Use of stimulating agents to assay the potency of immune cells

Abstract

A method for determining the potency of immune cells is described. The method includes contacting immune cells with an effective amount of a stimulant (e.g., phytohemagglutinin (PHA)) and detecting the amount of cytokines produced by the immune cells. A kit for assaying the potency of immune cells is also described. Potency assays are important for meeting FDA requirements for new biological agents, such as immunotherapeutic cells. A method for using potent immune cells as an immunotherapeutic treatment is described.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 805,349, filed February 14, 2019, which is incorporated by reference in its entirety.

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

[0003] Immunotherapy is the treatment of disease by activating or suppressing the immune system. Cells derived from the immune system can be used to improve immune function and characteristics. In recent years, immunotherapy has become of great interest to 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 less likelihood of creating resistance when treating microbial diseases.

[0004] Traditional cancer treatments focus on killing or eliminating cancer cells using chemotherapy, surgery, and / or radiation. However, the field of therapeutic immune cells is growing rapidly and can be used in combination with, or in some cases, instead of, traditional 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 work together to defend the body against cancer by targeting abnormal antigens expressed on the surface of tumor cells. Recent developments in cancer treatments focus on directing the patient's immune system to attack and destroy tumors. A variety of strategies are in use or are being researched and tested.

[0005] Adoptive cell transfer (ACT) is the transfer of cells into a patient and has shown promise against lung cancer, melanoma, and other cancers. The cells can be derived from the patient (autologous) or from another individual (allogeneic). Allogeneic therapy involves cells isolated and expanded from a donor other than the patient receiving the cells. Alternatively, adoptive cell transfer can be used to culture and expand autologous extracted cells in vitro for later transfusion. For example, autoimmune enhancement therapy involves the extraction of the subject's own peripheral blood-derived natural killer cells, cytotoxic T lymphocytes, epithelial cells, and other relevant immune cells, expanding these cells in vitro, and then reinjecting these cells into the subject's body.

[0006] In some therapies, cells (e.g., T cells) are genetically modified and expanded in vitro before being returned to the same patient. Chimeric antigen receptor T cell therapy (CAR-T) involves taking T cells from a subject and then infecting the T cells with a retrovirus that contains a copy of a T cell receptor (TCR) gene. The TCR gene is specialized to recognize a tumor antigen (e.g., a chimeric antigen receptor, or CAR). The virus integrates the receptor into the genome of the T cell. The cells are then non-specifically expanded and / or stimulated. The cells are then reinjected to generate an immune response against the tumor cells.

[0007] With the approval of the first CAR-T therapy and involvement of multiple commercial companies in multiple clinical trials, the field has expanded commercially, showing the promising future of immunotherapy. As the field advances with new clinical trials every other day, the need for reliable and reproducible efficacy testing for these therapeutic immune cells has subsequently increased. The industry's "gold standard" for testing immune cell effector function is the chromium release assay, developed in the 1960s, which is still in use despite concerns due to the use of radioactive materials and variability caused by the target tumor cells. An available alternative is the calcein-based assay, which still has a lot of variability caused by the use of different tumor targets, due to the capture of calcein in the apoptotic body of the tumor target.

[0008] Other efforts have been made to develop different methods to visually see the effector function of these immune cells, but these methods still use target tumor cells. Another surrogate way to check the effector function of immune cells is to check the cytokines produced by these cells, for which all conventional methods use target tumor cells to induce cytokine production from immune cells. The use of target tumor cells adds biological variability to all of these tests due to variability between tumor cell types. Also, these assays require a tedious setup that introduces batch effects to these assays. Batch effects are caused by the condition of the target cells, inter-individual variability in loading the plates, plate conditions, variability in various reagents, variability in readouts, etc. There is a clear need for an immune cell potency assay that can remove all of these variabilities and produce reliable and reproducible results.

[0009] Reliable and reproducible potency assays are needed to evaluate the quality of immune cell therapy products. The approval process is highly regulated and drug developers must submit a large amount of information about their drug to regulatory agencies in order to gain approval. This may include information about the potency of the drug product and assays to determine this potency. As required by the FDA (21 CFR 610.10), the potency of cell therapy products should be demonstrated by appropriate tests to demonstrate effector function and potency of these therapeutic immune cells, which would measure the relevant cytokine production by these immune cells. Summary of the Invention

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

[0011] In one aspect, a method for assaying efficacy of an immune cell (such as, for example, a T cell, a macrophage, an NK cell, an NK T cell, a CAR T cell, and / or a CAR NK cell) is provided, comprising contacting the immune cell with an effective amount of a stimulant (such as, for example, phytohemagglutinin (PHA), phorbol myristate acetate (PMA) / ionomycin, concanavalin A (Con A), lipopolysaccharide (LPS), and / or Pork Week mitogen (PWM)) and measuring the level of one or more cytokines produced by the immune cell (such as, for example, IL-2, IL-6, IFN-γ, TNF-α, BAFF / TNFSF13B, CD163, CD30 / TNFRSF8, chitinase 3-like 1, gp130, IFN-α2, IL-6Rα, IL-8, IL-10, IL-11, IL-12(p40 Disclosed herein is a method comprising detecting an amount of a cytokine produced from an immune cell (such as 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). In one embodiment, the method may further comprise comparing the amount of cytokine produced to a cytokine potency level required for use of the immune cell in immunotherapy.

[0012] Also disclosed herein is a method of assaying the efficacy of an immune cell of any preceding embodiment, wherein the amount of cytokine is detected using an immunoassay (e.g., ELISA, intracellular cytokine staining, ELISpot, flow cytometry, Luminex xMAP®, quantitative PCR (including but not limited to qRT-PCR), and / or a bead assay).

[0013]

[0023] In one embodiment, there is provided a method of assaying efficacy of an immune cell of any preceding embodiment, comprising: incubating the immune cell with an effective amount of a stimulant (such as, for example, PHA, PMA / ionomycin, Con A, LPS and / or PWM) for 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 ... , 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, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 45, 60, 61, 62 days, 3, 4, 5, or 6 months.

[0014] Also disclosed herein is a method of assaying the efficacy of an immune cell of any preceding embodiment, wherein a stimulant (e.g., PHA, PMA / ionomycin, Con A, LPS and / or PWM, etc.) is provided at a concentration of 1.0 μg / mL to 1000 μg / mL (including but not limited to, a concentration of 5 μg / mL to 15 μg / mL).

[0015] In one aspect, disclosed herein is a kit for assaying the efficacy of immune cells (such as, for example, T cells, macrophages, NK cells, NK T cells, CAR T cells, and / or CAR NK cells), comprising a container (such as, for example, a microcentrifuge tube) containing an effective amount of a stimulatory agent (such as, for example, PHA, PMA / ionomycin, Con A, LPS, and / or PWM) and a suitable buffer for the immune cells. In some aspects, the kit may further comprise instructions for using the kit to stimulate cytokine production by the immune cells.

[0016] Also disclosed herein is a kit for assaying efficacy of the immune cells of any preceding embodiment, wherein a stimulant (e.g., PHA, PMA / ionomycin, Con A, LPS and / or PWM, etc.) is provided at a concentration of 1.0 μg / mL to 1000 μg / mL (including but not limited to, a concentration of 5 μg / mL to 15 μg / mL).

