USE OF A STIMULATING AGENT TO EVALUATE THE POTENCY OF IMMUNE CELLS

Abstract

A method for determining the potency of an immune cell is described. The method includes the steps of exposing an immune cell to an effective amount of a stimulating agent (e.g., phytohemagglutinin [PHA]) and detecting the amount of a cytokine produced by the immune cell. Kits for assessing the potency of immune cells are also described. Potency assays are important for meeting FDA requirements for new biological agents, such as immunotherapeutic cells. Methods for using potent immune cells as immunotherapeutic treatment are described.

Description

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

[0002] This invention relates to immunotherapy, and more particularly to evaluating the effector function of immune cells. BACKGROUND OF THE INVENTION

[0003] Immunotherapy is the treatment of disease by activating or inhibiting the immune system. Cells derived from the immune system can be used to enhance immune characteristics and function. In recent years, immunotherapy has become a topic of great interest to researchers, physicians, and pharmaceutical companies, particularly because of its potential to treat various forms of cancer. Immunomodulatory regimens often have fewer side effects than existing drugs, and even less potential to create resistance when treating a microbial disease.

[0004] Conventional cancer treatments focus on inactivating or eliminating cancer cells with chemotherapy, surgery, and / or radiation. However, the field of immune cell therapy is rapidly growing and can be used in conjunction with, or in some cases instead of, conventional treatments to treat, prevent, or delay the onset of certain types of cancer. Immune effector cells, such as lymphocytes, macrophages, dendritic cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), and others, naturally work together to defend the body against cancer by targeting abnormal antigens expressed on the surface of tumor cells. Recent developments in cancer treatment have focused on directing the patient's immune system to attack and destroy tumors. Several strategies are currently in use or under investigation and evaluation.

[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 may have originated from the patient (autologous) or from another individual (allogeneic). Allogeneic treatments involve isolated and expanded cells 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, autologous immune enhancement therapy involves the extraction of the subject's own natural killer cells, cytotoxic T lymphocytes, epithelial cells, and other relevant immune cells from peripheral blood, the expansion of these cells in vitro, and then the reinfusion of these cells into the subject's body.

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

[0007] With the approval of the first CAR T-cell therapy and the participation of several commercial companies in various clinical trials, this field experienced explosive growth and has shown a promising future for immunotherapies. As the field advances with new and frequent clinical trials, the need for a reliable and reproducible potency test for these therapeutic immune cells has grown steadily. The industry gold standard for assessing the effector function of immune cells is the chromium release assay, which was developed in the 1960s and is still in use, despite concerns about the use of radioactive material and the variability caused by the target tumor cells. The available alternative is the calcein-based assay, which still has considerable variability due to the use of different tumor targets and the trapping of calcein in apoptotic bodies of the tumor targets.

[0008] There have been other efforts to develop different ways to visually observe the effector function of these immune cells, but these methods still target tumor cells. Another alternative method for assessing the effector function of immune cells is to evaluate the cytokines produced by these cells. All conventional methods for this use target tumor cells to induce cytokine production in the immune cells. The use of target tumor cells adds biological variability to all these tests due to the variability among tumor cell types. Furthermore, these assays require tedious setup, which introduces batch effects. Batch effects are caused by target cell conditions, person-to-person variability in plate loading, plate conditions, variability in different reagents, readout variability, and other factors.There is a clear need for an immune cell potency assay that can eliminate all these variabilities and produce reliable and reproducible results.

[0009] A reliable and reproducible potency assay is needed to evaluate the quality of immune cell therapy products. The approval process is highly regulated, and drug developers must submit a substantial amount of information about the drug product to regulatory agencies to obtain 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 the cell therapy product must be indicated by appropriate tests that demonstrate the effector function of these therapeutic immune cells, which would be done by measuring the relevant cytokine production by these immune cells. SUMMARY OF THE INVENTION

[0010] 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, the drawings, and the claims.

[0011] In one aspect, methods for assessing the potency of an immune cell (such as, for example, a T lymphocyte, macrophage, NK cell, T NK lymphocyte, CAR T lymphocyte, and / or CAR NK cell) are disclosed herein, comprising bringing an immune cell into contact with an effective amount of a stimulating agent (such as, for example, phytohemagglutinin (PHA), phorbol myristate acetate (PMA) / ionomicin, concanavalin A (Con A), lipopolysaccharide (LPS), and / or carmine herb mitogen (PWM)) and detecting the amount of one or more cytokines (such as, for example, IL-2, IL-6, IFN-γ, TNF-α, BAFF / TNFSF13B, CD163, CD30 / TNFRSF8, aquitinase-like protein 1 3, gp130, IFN-α2, IL-6Ra, IL-8, IL-10, IL-11, IL-12(p40), IL-12(p70), IL-20, IL-22, IL-26, IL-29 / IFN-I1, IL-32, IL-34, IL-35, MMP-1, osteocalcin, OPN, pentraxin-3, TNF-R1, TNF-R2, TSLP, GM-CSF, ΜΙΡ-1α, ΜΙΡ-1β, RANTES and / or TWEAK / TNFSF12) produced by the immune cell.In one aspect, the method may also involve comparing the amount of cytokine produced with the level of cytokine potency required to use the immune cell in immunotherapy.

[0012] Also disclosed herein are methods for assessing the potency of an immune cell according to any of the above aspects, wherein the amount of cytokine is detected by an immunoassay (such as, for example, ELISA, intracellular cytokine staining, ELISpot, flow cytometry, Luminex xMAP®, quantitative POR (including, among others, qRT-PCR) and / or microsphere array).

[0013] In one aspect, methods for evaluating the potency of an immune cell according to any of the foregoing aspects are disclosed herein, wherein the immune cell is brought into contact with an effective amount of a stimulating agent (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, 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, 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 are methods for assessing the potency of an immune cell according to any of the above aspects, wherein the stimulating agent (such as, for example, PHA, PMA / ionomycin, Con A, LPS and / or PWM) is provided at a concentration of 1.0 pg / mL to 1000 pg / mL, including, among others, a concentration of 5 pg / mL to 15 pg / mL.

[0015] In one aspect, kits for assessing the potency of an immune cell (such as, for example, a T lymphocyte, macrophage, NK cell, NK T lymphocyte, CAR T lymphocyte, and / or CAR NK cell) are disclosed herein, comprising a container (such as, for example, a microcentrifuge tube) containing an effective amount of a stimulating agent (such as, for example, PHA, PMA / ionomycin, Con A, LPS and / or PWM) and a suitable buffer for immune cells. In some aspects, the kit may also include instructions for using the kit to stimulate cytokine production by an immune cell.

[0016] Also disclosed herein are kits for assessing the potency of an immune cell according to any of the above aspects, wherein the stimulating agent (such as, for example, PHA, PMA / ionomycin, Con A, LPS and / or PWM) is provided at a concentration of 1.0 g / mL to 1000 pg / mL, including, among others, a concentration of 5 pg / mL to 15 pg / mL.

