阅读说明：本技术 刺激剂用于测定免疫细胞效力的用途 (Use of stimulators for determining the efficacy of immune cells ) 是由 D·A·李 A·塔卡 M·霍尔 J·穆辛斯基 于 2020-02-14 设计创作，主要内容包括：描述了一种测定免疫细胞的效力的方法。所述方法包含使免疫细胞与有效量的刺激剂(例如,植物凝集素(PHA))接触并检测由所述免疫细胞产生的细胞因子的量的步骤。还描述了用于测定免疫细胞效力的试剂盒。效力测定对于满足针对如免疫治疗性细胞等新型生物药剂的FDA要求来说非常重要。描述了使用强效免疫细胞作为免疫治疗性治疗的方法。(A method of determining the efficacy of an immune cell is described. The method comprises the steps of contacting an immune cell with an effective amount of a stimulating agent (e.g., Phytohemagglutinin (PHA)) and detecting the amount of cytokine produced by the immune cell. Kits for determining the efficacy of immune cells are also described. Potency assays are important to meet FDA requirements for new biopharmaceuticals such as immunotherapeutic cells. Methods of using potent immune cells as immunotherapeutic treatments are described.)

1. A method of determining the efficacy of an immune cell, the method comprising contacting an immune cell with an effective amount of a stimulating agent and detecting the amount of cytokine produced by the immune cell.

2. The method of claim 1, further comprising: a step of comparing the amount of cytokine produced with the level of cytokine potency required for using the immune cells in immunotherapy.

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

4. The method of any one of claims 1-4, wherein the immune cell is a T cell, a macrophage, a Natural Killer (NK) cell, an NK T cell, a Chimeric Antigen Receptor (CAR) T cell, or a CAR NK cell.

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

6. The method of claim 5, wherein the NK cells are IL-21 expanded NK cells.

7. The method of any one of claims 1 to 6, wherein the stimulant comprises Phytohemagglutinin (PHA), Phorbol Myristate Acetate (PMA)/ionomycin, concanavalin A (Con A), Lipopolysaccharide (LPS), and/or pokeweed mitogen (PWM).

8. The method of any one of claims 1 to 7, wherein the amount of cytokine is detected using an immunoassay.

9. The method of any one of claims 1 to 8, wherein the cytokine is selected from the group comprising: 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 protein 1(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-1 (MMP-1), osteocalcin, Osteopontin (Ostepontin, OPN), Pentraxin-3 (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 the chemokines Macrophage Inflammatory Protein (MIP) -1 alpha (MIP-1 alpha), MIP-1 beta, RANTES, and/or TNF-related weak apoptosis-inducing factor (TWEAK)/TNF superfamily member 12(TWEAK/TNFSF 12).

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

11. The method of claim 1, wherein the stimulating agent is provided at a concentration of 5 μ g/mL to 15 μ g/mL.

12. A kit for determining the potency of an immune cell, the kit comprising a container comprising an effective amount of a stimulating agent and a buffer suitable for an immune cell.

13. The kit of claim 11, wherein the stimulant is provided at a concentration of 5 μ g/mL to 15 μ g/mL.

14. The kit of claim 11, wherein the container is an Eppendorf microcentrifuge tube.

15. The kit of claim 11, wherein the kit further comprises instructions for using the kit to stimulate cytokine production by immune cells.

16. An immunotherapy method, comprising:

a. performing the method of any one of claims 1 to 14 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 cytokine detected; and

c. administering to a subject in need thereof a therapeutically effective amount of the potent immune cells as an immunotherapeutic agent.

17. The immunotherapy method of claim 15, further comprising: extracting the plurality of immune cells from an allogeneic or autologous donor prior to determining the potency of the immune cells.

18. The immunotherapy method of claim 15, 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 of claim 15, further comprising: directing the plurality of immune cells or the potent immune cells to respond to a specified antigen.

20. The immunotherapy method of claim 18, further comprising: genetically altering the plurality of 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, the method comprising:

a. obtaining one or more immune cells;

b. contacting an immune cell with an effective amount of a stimulating agent;

c. detecting the amount of cytokine produced by the immune cell;

d. selecting at least one potent immune cell based on the amount of cytokine detected; and

e. administering to the subject a therapeutically effective amount of the potent immune cells.

22. The method of treating, inhibiting, reducing, preventing and/or ameliorating 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.

23. The method of treating, inhibiting, reducing, preventing and/or ameliorating cancer and/or metastasis in a subject according to claim 20, further comprising extracting the plurality of immune cells from an allogeneic or autologous donor.

24. The method of treating, inhibiting, reducing, preventing and/or ameliorating cancer and/or metastasis in a subject according to any one of claims 20 to 22, wherein the immune cell is a T cell, a macrophage, a Natural Killer (NK) cell, an NK T cell, a Chimeric Antigen Receptor (CAR) T cell, or a CAR NK cell.

25. The method of treating, inhibiting, reducing, preventing and/or ameliorating cancer and/or metastasis in a subject according to any one of claims 20-23, 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.

Technical Field

The present invention relates to immunotherapy and more particularly to testing immune cells for effector function.

Background

Immunotherapy treats diseases by activating or suppressing the immune system. Cells derived from the immune system can be used to improve immune function and properties. In recent years, immunotherapy has received much attention from researchers, clinicians, and pharmaceutical companies, particularly in terms of its promise of treating various forms of cancer. Immunomodulatory regimens generally have fewer side effects than current drugs, including less potential for developing drug resistance in the treatment of microbial diseases.

Conventional cancer treatments have focused on killing or removing cancer cells using chemotherapy, surgery, and/or radiation. However, the field of therapeutic immune cells is rapidly evolving and can be used in conjunction with or in some cases in lieu of conventional therapy to treat, prevent or delay the onset of cancer. Immune effector cells such as lymphocytes, macrophages, dendritic cells, natural killer cells (NK cells), Cytotoxic T Lymphocytes (CTLs), etc., work together naturally to protect the body from cancer by targeting aberrant antigens expressed on the surface of tumor cells. Recent cancer treatment developments have focused on directing the patient's immune system to attack and destroy tumors. Various strategies are being used or being studied and tested.