[0017] In one embodiment, a method of immunotherapy includes a) performing a method of assaying the potency of an immune cell (such as T cells, macrophages, NK cells, NK T cells, CAR T cells, and / or CAR NK cells) of any preceding embodiment on a plurality of immune cells to determine the potency of each immune cell; and b) determining the potency of each immune cell by detecting cytokines detected (e.g., IL-2, IL-6, IFN-γ, TNF-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-α2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12(p40), IL-12(p70), IL-12(p70), IL-20, IL-22, IL-36, IL-38, IL-40, IL-42, IL-44, IL-46, IL-48, IL-49, IL-50, IL-51, IL-52, IL-53, IL-54, IL-55, IL-56, IL-57, IL-58, IL-59, IL-60, IL-61, IL-62, IL-63, IL-64, IL-65, IL-66, IL-67, IL-68, IL-69, IL-70, IL-81 Disclosed herein is a method of immunotherapy comprising: c) selecting at least one potent immune cell based on the amount of a potent immune cell that is a potent immune cell of a given marker, such as a potent immune cell-1, a potent immune cell-2, a potent immune cell-3, a potent immune cell-4, a potent immune cell-5, a potent immune cell-6, a potent immune cell-7, a potent immune cell-8, a potent immune cell-9, a potent immune cell-10, a potent immune cell-11, a potent immune cell-12, a potent immune cell-13, a potent immune cell-14, a potent immune cell-15, a potent immune cell-16, a potent immune cell-17, a potent immune cell-18, a potent immune cell-19, a potent immune cell-20, a potent immune cell-21, a potent immune cell-22, a potent immune cell-23, a potent immune cell-24, a potent immune cell-25, a potent immune cell-26, a potent immune cell-27, a potent immune cell-28, a potent immune cell-29, a potent immune cell-30, a potent immune cell-31, a potent immune cell-32, a potent immune cell-34, a potent immune cell-35, a potent immune cell-35, a potent immune cell-36, a potent immune cell-37, a potent immune cell-38, a potent immune cell-39, a potent immune cell-40, a potent immune cell-41, a potent immune cell-42, a potent immune cell-43, a potent immune cell-44, a potent immune cell-45, a potent immune cell-45, a potent immune cell-46, a potent immune cell-47, a potent immune cell-48, a potent immune cell-49, a potent immune cell-49, a potent immune cell-49, a potent immune cell

[0018] Also disclosed herein is the immunotherapy method of any preceding aspect, further comprising expanding at least one potent immune cell prior to delivering the therapeutically effective amount of the potent immune cell.

[0019] In one embodiment, disclosed herein is an immunotherapy method of any preceding embodiment, further comprising directing a plurality of immune cells or potent immune cells to respond to a specific antigen. In one embodiment, the method may further comprise modifying the cell line from which the exosomes are derived, or modifying the exosomes themselves, to include the specific antigen to which the immune cells of interest respond (e.g., adding CD19 to specifically determine the potency of CD19 CAR T cells in a heterogeneous sample).

[0020] Also disclosed herein is an immunotherapy method of any preceding aspect, further comprising genetically modifying the plurality of immune cells or potent immune cells to present a chimeric antigen receptor.

[0021] In one aspect, a method of treating, inhibiting, reducing, preventing, and / or ameliorating cancer and / or metastasis in a subject, comprising the steps of: a) obtaining one or more immune cells (e.g., T cells, macrophages, NK cells, NK T cells, CAR T cells, and / or CAR NK cells, etc.) obtained from an allogeneic or autologous donor; b) administering the immune cells to an effective amount of a stimulatory agent (e.g., PHA, PMA / ionomycin, Con A, LPS and / or PWM, and c) cytokines produced by immune cells (e.g., IL-2, IL-6, IFN-γ, TNF-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-40, IL-50, IL-60, IL-70, IL-81, IL-90, IL-102, IL-104, IL-106, IL-108, IL-109, IL-111, IL-112(p40), IL-112(p70), IL-113, IL-114, IL-115, IL-116, IL-117, IL-118, IL-119 ... Disclosed herein are methods for detecting the amount of a cytokine (such as 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) in a subject, d) selecting at least one potent immune cell based on the amount of the detected cytokine, and e) administering to the subject a therapeutically effective amount of the potent immune cell. In some embodiments, the method may further include extracting immune cells from an autologous or allogeneic donor.

[0022] Also disclosed herein is a method of treating, inhibiting, reducing, preventing, and / or ameliorating cancer and / or metastasis of any preceding aspect, further comprising expanding the at least one potent immune cell prior to delivering a therapeutically effective amount of the at least one potent immune cell.

[0023] Also disclosed herein are methods of determining the identity of at least one immune cell or cell population (e.g., differentiated Th1, Th2, Th3, Th9, Th17, effector memory T (Tem) cells, central memory T (Tcm) cells, γδ T cells, or regulatory T (Treg) cells, resting NK cells, expanded NK cells) based on a cytokine signature associated with that cell type. [Brief description of the drawings]

[0024] [Figure 1] 1 provides a plot showing the correlation between NK cell cytokine release induced by K-562 tumor cells and NK cell cytokine release induced by PHA (in pg / million cells / hr). [Diagram 2] Graphs are provided showing analysis of results based on incubation time. [Diagram 3] Graphs are provided showing analysis of results based on cell count. [Figure 4] 1 provides a graph showing the results of a PHA assay of NK cells expanded with mb-IL-21 and TGF-β. [Diagram 5] A plot showing the correlation between freshly isolated NK cell cytokine release (induced by PHA) and expanded NK cell cytokine release induced by PHA (in pg / million cells / hr) is provided. [Figure 6] 1 provides plots showing NK cell cytokine expression after 1 hour stimulation with PHA in expanded NK cells (N=18) and freshly procured NK cells (N=10). [Figure 7] A comparison of the concentrations of cytokines expressed between freshly procured and expanded NK cells following PHA stimulation is shown. [Figure 8] 1 provides plots showing the specificity and sensitivity in differentiating expanded NK cells based on IFN-γ and IL-2 expression. [Figure 9]We show that freshly procured and expanded NK cells can be differentiated based on the upregulation of IL-2 and downregulation of pentraxin-3 or chitinase 3-like 1. [Figure 10] We show that freshly procured and expanded NK cells can be differentiated based on the upregulation of IFN-γ and downregulation of pentraxin-3 or chitinase 3-like 1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] The present invention provides a method for determining the efficacy of immune cells, comprising contacting immune cells with an effective amount of a stimulant and detecting the amount of cytokines produced by immune cells.Although the present disclosure is given in the context of cancer immunotherapy, the concepts and innovations disclosed herein can be applied to the immunotherapy of other diseases and disorders.For example, immune cells used in immunotherapy for autoimmune diseases, inflammatory diseases or disorders, viral diseases and / or bacterial infections can also be tested for efficacy using the assays disclosed herein.

[0026] definition For clarity of understanding and ease of reference, a list of terms used throughout the Brief Description section and the remainder of the specification is compiled here. Some terms are well known throughout the field and are defined here for clarity, while some terms are unique to this application and therefore need to be defined for a proper understanding of this application.

[0027] As used in this specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells (including mixtures thereof). Plurals, when used herein, generally include the singular form.

[0028] As used herein, ranges may be expressed as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, it will be understood that by using the antecedent "about," the particular value forms another embodiment. It will be further understood that each of the endpoints of a range is significant both in relation to the other endpoint, and independently of the other endpoint. The recitation of numerical ranges by endpoints includes all numbers contained within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.). It is also understood that there are several values ​​disclosed herein, and that each value is also disclosed herein as "about" that particular value in addition to the value itself. For example, when the value "10" is disclosed, "about 10" is also disclosed. As would be well understood by one of ordinary skill in the art, when a value is disclosed to be "up to" that value, it is also understood that "up to" that value and the possible ranges between those values ​​are also disclosed, as "greater than or equal to" that value, as well as the ranges possible therebetween. For example, if the value "10" is disclosed, "less than or equal to 10" as well as "greater than or equal to 10" are also disclosed. It is also understood that throughout this application, data is provided in several different formats, and this data represents endpoints and starting points, as well as ranges of any combination of data points. For example, if a specific data point "10" and a specific data point 15 are disclosed, it is understood that values ​​between 10 and 15, as well as greater than 10, greater than or equal to 10, less than 10, less than or equal to 10, and equal to 10, greater than 15, greater than or equal to 15, less than 15, less than or equal to 15, and equal to 15 are considered to be disclosed. It is also understood that each unit between two specific units is also disclosed. For example, if 10 and 15 are disclosed, 11, 12, 13, and 14 are also disclosed.

[0029] As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements but do not exclude other elements. When used to define compositions and methods, "consisting essentially of" is intended to mean excluding other elements of any essential importance to the combination. Thus, a composition consisting essentially of the elements defined herein does not exclude trace contaminants and pharma- ceutically acceptable carriers, e.g., phosphate buffered saline, preservatives, etc., from the isolation and purification methods. "Consisting of" is intended to mean excluding more than trace elements of other components and substantial method steps for administering the compositions of the invention. Embodiments defined by each of these transition terms are within the scope of the invention.

[0030] "Increase" can refer to any change that results in a greater amount of a symptom, disease, composition, condition, or activity. An increase can be any individual, median, or average increase in a statistically significant amount of a condition, symptom, activity, or composition. Thus, an increase can be a 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, 95, or 100% increase, as long as the increase is statistically significant.