[0017] In one aspect, an immunotherapy method is disclosed herein comprising a) performing the method of assessing the potency of an immune cell (such as, for example, a T lymphocyte, macrophage, NK cell, T NK lymphocyte, CAR T lymphocyte and / or CAR NK cell) according to any of the above aspects in multiple immune cells to determine the potency of each immune cell; b) selecting at least one potent immune cell based on the detected amount of cytokine (such as, for example, IL-2, IL-6, IFN-γ, TNF-α, BAFF / TNFSF13B, CD163, CD30 / TNFRSF8, chitinase-like protein 3, gp130, IFN-α2, IL-6Ra, IL-8, IL-10, IL-11, IL-12(p40), IL-12(p70), IL-20, IL-22, IL-26, IL-29 / IFN-I1, IL-32, IL-34, IL-35, MMP-1, osteocalcin, OPN, pentraxin-3, TNF-R1, TNF-R2, TSLP, GMCSF, ΜΙΡ-1α, ΜΙΡ-1β, RANTES and / or TWEAK / TNFSF12);(yc) administering a therapeutically effective quantity of the potent immune cell to a subject in need as an immunotherapeutic agent. In one aspect, the method may further comprise extracting multiple immune cells from an allogeneic or autologous donor before assessing the potency of the immune cell.

[0018] Also disclosed herein are methods of immunotherapy in accordance with any of the above aspects which further comprise expanding at least one potent immune cell before administering a therapeutically effective amount of the potent immune cell.

[0019] In one aspect, immunotherapy methods are disclosed herein in accordance with any of the foregoing aspects, further comprising directing multiple immune cells or a potent immune cell to respond to a specified antigen. In one aspect, the method may further comprise modifying the cell line from which the exosomes are derived, or modifying the exosomes themselves, to include a specified antigen to which the immune cells of interest respond (such as, for example, adding CD19 to specifically determine the potency of CD19 CAR T lymphocytes in a heterogeneous sample).

[0020] Also disclosed herein are methods of immunotherapy in accordance with any of the above aspects which further comprise genetically altering multiple immune cells or the potent immune cell to present a chimeric antigen receptor.

[0021] In one aspect, methods for treating, inhibiting, reducing, preventing, and / or improving a type of cancer and / or metastasis in a subject are disclosed herein, comprising: a) obtaining one or more immune cells (such as, for example, a T lymphocyte, macrophage, NK cell, NK T lymphocyte, CAR T lymphocyte, and / or CAR NK cell obtained from an allogeneic or autologous donor); b) bringing an immune cell into contact with an effective amount of a stimulating agent (such as, for example, PHA, PMA / ionomycin, With A, LPS and / or PWM); c) detecting the amount of a cytokine (such as, for example, IL-2, IL-6, IFN-γ, TNF-α, BAFF / TNFSF13B, CD163, CD30 / TNFRSF8, chitinase-like protein 3, gp130, IFN-α2, IL6Ra, IL-8, IL-10, IL-11, IL-12(p40), IL-12(p70), IL-20, IL-22, IL-26, IL-29 / IFN-I1, IL-32, IL-34, IL-35, MMP1, osteocalcin, OPN, pentraxin-3, TNF-R1, TNF-R2, TSLP, GM-CSF, ΜΙΡ-1α, ΜΙΡ-1β, RANTES and / or TWEAK / TNFSF12) produced by the cell immune; d) selecting at least one potent immune cell based on the amount of cytokine detected; and e) administering a therapeutically effective amount of the potent immune cell to the subject. In some respects, the method may also involve extracting the immune cell from an autologous or allogeneic donor.

[0022] Also disclosed herein are methods for treating, inhibiting, reducing, preventing and / or improving a type of cancer and / or metastasis in accordance with any of the above aspects which further comprise expanding at least one potent immune cell before administering a therapeutically effective amount of at least one potent immune cell.

[0023] Also disclosed herein are methods for determining the identity (such as, for example, differentiating Th1, Th2, Th3, Th9, Th17, memory voter T cells (Tem), central memory T cells (Tcm), γδT cells or regulatory T cells (Treg), resting NK cells, expanded NK cells) of at least one immune cell or cell population based on the cytokine signature associated with that cell type. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 provides a graph showing the correlation between tumor cell-induced K562 NK cell cytokine release and PHA-induced NK cell cytokine release (in pg / million cells / h).

[0025] Figure 2 provides a graph showing an analysis of the results as a function of incubation time.

[0026] Figure 3 provides a graph showing the analysis of the results as a function of the number of cells.

[0027] Figure 4 provides a graph showing the results of a PHA assay of NK cells expanded with mb-IL-21 and TGF-β.

[0028] Figure 5 provides a graph showing the correlation between cytokine release from newly isolated NK cells (induced by PHA) and cytokine release from expanded NK cells induced by PHA (in pg / million cells / h).

[0029] Figure 6 provides a graph showing the cytokine expression of NK cells after 1 hour of PHA stimulation in expanded (N=18) and freshly obtained (N=10) NK cells.

[0030] Figure 7 shows a comparison of the concentration of cytokines expressed by freshly obtained and expanded NK cells after stimulation with PHA.

[0031] Figure 8 provides a graph showing the specificity and sensitivity of expanded NK cell differentiation as a function of IFN-γ and IL-2 expression.

[0032] Figure 9 shows that freshly obtained NK cells and expanded NK cells can differentiate based on the upregulation of IL-2 and the downregulation of pentraxin-3 or chitinase-like protein 1 3.

[0033] Figure 10 shows that freshly obtained NK cells and expanded NK cells can differentiate based on upregulation of IFN-γ and downregulation of pentraxin-3 or chitinase-like protein 1 3. DETAILED DESCRIPTION OF THE INVENTION

[0034] The present invention provides a method for determining the potency of an immune cell, which includes exposing an immune cell to an effective amount of a stimulating agent and detecting the amount of a cytokine produced by the immune cell. Although the disclosure is provided in the context of cancer immunotherapies, the concepts and innovations disclosed herein may be applied to immunotherapies for other diseases and disorders. For example, the potency of an immune cell used in immunotherapy against autoimmune diseases, inflammatory diseases or disorders, viral diseases, and / or bacterial infections may also be assessed using the assays disclosed herein. Definitions

[0035] To aid understanding and facilitate reference, a list of terms used throughout the brief description of the invention section and the rest of the application has been compiled herein. Some of the terms are well known in the field and are defined herein for clarity, while some terms are unique to this application and therefore must be defined for a proper understanding of the application.

[0036] As used in the descriptive memorandum and claims, the singular forms “a”, “an”, “the” and “the” include plural references unless the context clearly indicates otherwise. For example, the expression “a cell” includes a plurality of cells, even mixtures of them. When the plural form is used herein, it generally includes the singular.

[0037] Ranges may be expressed herein as "around" a particular value, and / or "around" another particular value. When such a range is expressed, another realization includes "from" a particular value and / or "to" the other particular value. Similarly, when values ​​are expressed as approximations by using the antecedent "around," the particular value is understood to form another realization. It is further understood that the boundary values ​​of each range are significant both in relation to and independently of the other boundary value. References to numerical ranges by boundary values ​​include all numbers subsumed within those ranges (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.). It is also understood that several values ​​are disclosed herein, and that each value is also disclosed herein as "around" that particular value, in addition to the value itself.For example, if the value “10” is disclosed, then “around 10” is also disclosed. It is also understood that when a value is disclosed as “less than or equal to” the value, “greater than or equal to” the value is also disclosed, as well as the possible ranges between the values, as appropriately understood by a person of the mid-level trade. For example, if the value “10” is disclosed, then “less than or equal to 10” and “greater than or equal to 10” are also disclosed. It is further understood that throughout the application, data is provided in several different formats, and that this data represents threshold values, starting points, and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed, as well as between 10 and 15.It is also understood that each unit between any two particular units is disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0038] As used herein, the expression “comprising” means that the compositions and methods include the elements mentioned, but do not exclude others. The meaning of “consisting essentially of,” when used to define compositions and methods, excludes other elements of any essential importance to the combination. Therefore, a composition consisting essentially of the elements defined herein would not exclude minimal amounts of contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate-buffered saline, preservatives, and the like. “Consisting of” means that it excludes more than one trace element from other ingredients and method steps substantial to administering the compositions of this invention. The embodiments defined by each of these transitional terms are within the scope of the present invention.

