein.

[0074] It is understood and contemplated herein that the disclosed methods of treatment can be used to treat any disease or condition in which uncontrolled cell proliferation occurs, including but not limited to cancer and metastases. Treating with the disclosed methods using potent immune cells​ The following is a list of representative, but not limited, cancers that can be treated: Lymphoma , B-cell lymphoma, T-cell lymphoma, mycosis fungoides, Hodgkin's disease, myeloid leukemia, bladder cancer, Brain cancer, nervous system cancer, head and neck cancer, squamous cell carcinoma of the head and neck, small cell lung cancer and non-small cell lung cancer. Which lung cancers, neuroblastoma / glioblastoma, ovarian cancer, skin cancer, liver cancer, melanoma, mouth, throat, larynx, and Squamous cell carcinoma of the lung, uterine cancer, uterine carcinoma, breast cancer, as well as epithelial carcinoma, kidney cancer, and genitourinary cancer. Lung cancer, esophageal cancer, head and neck cancer, colorectal cancer, hematopoietic cancer, testicular cancer, colon cancer, rectal cancer, prostate cancer, Or pancreatic cancer.

[0075] Examples of autoimmune diseases that can be treated using the disclosed methods include, but are not limited to, However, there are achalasia, acute disseminated encephalomyelitis, acute motor axonal neuropathy, and Addison's disease. Diseases, painful steatosis, adult Still's disease, agammaglobulinemia, alopecia areata, Alzheimer's disease – disease, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome, Aplastic anemia, autoimmune angioedema, autoimmune autonomic dysregulation, autoimmune cerebrospinal fluid inflammation, autoimmune intestinal disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease ( AIED, autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis Autoimmune polyendocrine syndrome, autoimmune retinopathy, autoimmune urticaria, axons and nerves Neuropathies (AMAN), Baro disease, Behçet's disease, benign mucosal pemphigoid (Ben IGN (mucosal emphigoid), Vickerstaff's encephalitis, bullous pemphigoid Acne, Castleman disease (CD), celiac disease, Chagas disease, chronic fatigue syndrome, chronic inflammation Symptomatic demyelinating polyneuropathy (CIDP), chronic relapsing polymyelitis (CRMO), Char Gustavus syndrome (CSS), eosinophilic granulomatosis (EGPA), pemphigoid scarring, -Cancer syndrome, cold agglutinin disease, congenital heart block, coxsackie myocarditis, CREST syndrome Group, Crohn's disease, herpetiform dermatitis, dermatomyositis, Devic's disease (neuromyelitis optica), type 1 diabetes, Discoid erythema, Dressler syndrome, endometriosis, enthesitis, eosinophilic esophagitis (EoE), Eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, F Jerty syndrome, fibromyalgia, fibrotic alveolitis, giant cell arteritis (temporal arteritis), giant cell heart Myositis, glomerulonephritis, Goodpasture syndrome, granulomatous ulcers with polyangiitis, Graves' disease Guillain-Barré syndrome, Hashimoto's encephalopathy, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schönla syndrome Hemopura purpura (HSP), herpes zoster of pregnancy or bullous pemphigoid of pregnancy (PG), sweat gland abscess (HS) (Anti-acne), hypogammaglobulinemia, IgA nephropathy, IgG4-related sclerosing disease, immunological Thrombocytopenic purpura (ITP), inclusion body myositis (IBM), interstitial cystitis (IC), inflammatory Intestinal diseases (IBD), juvenile arthritis, juvenile diabetes (Type 1 diabetes), juvenile myositis (JM), Kawasaki disease, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosing, wood Conjunctival conjunctivitis, linear IgA disease (LAD), lupus nephritis, lupus vasculitis, chronic Lyme disease, Nyer's disease, microscopic polyangiitis (MPA), mixed connective tissue disease (MCTD), Mollen's disease Ulcers, Mucha-Habermann disease, multifocal motor neuropathy (MMN) or MMNCB Multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neonatal lupus, neuromyelitis optica Neutropenia, benign mucous membrane pemphigoid of the eye, optic neuritis, Ord's thyroiditis, relapsing rheumatoid arthritis (P R), PANDAS, paraneoplastic cerebellar degeneration (PCD), paroxysmal nocturnal hemoglobinuria (P NH), Parry-Romberg syndrome, pars planitis (peripheral uveitis), Personage-Ta yner syndrome, pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia (PA) , POEMS syndrome, polyarteritis nodosa, polyglandular syndrome type I, II, III, rheuma tic polymyalgia, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, primary biliary cirrhosis , primary sclerosing cholangitis, progesterone dermatitis, psoriasis, psoriatic arthritis, erythroid myelosis (PRC A), pyoderma gangrenosum, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, relapsing polychondritis, restless legs syndrome (RLS), retroperitoneal fibrosis, rheumatism fever, rheumatoid arthritis, rheumatoid vasculitis, sarcoidosis, Schmidt syndrome, Schu nnler syndrome, episcleritis, scleroderma, Sjögren's syndrome, semen and testicular autoimmunity, Ste iff-Paterson syndrome (SPS), subacute bacterial endocarditis (SBE), Sazak syndrome, Sydenham chorea, sympathetic ophthalmia (SO), systemic lupus erythematosus, systemic sclerosis, hyper tensive arteritis, temporal arteritis / giant cell arteritis, thrombotic thrombocytopenic purpura (TTP), Troja Han t syndrome (THS), transverse myelitis, type 1 diabetes, ulcerative colitis (UC), undifferentiated connective tive tissue disease (UCTD), urticaria, urticarial vasculitis, uveitis, vasculitis, vitiligo, Vogt -Koyanagi-Harada disease, and Wegener granulomatosis (or granulomatosis with polyangiitis (GPA )) are included.