Adoptive Cell Transfer (ACT) is a means of transferring cells into patients and has shown promise against lung, melanoma, and other cancers. These cells may be derived from the patient (autologous) or from another individual (allogeneic). Allotherapy involves cells isolated and expanded from a donor separate from the patient receiving the cells. Alternatively, adoptive cell transfer can be used to culture autologous extracted cells in vitro and expand them for later transfusion. For example, autoimmune-enhanced therapy involves extracting 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 re-infusing these cells into the subject.

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 collecting T cells from a subject and then infecting the T cells with a retrovirus containing a copy of the T Cell Receptor (TCR) gene. TCR genes are specialized for recognizing tumor antigens (e.g., chimeric antigen receptors or CARs). The virus integrates the receptor into the genome of the T cell. Cells are non-specifically expanded and/or stimulated. The cells are then reinfused and an immune response is generated against the tumor cells.

With the approval of the first CAR-T therapy and the participation of multiple commercial companies in multiple clinical trials, this field has proliferated commercially and shown promise for immunotherapy. The need for reliable and reproducible efficacy testing of these therapeutic immune cells has since increased as new clinical trials in the field have advanced every other day. The industry "gold standard" for testing effector functions of immune cells is the chromium release assay, which was developed in the 60's of the 20 th century and is still in use, even though there are concerns due to the use of radioactive substances and variability caused by target tumor cells. A useful alternative is an assay based on calcein, which still has a lot of variability due to the use of different tumor targets and the entrapment of calcein in the apoptotic bodies of the tumor targets.

There have been other efforts to develop different ways to visually look at the effector functions of these immune cells, but these methods still use target tumor cells. An alternative method for examining effector functions of immune cells is to examine cytokines produced by these cells, wherein all conventional methods use target tumor cells to induce cytokine production from immune cells. The use of target tumor cells increases the biological variability of all of these tests due to variability between tumor cell types. Moreover, these assays require cumbersome setup, which introduces batch effects in these assays. Batch effects are caused by target cell conditions, human-to-human variability in plate loading, plate conditions, variability of various reagents, variability in readings, and the like. There is clearly a need for an immune cell potency assay that is able to remove all of these variability and produce reliable and reproducible results.

Assessing the quality of immune cell therapy products requires reliable and reproducible potency assays. The approval process is strictly regulated and in order to obtain approval, a drug developer will need to submit a large amount of information about the drug product to a regulatory body. This may include information about the potency of the pharmaceutical product and assays for determining this potency. The efficacy of cell therapy products should be indicated by appropriate tests to show the effector function of these therapeutic immune cells showing efficacy, according to the requirements of the FDA (21CFR610.10), which would be to measure relevant cytokines produced by these immune cells.

Disclosure of Invention

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.

In one aspect, disclosed herein is a method of determining the efficacy of an immune cell (e.g., a T cell, a macrophage, an NK cell, an NK T cell, a CAR T cell, and/or a CAR NK cell), the method comprising contacting an immune cell with an effective amount of a stimulating agent (e.g., Phytohemagglutinin (PHA), Phorbol Myristate Acetate (PMA)/ionomycin, concanavalin a (con a)), Lipopolysaccharide (LPS), and/or pokeweed mitogen (PWM)) and detecting one or more cytokines produced by the immune cell (e.g., IL-2, IL-6, IFN- γ, TNF- α, BAFF/TNFSF13B, CD163, CD30/TNFRSF8, Chitinase 3-like protein 1(Chitinase 3-li 1), gp130, IFN- α 2, IL-6ra, IL-8, and/or IL-b), and detecting one or more cytokines produced by the immune cell, IL-10, IL-11, IL-12(p40), IL-12(p70), IL-20, IL-22, IL-26, IL-29/IFN-l1, IL-32, IL-34, IL-35, MMP-1, osteocalcin, OPN, pentraxin-3, TNF-R1, TNF-R2, TSLP, GM-CSF, MIP-1 α, MIP-1 β, RANTES, and/or TWEAK/TNFSF 12). In one aspect, the method can further comprise comparing the amount of cytokine produced to a level of cytokine potency required for use of the immune cell in immunotherapy.

Also disclosed herein is a method of determining the efficacy of an immune cell according to any of the preceding aspects, wherein an immunoassay (e.g., ELISA, intracellular cytokine staining, ELISpot, flow cytometry, Luminex) is usedQuantitative PCR (including but not limited to qRT-PCR) and/or bead arrays) detects the amount of cytokines.

In one aspect, disclosed herein is a method of determining the efficacy of an immune cell according to any of the preceding aspects, wherein the immune cell is contacted with an effective amount of a stimulating agent (e.g., PHA, PMA/ionomycin, Con a, LPS, and/or PWM) for at least 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 65 minutes, 70 minutes, 75 minutes, 80 minutes, 85 minutes, 90 minutes, 95 minutes, 100 minutes, 105 minutes, 110 minutes, 115 minutes, 120 minutes, 150 minutes, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, a, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 30 hours, 32 hours, 36 hours, 42 hours, 48 hours, 60 hours, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 45 days, 60 days, 61 days, 62 days, 3 months, 4 months, 5 months, or 6 months.

Also disclosed herein is a method of determining the efficacy of an immune cell according to any preceding aspect, wherein the stimulating agent (e.g., PHA, PMA/ionomycin, Con a, LPS, and/or PWM) is provided at a concentration of 1.0 μ g/mL to 1000 μ g/mL, including but not limited to a concentration of 5 μ g/mL to 15 μ g/mL.

In one aspect, disclosed herein is a kit for determining the efficacy of an immune cell (e.g., T cell, macrophage, NK cell, NK T cell, CAR T cell, and/or CAR NK cell), the kit comprising a container (e.g., a microcentrifuge tube) comprising an effective amount of a stimulating agent (e.g., PHA, PMA/ionomycin, Con a, LPS, and/or PWM) and a buffer suitable for an immune cell. In a certain aspect, the kit can further include instructions for using the kit to stimulate cytokine production by an immune cell.

Also disclosed herein is a kit for determining the potency of an immune cell according to any preceding aspect, wherein the stimulating agent (e.g., PHA, PMA/ionomycin, Con a, LPS, and/or PWM) is provided at a concentration of 1.0 μ g/mL to 1000 μ g/mL, including but not limited to a concentration of 5 μ g/mL to 15 μ g/mL.