[0039] An “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 of a condition, symptom, activity, or composition by a statistically significant amount. Thus, the increase can be an increase of 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%, provided the increase is statistically significant.

[0040] A “decrease” can refer to any change that results in a lesser amount of a symptom, disease, composition, condition, or activity. A substance is also understood to decrease the gene output of a gene when the gene output with the substance is less compared to the output without the substance. Also, for example, a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed. A decrease can be any individual, median, or average decrease in a condition, symptom, activity, or composition by a statistically significant amount. Therefore, the decrease can be a decrease of 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% as long as the decrease is statistically significant.

[0041] “Inhibit,” “inhibiting,” and “inhibition” mean to decrease an activity, response, condition, disease, or other biological parameter. This may include, among other things, the complete elimination of the activity, response, condition, or disease. This may also include, for example, a 10% reduction of the activity, response, condition, or disease compared to the native or control level. Thus, the reduction may be 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any intermediate amount of reduction compared to the native or control levels.

[0042] By “reduce” or other forms of the word, such as “reducing” or “reduction,” it is understood that an event or characteristic (e.g., the growth of a tumor) is lessened. It is generally understood that this is relative to some standard or expected value, but it is not always necessary to refer to the standard or relative value. For example, “reducing the growth of a tumor” means that the rate of tumor growth is reduced relative to a standard or control.

[0043] By “prevent” or other forms of the word, such as “preventing” or “prevention,” it is understood that it stops a particular event or feature, stabilizes or delays the development or progression of a particular event or feature, or minimizes the chances of a particular event or feature occurring. “Prevent” does not require comparison to a control, as it is generally more absolute than, for example, “reduce.” As used herein, something could be reduced but not prevented, but something that is reduced could also be prevented. Likewise, something could be prevented but not reduced, but something that is prevented could also be reduced. It is understood that where reduce or prevent is used, unless specifically stated otherwise, the use of the other words is also expressly disclosed.

[0044] The term “therapeutically effective” is intended to describe the amount of an active agent (such as immunotherapeutic cells) that will achieve the goal of reducing disease severity while avoiding adverse side effects such as those typically associated with alternative treatments. A therapeutically effective amount may be administered in one or more doses. Treatments that are therapeutically effective include those that improve a person's quality of life even if they do not improve the outcome of the disease itself.

[0045] An “effective amount” generally means an amount that provides the desired local or systemic effect, for example, effective in stimulating cytokine formation, including achieving the specific desired effects described in this application. For example, an effective amount is an amount sufficient to produce a beneficial or desired clinical result.

[0046] The term “subject” refers to any individual that is the target of administration or treatment. The subject may be a vertebrate, for example, a mammal. In one respect, the subject may be a human, a non-human primate, a bovine, an equine, a porcine, a canine, or a feline. The subject may be a guinea pig, rat, hamster, rabbit, mouse, or mole. Therefore, the subject may be a human or veterinary patient. The term “patient” refers to a subject treated by a physician, for example, a clinician.

[0047] The term “therapeutically acceptable carrier” means a carrier or excipient that is useful in preparing a composition that is generally safe and non-toxic, and includes a carrier that is acceptable for veterinary and / or human use. Methods of administration (e.g., more than 50% of the body), for example, by entry into the circulatory or lymphatic systems. In contrast, “local administration” refers to introducing or administering an agent to a subject by a route that introduces or delivers the agent to the area or the area immediately adjacent to the site of administration and does not introduce the agent systemically in a therapeutically significant amount. For example, locally administered agents are readily detectable in the vicinity of the site of administration but are undetectable or detectable only in negligible amounts in distant parts of the subject’s body.Administration includes self-administration and administration by another.

[0050] “Treat,” “treating,” “treatment,” and grammatical variations thereof as used herein, include the administration of a composition with the intent or purpose of preventing, delaying, curing, healing, alleviating, altering, remedying, improving, stabilizing, mitigating, and / or partially or completely reducing the intensity or frequency of one or more diseases or conditions, a symptom of a disease or condition, or an underlying cause of a disease or condition. Treatments according to the invention may be applied preventively, prophylactically, palliatively, or curatively. Prophylactic treatments are administered to a subject before onset (e.g., before obvious signs of cancer), during early onset (e.g., after initial signs and symptoms of cancer), or after established cancer development. Prophylactic administration may occur for days to years before the manifestation of symptoms of a disease or infection.

[0051] The methods and kits disclosed herein utilize a stimulating agent. As used herein, a stimulating agent may be any molecule, peptide, polypeptide, protein, lectin, and / or mitogen that can act as an antigen and / or immunogen to stimulate an immune cell to secrete cytokines. Stimulating agents include, but are not limited to, phytohemagglutinin (PHA), phorbol myristate acetate (PMA) / ionomicin, concanavalin A (Con A), lipopolysaccharide (LPS) and / or carmine herb mitogen (PWM), peanut agglutinin (PNA), wheat germ agglutinin, and ricin.

[0052] Reference is made to various publications throughout this application. The disclosures in these publications are incorporated herein by reference in their entirety to more fully describe the prior art to which this application belongs. The disclosed references are also incorporated individually and specifically by reference herein for the material contained therein that is discussed in the sentence in which the reference is used. Immune potency assay

[0053] In one aspect, the invention provides a method for determining the potency of an immune cell. The method includes the steps of bringing an immune cell into contact with an effective amount of a stimulating agent (such as, for example, PHA, PMA / ionomycin, Con A, LPS, and / or PWM) and detecting the amount of a cytokine produced by the immune cell. For example, the immune cell can be brought into contact with a stimulating agent (such as, for example, PHA, PMA / ionomycin, Con A, LPS, and / or PWM) by suspending the stimulating agent in a cellular medium and ΜλΌΖϋΖ 1 / UUM / OD expose immune cells to the cellular environment.

[0054] In some embodiments, the method includes the step of comparing the amount of cytokine produced with the level of cytokine potency required to use the immune cell in immunotherapy. A potency assay serves to characterize the product (i.e., immune cells), monitor batch-to-batch consistency, and ensure product stability. Therefore, it must be sensitive enough to detect differences that could affect the product's mechanism of action and function and thus have potential clinical significance. The assay can also be used as a predictive biomarker or a pharmacodynamic assay for cell-mediated immunotherapy. Ideally, the potency assay should have the closest possible correlation with the putative physiological / pharmacological activity of the product.The potency assay described herein provides the ability to measure potency within product specifications; high sensitivity for detecting potentially clinically significant differences; and a close relationship with the putative physiological / pharmacological activity and mechanism of action of the product. Preferably, the potency assay also meets the following secondary criteria: sufficiently low intra- and inter-assay variation (to achieve the precision required to support product specifications); sufficient robustness; and amenability to high-throughput analysis. In some embodiments, the assay is used as a clinical assay to quantify the function of T lymphocytes, macrophages, NK cells, NK T lymphocytes, CAR T lymphocytes, and / or CAR NK cells (diagnostic assessment of NK cell immunodeficiency, biomarker for monitoring immunosuppressive or immunoactivating efficacy).