[0031] "Reduction" can refer to any change that results in a lower amount of symptoms, disease, composition, condition, or activity. A substance is also understood to reduce the genetic output of a gene when the genetic output of the gene product containing the substance is less compared to the output of the gene product without the substance. Also, for example, a reduction can be a change in the symptoms of a disorder, such that the symptoms are less than those previously observed. A reduction can be any individual value, median, or average reduction in a statistically significant amount of a condition, symptom, activity, or composition. Thus, a reduction can be a 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, 95, or 100% reduction, as long as the reduction is statistically significant.

[0032] "Inhibit", "inhibiting" and "inhibition" refer to a decrease in an activity, response, condition, disease, or other biological parameter. This can include, but is not limited to, the complete elimination of the activity, response, condition, or disease. This can also include, for example, a 10% reduction in the activity, response, condition, or disease compared to native or control levels. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100% reduction, or any amount in between, compared to native or control levels.

[0033] "Reduce" or other forms of the word, such as "reducing" or "reduction", refer to the reduction of an event or characteristic (e.g., tumor growth). This is typically relative to some standard or expected value, in other words, it is relative, but it is understood that it is not necessarily to refer to a standard or relative value. For example, "reducing tumor growth" refers to reducing the growth rate of a tumor compared to a standard or control.

[0034] "Prevent" or other forms of the word, such as "preventing" or "prevention," means to stop a particular event or characteristic, stabilize or slow the development or progression of a particular event or characteristic, or minimize the likelihood of a particular event or characteristic occurring. Prevention is typically more absolute than, for example, reduction, and does not require a comparison to a control. As used herein, something can be reduced, but may not be prevented, but something that is reduced can be prevented. Similarly, something can be prevented, but may not be reduced, but something that is prevented can be reduced. When reduction or prevention is used, it is understood that the use of other words is expressly disclosed unless specifically specified otherwise.

[0035] The term "therapeutically effective" is intended to qualify the number or amount of active agents (such as immunotherapy cells) that achieve the goal of reducing the severity of disease while avoiding side effects (e.g., side effects typically associated with alternative therapies). A therapeutically effective amount may be administered in one or more doses. A therapeutically effective treatment includes a treatment that improves the quality of life of a subject, even if it does not improve the outcome of the disease itself.

[0036] "Effective amount" generally refers to an amount that provides a desired local or systemic effect, e.g., effective to stimulate cytokine formation, including achieving a particular desired effect described in this application. For example, an effective amount is an amount sufficient to achieve a beneficial or desired clinical result.

[0037] The term "subject" refers to any individual who is the target of administration or treatment. The subject may be a vertebrate, e.g., a mammal. In one aspect, the subject may be a human, a non-human primate, a cow, a horse, a pig, a dog, or a cat. The subject may also be a guinea pig, a rat, a hamster, a rabbit, a mouse, or a mole. Thus, the subject may be a human or a veterinary patient. The term "patient" refers to a subject under the care of a clinician, e.g., a physician.

[0038] The term "therapeutically acceptable carrier" refers to a carrier or excipient that is generally safe and useful for preparing a non-toxic composition, including carriers that are acceptable for veterinary and / or human use. Intravenous delivery methods utilize a therapeutically acceptable carrier that is physiologically balanced (e.g., at osmolality and pH levels that are safe for intravenous use). As used herein, the term "therapeutically acceptable carrier" encompasses any of the standard carriers, such as saline, Ringer's, phosphate buffered saline, water, aqueous dextrose, and emulsions (e.g., oil / water or water / oil emulsions), as well as various types of wetting agents. As used herein, the term "carrier" encompasses any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in therapeutic formulations. Therapeutically acceptable carriers may also include preservatives (including cryopreservatives), such as those that maintain immune cell viability and / or efficacy. A "therapeutically acceptable carrier" as used in the specification and claims includes both one such carrier and more than one such carrier.

[0039] The term "treatment" refers to the medical management of a patient with the intent to cure, alleviate, stabilize or prevent a disease, pathological condition or disorder. This term includes active treatment, i.e., treatment specifically directed at ameliorating a disease, pathological condition or disorder, and also includes causal treatment, i.e., treatment directed at eliminating the cause of an associated disease, pathological condition or disorder. In addition, this term includes palliative treatment, i.e., treatment designed for the relief of symptoms rather than a cure of a disease, pathological condition or disorder, preventive treatment, i.e., treatment directed at minimizing or partially or completely inhibiting the onset of an associated disease, pathological condition or disorder, and adjunctive treatment, i.e., treatment used to supplement another specific therapy directed at ameliorating an associated disease, pathological condition or disorder.

[0040] "Administration" to a subject includes any route of introducing or delivering an agent to a subject. Administration can be performed by any suitable route, including oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intraarticular, parenteral, intraarterial, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation, via an implanted reservoir, parenteral (e.g., subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intraperitoneal, intrahepatic, intralesional, and intracranial injection or infusion techniques), and the like. "Concurrent administration" as used herein means that the compounds are administered at the same time in time or essentially immediately after each other. In the latter case, the two compounds are administered close enough in time that the results observed are indistinguishable from those obtained when administered at the same time in time. "Systemic administration" refers to the introduction or delivery of an agent to a subject via a route that introduces or delivers the agent to a wide area of ​​the subject's body (e.g., more than 50% of the body), for example, through an entrance into the circulatory or lymphatic system. In contrast, "local administration" refers to the introduction or delivery of an agent to a subject via a route that introduces or delivers the agent to the area of ​​the point of administration or an area immediately adjacent thereto, and does not introduce the agent systemically in a therapeutically significant amount. For example, a locally administered agent is readily detectable in the local vicinity of the point of administration, but is undetectable or detectable in negligible amounts in distal parts of the subject's body. Administration includes self-administration and administration by another person.

[0041] "Treat", "treating", "treatment" and grammatical variations thereof, as used herein, include administration of a composition intended or intended to partially or completely prevent, delay, cure, comfort, palliate, alleviate, alter, treat, mitigate, improve, stabilize, soothe, and / or reduce the intensity or frequency of one or more diseases or conditions, symptoms of a disease or condition, or the underlying cause of a disease or condition. Treatment according to the present invention may be applied prophylactically, preventatively, palliatively, or symptomatically. Preventative treatment is administered to a subject before onset (e.g., before obvious signs of cancer), during early onset (e.g., at the earliest signs and symptoms of cancer), or after the development of established cancer. Preventative administration may occur one (a few) days to several years before the onset of symptoms of a disease or infection.

[0042] The methods and kits disclosed herein utilize stimulating agents. As used herein, stimulating agents can be any molecule, peptide, polypeptide, protein, lectin, and / or mitogen that can act as an antigen and / or immunogen to stimulate immune cells to secrete cytokines. Stimulating agents include, but are not limited to, phytohemagglutinin (PHA), phorbol myristate acetate (PMA) / ionomycin, concanavalin A (Con A), lipopolysaccharide (LPS), and / or porcine week mitogen (PWM), peanut agglutinin (PNA), wheat germ agglutinin, and ricin.

[0043] Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also discussed in the sentence in which they are relied upon, and are individually and specifically incorporated by reference herein for the material contained therein.

[0044] Immunopotency assay In one aspect, the present invention provides a method for determining the efficacy of immune cells. The method includes contacting immune cells with an effective amount of a stimulant (e.g., PHA, PMA / ionomycin, Con A, LPS and / or PWM, etc.) and detecting the amount of cytokines produced by immune cells. For example, the immune cells can be contacted with a stimulant (e.g., PHA, PMA / ionomycin, Con A, LPS and / or PWM, etc.) by suspending the stimulant in cell culture medium and exposing the immune cells to the cell culture medium.

[0045] In some embodiments, the method includes comparing the amount of cytokine produced with a cytokine potency level required for the use of the immune cells in immunotherapy. Potency assays serve to characterize the product (i.e., immune cells), monitor lot-to-lot consistency, and ensure product stability, and should therefore be sensitive enough to detect differences that may affect the product's mechanism of action and function, and thereby have potential clinical significance. The assays can also be used as predictive biomarkers or pharmacodynamic assays for cell-mediated immunotherapy. Preferably, the potency assay has the closest possible relationship to the estimated physiological / pharmacological activity of the product. The potency assays described herein provide the ability to measure potency values ​​within product specifications, high sensitivity to detect differences of potential clinical significance, and a close relationship to the product's mechanism of action and estimated physiological / pharmacological activity. Preferably, the potency assay also meets the following secondary criteria: sufficiently low intra- and inter-assay variability (to obtain the precision required to support product specifications), sufficient robustness, and amenable to high-throughput analysis. In some embodiments, the assay is used as a clinical assay to quantify T cell, macrophage, NK cell, NK T cell, CAR T cell, and / or CAR NK cell function (diagnosis of NK cell immunodeficiency, biomarker for monitoring efficacy of immunosuppressants or immune activators).