In one aspect, disclosed herein is a method of immunotherapy, comprising: a) performing the method of determining the efficacy of immune cells (e.g., T cells, macrophages, NK cells, NK T cells, CAR T cells, and/or CAR NK cells) according to any preceding aspect on a plurality of immune cells to determine the efficacy of each immune cell; b) selecting at least one potent immune cell based on the detected amount of cytokine (e.g., IL-2, IL-6, IFN-. gamma., TNF-. alpha., BAFF/TNFSF13B, CD163, CD30/TNFRSF8, chitinase 3-like protein 1, gp130, IFN-. alpha.2, 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, MMP-1, osteocalcin, OPN, pentraxin-3, TNF-R1, TNF-R2, TSLP, GM-CSF, MIP-1. alpha., MIP-1. beta., RANTES, and/or TWEAK/TNFSF 12); and c) administering to a subject in need thereof a therapeutically effective amount of the potent immune cells as an immunotherapeutic agent. In one aspect, the method may further comprise extracting the plurality of immune cells from an allogeneic or autologous donor prior to determining the potency of the immune cells.

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

In one aspect, disclosed herein is a method of immunotherapy according to any preceding aspect, further comprising directing the plurality of immune cells or the potent immune cells to respond to a specified antigen. In one aspect, the method may further comprise modifying the cell line derived from the exosome or modifying the exosome itself to comprise a designated antigen to which the immune cell of interest is responsive (e.g., adding CD19 to specifically determine the potency of CD19CAR T cells in a heterogeneous sample).

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

In one aspect, disclosed herein is a method of treating, inhibiting, reducing, preventing and/or ameliorating cancer and/or metastasis in a subject, the method comprising: 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 obtained from an allogeneic or autologous donor); b) contacting the immune cell with an effective amount of a stimulating agent (e.g., PHA, PMA/ionomycin, Con a, LPS, and/or PWM); c) detecting the amount of a cytokine (e.g., IL-2, IL-6, IFN- γ, TNF- α, BAFF/TNFSF13B, CD163, CD30/TNFRSF8, chitinase 3-like protein 1, gp130, IFN- α 2, 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, MMP-1, osteocalcin, OPN, pentraxin-3, TNF-R1, TNF-R2, TSTSCSF, LP-CSF, MIP-1 α, MIP-1 β, RANTES, and/or TWEAK/TNFSF12) produced by the immune cell; d) selecting at least one potent immune cell based on the amount of cytokine detected; and e) administering to the subject a therapeutically effective amount of the potent immune cells. In a certain aspect, the method may further comprise extracting the immune cells from an autologous or allogeneic donor.

Also disclosed herein is a method of treating, inhibiting, reducing, preventing, and/or ameliorating cancer and/or metastasis according to any of the preceding aspects, 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.

Also disclosed herein are methods of determining the identity of at least one immune cell or cell population based on cytokine characteristics associated with the cell type (e.g., distinguishing between Th1, Th2, Th3, Th9, Th17, effector memory T (tem) cells, central memory T (tcm) cells, γ δ T cells, or regulatory T (treg) cells, resting NK cells, expanded NK cells).

Drawings

Figure 1 provides a plot showing the correlation between NK cytokine release induced by K-562 tumor cells versus PHA-induced NK cytokine release (in picograms/million cells/hour).

Figure 2 provides a graph showing analysis of results based on incubation time.

Figure 3 provides a graph showing analysis of results based on cell number.

FIG. 4 provides a graph showing PHA assay results for NK cells expanded with mb-IL-21 and TGF- β.

Figure 5 provides a plot showing the correlation between freshly isolated NK cytokine release (induced by PHA) versus PHA-induced expanded NK cytokine release (in picograms/million cells/hour).

Figure 6 provides a plot showing cytokine expression of NK cells after 1 hour stimulation with PHA in expanded (N-18) and freshly obtained (N-10) NK cells.

Figure 7 shows a comparison of the concentration of cytokines expressed between freshly obtained NK cells and expanded NK cells after PHA stimulation.

FIG. 8 provides a plot showing specificity and sensitivity in differentiating expanded NK cells based on IFN- γ and IL-2 expression.

Figure 9 shows that freshly obtained NK cells and expanded NK cells can be distinguished based on IL-2 up-regulation and pentraxin-3 or chitinase 3-like protein 1 down-regulation.

Figure 10 shows that freshly obtained NK cells and expanded NK cells can be distinguished based on IFN- γ up-regulation and pentraxin-3 or chitinase 3-like protein 1 down-regulation.

Detailed Description

The present invention provides a method of determining the efficacy of an immune cell, the method comprising contacting an immune cell with an effective amount of a stimulating agent and detecting the amount of cytokine produced by the immune cell. While the present disclosure is given in the context of cancer immunotherapy, the concepts and innovations disclosed herein may be applied to immunotherapy for other diseases and disorders. For example, the assays disclosed herein can also be used to test the efficacy of immune cells used in immunotherapy for autoimmune diseases, inflammatory diseases or disorders, viral diseases, and/or bacterial infections.

Definition of

For clarity of understanding and ease of reference, glossaries have been compiled herein for use throughout the brief description and the remainder of the application. Some of the terms are well known in the art and are defined herein for clarity, and some of the terms are specific to this application and must therefore be defined for proper understanding of the application.

As used in the specification and in the claims, the singular form of "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "cell" encompasses a plurality of cells, including mixtures thereof. Where the plural is used herein, it typically encompasses the singular.

Ranges can be expressed herein 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, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. The recitation of numerical ranges by endpoints includes all numbers subsumed within that range (e.g. 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.). It will also be understood that a plurality of values are disclosed herein, and that each value is also disclosed herein as "about" that particular value in addition to the value itself. For example, if the value "10" is disclosed, then "about 10" is also disclosed. It is also understood that when a disclosed value is "less than or equal to" the recited value, it is also disclosed that "greater than or equal to the recited value" and possible ranges between the recited values, as suitably understood by one of skill in the art. 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 should also be understood that throughout this application, data is provided in a number of different formats, and that this data represents the range of endpoints and starting points, and any combination of data points. For example, if a particular data point "10" and a particular data point 15 are disclosed, it should be understood that greater than, greater than or equal to, less than or equal to, and equal to 10 and 15 and between 10 and 15 are considered disclosed. It is also understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, 11, 12, 13 and 14 are also disclosed.