[0055] As previously stated, the disclosed methods allow for the determination of the potency of an immune cell. 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 lymphocytes and T lymphocytes (including CD4 and CD8 T lymphocytes). In one respect, an immune cell may comprise a tumor-infiltrating lymphocyte (TIL), T lymphocyte, macrophage, natural killer (NK) cell, NK T lymphocyte, chimeric antigen receptor (CAR) T lymphocyte, and / or CAR NK cell. Immune cells may be obtained from cell culture or from a subject (such as, for example, an allogeneic or autologous donor).

[0056] In some embodiments, the immune cell is a T lymphocyte. T lymphocytes play a central role in cell-mediated immunity and can be distinguished from other lymphocytes, such as B lymphocytes and natural killer cells, by the presence of a T cell receptor on their cell surface. Examples of T lymphocytes include helper T lymphocytes (TH lymphocytes), cytotoxic T lymphocytes (TC lymphocytes), memory T lymphocytes, regulatory or “inhibitory” T lymphocytes, and natural killer T lymphocytes (NKT lymphocytes, which are different from NK cells and recognize a glycolipid antigen rather than peptides presented by the MHC molecule. Different types of T lymphocytes differ from one another in their cytokine production pattern). T lymphocytes can be either CD4+ or CD8+ T lymphocytes. In addition, T lymphocytes may comprise chimeric antigen receptor (CAR) T lymphocytes or tumor-infiltrating lymphocytes (TILs).

[0057] In some embodiments, the immune cell is an NK cell. Natural killer cells are a type of cytotoxic lymphocyte of the immune system. NK cells provide rapid responses to virally infected cells and respond to transformed cells. Typically, immune cells detect pathogen peptides presented by major histocompatibility complex (MHC) molecules on the surface of infected cells, triggering the release of cytokines, which leads to lysis or apoptosis. However, NK cells are unique in that they have the ability to recognize stressed cells regardless of whether pathogen peptides are present on MHC molecules. They were termed “natural killers” because of the initial notion that they do not require prior activation to inactivate the target.NK cells are large granular lymphocytes (LGLs) and are known to differentiate and mature in the bone marrow, from where they then enter the circulation. In some respects, an NK cell can be a CAR NK cell.

[0058] Therefore, in one aspect, methods for assessing the potency of an immune cell (such as, for example, a T lymphocyte, macrophage, NK cell, T NK lymphocyte, CAR T lymphocyte, and / or CAR NK cell) are disclosed herein. These methods comprise exposing an immune cell to an effective amount of a stimulating agent (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 with the level of cytokine potency required for the use of the immune cell in immunotherapy.

[0059] The assay includes the step of detecting the amount of a cytokine produced by the immune cell after the immune cells are stimulated with the stimulating agent. As used herein, the term “cytokine” refers to a small protein (-5-20 kDa) that is important in cell signaling and, in particular, immunomodulation, which can be produced by an immune cell. Examples of cytokines include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors. The cytokines detected may include cytokines known to be produced by the immune cells under evaluation, or the detection may encompass a broader range of cytokines, including cytokines not known to be produced by the immune cells.

[0060] In some embodiments, the detected cytokines include cytokines known to be produced by T lymphocytes or natural killer cells. In some embodiments, the cytokines include those known to be produced by T lymphocytes. T lymphocytes include Th1 and Th2 lymphocytes; Th1 lymphocytes predominantly produce interferon (IFN)-γ, tumor necrosis factor (TNF)-γ, and IL-2; Th2 lymphocytes produce interleukin (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-1, 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 macrophage inflammatory protein (MIP)-1α (MIRP-1α), MIRP-1β, and RANTES chemokines.Other cytokines useful for determining the potency of an immune cell include, among others, B-cell activating factor / tumor necrosis factor (TNF) ligand superfamily member 13B (BAFF / TNFSF13B), cluster of differentiation (CD) 163 (CD163), CD30 / TNFRSF8, chitinase-like protein 1 3, gp130, IFN-α2, IL6Ra, IL-11, IL-12(p40), IL-12(p70), IL-20, IL-26, IL-29 / IFN41, 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, and lymphopoietin. thymic stromal (TSLP), or TNF-related weak inducer of apoptosis (TWEAK) / TNF superfamily member 12 (TWEAK / TNFSF12).Therefore, in one aspect, methods for assessing the potency of an immune cell (such as, for example, a T lymphocyte, macrophage, NK cell, T NK lymphocyte, CAR T lymphocyte, and / or CAR NK cell) are disclosed herein. These methods comprise bringing an immune cell into contact with an effective amount of a stimulating agent (such as, for example, phytohemagglutinin (PHA), phorbol myristate acetate (PMA) / ionomicin, concanavalin A (Con A), lipopolysaccharide (LPS), and / or carmine herb mitogen (PWM)) and detecting the amount of one or more cytokines (such as, for example, IL-2, IL-6, IFN-γ, TNF-α, BAFF / TNFSF13B, CD163, CD30 / TNFRSF8, chitinase-like protein 1 3, gp130, IFN-α2, IL-6Ra, IL-8, IL-10, IL-11, IL-12(p40), IL-12(p70), IL-20, IL-22, IL-26, IL-29 / IFN-I1, IL-32, IL-34, IL-35, MMP-1, osteocalcin, OPN, pentraxin-3, TNF-R1, TNF-R2, TSLP, GM-CSF, LIF, ΜΙΡ-1α, ΜΙΡ-1β, RANTES and / or TWEAK / TNFSF12) produced by the immune cell.Methods for assessing the potency of an immune cell (such as, for example, a T lymphocyte, macrophage, NK cell, T NK lymphocyte, CAR T lymphocyte, and / or CAR NK cell) are disclosed herein, comprising exposing an immune cell to an effective amount of a stimulating agent (such as, for example, PHA, PMA / ionomycin, Con A, LPS, and / or PWM) and detecting the amount of one or more cytokines (such as, for example, IL-2, IL-6, IFN-γ, TNFα, BAFF / TNFSF13B, CD163, CD30 / TNFRSF8, chitinase-like protein 1 3, gp130, IFN-α2, IL-6Ra, IL-8, IL-10, IL-11, IL-12(p40), IL-12(p70), IL-20, IL-22, IL-26, IL-29 / IFN-I1, IL-32, IL-34, IL-35, MMP-1, osteocalcin, OPN, pentraxin-3, TNF-R1, TNF-R2, TSLP, GM-CSF, ΜΙΡ-1α, ΜΙΡ-1β, RANTES and / or TWEAK / TNFSF12) produced by the immune cell.In one aspect, the method may further involve comparing the amount of cytokine produced with the level of cytokine potency required for use in immunotherapy with the immune cell. In some embodiments, the levels of multiple cytokines are determined. In additional embodiments, the cytokine is selected from the group consisting of interleukin-2, interleukin-6, and interferon-gamma.