[0046] As mentioned above, the disclosed method is provided for determining the efficacy of immune cells. Immune cells, as defined herein, are any cells of the immune system that produce cytokines (i.e., cytokine-producing immune cells). Examples of cytokine-producing immune cells include lymphocytes, neutrophils, macrophages, and natural killer cells. Lymphocytes include both B cells and T cells (including CD4 and CD8 T cells). In one embodiment, immune cells can include tumor infiltrating lymphocytes (TILs), T cells, macrophages, natural killer (NK) cells, NK T cells, chimeric antigen receptor (CAR) T cells, and / or CAR NK. Immune cells can be obtained from cell culture or from a subject (such as, for example, an allogeneic or autologous donor).

[0047] In some embodiments, the immune cell is a T cell. T cells play a central role in cell-mediated immunity and can be distinguished from other lymphocytes, such as B cells and natural killer cells, by the presence of T cell receptors on the cell surface. Examples of T cells include 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, which are distinct from NK cells and recognize glycolipid antigens rather than peptides presented by MHC molecules. Different types of T cells differ from each other in their patterns of cytokine production). T cells can be CD4 T cells or CD8 T cells. Additionally, T cells can include chimeric antigen receptor (CAR) T cells or tumor infiltrating lymphocytes (TILs).

[0048] In some embodiments, the immune cells are NK cells. Natural killer cells are a type of cytotoxic lymphocyte of the immune system. NK cells provide a rapid response to virus-infected cells and respond to transformed cells. Typically, immune cells detect peptides from pathogens presented by major histocompatibility complex (MHC) molecules on the surface of infected cells, triggering cytokine release and causing lysis or apoptosis. However, NK cells are unique because they have the ability to recognize stressed cells regardless of whether pathogen-derived peptides are present on the MHC molecule. These cells were named "natural killer" due to the early idea that they do not require prior activation to kill targets. NK cells are large granular lymphocytes (LGLs) that are known to differentiate and mature in the bone marrow, from where they enter the circulation. In some aspects, the NK cells can be CAR NK cells.

[0049] Thus, in one aspect, disclosed herein is a method of assaying the potency of an immune cell (such as, for example, a T cell, a macrophage, an NK cell, an NK T cell, a CAR T cell, and / or a CAR NK cell), comprising contacting the immune cell with an effective amount of a stimulant (such as, for example, PHA, PMA / ionomycin, Con A, LPS, and / or PWM) and detecting the amount of one or more cytokines produced by the immune cell. In one aspect, the method may further comprise comparing the amount of cytokine produced to a cytokine potency level required for use of the immune cell in immunotherapy.

[0050] The assay involves detecting the amount of cytokines produced by immune cells after stimulating the immune cells with a stimulant. As used herein, the term "cytokine" refers to small proteins (approximately 5-20 kDa) that are important in cell signaling, particularly immune regulation, that may be produced by immune cells. Examples of cytokines include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors. The detected cytokines may include cytokines known to be produced by the immune cells being evaluated, or detection may encompass a broader variety of cytokines, including cytokines not known to be produced by immune cells.

[0051] In some embodiments, the detected cytokines include cytokines known to be produced by T cells or natural killer cells. In some embodiments, the cytokines include cytokines known to be produced by T cells. T cells include Th1 and Th2 cells, with Th1 cells primarily producing interferon (IFN)-gamma (IFN-γ), tumor necrosis factor (TNF)-alpha (TNF-α), and IL-2, and Th2 cells producing interleukin (IL)-2 (IL-2), IL-4, IL-5, IL-6, IL-9, IL-13, and IL-22. Examples of cytokines produced by stimulated natural killer cells include IL-lα, IL-1β, IL-2, IL-5, IL-8, IL-10, IL-13, IFN-γ, TNF-α, granulocyte-macrophage colony-stimulating factor (GM-CSF), leukemia inhibitory factor (LIF), and the chemokines macrophage inflammatory protein (MIP)-1α (MIP-1α), MIP-1β, and RANTES. Other cytokines useful in determining immune cell potency include, but are not limited to, B cell activating factor / tumor necrosis factor (TNF) ligand superfamily member 13B (BAFF / TNFSF13B), cluster of differentiation (CD) 163 (CD163), CD30 / TNFRSF8, chitinase 3-like 1, gp130, IFN-α2, IL-6Rα, IL-11, IL-12(p40), IL-12(p70), IL-20, IL-26, IL-29 / I These include FN-l1, IL-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 stromal lymphopoietin (TSLP), or TNF-related weak inducer of apoptosis (TWEAK) / TNF superfamily member 12 (TWEAK / TNFSF12).Thus, in one aspect, a method is provided for assaying the efficacy of an immune cell (such as, for example, a T cell, a macrophage, an NK cell, an NK T cell, a CAR T cell, and / or a CAR NK cell), comprising contacting the immune cell with an effective amount of a stimulant (such as, for example, phytohemagglutinin (PHA), phorbol myristate acetate (PMA) / ionomycin, concanavalin A (Con A), lipopolysaccharide (LPS), and / or Pork Week mitogen (PWM)) and measuring the level of one or more cytokines produced by the immune cell (such as, for example, IL-2, IL-6, IFN-γ, TNF-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-20, IL-30, IL-40, IL-50, IL-60, IL-70, IL-80, IL-90, IL-110, IL-12(p40), IL-112(p50), IL-113, IL-114, IL-115, IL-116, IL-117, IL-118, IL-119, IL-12(p50), ...9, IL-12(p50), IL-119, IL-12(p50), IL-119, IL-12( and detecting an amount of a 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, LIF, MIP-1α, MIP-1β, RANTES, and / or TWEAK / TNFSF12.A method for assaying efficacy of immune cells (such as, for example, T cells, macrophages, NK cells, NK T cells, CAR T cells, and / or CAR NK cells), comprising contacting the immune cells with an effective amount of a stimulant (such as, for example, PHA, PMA / ionomycin, Con A, LPS, and / or PWM) and measuring the level of one or more cytokines (e.g., IL-2, IL-6, IFN-γ, TNF-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-12(p80), IL-12(p90), IL-12(p100), IL-12(p110), IL-12(p120), IL-12(p130), IL-12(p140), IL-12(p150), IL-12(p160), IL-12(p170), IL-12(p180), IL-12(p190), IL-12(p200), IL-12(p210), IL-12(p220), IL-12(p230), IL-12(p240), IL-12(p250), IL-12(p260), IL-12(p270), IL-12(p280), IL-12(p290), IL-12(p300), IL-12(p310), IL-12(p320), IL-12(p330), IL-12( Disclosed herein is a method comprising detecting an amount of a cytokine (such as IL-20, IL-22, IL-26, IL-29 / IFN-11, 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) in a cell culture medium. In one aspect, the method may further comprise comparing the amount of cytokine produced to a cytokine potency level required for use of the immune cells in immunotherapy. In some embodiments, the levels of multiple cytokines are determined. In further embodiments, the cytokine is selected from the group consisting of interleukin-2, interleukin-6, and interferon-γ.

[0052] The assay includes detecting the amount of cytokines produced by immune cells. A wide variety of methods for detecting cytokines are known to those skilled in the art and may vary depending on the cytokine to be detected. In some embodiments, one or more methods may be used to detect and / or quantify the presence of multiple different cytokines. Cytokines may be detected, for example, by the use of specific reagent kits or immunoassays. Cytokines may be detected using kits available from commercial providers such as Miltenyi Biotec™, Luminex, and Thermo Fisher scientific™. Examples of kits suitable for detecting cytokines are the Rapid Cytokine Inspector (CD4 / CD8) kit, or the MACSPlex Cytokine T / NK kit, which can detect cytokines formed by either T cells or NK cells, both of which are sold by Miltenyi Biotec™.