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 … …" shall mean excluding other elements that have any significance to the combination. Thus, a composition consisting essentially of the elements as defined herein will not exclude trace contaminants from the isolation and purification process and pharmaceutically acceptable carriers (e.g., phosphate buffered saline, preservatives, etc.). "consisting of … … (of) shall mean excluding other ingredients more than trace elements and a large number of process steps for applying the composition of the invention. Embodiments defined by each of these transition terms are within the scope of the present invention.

"increase" may 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 the condition, symptom, activity, composition in a statistically significant amount. Thus, the 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.

"reducing" 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 reduce the genetic output of a gene when the genetic output of the gene product utilizing the substance is low relative to the output of the gene product not utilizing the substance. Also for example, a reduction may be a change in symptoms of a disorder such that the symptoms are less than previously observed. A reduction can be any individual, median or average reduction in condition, symptom, activity, composition in a statistically significant amount. Thus, the 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.

"inhibit", "inhibiting" and "inhibition" mean to reduce activity, response, condition, disease or other biological parameter. This may include, but is not limited to, complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in activity, response, condition, or disease as compared to native or control levels. Thus, the reduction may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or any reduction therebetween as compared to the native or control level.

By "reducing" or other forms of the word (such as "reducing" or "reduction") is meant reducing an event or characteristic (e.g., tumor growth). It will be appreciated that this is typically related to a certain standard or expected value, in other words it is relative, but it is not always necessary to refer to a standard or relative value. For example, "reducing tumor growth" means reducing the rate of tumor growth relative to a standard or control.

"preventing" or other forms of the words (e.g., "preventing" or "prevention") means to block a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the likelihood of the occurrence of a particular event or characteristic. Prevention does not require comparison to a control as it is generally more absolute than, for example, a reduction. As used herein, something can be reduced but cannot be prevented, but something that is reduced can also be prevented. Likewise, something can be prevented but not reduced, but something that is prevented can also be reduced. It is to be understood that the use of other words is also expressly disclosed, unless expressly stated otherwise, in the context of such reduction or prevention.

The term "therapeutically effective" is intended to quantify the amount or quantity of an active agent (e.g., immunotherapeutic cells) that will achieve the goal of reducing the severity of the disease while avoiding adverse side effects, such as those typically associated with alternative therapies. A therapeutically effective amount may be administered in one or more doses. Therapeutically effective treatments include those that improve the quality of life of the subject, even if the treatment itself does not improve disease outcome.

An "effective amount" generally means an amount that provides a desired local or systemic effect, e.g., an amount effective to stimulate cytokine formation, including an amount that achieves the particular desired effect described herein. For example, an effective amount is an amount sufficient to achieve a beneficial or desired clinical result.

The term "subject" refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, e.g., a mammal. In one aspect, the subject can be a human, a non-human primate, a bovine, equine, porcine, canine, or feline. The subject may also be a guinea pig, rat, hamster, rabbit, mouse, or mole. Thus, the subject may be a human or veterinary patient. The term "patient" refers to a subject under the treatment of a clinician (e.g., physician).

The term "therapeutically acceptable carrier" means a carrier or excipient that can be used to prepare a generally safe and non-toxic composition and includes acceptable carriers for veterinary and/or human use. Intravenous delivery methods will utilize physiologically balanced therapeutically acceptable carriers (e.g., at permeation levels and pH levels that are safe for intravenous use). As used herein, the term "therapeutically acceptable carrier" encompasses any carrier as in standard carriers, such as saline, ringer's (Ringers), phosphate buffered saline solution, water, aqueous dextrose solution, and emulsions such as 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 known in the art for use in therapeutic formulations. The therapeutically acceptable carrier may also comprise preservatives (including frozen preservatives), such as those that will maintain the viability and/or efficacy of the immune cells. As used in the specification and claims, a "therapeutically acceptable carrier" includes both one and more than one such carrier.

The term "treatment" refers to the medical management of a patient, intended to cure, ameliorate, stabilize or prevent a disease, pathological condition or disorder. This term includes active treatment, that is, treatment specific to the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed to the removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed to alleviate symptoms rather than cure the disease, pathological condition, or disorder; prophylactic treatment, that is, treatment is directed to minimizing the development of, or partially or completely inhibiting the development of, the relevant disease, pathological condition, or disorder; and supportive treatment, that is, treatment to supplement another specific therapy for improvement of the associated disease, pathological condition, or disorder.

"administration" to a subject includes any route of introducing or delivering an agent to a subject. Administration can be by any suitable route, including oral, topical, intravenous, subcutaneous, transdermal, intramuscular, intraarticular (intra-joint), parenteral, intraarteriolar, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation, by implantable drug reservoir, parenteral (e.g., subcutaneous, intravenous, intramuscular, intraarticular (intra-articular), intrasynovial, intrasternal, intrathecal, intraperitoneal, intrahepatic, intralesional, and intracranial injection or infusion techniques), and the like. As used herein, "simultaneous administration," "combined administration," "simultaneous administration," or "administered simultaneously" means that the compounds are administered at the same time point or substantially one after the other. In the latter case, the administration times of the two compounds are close enough that the observed results are indistinguishable from those obtained when the compounds are administered at the same time point. By "systemic administration" is meant introducing or delivering an agent to a subject by a route that introduces or delivers the agent to a broad area of the subject's body (e.g., greater than 50% of the body), such as through an entrance into the circulatory or lymphatic systems. In contrast, "topical administration" refers to introducing or delivering an agent to a subject by a route that introduces or delivers the agent to the area of or immediately adjacent to the point of administration and does not introduce the agent systemically in therapeutically significant amounts. For example, a topically applied agent can be easily detected in the local vicinity of the point of application, but in a non-detectable or detectable amount in the distal portion of the subject's body, can be negligible. Administration includes self-administration and administration by others.

As used herein, "treating" and grammatical variations thereof includes administering a composition intended or intended to partially or completely prevent, delay, cure, heal, alleviate, alter, remedy, ameliorate, improve, stabilize, alleviate (missing), and/or reduce the intensity or frequency of one or more diseases or conditions, symptoms of a disease or condition, or underlying cause of a disease or condition. The treatment according to the invention can be applied prophylactically, palliatively or remedially. The prophylactic treatment is administered to the subject prior to the onset of cancer (e.g., prior to overt signs of cancer), during early onset (e.g., at the time of initial signs and symptoms of cancer), or after established cancer progression. Prophylactic administration can be performed days to years before symptoms of the disease or infection manifest.