[0076] To illustrate preferred embodiments of the present invention, the following examples are included. The techniques disclosed in the following examples were discovered by the inventors to function well in the implementation of the present invention. It can be considered to represent the technology and, therefore, constitute a preferred embodiment for its implementation. This should be understood by those skilled in the art. However, those skilled in the art will understand this in light of the present disclosure. Furthermore, disclosed and without departing from the spirit and scope of the present invention, similar or comparable inventions are available. It should be understood that many modifications can be made to a particular embodiment to obtain similar results. That is the case. [Examples]

[0077] The assays disclosed herein address the problems present in current standard methods. The intention is to test the efficacy of therapeutic immune cells that meet FDA requirements. To achieve this, K562-derived exosomes induce cytokine production in immune cells. It is used as a substitute substance for immune cells. K562 (chronic myeloid leukemia cell line) is an immune cell These are widely used as universal control target cell lines in cytotoxic assays. K562 cells are exosome-multivesicular cells formed by budding inside the endosomal membrane. They periodically release exosomes. Exosomes induce cytokine production, such as in K562 cells. However, this eliminates the variability caused by the use of target tumor cells. This assay is We have a fully functional laboratory to test the efficacy of therapeutic immune cells at multiple clinical injection sites. This eliminates the need to do so and provides a faster turnaround time for such tests. ru.

[0078] We will use K562-derived exosomes as a stimulant to test the efficacy of therapeutic immune cells. thing The ability of exosomes to assay the efficacy of immune cells is shown in Figures 1 and 2. Figure 1 shows therapeutic NK cells in efficacy assays - PHA or exosome-efficacy assays. Alternatively, IL-2 or IFN-γ can be produced via either exosomes. This provides a graph showing the use of exosome efficacy assays for therapeutic NK cells. This demonstrates that high IL can be detected using an exosome efficacy assay. Figure 2 shows that high IL can be detected using an exosome efficacy assay. It is also possible to identify proliferating therapeutic NK cells via -2 or IFN-g production. A graph is provided showing this. Furthermore, NK cells newly isolated from a healthy donor exhibit this effect. Stimulated by force assay, it can be used to diagnose NK cell deficiency in patients. APRIL / TNSF13, CD163, and other cytokines such as BAFF are separated. To secrete.