[0053] In some embodiments, the amount of cytokines is detected using an immunoassay. There are many different formats and variations of immunoassays. Immunoassays may be performed in multiple steps where reagents are added and washed away or separated at different points in the assay. Immunoassays include heterogeneous immunoassays, which include multiple steps, and homogeneous immunoassays, which involve simply mixing reagents with the sample and taking a physical measurement. Immunoassays often utilize calibrators, which are solutions known to contain the analyte of interest, the concentration of which is generally known. Comparing the response of the assay generated by the calibrator to the response of the assay to the actual sample allows the signal strength to be interpreted in terms of the presence or concentration of the analyte in the sample. Types of immunoassays include competitive homogeneous immunoassays, competitive heterogeneous immunoassays, one-site non-competitive immunoassays, and two-site non-competitive immunoassays. Immunoassays also include enzyme-linked immunosorbent assays (ELISAs), lateral flow immunoassays, enzyme-linked immunosorbent spot (ELIspot) assays, flow cytometry, intracellular cytokine staining, antibody array assays and bead-based assays, magnetic immunoassays, radioimmunoassays, and quantitative PCR (including, but not limited to, qRT-PCR). In one embodiment, the assay comprises Luminex xMAP®.

[0054] Methods for determining the potency of immune cells include contacting the immune cells with an effective amount of a stimulant (eg, PHA, PMA / ionomycin, Con A, LPS and / or PWM, etc.).

[0055] It is understood and contemplated herein that immune cells must be exposed to a stimulant for a period of time to be induced to produce cytokines. In one aspect, a method of assaying efficacy of immune cells includes exposing immune cells to an effective amount of a stimulant (e.g., PHA, PMA / ionomycin, Con A, LPS, and / or PWM, etc.) for 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 ... Disclosed herein are methods of contacting the cells for 0, 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, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 45, 60, 61, 62 days, 3, 4, 5, or 6 months.

[0056] Also disclosed herein is a method of assaying the efficacy of an immune cell of any preceding embodiment, wherein a stimulant (e.g., PHA, PMA / ionomycin, Con A, LPS, and / or PWM, etc.) is provided at a concentration between 5 μg / mL and 1000 μg / mL. In one embodiment, the concentration of the stimulant is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 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, 800, 850, 900, 950, or 1000 μg / mL. In one embodiment, the concentration of the stimulant is about 1 μg / mL to 100 μg / mL, 1 μg / mL to 50 μg / mL, 1 μg / mL to 15 μg / mL, or 5 μg / mL to 15 μg / mL.

[0057] In one aspect, it is understood and contemplated herein that the same cytokines produced to determine the potency of immune cells can also be used to identify the cells that produce the cytokines. Immune cells have distinct expression profiles that are well known in the art. Also disclosed herein is a method of determining the identity of at least one immune cell or cell population (e.g., differentiated Th1, Th2, Th3, Th9, Th17, effector memory T (Tem) cells, central memory T (Tcm) cells, gamma delta T cells, or regulatory T (Treg) cells, resting NK cells, expanded NK cells) based on the cytokine signature associated with that cell type. Thus, a method of identifying immune cells (e.g., T cells, macrophages, NK cells, NK T cells, CAR T cells, and / or CAR NK cells, etc.), comprising inducing the immune cells to react with an effective amount of a stimulant (e.g., PHA, PMA / ionomycin, Con A, LPS, and / or PWM) and one or more cytokines produced by the immune cells (e.g., IL-2, IL-6, IFN-γ, TNF-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, and detecting the amount of cytokines expressed by the immune cells (such as 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), wherein the identity of the immune cells is revealed based on the profile of expressed cytokines.

[0058] Kits for assessing immune cell potency Another aspect of the invention provides a kit for determining the potency of immune cells (e.g., T cells, macrophages, NK cells, NK T cells, CAR T cells, and / or CAR NK cells, etc.), comprising a container containing an effective amount of a stimulant (e.g., PHA, PMA / ionomycin, Con A, LPS, and / or PWM, etc.) and a suitable buffer for the immune cells. In some embodiments, the stimulant in the kit is provided at a concentration of 5 μg / mL to 1000 μg / mL. In one embodiment, the concentration of the stimulant is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 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, 800, 850, 900, 950, or 1000 μg / mL. In one aspect, the concentration of the stimulating agent is about 1 μg / mL to 100 μg / mL, 1 μg / mL to 50 μg / mL, 1 μg / mL to 15 μg / mL, or 5 μg / mL to 15 μg / mL. In some embodiments, the container is a microcentrifuge tube (e.g., an Eppendorf microcentrifuge tube, etc.). The kit can also include a tool for obtaining a sample from a subject, such as a syringe for obtaining a sample containing one or more immune cells. A suitable buffer is RPMI.

[0059] The kit may also include components necessary for performing the immunoassay (e.g., a solid phase) to which the antibody that functions as the capture antibody and / or the detection antibody in a sandwich immunoassay format is bound. The solid phase may be a material such as a magnetic particle, a bead, a test tube, a microtiter plate, a cuvette, a membrane, a scaffold molecule, a quartz crystal, a film, a filter paper, a disk or a chip. The kit may also include a detectable label that may be conjugated or conjugated to an antibody such as the antibody that functions as the detection antibody. The detectable label may be a direct label, which may be, for example, an enzyme, an oligonucleotide, a nanoparticle chemiluminophore, a fluorophore, a fluorescence quencher, a chemiluminescence quencher, or biotin. The test kit may optionally include any additional reagents necessary to detect the label.

[0060] The kit may further include instructions for using the kit to stimulate cytokine production by immune cells to assess the efficacy of the immune cells. In some embodiments, the kit further includes instructions for using the amount of cytokine to determine the efficacy of the cells. The instructions included in the kit may be affixed to the packaging material or included as a package insert. The instructions are typically written or printed, but are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. As used herein, the term "instructions" may include the address of an internet site that provides the instructions.

[0061] Immunotherapy methods The method of determining the potency of immune cells can be performed prior to the use of the immune cells as an immunotherapeutic agent. For example, the method of determining the potency of one or more immune cells can be performed as described above, after which at least one potent immune cell can be selected (based on the amount of cytokines detected), and a therapeutically effective amount of the potent immune cell can be delivered to a subject as an immunotherapeutic drug. Thus, in one aspect, there is provided an immunotherapy method, comprising: a) administering to a subject an immune cell (e.g., T cells, macrophages, NK cells, NK T cells, CAR T cells, and / or CAR T cells) disclosed herein; and b) performing a method of assaying the potency of a cytokine (e.g., IL-2, IL-6, IFN-γ, TNF-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-γ, IL-30, IL-40, IL-50, IL-60, IL-70, IL-81, IL-90, IL-102, IL-92, IL-94, IL-96, IL-98, IL-99, IL-103, IL-99, IL-104, IL-105, IL-106, IL-107, IL-108, IL-109, IL-110, IL-111, IL-112(p40), IL-112(p70), IL-113, IL-114, IL-115, IL-116, IL-117, IL-118, IL-119, IL-120, IL-121, IL-122, IL-123, IL-124, IL-125, IL-126, IL-127, IL-128, IL-129, IL-130, IL-131, IL-132, IL-133, IL-134, IL-135, IL-136, IL-137, IL- Disclosed herein is a method of immunotherapy comprising: c) selecting at least one potent immune cell based on the amount of a potent immune cell that is a potent immune cell of a given marker, such as a potent immune cell-1, a potent immune cell-2, a potent immune cell-3, a potent immune cell-4, a potent immune cell-5, a potent immune cell-6, a potent immune cell-7, a potent immune cell-8, a potent immune cell-9, a potent immune cell-10, a potent immune cell-11, a potent immune cell-12, a potent immune cell-13, a potent immune cell-14, a potent immune cell-15, a potent immune cell-16, a potent immune cell-17, a potent immune cell-18, a potent immune cell-19, a potent immune cell-20, a potent immune cell-21, a potent immune cell-22, a potent immune cell-23, a potent immune cell-24, a potent immune cell-25, a potent immune cell-26, a potent immune cell-27, a potent immune cell-28, a potent immune cell-29, a potent immune cell-30, a potent immune cell-31, a potent immune cell-32, a potent immune cell-34, a potent immune cell-35, a potent immune cell-35, a potent immune cell-36, a potent immune cell-37, a potent immune cell-38, a potent immune cell-39, a potent immune cell-40, a potent immune cell-41, a potent immune cell-42, a potent immune cell-43, a potent immune cell-44, a potent immune cell-45, a potent immune cell-45, a potent immune cell-46, a potent immune cell-47, a potent immune cell-48, a potent immune cell-49, a potent immune cell-49, a potent immune cell-49, a potent immune cell