The methods and kits disclosed herein utilize stimulants. As used herein, a stimulating agent can 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. Stimulants include, but are not limited to, Phytohemagglutinin (PHA), Phorbol Myristate Acetate (PMA)/ionomycin, concanavalin a (con a), Lipopolysaccharide (LPS) and/or pokeweed mitogen (PWM), peanut agglutinin (PNA), wheat germ agglutinin and ricin.

Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this application pertains. The disclosed references are also individually and specifically incorporated by reference herein for the material contained in the document that is discussed in the sentence in which the document depends.

Immunopotential assay

In one aspect, the invention provides a method of determining the efficacy of an immune cell. The method comprises the steps of contacting an immune cell with an effective amount of a stimulating agent (e.g., PHA, PMA/ionomycin, Con a, LPS and/or PWM) and detecting the amount of cytokine produced by the immune cell. For example, the immune cells can be contacted with a stimulating agent (e.g., PHA, PMA/ionomycin, Con a, LPS, and/or PWM) by suspending the stimulating agent in a cell culture medium and exposing the immune cells to the cell culture medium.

In some embodiments, the method comprises the step of comparing the amount of cytokine produced to a level of cytokine potency required for use of the immune cell in immunotherapy. Potency assays are used to characterize products (i.e. immune cells) to monitor batch-to-batch consistency and ensure stability of the product, and therefore should be sensitive enough to detect differences that may affect the mechanism of action and function of the product and are thus of 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 putative physiological/pharmacological activity of the product. The potency assay described herein provides the ability to measure potency values within product specifications; high sensitivity to detect differences of potential clinical significance; closely related to the mechanism of action and putative physiological/pharmacological activity of the product. Preferably, the potency assay also meets the following minor criteria: sufficiently low intra-and inter-assay variation (to achieve the accuracy required to support product specifications); sufficient robustness; and is suitable for 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 the effectiveness of immunosuppressive or immune activator).

As described above, the disclosed methods provide for determining the efficacy of immune cells. As defined herein, an immune cell is any cell of the immune system that produces cytokines (i.e., an immune cell that produces cytokines). Examples of cytokine-producing immune cells include lymphocytes, neutrophils, macrophages, and natural killer cells. Lymphocytes comprise both B cells and T cells (including CD4 and CD 8T cells). In one aspect, the 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. The immune cells can be obtained from a cell culture or can be obtained from a subject (e.g., an allogeneic donor or an autologous donor).

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 or "inhibitory" 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. The T cell may be a CD4 or CD 8T cell. In addition, the T cells may include Chimeric Antigen Receptor (CAR) T cells or Tumor Infiltrating Lymphocytes (TILs).

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 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, leading to lysis or apoptosis. However, NK cells are unique in that they are able to recognize stimulated cells regardless of the presence of peptides from pathogens on MHC molecules. NK cells were named "natural killers" because the original idea was that these cells did not need to be activated beforehand to kill the target. NK cells are Large Granular Lymphocytes (LGLs) and are known to differentiate and mature in the bone marrow and then enter the circulation from the bone marrow. In a certain aspect, the NK cell can be a CAR NK cell.

Thus, in one aspect, disclosed herein is a method of determining the efficacy of an immune cell (e.g., a T cell, a macrophage, an NK cell, an NK T cell, a CAR T cell, and/or a CAR NK cell), the method comprising contacting an immune cell with an effective amount of a stimulating agent (e.g., 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 can further comprise comparing the amount of cytokine produced to a level of cytokine potency required for use of the immune cell in immunotherapy.

The assay comprises the step of detecting the amount of cytokine produced by the immune cell after stimulation of the immune cell with a stimulating agent. As used herein, the term "cytokine" refers to a small protein (about 5-20kDa) that is important in cell signaling and in particular immune regulation that can be produced by immune cells. Examples of cytokines include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors. The detected cytokines may comprise cytokines known to be produced by the immune cells being evaluated, or the detection may encompass a broader range of individual cytokines, including cytokines not known to be produced by immune cells.

In some embodiments, the cytokines being detected comprise cytokines known to be produced by T cells or natural killer cells. In some embodiments, the cytokines comprise those cytokines known to be produced by T cells. T cells include Th1 cells and Th2 cells; th1 cells produce mainly Interferon (IFN) -gamma (IFN-gamma), Tumor Necrosis Factor (TNF) -alpha (TNF-alpha) and IL-2; th2 cells produce 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-l α, IL-1 β, 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) -1 α (MIP-1 α), MIP-1 β, and RANTES. Other cytokines that may be used to determine the potency of immune cells 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 protein 1, gp130, IFN-alpha 2, IL-6Ra, IL-11, IL-12(p40), IL-12(p70), IL-20, IL-26, IL-29/IFN-l1, IL-32, IL-34, IL-35, matrix metalloproteinase-1 (MMP-1), osteocalcin, Osteopontin (OPN), Pentraxin-3 (Pentraxin-3), Tumor Necrosis Factor (TNF) -receptor 1(TNF-R1), TNF-R2, Thymic Stromal Lymphopoietin (TSLP), or TNF-related weak apoptosis-inducing factor (TWEAK)/TNF superfamily member 12(TWEAK/TNFSF 12). Thus, in one aspect, disclosed herein is a method of determining the efficacy of an immune cell (e.g., a T cell, a macrophage, an NK cell, an NK T cell, a CAR T cell, and/or a CAR NK cell), the method comprising contacting an immune cell with an effective amount of a stimulating agent (e.g., Phytohemagglutinin (PHA), Phorbol Myristate Acetate (PMA)/ionomycin, concanavalin a (con a), Lipopolysaccharide (LPS), and/or pokeweed mitogen (PWM)) and detecting one or more cytokines produced by the immune cell (e.g., IL-2, IL-6, IFN- γ, TNF- α, BAFF/TNFSF13B, CD163, CD30/TNFRSF8, chitinase 3-like protein 1, gp130, IFN- α 2, IL-6ra, IL-8, IL-10, IL-11, IL-6, IL- γ, TNF- α, and/or phse mitogen-1, gp130, IFN- α 2, IL-6, IL-8, IL-10, IL-11, and/or a pharmaceutically acceptable salt thereof, IL-12(p40), IL-12(p70), IL-20, IL-22, IL-26, IL-29/IFN-l1, IL-32, IL-34, IL-35, MMP-1, osteocalcin, OPN, pentraxin-3, TNF-R1, TNF-R2, TSLP, GM-CSF, LIF, MIP-1 α, MIP-1 β, RANTES, and/or TWEAK/TNFSF 12). Disclosed herein are methods of determining the efficacy of an immune cell (e.g., a T cell, a macrophage, an NK cell, an NK T cell, a CAR T cell, and/or a CAR NK cell), the method comprising contacting an immune cell with an effective amount of a stimulating agent (e.g., PHA, PMA/ionomycin, Con a, LPS, and/or PWM) and detecting one or more cytokines produced by the immune cell (e.g., IL-2, IL-6, IFN- γ, TNF- α, BAFF/TNFSF13B, CD163, CD30/TNFRSF8, chitinase 3-like protein 1, gp130, IFN- α 2, 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-10, IL-11, IL-12(p70), IL-20, IL-22, IL-26, IL-29/IFN-l1, and/l 1, IL-32, IL-34, IL-35, MMP-1, osteocalcin, OPN, pentraxin-3, TNF-R1, TNF-R2, TSLP, GM-CSF, MIP-1 α, MIP-1 β, RANTES, and/or TWEAK/TNFSF 12). In one aspect, the method can further comprise comparing the amount of cytokine produced to a level of cytokine potency required for use of the immune cell in immunotherapy. In some embodiments, the level of a plurality of cytokines is determined. In further embodiments, the cytokine is selected from the group consisting of interleukin-2, interleukin-6, and interferon- γ.