[0079] The inventors of this application have developed nine different donors in response to various concentrations of CSTX002 exosomes. We tested the production of 29 different cytokines and chemokines by NK cells from this study. There was no difference in expression patterns across different exosome concentrations (Figure 3). To specifically evaluate the reproducibility and variability of the enzyme, NK cells from nine different donors were used. Across 29 cytokines and chemokines, low (50 ug / mL) and high ( The correlation of exosomes at a concentration of 400 ug / mL was tested. A very high correlation (r² = 0) was observed. 964) and less than 2% variation (slope -1) indicates a wide range of exosomes. This shows that the same concentration yields the same result (Figure 4).

[0080] Unless otherwise defined, all technical and scientific terms used herein are disclosed. The term has the same meaning as that generally understood by those skilled in the art to which the invention belongs. Publications cited herein and materials from which they are cited are, specifically, by reference. It is invoked herein by reference. However, patents, publications, and Or all or part of other disclosure materials, or materials that are referenced, are existing terms described in this disclosure. You understand that it is incorporated herein only to the extent that it does not conflict with any other disclosures, statements, or other materials. Please do so. Therefore, to the extent necessary, the disclosures expressly set forth herein shall be referred to by reference. This supersedes any conflicting material incorporated herein by reference. However, this may be inconsistent with existing definitions, descriptions, or other disclosures described herein. Any material, or any part thereof, may cause inconsistencies between the material it references and existing disclosures. It is only used to the extent that it does not apply.

[0081] Those skilled in the art will recognize many equivalents to the specific embodiments of the present invention described herein. This can be observed or confirmed using experiments that do not exceed the standards of normal experimentation. The present invention has been described with reference to specific embodiments and examples, but the scope of the present invention or this invention may not apply. Without deviating from the original inventive concept, various modifications and additional variations can be made, It should be understood that equivalents can be substituted for the elements of this invention. In addition, the present invention Without departing from the qualitative scope, adapt a specific situation or device to the teachings of the present invention. Many modifications can be made to achieve this. Such equivalents are described in the following claims. The present invention is intended to encompass the specific embodiments disclosed herein. Although not specified, the present invention is intended to include all embodiments within the scope of the appended claims. It will be done.

Claims

1. A method for assaying the efficacy of immune cells, comprising: immune cells in an effective amount of plasma membrane particles, lipo Contacting the sosome or exosome, and the cytoplasm produced by the immune cells. A method comprising detecting the amount of tokine.

2. The amount of cytokines produced is determined to be the amount necessary for the use of the immune cells in immunotherapy. The method according to claim 1, further comprising the step of comparing with the itokine potency level.

3. The method according to claim 1, wherein the amounts of multiple cytokines are determined.

4. The aforementioned immune cells include T cells, macrophages, natural killer (NK) cells, and NK T cells. The cell is a chimeric antigen receptor (CAR) T cell, or a CAR NK cell, according to claim 1. Method of description.

5. The method according to claim 1, wherein the immune cells are NK cells.

6. The method according to claim 1, wherein the exosome is a cancer cell exosome.

7. The amount of the cytokine is detected using an immunoassay, according to claim 1. Law.

8. The aforementioned cytokines include interleukin (IL)-2 (IL-2), IL-6, and inter -feron (IFN)-γ (IFN-γ), B cell activating factor / tumor necrosis factor (TNF) Ligand superfamily member 13B (BAFF / TNFSF13B), TNF- α, Differentiation Cluster (CD) 163 (CD163), CD30 / TNFRSF8, Kichina -ase3-like 1, gp130, IFN-a2, IL-6Ra, IL-8, IL-10, IL- 11, IL-12 (p40), IL-12 (p70), IL-20, IL-22, IL- 26. IL-29 / IFN-1, IL-32, IL-34, IL-35, Matrix Metalloproteinase-1 (MMP-1), osteocalcin, osteopontin (OP N), Pentraxin-3, Tumor Necrosis Factor (TNF) Receptor 1 (TNF-R1), TN F-R2, thymic interstitial lymphopoietin (TSLP), granulocyte-macrophage colony stimulation Factors (GM-CSF), leukemia-inhibiting factor (LIF), and chemokine macrophage inflammation Symptomatic protein (MIP)-1α (MIP-1α), MIP-1β, RANTES, and B / or TNF-related weak apoptosis-inducing factors (TWEAKs) / TNF super A claim is selected from a group that includes Family Member 12 (TWEAK / TNFSF12). The method described in item 1.