[0062] In some embodiments, the immune cells are immunotherapeutic immune cells. Immunotherapeutic immune cells are those that are useful for treating diseases such as cancer. Becker et al., Cancer Immunol. Immunother 65, 477-484 (2016). The use of expanded NK cells for the treatment of cancer has been described. Rezvani et al., Front Immunol., 6, 578 (2015). In some embodiments, the immune cells are expanded immune cells, since it is useful to be able to administer large numbers of immune cells during immunotherapy. The expanded immune cells are immune cells that are grown ex vivo to grow large numbers of immune cells. In some embodiments, the expanded immune cells are autologous cells that can be easily administered to a subject without eliciting an immune response. However, in some embodiments, the expanded immune cells are allogeneic immune cells, and their inherent alloreactivity can be beneficial. In further embodiments, the expanded immune cells are genetically engineered to contain chimeric antigen receptors to help the immune cells target diseased tissue. The preparation of expanded immune cells includes activating and expanding immune cells. Koepsell et al., Transfusion, 53(2): 404-10 (2013). Several cytokines (IL-2, IL-12, IL-15, IL-18, IL-21, type I IFN, and TGF-β) have been shown to be useful for activating and expanding immune cells ex vivo. For example, in some embodiments, the NK cells evaluated are NK cells expanded by IL-21. Thus, in one aspect, an immunotherapy method is disclosed herein, further comprising expanding at least one potent immune cell prior to delivering a therapeutically effective amount of the potent immune cell.

[0063] Expansion refers to the ex vivo expansion of NK cells, such that the population of NK cells is increased. NK cells can be expanded, for example, from peripheral blood mononuclear cells. However, NK cells can also be expanded from other types of cells, such as hematopoietic stem or progenitor cells. Early blood or stem cells can be isolated from a variety of different sources, such as placenta, umbilical cord blood, placental blood, peripheral blood, spleen or liver. Expansion occurs in cell culture medium. Suitable cell culture media are known to those skilled in the art. The expanded cells can be provided as a cell line, where a cell line is a plurality of cells that can be maintained in cell culture. Thus, in one aspect, a method of immunotherapy is disclosed herein, which further comprises expanding at least one potent immune cell before delivering a therapeutically effective amount of the potent immune cell. In some aspects, the immune cell has been extracted from a subject using known methods before carrying out the method of determining the efficacy of the immune cell. Alternatively, the immune cell can be sourced from cell culture expansion.

[0064] In some aspects, the immune cells are directed to respond to a particular antigen (e.g., CD19). The immune cells can be directed to respond before the method of determining their efficacy or after the method of determining their efficacy. In some embodiments, the immune cells are genetically modified to respond to a particular antigen. The antigen can be, for example, a tumor-specific antigen. In some aspects, the immunotherapy method includes genetically modifying the immune cells (either before or after determining the efficacy of the immune cells) to present a chimeric antigen receptor. In one aspect, the method can further include modifying the cell line from which the exosomes are derived, or modifying the exosomes themselves, to include the particular antigen to which the immune cells of interest respond (e.g., adding CD19 to specifically determine the efficacy of CD19 CAR T cells in a heterogeneous sample).

[0065] As pointed out throughout, the method of determining the efficacy of immune cells can be used as part of adoptive cell transfer therapy.Potent immune cells can be delivered to a subject using a therapeutically acceptable carrier.Intravenous delivery is traditionally used to deliver immunotherapy cells, but other methods can also be considered (e.g., direct transplantation into the local area of ​​the body that requires immunotherapy).

[0066] The therapeutically effective amount can be determined by comparing the amount of cytokine produced by immune cells with the cytokine potency level required for the use of immune cells in immunotherapy.It is understood and contemplated herein that the therapeutically effective amount depends on the immune cells administered, the subject being treated, and the disease, disorder, and / or condition being treated.Those skilled in the art will know how to use the appropriate dose of immune cells that will be therapeutically effective for the subject being treated.

[0067] A therapeutically effective amount of a potent immune cell includes a plurality of potent immune cells. For example, after selecting at least one potent immune cell, the selected cells can be expanded in vitro to produce a plurality of potent immune cells.

[0068] The subject receiving the potent immune cells may be any subject that may benefit from immunotherapy, such as a subject with an autoimmune disease, an inflammatory disease or disorder, a viral disease, and / or a bacterial infection. In some embodiments, the subject may be a cancer patient. In some embodiments, the subject may be an individual with a high risk of developing cancer, an individual diagnosed with cancer, an individual being treated for cancer, or an individual recovering from cancer after surgery. In some embodiments, the potent immune cells may be delivered to the subject as a prophylactic agent to prevent, inhibit, or delay the onset of cancer or metastasis.

[0069] Methods for Treating Disease It is understood and contemplated herein that the potent immune cells identified herein can be used in the treatment of any disease or disorder for which adoptive immunotherapy can be used for treatment, including, but not limited to, autoimmune diseases, inflammatory diseases or disorders, viral diseases and / or bacterial infections. Thus, in one aspect, a method of treating, inhibiting, reducing, preventing, and / or ameliorating cancer and / or metastasis in a subject comprises the steps of: a) obtaining one or more immune cells (e.g., T cells, macrophages, NK cells, NK T cells, CAR T cells, and / or CAR NK cells, etc.) obtained from an allogeneic or autologous donor; b) administering the immune cells to an effective amount of a stimulatory agent (e.g., PHA, PMA / ionomycin, Con A, LPS and / or PWM, and c) cytokines produced by immune cells (e.g., IL-2, IL-6, IFN-γ, TNF-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-40, IL-50, IL-60, IL-70, IL-81, IL-90, IL-102, IL-104, IL-106, IL-108, IL-109, IL-111, IL-112(p40), IL-112(p70), IL-113, IL-114, IL-115, IL-116, IL-117, IL-118, IL-119 ... Disclosed herein are methods for detecting the amount of a cytokine (such as 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) in a subject, d) selecting at least one potent immune cell based on the amount of the detected cytokine, and e) administering to the subject a therapeutically effective amount of the potent immune cell. In some embodiments, the method may further include extracting immune cells from an autologous or allogeneic donor.

[0070] It is useful to be able to administer a large number of immune cells during immunotherapy, and in some embodiments, it is understood and contemplated herein that the immune cells are expanded immune cells. Expanded immune cells are immune cells that are grown ex vivo to grow a large number of immune cells. Thus, disclosed herein is a method for treating, inhibiting, reducing, preventing, and / or alleviating an autoimmune disease, an inflammatory disease or disorder, a viral disease, a bacterial infection, cancer, and / or metastasis, further comprising expanding at least one potent immune cell before delivering a therapeutically effective amount of at least one potent immune cell.

[0071] It is understood and contemplated herein that the disclosed therapeutic method can be used to treat any disease or condition in which uncontrolled cell proliferation occurs, including but not limited to cancer and metastasis.The following is a representative but non-limiting list of cancers that can be treated using the disclosed method that uses powerful immune cells: 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 head and neck, lung cancer such as small cell lung cancer and non-small cell lung cancer, neuroblastoma / glioblastoma, ovarian cancer, skin cancer, liver cancer, melanoma, squamous cell carcinoma of mouth, throat, larynx, and lung, uterine cancer, uterine carcinoma, breast cancer, and epithelial cancer, kidney cancer, genitourinary cancer, lung cancer, esophageal carcinoma, head and neck carcinoma, colon cancer, hematopoietic cancer, testicular cancer, colon cancer, rectal cancer, prostate cancer, or pancreatic cancer.