The assay comprises the step of detecting the amount of cytokine produced by the immune cell. A wide variety of methods for detecting cytokines are known to those skilled in the art and may vary depending on the cytokine being detected. In some embodiments, one or more methods may be used to detect and/or quantify the presence of a plurality of different cytokines. For example, cytokines can be detected by using a specific kit or immunoassay. Available from, for example, Miltenyi Biotec, can be used^TMLuminex and Thermo Fisher Scientific^TMAnd the like, obtained from commercial suppliers. Examples of kits suitable for detecting cytokines are the rapid cytokine tester (CD4/CD8) kit or the MACSPlex cytokine T/NK kit, which can detect cytokines formed by T cells or NK cells, both of which are from Miltenyi Biotec^TMAnd (5) selling.

In some embodiments, the amount of cytokine is detected using an immunoassay. There are many different forms and variations of immunoassays. Immunoassays can be run in multiple steps, where reagents are added and washed away or separated at different points of the assay. Immunoassays include heterogeneous immunoassays, which comprise multiple steps, and homogeneous immunoassays, which involve simply mixing reagents and samples and performing physical measurements. Immunoassays typically utilize a calibrator, which is a solution known to contain the analyte in question, and the concentration of the analyte is typically known. Comparing the measured response to the authentic sample with the measured response produced by the calibrator allows interpretation of the signal intensity in terms of the presence or concentration of 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. The immunoassay also comprises enzyme-linked immunosorbent assay(ELISA), lateral flow immunoassay, enzyme-linked immunosorbent spot (ELIspot) assay, flow cytometry, intracellular cytokine staining, antibody array assay and bead-based assay, magnetic immunoassay, radioimmunoassay and quantitative PCR (including but not limited to qRT-PCR). In one aspect, the assay comprises Luminex

A method of determining the efficacy of an immune cell comprises the step of contacting the immune cell with an effective amount of a stimulating agent (e.g., PHA, PMA/ionomycin, Con a, LPS, and/or PWM).

It is understood and contemplated herein that immune cells must be exposed to a stimulating agent for a period of time to be induced to produce cytokines. In one aspect, disclosed herein are methods of determining the efficacy of an immune cell, wherein the immune cell is contacted with an effective amount of a stimulating agent (e.g., PHA, PMA/ionomycin, Con a, LPS, and/or PWM) for at least 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 65 minutes, 70 minutes, 75 minutes, 80 minutes, 85 minutes, 90 minutes, 95 minutes, 100 minutes, 105 minutes, 110 minutes, 115 minutes, 120 minutes, 150 minutes, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, a, 21 hours, 22 hours, 23 hours, 24 hours, 30 hours, 32 hours, 36 hours, 42 hours, 48 hours, 60 hours, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 45 days, 60 days, 61 days, 62 days, 3 months, 4 months, 5 months, or 6 months.

Also disclosed herein is a method of determining the efficacy of an immune cell according to any of the foregoing aspects, wherein the stimulating agent (e.g., PHA, PMA/ionomycin, Con a, LPS, and/or PWM) is provided at a concentration of 5 μ g/mL to 1000 μ g/mL. In one aspect, the concentration of the stimulating agent is 1. mu.g/mL, 2. mu.g/mL, 3. mu.g/mL, 4. mu.g/mL, 5. mu.g/mL, 6. mu.g/mL, 7. mu.g/mL, 8. mu.g/mL, 9. mu.g/mL, 10. mu.g/mL, 11. mu.g/mL, 12. mu.g/mL, 13. mu.g/mL, 14. mu.g/mL, 15. mu.g/mL, 20. mu.g/mL, 25. mu.g/mL, 30. mu.g/mL, 35. mu.g/mL, 40. mu.g/mL, 45. mu.g/mL, 50. mu.g/mL, 55. mu.g/mL, 60. mu.g/mL, 65. mu.g/mL, 70. mu.g/mL, 75. mu.g/mL, 80. mu.g/mL, 85. mu.g/mL, 90. mu.g/mL, 95. mu.g/mL, or, 100. mu.g/mL, 110. mu.g/mL, 120. mu.g/mL, 125. mu.g/mL, 130. mu.g/mL, 140. mu.g/mL, 150. mu.g/mL, 160. mu.g/mL, 170. mu.g/mL, 175. mu.g/mL, 180. mu.g/mL, 190. mu.g/mL, 200. mu.g/mL, 225. mu.g/mL, 250. mu.g/mL, 275. mu.g/mL, 300. mu.g/mL, 325. mu.g/mL, 350. mu.g/mL, 375. g/mL, 400. mu.g/mL, 425. mu.g/mL, 450. mu.g/mL, 475. mu.g/mL, 500. mu.g/mL, 550. mu.g/mL, 600. mu.g/mL, 650. mu.g/mL, 700. mu.g/mL, 750. mu.g/mL, 800. mu.g/mL, 850. mu.g/mL, 900. mu.g/mL, 950. mu.g/mL or 1000. mu.g/mL. In one aspect, the concentration of the stimulating agent is about 1 μ g/mL to 100 μ g/mL, 1 μ g/mL to 50 μ g/mL, 1 μ g/mL to 15 μ g/mL, or 5 μ g/mL to 15 μ g/mL.