[0061] The assay includes the step of detecting the amount of a cytokine produced by the immune cell. People of intermediate skill are familiar with a wide variety of methods for detecting cytokines, which can vary depending on the cytokine being detected. In some embodiments, methods can be used to detect and / or quantify the presence of multiple different cytokines. For example, cytokines can be detected using specific immunoassays or reagent kits. Cytokines can be detected using kits available from commercial suppliers such as Miltenyi Biotec™, Luminex, and Thermo Fisher Scientific™. Examples of suitable kits 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 lymphocytes or NK cells, both marketed by Miltenyi Biotec™.

[0062] In some embodiments, the amount of cytokine is detected by an immunoassay. Immunoassays have many different formats and variations. They can be multi-stage, involving the addition, removal by washing, or separation of reagents at different points in the assay. Immunoassays include heterogeneous immunoassays, which involve multiple stages, and homogeneous immunoassays, which simply involve mixing the reagents and sample and performing a physical measurement. Immunoassays often use a calibrator, which is a solution known to contain the analyte in question, and the concentration of that analyte is usually known. Comparing the assay response to an actual sample with the assay response produced by calibrators allows the signal strength to be interpreted in terms of the presence or concentration of the analyte in the sample.The types of immunoassays include homogeneous competitive immunoassays, heterogeneous competitive 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 assays (ELIspots), flow cytometry, intracellular cytokine staining, antibody array assays and microbead assays, magnetic immunoassays, radioimmunoassays, and quantitative PCR (including, but not limited to, qRT-PCR). In one aspect, the assay comprises a Luminex xMAP®.

[0063] The method for determining the potency of an immune cell includes the step of putting an immune cell in contact with an effective amount of a stimulating agent (such as, for example, PHA, PMA / ionomycin, Con A, LPS and / or PWM).

[0064] It is understood and contemplated herein that immune cells must be exposed to the stimulating agent for a period of time to induce cytokine production. In one aspect, methods for evaluating the potency of an immune cell are disclosed herein, wherein the immune cell is brought into contact with an effective amount of a stimulating agent (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, 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, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 45, 60, 61, 62 days, 3, 4, 5 or 6 months.

[0065] Also disclosed herein are methods for assessing the potency of an immune cell according to any of the above aspects, wherein the stimulating agent (such as, for example, PHA, PMA / ionomycin, Con A, LPS and / or PWM) is provided at a concentration of 5 pg / mL to 1000 pg / mL. In one aspect, 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 pg / mL. In one aspect, the concentration of the stimulating agent is around 1 pg / mL to 100 pg / mL, 1 pg / mL to 50 pg / mL, 1 pg / mL to 15 pg / mL or 5 pg / mL to 15 pg / mL.

[0066] In one respect, it is understood and contemplated herein that the same cytokines produced to determine the potency of an immune cell can also be used to identify the cells that produce the cytokines. Immune cells have distinct expression profiles that are known in the art. Methods for determining the identity of at least one immune cell or cell population (such as, for example, differentiating Th1, Th2, Th3, Th9, Th17, effector memory T cells (Tem), central memory T cells (Tcm), γδT cells or regulatory T cells (Treg), resting NK cells, and expanded NK cells) are also disclosed herein.Accordingly, methods for identifying an immune cell (such as, for example, a T lymphocyte, macrophage, NK cell, NK T lymphocyte, CAR T lymphocyte, and / or CAR NK cell) are disclosed herein, comprising exposing an immune cell to an effective amount of a stimulating agent (such as, for example, PHA, PMA / ionomycin, Con A, LPS, and / or PWM) and detecting the amount of one or more cytokines (such as, for example, IL-2, IL-6, IFN-γ, TNF-α, BAFF / TNFSF13B, CD163, CD30 / TNFRSF8, chitinase-like protein 1 3, gp130, IFNa2, IL-6Ra, IL-8, IL-10, IL-11, IL-12(p40), IL-12(p70), IL-20, IL-22, IL-26, IL-29 / IFN-I1, IL-32, IL-34, IL-35, MMP-1, osteocalcin, OPN, pentraxin-3, TNF-R1, TNF-R2, TSLP, GM-CSF, MIP-1α, MIP-1 β, RANTES and / or TWEAK / TNFSF12) produced by the immune cell; wherein the identity of the immune cell is revealed based on the profile of the expressed cytokines. Kits for evaluating the potency of immune cells

[0067] Another aspect of the invention provides a kit for determining the potency of an immune cell (such as, for example, a T lymphocyte, macrophage, NK cell, T NK lymphocyte, CAR T lymphocyte, and / or CAR NK cell), comprising a container that includes an effective amount of a stimulating agent (such as, for example, PHA, PMA / ionomycin, Con A, LPS, and / or PWM) and a buffer suitable for immune cells. In some embodiments, the stimulating agent in the kit is provided at a concentration of 5 pg / mL to 1000 pg / mL. In one aspect, 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 pg / mL.In one aspect, the concentration of the stimulating agent is approximately 1 pg / mL to 100 pg / mL, 1 pg / mL to 50 pg / mL, 1 pg / mL to 15 pg / mL, or 5 pg / mL to 15 pg / mL. In some embodiments, the container is a microcentrifuge tube (such as, for example, an Eppendorf microcentrifuge tube). The kits may 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.

[0068] The kits may also include the components necessary to perform an immunoassay, such as a solid phase to which antibodies functioning as capture and / or detection antibodies are attached in a sandwich immunoassay format. The solid phase may be a material such as a magnetic particle, microsphere, test tube, microtiter plate, cuvette, membrane, scaffold molecule, quartz crystal, film, filter paper, disk, or chip. The kit may also include a detectable label that may be on or conjugated to an antibody, such as an antibody functioning as a detection antibody. The detectable label may be, for example, a direct label, which may be an enzyme, oligonucleotide, nanoparticle chemiluminescent agent, fluorophore, fluorescence inhibitor, chemiluminescence inhibitor, or biotin.Test kits may optionally include any additional reagents needed to detect the label.

[0069] The kit may also include instructions for using the kit to stimulate cytokine production from an immune cell in order to assess the potency of the immune cell. In some embodiments, the kit also includes instructions for using the amount of cytokine to determine the potency of the cell. The instructions included in kits may be affixed to the packaging material or included as a package insert. While instructions are typically written or printed materials, they are not limited to these. This disclosure covers any medium capable of storing such instructions and communicating them to an end user. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, tapes, cartridges, chips), optical media (e.g., CD-ROMs), and the like. As used herein, the term “instructions” may include the address of a website that provides the instructions. Immunotherapy methods

[0070] The method for determining the potency of an immune cell can be performed prior to the use of the immune cell as an immunotherapeutic agent. For example, the method for determining the potency of one or multiple immune cells can be performed as described above, after which at least one potent immune cell can be selected (based on the amount of cytokine detected) and a therapeutically effective amount of the potent immune cell can be administered to a subject as an immunotherapeutic agent. Accordingly, in one aspect, immunotherapy methods comprising a) performing the method of assessing the potency of an immune cell (such as, for example, a T lymphocyte, macrophage, NK cell, T NK lymphocyte, CAR T lymphocyte, and / or CAR NK cell) as disclosed herein on multiple immune cells to determine the potency of each immune cell;b) selecting at least one potent immune cell based on the detected amount of cytokine (such as, for example, IL-2, IL-6, IFNa, TNF-a, BAFF / TNFSF13B, CD163, CD30 / TNFRSF8, chitinase 3-like protein 1, gp130, IFN-a2, IL6Ra, IL-8, IL-10, IL-11, IL-12(p40), IL-12(p70), IL-20, IL-22, IL-26, IL-29 / IFN-I1, IL-32, IL-34, IL-35, MMP1, osteocalcin, OPN, pentraxin-3, TNF-R1, TNF-R2, TSLP, GM-CSF, ΜΙΡ-1α, ΜΙΡ-1β, RANTES and / or TWEAK / TNFSF12); and c) administering a therapeutically effective quantity of the potent immune cell to a subject in need as an immunotherapeutic agent. In one aspect, the method may further comprise extracting multiple immune cells from an allogeneic or autologous donor prior to assessing the potency of the immune cell.