9. The immune cells then absorb an effective amount of the plasma membrane particles, liposomes, or exosole The method according to claim 1, wherein contact with the substance is maintained for at least four hours.

10. The plasma membrane particles, liposomes, or exosomes are 50 μg to 40 The method according to claim 1, provided at a concentration of 0 μg / mL.

11. A kit for assaying the efficacy of immune cells, comprising an effective amount of plasma membrane particles and / Alternatively, a kit including a container containing exosomes and a buffer suitable for immune cells.

12. The plasma membrane particles, liposomes, or exosomes are 50 μg to 40 The kit according to claim 11, provided at a concentration of 0 μg / mL.

13. The kit according to claim 11, wherein the container is an Eppendorf microcentrifuge tube. 。

14. The aforementioned kit is used to stimulate cytokine production by immune cells. The kit according to claim 11, further comprising instructions for doing so.

15. It is an immunotherapy method, a. Perform the method according to any one of claims 1 to 14 on a plurality of immune cells, Determining the efficacy of the cells, b. Based on the amount of cytokines detected, select at least one potent immune cell. to, c. A therapeutically effective amount of the aforementioned potent immune cells, to the target that requires them as an immunotherapy agent. An immunotherapy method that includes administering to [a specific body part].

16. Before assaying the efficacy of the immune cells, multiple immune cells from an allogeneic or autologous donor may be obtained. The immunotherapy method according to claim 15, further comprising extracting disease cells.

17. Before delivering a therapeutically effective amount of the potent immune cells, at least one potent immune The immunotherapy method according to claim 15, further comprising causing the proliferation of disease cells.

18. The plurality of immune cells or the potent immune cells are instructed to respond to a specific antigen. The immunotherapy method according to claim 15, further comprising the following:

19. The plurality of immune cells or the potent immune cells present chimeric antigen receptors The immunotherapy method according to claim 18, further comprising genetic modification.

20. In the target population, to treat, inhibit, reduce, prevent, and / or treat cancer and / or metastasis. or a method of mitigation, a. Obtaining one or more immune cells, b. Contact immune cells with an effective amount of plasma membrane particles, liposomes, or exosomes. That thing, c. To detect the amount of cytokines produced by the immune cells, d. Based on the amount of cytokines detected, select at least one potent immune cell. To choose, e. A method comprising administering to the subject a therapeutically effective amount of the potent immune cells. 。

21. The claim 20 states that one or more immune cells are obtained from an allogeneic or autologous donor. In the target, treat, inhibit, reduce, prevent, and / or treat cancer and / or metastasis. This is a way to alleviate the problem.

22. Claim 2 further comprises extracting the plurality of immune cells from an identical or autologous donor. In the subjects described in 0, the treatment, inhibition, reduction, and prevention of cancer and / or metastasis A way to call for / reduce it.

23. The aforementioned immune cells include T cells, macrophages, natural killer (NK) cells, and NK T cells. Claim 20, which is a cell, a chimeric antigen receptor (CAR) T cell, or a CAR NK cell. Treat, inhibit, or reduce cancer and / or metastasis in any of the subjects described in any of the following 22. Methods of preventing, mitigating, and / or reducing.

24. Before delivering a therapeutically effective amount of the at least one potent immune cell, the at least Any of claims 20 to 23 further comprises increasing another powerful immune cell. In the subjects described, the treatment, inhibition, reduction, prevention, and / or ways to mitigate it.

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