In one aspect, it is understood and contemplated herein that the same cytokines produced for determining the efficacy of immune cells may also be used to identify cytokine producing cells. Immune cells have different expression profiles known in the art. Also disclosed herein are methods of determining the identity of at least one immune cell or cell population based on cytokine characteristics associated with the cell type (e.g., distinguishing between Th1, Th2, Th3, Th9, Th17, effector memory T (tem) cells, central memory T (tcm) cells, γ δ T cells, or regulatory T (treg) cells, resting NK cells, expanded NK cells). Thus, disclosed herein are methods of identifying an immune cell (e.g., a T cell, a macrophage, an NK cell, an NK T cell, a CAR T cell, and/or a CAR NK cell) comprising contacting an immune cell with an effective amount of a stimulating agent (e.g., PHA, PMA/ionomycin, Con a, LPS, and/or PWM) and detecting one or more cytokines produced by the immune cell (e.g., IL-2, IL-6, IFN- γ, TNF- α, BAFF/TNFSF13B, CD163, CD30/TNFRSF8, chitinase 3-like protein 1, gp130, IFN- α 2, 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-10, IL-11, 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-1 α, MIP-1 β, RANTES, and/or TWEAK/TNFSF 12); wherein the identity of the immune cell is revealed based on the profile of the expressed cytokine.

Kit for evaluating the efficacy of immune cells

Another aspect of the invention provides a kit for determining the efficacy of an immune cell (e.g., T cell, macrophage, NK cell, NK T cell, CAR T cell, and/or CAR NK cell), the kit comprising a container comprising an effective amount of a stimulating agent (e.g., PHA, PMA/ionomycin, Con a, LPS, and/or PWM) and a buffer suitable for an immune cell. In some embodiments, the stimulating agent in the kit is provided at a concentration of 5 μ g/mL to 1000 μ g/mL. In one aspect, the concentration of the stimulating agent is 1. mu.g/mL, 2. mu.g/mL, 3. mu.g/mL, 4. mu.g/mL, 5. mu.g/mL, 6. mu.g/mL, 7. mu.g/mL, 8. mu.g/mL, 9. mu.g/mL, 10. mu.g/mL, 11. mu.g/mL, 12. mu.g/mL, 13. mu.g/mL, 14. mu.g/mL, 15. mu.g/mL, 20. mu.g/mL, 25. mu.g/mL, 30. mu.g/mL, 35. mu.g/mL, 40. mu.g/mL, 45. mu.g/mL, 50. mu.g/mL, 55. mu.g/mL, 60. mu.g/mL, 65. mu.g/mL, 70. mu.g/mL, 75. mu.g/mL, 80. mu.g/mL, 85. mu.g/mL, 90. mu.g/mL, 95. mu.g/mL, or, 100. mu.g/mL, 110. mu.g/mL, 120. mu.g/mL, 125. mu.g/mL, 130. mu.g/mL, 140. mu.g/mL, 150. mu.g/mL, 160. mu.g/mL, 170. mu.g/mL, 175. mu.g/mL, 180. mu.g/mL, 190. mu.g/mL, 200. mu.g/mL, 225. mu.g/mL, 250. mu.g/mL, 275. mu.g/mL, 300. mu.g/mL, 325. mu.g/mL, 350. mu.g/mL, 375. g/mL, 400. mu.g/mL, 425. mu.g/mL, 450. mu.g/mL, 475. mu.g/mL, 500. mu.g/mL, 550. mu.g/mL, 600. mu.g/mL, 650. mu.g/mL, 700. mu.g/mL, 750. mu.g/mL, 800. mu.g/mL, 850. mu.g/mL, 900. mu.g/mL, 950. mu.g/mL or 1000. mu.g/mL. In one aspect, the concentration of the stimulating agent is about 1 μ g/mL to 100 μ g/mL, 1 μ g/mL to 50 μ g/mL, 1 μ g/mL to 15 μ g/mL, or 5 μ g/mL to 15 μ g/mL. In some embodiments, the container is a microcentrifuge tube (e.g., an Eppendorf microcentrifuge tube). The kit may further comprise means for obtaining a sample from a subject, such as a syringe for obtaining a sample comprising one or more immune cells. A suitable buffer is RPMI.

The kit may also comprise components required for performing an immunoassay, such as a solid phase, said combination being bound to an antibody which serves as a capture antibody and/or a detection antibody in a sandwich immunoassay format. The solid phase may be a material such as magnetic particles, beads, test tubes, microtiter plates, cuvettes, membranes, scaffold molecules, quartz crystals, membranes, filter papers, discs or chips. The kit may also comprise a detectable label which may be conjugated to or with an antibody such as an antibody used as a detection antibody. The detectable label may, for example, be a direct label, which may be an enzyme, an oligonucleotide, a nanoparticle chemiluminescer, a fluorophore, a fluorescence quencher, a chemiluminescent quencher, or biotin. The test kit may optionally comprise any additional reagents required to detect the label.

The kit can further comprise instructions for using the kit to stimulate cytokine production by an immune cell to assess the efficacy of the immune cell. In some embodiments, the kit further comprises instructions for using the amount of the cytokine to determine the potency of the cell. The instructions contained in the kit may be attached to a packaging material or may be contained as a packaging insert. Although the description is generally written or printed material, it is not limited thereto. The present disclosure contemplates any medium that is capable of storing such instructions and transmitting them to an end user. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, magnetic tape, cassettes, chips), optical media (e.g., CD ROM), and the like. As used herein, the term "specification" may include the address of the internet site that provides the specification.