[0071] In some embodiments, the immune cells are immunotherapeutic immune cells. Immunotherapeutic immune cells are those that are useful for the treatment of diseases such as cancer. Becker et al., Cancer Immunol. Immunother 65, 477-484 (2016). The use of expanded NK cells for cancer treatment has been described. Rezvani et al., Front Immunol., 6, 578 (2015). Because it is useful to be able to administer large quantities of immune cells during immunotherapy, in some embodiments the immune cells are expanded immune cells. Expanded immune cells are those that are cultured ex vivo to develop a large number of immune cells. In some embodiments, the expanded immune cells are autologous cells that can be readily administered to a subject without eliciting an immune response.However, in some embodiments, expanded immune cells are allogeneic immune cells, in which their inherent alloreactivity may be beneficial. In further embodiments, expanded immune cells are genetically engineered to include chimeric antigen receptors to help the immune cells target diseased tissue. Preparation of expanded immune cells involves activating and expanding the 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 being evaluated are IL-21-expanded NK cells.Accordingly, in one aspect, immunotherapy methods are disclosed herein, which further comprise expanding at least one potent immune cell before administering a therapeutically effective amount of the potent immune cell.

[0072] Expansion refers to the ex vivo proliferation of NK cells, thereby increasing the NK cell population. NK cells can be expanded, for example, from peripheral blood mononuclear cells. However, NK cells can also be expanded from other cell types, such as hematopoietic stem cells or progenitor cells. Blood cells or initial 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 takes place in a cell culture medium. People of intermediate skill are familiar with suitable cell culture media. The expanded cells can be provided as a cell line, which is a plurality of cells that can be maintained in cell culture.Therefore, in one respect, immunotherapy methods are disclosed herein, which further comprise expanding at least one potent immune cell before administering a therapeutically effective amount of the potent immune cell. In some respects, the immune cell has been extracted from a subject using known methods before performing the method to determine the potency of the immune cell. Alternatively, the immune cell may be obtained from the expansion of a cell culture.

[0073] In some aspects, an immune cell is directed to respond to a specific antigen (e.g., CD19). The immune cell may be directed to respond before or after the method used to determine its potency. In some embodiments, the immune cell is genetically engineered to respond to a specific antigen. The antigen may be a tumor-specific antigen, for example. In some aspects, immunotherapy methods include genetically altering immune cells to present a chimeric antigen receptor (either before or after determining the potency of the immune cell).In one aspect, the method may further comprise the modification of the cell line from which the exosomes are derived, or the modification of the exosomes themselves, to include a specified antigen to which the immune cells of interest respond (such as, for example, the addition of CD19 to specifically determine the potency of CAR CD19 T lymphocytes in a heterogeneous sample).

[0074] As discussed throughout, the method for determining the potency of an immune cell can be used as part of an adoptive cell transfer therapy. The potent immune cell can be administered to a subject via a therapeutically acceptable carrier. Intravenous administration is conventionally used for administering immunotherapeutic cells, but other methods (such as direct transplantation to a localized area of ​​the body requiring immunotherapy) may also be considered.

[0075] The therapeutically effective amount can be determined by comparing the amount of cytokine produced by the immune cell with the level of cytokine potency required for the use of the immune cell in immunotherapy. It is understood and contemplated herein that the therapeutically effective amount depends on the immune cell being administered, the subject being treated, and the disease, disorder, and / or condition being treated. Persons of intermediate skill will know the appropriate dose of immune cells to use that will be therapeutically effective for the subject being treated.

[0076] A therapeutically effective quantity of a potent immune cell comprises a plurality of potent immune cells. For example, after selecting at least one potent immune cell, the selected cell can be expanded in vitro to produce a plurality of potent immune cells.

[0077] The subject receiving the potent immune cells may be any subject who would benefit from immunotherapy (such as, for example, a subject with an autoimmune disease, inflammatory diseases or disorders, viral diseases, and / or bacterial infections). In some embodiments, the subject may be a cancer patient. In some embodiments, the subject may be an individual at high risk of developing cancer, diagnosed with cancer, undergoing cancer treatment, or recovering from cancer after surgery. In some embodiments, the potent immune cells may be administered to a subject as a prophylactic agent to prevent, inhibit, or delay the onset of cancer or metastasis. Methods for treating a disease

[0078] It is understood and contemplated herein that the potent immune cells identified herein may be used in the treatment of any disease or disorder for which adoptive immunotherapy could be used for treatment, including, but not limited to, autoimmune diseases, inflammatory diseases or disorders, viral diseases, and / or bacterial infections. Accordingly, in one aspect, methods for treating, inhibiting, reducing, preventing, and / or improving a type of cancer and / or metastasis in a subject are disclosed herein, comprising: a) obtaining one or more immune cells (such as, for example, a T lymphocyte, macrophage, NK cell, NK T lymphocyte, CAR T lymphocyte, and / or CAR NK cell obtained from an allogeneic or autologous donor); b) exposing an immune cell to an effective amount of a stimulating agent (such as, for example, PHA, PMA / ionomycin, Con A, LPS, and / or PWM);c) detecting the amount of a cytokine (such as, for example, IL2, IL-6, IFN-γ, TNF-α, BAFF / TNFSF13B, CD163, CD30 / TNFRSF8, chitinase-like protein 3, gp130, IFN-α2, IL-6Ra, IL-8, IL-10, IL-11, IL-12(p40), IL-12(p70), IL-20, IL-22, IL-26, IL-29 / IFN-I1, IL-32, IL-34, IL35, MMP-1, osteocalcin, OPN, pentraxin-3, TNF-R1, TNF-R2, TSLP, GM-CSF, ΜΙΡ-1α, ΜΙΡ-1β, RANTES and / or TWEAK / TNFSF12) produced by the cell immune; d) selecting at least one potent immune cell based on the amount of cytokine detected; e) administering a therapeutically effective amount of the potent immune cell to the subject. In some respects, the method may further involve extracting the immune cell from an autologous or allogeneic donor.

[0079] It is understood and contemplated herein that it is useful to be able to administer large quantities of immune cells during immunotherapy. In some embodiments, the immune cells are expanded immune cells. Expanded immune cells are those that are cultured ex vivo to develop a large number of immune cells. Accordingly, methods for treating, inhibiting, reducing, preventing, and / or improving an autoimmune disease, inflammatory disease or disorder, viral disease, bacterial infection, type of cancer, and / or metastasis are disclosed herein, which further comprise expanding at least one potent immune cell before administering a therapeutically effective quantity of that at least one potent immune cell.