[0072] Examples of autoimmune diseases that can be treated using the disclosed methods include, but are not limited to, achalasia, acute disseminated encephalomyelitis, acute motor axonal neuropathy, Addison's disease, adiposity, adult Still's disease, agammaglobulinemia, alopecia areata, Alzheimer's disease, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome, aplastic anemia, autoimmune angioedema, autoimmune pulmonary edema, and autoimmune pulmonary edema. Autonomic dysfunction, autoimmune encephalomyelitis, autoimmune enteropathy, 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, axonal and nerve neuropathy (AMAN), Baro's disease, Behçet's disease, benign mucosal pemphigoid emphigoid), Bickerstaff encephalitis, bullous pemphigoid, Castleman disease (CD), celiac disease, Chagas disease, chronic fatigue syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic relapsing multifocal osteomyelitis (CRMO), Churg-Strauss syndrome (CSS), eosinophilic granulomatosis (EGPA), cicatricial pemphigoid, Cogan syndrome, cold agglutinin disease, congenital heart block, Coxsackie myocarditis, CREST syndrome, Crohn's disease, dermatitis herpetiformis, 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, Felty's disease syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture's syndrome, granulomatosis with polyangiitis, Graves' disease, Guillain-Barre syndrome, Hashimoto's encephalopathy, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schönlein purpura (HSP), herpes gestationis or pemphigoid of gestationis (PG), hidradenitis suppurativa (HS) (acne contralateralis), hypogammaglobulinemia, IgA nephropathy, IgG4-related sclerosing disease, immune thrombocytopenic purpura (ITP), inclusion body myositis (IBM), interstitial cystitis (IC), inflammatory bowel disease (IBD), juvenile arthritis, juvenile diabetes mellitus (type 1 diabetes), juvenile myositis (JM), Kawasaki disease, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus,Lignin conjunctivitis, Linear IgA Disease (LAD), Lupus Nephritis, Lupus Vasculitis, Chronic Lyme Disease, Meniere's Disease, Microscopic Polyangiitis (MPA), Mixed Connective Tissue Disease (MCTD), Mooren's Ulcer, Much-Habermann Disease, Multifocal Motor Neuropathy (MMN) or MMNCB, Multiple Sclerosis, Myasthenia Gravis, Myositis, Narcolepsy, Neonatal Lupus, Neuromyelitis Optica, Neutropenia, Ocular Benign Mucous Membrane Pemphigoid, Optic Neuritis, Ord's Thyroiditis, Relapsing Rheumatism (PR), PANDAS, Paraneoplastic Syndrome (PANDAS), Tumor-prone cerebellar degeneration (PCD), paroxysmal nocturnal hemoglobinuria (PNH), Parry-Romberg syndrome, pars planitis (peripheral uveitis), Parsonage-Turner syndrome, pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia (PA), POEMS syndrome, polyarteritis nodosa, polyglandular syndrome type I, II, III, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, progesterone dermatitis, psoriasis , psoriatic arthritis, pure red cell aplasia (PRCA), pyoderma gangrenosum, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, relapsing polychondritis, restless legs syndrome (RLS), retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, rheumatic vasculitis, sarcoidosis, Schmidt's syndrome, Schnitzler's syndrome, scleritis, scleroderma, Sjogren's syndrome, semen and testicular autoimmunity, stiff person syndrome (SPS), subacute bacterial endocarditis (SBE), SAZAC Syndrome, Sydenham chorea, sympathetic ophthalmia (SO), systemic lupus erythematosus, systemic dermatosis, Takayasu arteritis, temporal arteritis / giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome (THS), transverse myelitis, type 1 diabetes mellitus, ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), urticaria, urticarial vasculitis, uveitis, vasculitis, vitiligo, Vogt-Koyanagi-Harada disease, and Wegener's granulomatosis (or granulomatosis with polyangiitis (GPA)).

[0073] The following examples are included to illustrate preferred embodiments of the invention. It should be understood by those skilled in the art that the techniques disclosed in the examples below represent techniques discovered by the inventors to work well in the practice of the invention, and therefore can be considered to constitute preferred modes for its practice. However, those skilled in the art should understand in light of this disclosure that many changes can be made in the specific embodiments that are disclosed and still obtain the same or similar results without departing from the spirit and scope of the invention. EXAMPLES

[0074] The assay disclosed herein addresses the problems that exist with current standard methods and is intended to test the efficacy of therapeutic immune cells that meets FDA requirements. To accomplish this, phytohemagglutinin (PHA) is used as a surrogate to induce cytokine production in immune cells. PHA is used in clinical settings to test immune responses in transplant patients. By using PHA to induce cytokine production in therapeutic immune cells, it removes all of the biological and batch variability from the immune cell efficacy assay. This assay eliminates the need to have a fully functional laboratory to test the efficacy of therapeutic immune cells at multiple clinical infusion sites and provides a faster turnaround time for such testing. A method to test the effector function of therapeutic immune cells is provided as part of the efficacy testing requirements set by the FDA.

[0075] Testing the efficacy of therapeutic immune cells using PHA as a stimulatory agent The use of tumor cells to test effector functions of therapeutic immune cells has been a standard technique. These target cells add biological variability to the results of the assay. Also, the setup of this assay is more laborious and complex, with added variability due to inter-individual differences in target conditions, plate setup, and technique. NK cell therapeutic response by comparison of PHA cytokine assay to the widely used tumor cell (K562)-mediated cytokine assay. The level of cytokines indicates the potency of the response from NK cells.

[0076] Figure 1 shows the correlation between the PHA-induced cytokine assay and the standard K562-induced cytokine assay. The outliers present in Figure 1 are the result of the variability introduced by using tumor cells. Higher GM-CSF secretion was detected in the tumor cell-mediated assay (GC-CSF is normally secreted by tumor cells). This demonstrated a clear advantage of the PHA assay due to the lack of variability of the introduced tumor cells.

[0077] Figure 2 shows a histogram of cytokine levels achieved using a 4 hour incubation versus a 24 hour incubation (with 1 million NK cells). Figure 3 shows a histogram of cytokine levels achieved using less than 1 million cells and more than 1 million cells. These conditions were tested to standardize the assay. Figure 4 shows the results of a PHA assay of NK cells expanded with mb-IL-21 and TGF-β. Figure 5 shows the difference in cytokine profile between donor immune cells and expanded therapeutic NK cell product when induced by PHA.

[0078] Optimal cell number for the assay (10 6After establishing a total of 100 donors (N = 10), fresh peripheral blood NK cells (N = 18) and expanded NK cells (N = 18) from multiple donors were stimulated with PHA to produce cytokines, and cytokines in the supernatants were evaluated after 1 h. Although the expression of most cytokines was similar between the two types of NK cells, several cytokines and chemokines related to NK cell function were highly differentially expressed in response to stimulation (Figure 6). Next, the expression of six cytokines in fresh and expanded NK cells was measured after 1 h of stimulation with PHA. Pentraxin-3 (average 1,062 vs. 7), IL-8 (average 821 vs. 11), and chitinase 3-like 1 (average 620 vs. 7) were highly overexpressed in fresh NK cells compared to expanded NK cells, while IFN-γ (average 15 vs. 105), IL-2 (average 3 vs. 141), and CD30 (average 9 vs. 156) were highly overexpressed in expanded NK cells compared to fresh NK cells (Figure 7). Based on overexpression alone, a cutoff of 10 pg / mL expression of both IFN-γ and IL-2 has 94% specificity (16 of 17) and 89% specificity (16 of 18) in expanded NK cells differentiated from fresh peripheral blood NK cells (Figure 8). Using IL-2 (upregulated) together with downregulated cytokines (Pentraxin-3 or Chitinase 3-like 1) was 100% sensitive and specific for expanded NK cells differentiated from fresh peripheral blood NK cells, with an expression ratio (IL-2 downregulated) greater than 1 for the fresh cells and less than 1 for the expanded cells (Figure 9). Using IFNg (upregulated) together with downregulated cytokines (Pentraxin-3 or Chitinase 3-like 1) was 95% effective in expanded NK cells differentiated from fresh peripheral blood NK cells. Combinations of multiple upregulated and downregulated cytokines could be used to increase the sensitivity and specificity of the defined types (Figure 10).

[0079] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the disclosed invention belongs. Publications cited herein and the materials to which they are cited are specifically incorporated by reference. However, it should be understood that all or part of a patent, publication, or other disclosure material that is said to be incorporated herein by reference is incorporated herein only to the extent that the incorporated material does not conflict with existing definitions, descriptions, or other disclosure material set forth in this disclosure. Thus, to the extent necessary, the disclosure expressly set forth herein takes precedence over any conflicting material incorporated herein by reference. Any material, or part thereof, that is said to be incorporated herein by reference but that conflicts with existing definitions, descriptions, or other disclosure material set forth herein is incorporated herein only to the extent that no conflict occurs between the incorporated material and the existing disclosure material.