[0080] It is understood and contemplated herein that the disclosed treatment methods may be used to treat any disease or condition in which uncontrolled cell proliferation occurs, including, but not limited to, cancer and metastasis. A representative but not exhaustive list of cancer types for which the disclosed methods of using potent immune cells may be used as treatment is as follows: lymphoma, B-cell lymphoma, T-cell lymphoma, mycosis fungoides, Hodgkin's disease, myeloid leukemia, bladder cancer, brain cancer, cancer of the nervous system, head and neck cancer, squamous cell carcinoma of the head and neck, types of 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 carcinomas of the mouth, throat, larynx, and lung,Cervical cancer, breast cancer and epithelial cancer, kidney cancer, genitourinary cancer, lung cancer, esophageal carcinoma, head and neck carcinoma, colon cancer, hematopoietic cancers; testicular cancer; colon cancer, rectal cancer, prostate cancer or pancreatic cancer.

[0081] Examples of autoimmune diseases that can be treated with the disclosed methods include, but are not limited to, achalasia, acute disseminated encephalomyelitis, acute motor axonal neuropathy, Addison's disease, adiposis dolorosa, adult-onset Still's disease, agammaglobulinemia, alopecia areata, Alzheimer's disease, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome, aplastic anemia, autoimmune angioedema, autoimmune dysautonomia, 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, and autoimmune urticaria. axonal and neuronal neuropathy (AMAN), Baló's disease, Behcet's disease, benign mucosal pemphigoid,Bickerstaff encephalitis, bullous pemphigoid, Castleman disease (CD), celiac disease, Chagas disease, chronic fatigue syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic recurrent multifocal osteomyelitis (CRMO), Churg-Strauss syndrome (CSS), eosinophilic granulomatosis (EGPA), cicatricial pemphigoid, Cogan syndrome, cryoagglutinin disease, congenital heart block, Coxsackie myocarditis, CREST syndrome, Crohn's disease, dermatitis herpetiformis, dermatomyositis, Devic's disease (neuromyelitis optica), type 1 diabetes mellitus, discoid lupus, Dressler's syndrome, endometriosis, eosinophilic esophagitis (EoE), eosinophilic fasciitis, erythema nodosum, mixed essential cryoglobulinemia, Evans syndrome, Felty's syndrome, fibromyalgia, alveolitis fibrosing, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture syndrome,granulomatosis with polyangiitis, Graves' disease, Guillain-Barré syndrome, Hashimoto's encephalopathy, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schönlein purpura (HSP), gestational herpes or gestational pemphigoid (PG), hidradenitis suppurativa (HS) (acne inverso), 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 (type 1 diabetes), juvenile myositis (JM), Kawasaki disease, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, woody conjunctivitis, linear IgA disease (LAD), lupus nephritis, lupus vasculitis, disease of Chronic Lyme disease, Meniere's disease, microscopic polyangiitis (MPA), mixed connective tissue disease (MCTD), Mooren's ulcer, Mucha-Habermann disease,multifocal motor neuropathy (MMN) or MMNCB, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neonatal lupus, neuromyelitis optica, neutropenia, ocular cicatricial pemphigoid, optic neuritis, Ord's thyroiditis, palindromic rheumatism (PR), PANDAS, paraneoplastic 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 syndromes type I, II, III, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, dermatitis due to progesterone, 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, rheumatoid vasculitis, sarcodosis, Schmidt's syndrome, Schnitzler's syndrome, scleritis, scleroderma, Sjögren's syndrome, spermatic testicular autoimmunity, stiff person syndrome (SPS), subacute bacterial endocarditis (SBE), Susac's syndrome, Sydenham's chorea, sympathetic ophthalmia (SO), systemic lupus erythematosus, systemic scleroderma, Takayasu arteritis, temporal arteritis / giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome (THS), transverse myelitis, type 1 diabetes, ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), urticaria, urticarial vasculitis, uveitis, vasculitis, vitiligo, disease of Vogt-Koyanagi-Harada and Wegener's granulomatosis (or granulomatosis with polyangiitis (GPA)).,

[0082] The following example is included to demonstrate preferred embodiments of the invention. Persons of the middle trade will understand that the techniques disclosed in the following examples represent techniques that the inventor has found to work well in carrying out the invention and, therefore, may be considered preferred modes of its implementation. However, persons of the middle trade will observe, in light of this disclosure, that many changes can be made to the specific embodiments described and still achieve a similar or like result without departing from the spirit and scope of the invention. EXAMPLE

[0083] The assays disclosed herein are intended to evaluate the potency of therapeutic immune cells, addressing existing problems with current standard methods and meeting FDA requirements. To achieve this, phytohemagglutinin (PHA) is used as a surrogate to induce cytokine production in immune cells. PHA is used in clinical settings to evaluate the immune response in transplant patients. Inducing cytokine production in therapeutic immune cells using PHA would eliminate all biological and batch-to-batch variability from the immune cell potency assay. The assay would eliminate the need for a fully operational research laboratory to evaluate the potency of therapeutic immune cells at multiple clinical infusion sites and would provide a faster turnaround time for such testing.It would provide a method for evaluating the effector function of therapeutic immune cells as part of a potency testing requirement established by the FDA. Evaluation of the potency of therapeutic immune cells using PHA as a stimulating agent

[0084] The use of tumor cells to assess the effector function of therapeutic immune cells has been standard practice. These target cells add biological variability to the assay results. Furthermore, setting up this assay is more tedious and complicated, and it adds variability related to the target condition, plate configuration, and person-to-person differences in techniques. The therapeutic response of NK cells is assessed by comparing a PHA cytokine assay with a widely used tumor cell-mediated cytokine assay (K562). Cytokine levels indicate the potency of the NK cell response.

[0085] Figure 1 shows the correlation of the PHA-induced cytokine assay with the standard K562-induced cytokine assay. The outliers in Figure 1 are a result of the variability introduced by the use of tumor cells. Increased GM-CSF secretion was detected in the tumor cell-mediated assay (GC-CSF is generally secreted by tumor cells). This demonstrated a clear advantage of the PHA assay due to the lack of variability introduced by tumor cells.

[0086] Figure 2 shows a histogram of cytokine levels achieved using a four-hour incubation compared to a 24-hour incubation (with 1 million NK cells). Figure 3 shows a histogram of cytokine levels achieved using fewer than 1 million cells and more than 1 million cells. These conditions were evaluated 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 the cytokine profile between donor immune cells and expanded therapeutic NK cell products when induced by PHA.

[0087] After establishing an optimal number of cells (10⁶) for the assay, multiple donors of fresh peripheral blood NK cells (N=10) and expanded NK cells (N=18) were stimulated with PHA to produce cytokines, and cytokine expression in the supernatant was assessed after 1 hour. The expression of most cytokines was similar between the two NK cell types, but several cytokines and chemokines related to NK cell function were expressed with high differentiation in response to stimulation (Figure 6). The expression of 6 cytokines in fresh and expanded NK cells was then measured after 1 hour of PHA stimulation.Pentraxin-3 (mean 1,062 compared to 7), IL-8 (mean 821 compared to 11) and chitinase-like protein 1 3 (mean 620 compared to 7) were highly overexpressed in fresh NK cells compared to expanded NK cells, and IFN-γ (mean 15 compared to 105), IL-2 (mean 3 compared to 141) and CD30 (mean 9 compared to 156) were highly overexpressed in expanded NK cells compared to fresh NK cells (Figure 7). Based solely on overexpression, an expression cutoff of 10 pg / mL of both IFN-γ and IL-2 has 94% specificity (16 of 17) and 89% sensitivity (16 of 18) for differentiating between expanded NK cells and fresh peripheral blood NK cells (Figure 8).The use of IL-2 (upregulated) in conjunction with a downregulated cytokine (pentraxin-3 or chitinase-like protein 1 3) was 100% sensitive and specific for differentiating between expanded NK cells and fresh peripheral blood NK cells with expression ratios (downregulation:IL-2) >1 in fresh cells and <1 in expanded cells (Figure 9). The use of IFNγ (upregulated) in conjunction with a downregulated cytokine (pentraxin-3 or chitinase-like protein 1 3) was 95% effective for differentiating between expanded NK cells and fresh peripheral blood NK cells. Combinations of multiple upregulated and downregulated cytokines could be used to increase sensitivity and specificity for defined cell types (Figure 10).