[0080] Those skilled in the art will recognize many equivalents to the specific embodiments of the invention described herein. It will be possible to recognize or ascertain using no more than routine experimentation. Although the invention has been described with reference to specific embodiments and implementations, the scope or scope of the invention is not to be construed as limiting the scope of the invention. Various modifications and additional variations may be made without departing from the inventive concept of the present invention. It is understood that equivalents may be substituted for the elements of the present invention. Certain modifications and variations may be made to adapt a particular situation or device to the teachings of the present invention without departing from its scope. Many modifications may be made to the present invention to achieve the same object. Such equivalents are encompassed by the following claims. The present invention is not limited to the specific embodiments disclosed herein. However, the present invention is intended to include all embodiments falling within the scope of the appended claims. For example, the present specification provides the following inventions. [1] A method for assaying efficacy of an immune cell, comprising contacting an immune cell with an effective amount of a stimulant and detecting the amount of a cytokine produced by the immune cell. [2] The method according to [1] above, further comprising the step of comparing the amount of cytokine produced with a cytokine potency level required for use of the immune cells in immunotherapy. [3] The method according to any one of [1] to [3] above, wherein the amounts of multiple cytokines are determined. [4] The method according to any one of [1] to [4] above, wherein the immune cells are T cells, macrophages, natural killer (NK) cells, NK T cells, chimeric antigen receptor (CAR) T cells, or CAR NK cells. [5] The method according to [4] above, wherein the immune cells are NK cells. [6] The method according to [5] above, wherein the NK cells are NK cells proliferated by IL-21. [7] The method according to any of the above [1] to [6], wherein the stimulating agent comprises phytohemagglutinin (PHA), phorbol myristate acetate (PMA) / ionomycin, concanavalin A (Con A), lipopolysaccharide (LPS), and / or pokeweak mitogen (PWM). [8] The method according to any one of [1] to [7] above, wherein the amount of the cytokine is detected using an immunoassay. [9] The cytokine is selected from the group consisting of interleukin (IL)-2 (IL-2), IL-6, interferon (IFN)-gamma (IFN-γ), B-cell activating factor / tumor necrosis factor (TNF) ligand superfamily member 13B (BAFF / TNFSF13B), TNF-α, cluster of differentiation (CD) 163 (CD163), CD30 / TNFRSF8, chitinase 3-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-l1, IL-32, IL-34, IL-35, and matrix metalloproteinases. The method according to any one of the above [1] to [8], wherein the protein is selected from the group including MMP-1, osteocalcin, osteopontin (OPN), pentraxin-3, tumor necrosis factor (TNF)-receptor 1 (TNF-R1), TNF-R2, thymic stromal lymphopoietin (TSLP), granulocyte-macrophage colony-stimulating factor (GM-CSF), leukemia inhibitory factor (LIF), and chemokines macrophage inflammatory protein (MIP)-1α (MIP-1α), MIP-1β, RANTES, and / or TNF-related weak inducer of apoptosis (TWEAK) / TNF superfamily member 12 (TWEAK / TNFSF12).

[10] The method according to [1] above, wherein the immune cells are contacted with an effective amount of the stimulating agent for at least 4 hours.

[11] The method according to [1] above, wherein the stimulant is provided at a concentration of 5 μg / mL to 15 μg / mL.

[12] A kit for assaying the efficacy of immune cells, comprising a container containing an effective amount of a stimulant and a buffer suitable for the immune cells.

[13] The kit described in

[12] above, wherein the stimulant is provided at a concentration of 5 μg / mL to 15 μg / mL.

[14] The kit described in

[12] above, wherein the container is an Eppendorf microcentrifuge tube.

[15] The kit described in

[12] above, further comprising instructions for using the kit to stimulate cytokine production by immune cells.

[16] A method of immunotherapy comprising: a. Carrying out the method according to any one of the above [1] to

[11] on a plurality of immune cells to determine the potency of each immune cell; b. selecting at least one potent immune cell based on the amount of cytokines detected; c. Administering a therapeutically effective amount of said potent immune cells as an immunotherapeutic drug to a subject in need thereof.

[17] The immunotherapy method of

[16] above, further comprising extracting the plurality of immune cells from an allogeneic or autologous donor prior to assaying the efficacy of the immune cells.

[18] The immunotherapy method described in

[16] above, further comprising expanding the at least one potent immune cell prior to delivering a therapeutically effective amount of the potent immune cell.

[19] The immunotherapy method described in

[16] above, further comprising instructing the multiple immune cells or the potent immune cells to respond to a specific antigen.

[20] The immunotherapy method described in

[19] above, further comprising genetically modifying the multiple immune cells or the potent immune cells to present a chimeric antigen receptor.

[21] A method of treating, inhibiting, reducing, preventing, and / or ameliorating cancer and / or metastasis in a subject, comprising: a. obtaining one or more immune cells; b. contacting an immune cell with an effective amount of a stimulatory agent; c. detecting the amount of cytokines produced by the immune cells; d. selecting at least one potent immune cell based on the amount of said cytokine detected; and e. administering to said subject a therapeutically effective amount of said potent immune cells.

[22] The method according to

[21] above, wherein the one or more immune cells are obtained from an allogeneic or autologous donor.

[23] The method according to

[21] above, further comprising extracting the plurality of immune cells from an allogeneic or autologous donor.

[24] The method according to any one of

[21] to

[23] above, wherein the immune cells are T cells, macrophages, natural killer (NK) cells, NK T cells, chimeric antigen receptor (CAR) T cells, or CAR NK cells.

[25] The method according to any one of

[21] to

[24] above, further comprising expanding the at least one potent immune cell prior to delivering a therapeutically effective amount of the at least one potent immune cell.

Claims

1. A method for assaying the potency of natural killer (NK) cells expanded by interleukin 21 (IL-21) under ex vivo conditions (IL-21 expanded NK cells), comprising: (i) contacting the IL-21 expanded NK cells with an effective amount of a stimulatory agent; (ii) detecting the amount of cytokines produced by the IL-21 expanded NK cells of step (i); (iii) comparing the amount of cytokine produced by the IL-21-expanded NK cells of step (i) with a potency level of cytokine required for using the IL-21-expanded NK cells in immunotherapy to indicate effector function of the IL-21-expanded NK cells; (iv) selecting the potent IL-21 expanded NK cells of step (iii) based on the amount of cytokines measured in step (ii); A method comprising:

2. The method of claim 1 , wherein the amounts of multiple cytokines are determined.

3. 3. The method of claim 1 or 2, wherein the stimulant comprises phytohemagglutinin (PHA), phorbol myristate acetate (PMA) / ionomycin, concanavalin A (Con A), lipopolysaccharide (LPS), and / or pokeweed mitogen (PWM).

4. The method according to any one of claims 1 to 3, wherein the amount of the cytokine is detected using an immunoassay.

5. The cytokines include interleukin (IL)-2 (IL-2), IL-6, interferon (IFN)-γ (IFN-γ), B-cell activating factor / tumor necrosis factor (TNF) ligand superfamily member 13B (BAFF / TNFSF13B), TNF-α, cluster of differentiation (CD) 163 (CD163), CD30 / TNFRSF8, chitinase 3-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-l1, IL-32, IL-34, IL-35, matrix metalloproteinase (MMP), and / or IL-16.

2. The method of claim 1, wherein the IL-16 receptor is one or more of: proteinase-1 (MMP-1), osteocalcin, osteopontin (OPN), pentraxin-3, tumor necrosis factor (TNF)-receptor 1 (TNF-R1), TNF-R2, thymic stromal lymphopoietin (TSLP), granulocyte-macrophage colony-stimulating factor (GM-CSF), leukemia inhibitory factor (LIF), chemokine macrophage inflammatory protein (MIP)-1 alpha (MIP-1 alpha), MIP-1 beta, RANTES, and / or TNF-related weak inducer of apoptosis (TWEAK) / TNF superfamily member 12 (TWEAK / TNFSF12).

6. The method of claim 1, wherein the IL-21 expanded NK cells are contacted with an effective amount of the stimulatory agent for at least 4 hours.

7. The method of claim 1, wherein the stimulatory agent is provided at a concentration of 5 μg / mL to 15 μg / mL.

8. The method of claim 1, 5 or 6, wherein the cytokine is selected from interferon (IFN)-gamma (IFN-γ), CD30 / TNFRSF8 and IL-2.

9. The method of claim 6 , wherein the stimulant comprises phytohemagglutinin (PHA).

10. 13. A kit for assaying the efficacy of IL-21 expanded NK cells, comprising a container containing an effective amount of a stimulatory agent and a buffer suitable for NK cells, and further comprising instructions for use in accordance with the method of claim 1.

11. The kit of claim 10, wherein the stimulatory agent is provided at a concentration of 5 μg / mL to 15 μg / mL.

12. The kit of claim 10 , wherein the container is a microcentrifuge tube.

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