[0088] Unless otherwise defined, all technical and scientific terms used herein have the same meanings commonly understood by a person of average skill in the trade to which the disclosed invention pertains. Publications cited herein and materials from which they are cited are specifically incorporated by reference. However, it should be appreciated that any patent, publication, or other disclosure material, in whole or in part, 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, assertions, or other disclosure material set forth in this disclosure. As such, and to the extent necessary, the disclosure as explicitly set forth herein supersedes any conflicting material incorporated herein by reference.Any material, or part thereof, that is said to be incorporated herein by reference, but which conflicts with the definitions, statements, or other disclosure material indicated herein, shall be incorporated only to the extent that no conflict arises between the incorporated material and the existing disclosure material.

[0089] Persons of average skill will recognize, or be able to determine simply through routine experimentation, many equivalents to the specific embodiments of the invention described herein. Although the invention has been described with reference to particular embodiments and implementations, it is understood that various further changes and variations may be made and that equivalents may be substituted for elements thereof without departing from the scope of the invention or its inventive concept. Furthermore, many modifications may be made to adapt a particular situation or device to the teachings of the invention without departing from its essential scope. It is intended that such equivalents are covered by the following claims.It is intended that the invention shall not be limited to the particular implementations disclosed herein, but that the invention shall include all implementations that fall within the scope of the appended claims.

Claims

1. A method for assessing the potency of an immune cell, comprising placing an immune cell in contact with an effective amount of a stimulating agent and detecting the amount of a cytokine produced by the immune cell.

2. The method according to claim 1, further comprising the step of comparing the amount of cytokine produced with the level of cytokine potency required to use the immune cell in immunotherapy.

3. The method according to any of claims 1-3, wherein the amount of a plurality of cytokines is determined.

4. The method according to any of claims 1-4, wherein the immune cell is a T lymphocyte, macrophage, natural killer (NK) cell, NK T lymphocyte, chimeric antigen receptor (CAR) T lymphocyte, or CAR NK cell.

5. The method according to claim 4, wherein the immune cell is an NK cell.

6. The method according to claim 5, wherein the NK cell is an IL-21 expanded NK cell.

7. The method according to any of claims 1-6, wherein the stimulating agent comprises phytohemagglutinin (PHA), phorbol myristate acetate (PMA) / ilonomicin, concanavalin A (Con A), lipopolysaccharide (LPS) and / or carmine herb mitogen (PWM).

8. The method according to any of claims 1-7, wherein the amount of cytokine is detected by an immunoassay.

9. The method of any one of claims 1 -8, wherein the cytokine is selected from the group comprising interleukin (IL)-2 (IL-2), IL-6, interferon (IFN)-y (IFN-γ), B lymphocyte activating factor / member 13B of the tumor necrosis factor ligand superfamily (TNF) (BAFF / TNFSF13B), TNF-α, cluster of differentiation (CD) 163 (CD163), CD30 / TNFRSF8, chitinase-like protein 13, 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-I1, IL-32, IL-34, IL-35, matrix metalloproteinase-1 (MMP-1), osteocalcin, osteopontin (OPN), pentraxin-3, tumor necrosis factor receptor (TNF) (TNF-R1), TNF-R2, thymic stromal lymphopoietin (TSLP), granulocyte and macrophage colony-stimulating factor (GM-CSF), leukemia inhibitory factor (LIF) and macrophage inflammatory protein (MIP)-1α (ΜΙΙ-1α), MIP-1β,RANTES and / or TNF-related weak inducer of apoptosis (TWEAK) / TNF superfamily member 12 (TWEAK / TNFSF12).

9. The method according to claim 1, wherein the immune cell is brought into contact with an effective amount of the stimulating agent for at least 4 hours.

10. The method according to claim 1, wherein the stimulating agent is provided at a concentration of 5 pg / mL to 15 pg / mL.

11. A kit for assessing the potency of an immune cell, comprising a container that includes an effective amount of a stimulating agent and a buffer suitable for immune cells.

12. The kit according to claim 11, wherein the stimulating agent is provided at a concentration of 5 pg / mL to 15 pg / mL.

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

14. The kit according to claim 11, wherein the kit further comprises instructions for using the kit to stimulate the production of cytokines from an immune cell.

15. An immunotherapy method comprising: a. performing the method according to any of claims 1-14 on multiple immune cells to determine the potency of each immune cell; b. selecting at least one potent immune cell based on the amount of cytokine detected; and c. administering a therapeutically effective amount of the potent immune cell to a subject in need as an immunotherapeutic agent.

16. The immunotherapy method according to claim 15, further comprising extracting multiple immune cells from an allogeneic or autologous donor prior to evaluating immune cell potency.

17. The immunotherapy method according to claim 15, further comprising expanding at least one potent immune cell before administering a therapeutically effective amount of the potent immune cell.

18. The immunotherapy method according to claim 15, further comprising directing multiple immune cells or the potent immune cell to respond to a specified antigen.

19. The immunotherapy method according to claim 18, further comprising genetically altering multiple immune cells or the potent immune cell to present a chimeric antigen receptor.

20. A method for treating, inhibiting, reducing, preventing and / or improving a type of cancer and / or metastasis in a subject comprising: a. obtaining one or more immune cells; b. exposing an immune cell to an effective amount of stimulating agent; c. detecting the amount of a cytokine produced by the immune cell; d. selecting at least one potent immune cell based on the amount of cytokine detected; and e. administering a therapeutically effective amount of the potent immune cell to the subject.

21. The method for treating, inhibiting, reducing, preventing and / or improving a type of cancer and / or metastasis in a subject according to claim 20, wherein the one or more immune cells are obtained from an allogeneic or autologous donor.

22. The method for treating, inhibiting, reducing, preventing and / or improving a type of cancer and / or 5 metastases in a subject according to claim 20, further comprising extracting multiple immune cells from an allogeneic or autologous donor.

23. The method for treating, inhibiting, reducing, preventing and / or improving a type of cancer and / or metastasis in a subject according to any of claims 20-22, wherein the immune cell is a T lymphocyte, macrophage, natural killer (NK) cell, NK T lymphocyte, chimeric antigen receptor (CAR) T lymphocyte or CAR NK cell.

24. The method for treating, inhibiting, reducing, preventing and / or improving a type of cancer and / or metastasis in a subject according to any of claims 20-23, further comprising expanding at least one potent immune cell prior to administering a therapeutically effective amount of the at least one potent immune cell.