Immunotherapy method

The method of determining the efficacy of immune cells may be performed prior to using the immune cells as an immunotherapeutic agent. For example, a method of determining the efficacy 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 cytokine detected) and a therapeutically effective amount of the potent immune cell can be delivered to the subject as an immunotherapeutic agent. Thus, in one aspect, disclosed herein is a method of immunotherapy, comprising: a) performing a method of determining the efficacy of immune cells (e.g., T cells, macrophages, NK cells, NK T cells, CAR T cells, and/or CAR NK cells) as disclosed herein on a plurality of immune cells to determine the efficacy of each immune cell; b) selecting at least one potent immune cell based on the detected amount of cytokine (e.g., IL-2, IL-6, IFN-. gamma., TNF-. alpha., BAFF/TNFSF13B, CD163, CD30/TNFRSF8, chitinase 3-like protein 1, gp130, IFN-. alpha.2, 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, MMP-1, osteocalcin, OPN, pentraxin-3, TNF-R1, TNF-R2, TSLP, GM-CSF, MIP-1. alpha., MIP-1. beta., RANTES, and/or TWEAK/TNFSF 12); and c) administering to a subject in need thereof a therapeutically effective amount of the potent immune cells as an immunotherapeutic agent. In one aspect, the method may further comprise extracting the plurality of immune cells from an allogeneic or autologous donor prior to determining the potency of the immune cells.

In some embodiments, the immune cell is an immunotherapeutic immune cell. Immunotherapeutic immune cells are cells that can be used to treat diseases such as cancer. Becker et al, Cancer immunological immunotherapy 65,477-484 (2016). The use of expanded NK cells for cancer therapy has been described. Rezvani et al, "Front Immunol," 6,578 (2015). Because it is helpful to be able to administer large numbers of immune cells during immunotherapy, in some embodiments, the immune cells are expanded immune cells. The expanded immune cells are 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, where their inherent alloreactivity may be beneficial. In further embodiments, the expanded immune cells are genetically engineered to comprise a chimeric antigen receptor to help target the immune cells to diseased tissue. The preparation of the expanded immune cells comprises activating and expanding the immune cells. Koepsell et al, "Transfusion," 53(2):404-10 (2013). A number of cytokines (IL-2, IL-12, IL-15, IL-18, IL-21, type I IFN and TGF-. beta.) have been shown to be useful for the activation and ex vivo expansion of immune cells. For example, in some embodiments, the NK cells evaluated are IL-21 expanded NK cells. Thus, in one aspect, disclosed herein are immunotherapy methods further comprising expanding the at least one potent TGF immune cell prior to delivering a therapeutically effective amount of the potent immune cell.

Expansion refers to ex vivo proliferation of NK cells, such that the population of NK cells increases. For example, NK cells can be expanded from peripheral blood mononuclear cells. However, NK cells can also be expanded from other types of cells such as hematopoietic stem or progenitor cells. The starting blood cells or stem cells can be isolated from a variety of different sources (e.g., placenta, umbilical cord blood, placental blood, peripheral blood, spleen, or liver). Amplification occurs in cell culture media. Suitable cell culture media are known to those skilled in the art. The expanded cells can be provided as a cell line, which is a plurality of cells that can be maintained in cell culture. Thus, in one aspect, disclosed herein are immunotherapy methods further comprising expanding the at least one potent immune cell prior to delivering a therapeutically effective amount of the potent immune cell. In some aspects, the immune cells have been extracted from the subject using known methods prior to performing the method of determining the efficacy of the immune cells. Alternatively, the immune cells may be derived from the expansion of a cell culture.

In some aspects, the immune cells are directed to respond to a specified antigen (e.g., CD 19). The immune cells may be directed to respond before or after the method of determining the efficacy of the immune cells. In some embodiments, the immune cells are genetically altered to respond to a given antigen. For example, the antigen may be a tumor specific antigen. In some aspects, the immunotherapy method comprises genetically altering (before or after determining the potency of the immune cell) the immune cell to present a chimeric antigen receptor. In one aspect, the method may further comprise modifying the exosome-derived cell line or modifying the exosome itself to comprise a designated antigen to which the immune cell of interest is responsive (e.g., adding CD19 to specifically determine the potency of CD19CAR T cells in a heterogeneous sample).

As indicated throughout the method of determining the efficacy of immune cells, the method may be used as part of an adoptive cell transfer therapy. A therapeutically acceptable carrier can be used to deliver potent immune cells to a subject. Intravenous delivery is commonly used to deliver immunotherapeutic cells, but other methods are also contemplated (e.g., guiding a graft to a localized area of the body in need of immunotherapy).

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

A therapeutically effective amount of a potent immune cell encompasses 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.

The subject receiving the potent immune cells can be any subject that would benefit from immunotherapy (e.g., a subject having 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 at high risk for developing cancer, diagnosed with cancer, treated for cancer, or recovered from cancer following surgery. In some embodiments, the potent immune cells can be delivered to a subject as a prophylactic agent to prevent, inhibit, or delay the onset of cancer or metastasis.

Methods of treating diseases

It is to be understood and contemplated herein that the potent immune cells identified herein may be used to treat any disease or disorder, wherein adoptive immunotherapy may be used to treat diseases including, but not limited to, autoimmune diseases, inflammatory diseases or disorders, viral diseases, and/or bacterial infections. Thus, in one aspect, disclosed herein is a method of treating, inhibiting, reducing, preventing and/or ameliorating cancer and/or metastasis in a subject, the method comprising: 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 obtained from an allogeneic or autologous donor); b) contacting the immune cell with an effective amount of a stimulating agent (e.g., PHA, PMA/ionomycin, Con a, LPS, and/or PWM); c) detecting the amount of a cytokine (e.g., IL-2, IL-6, IFN- γ, TNF- α, BAFF/TNFSF13B, CD163, CD30/TNFRSF8, chitinase 3-like protein 1, gp130, IFN- α 2, 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, MMP-1, osteocalcin, OPN, pentraxin-3, TNF-R1, TNF-R2, TSTSCSF, LP-CSF, MIP-1 α, MIP-1 β, RANTES, and/or TWEAK/TNFSF12) produced by the immune cell; d) selecting at least one potent immune cell based on the amount of cytokine detected; and e) administering to the subject a therapeutically effective amount of the potent immune cells. In a certain aspect, the method may further comprise extracting the immune cells from an autologous or allogeneic donor.

It is to be understood and contemplated herein that it is helpful to be able to administer a large number of immune cells during immunotherapy, in some embodiments the immune cells are expanded immune cells. The expanded immune cells are cells that are grown ex vivo to grow large numbers of immune cells. Thus, disclosed herein are methods of treating, inhibiting, reducing, preventing, and/or ameliorating an autoimmune disease, an inflammatory disease or disorder, a viral disease, a bacterial infection, a cancer, and/or a metastasis, the method 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.

It is to be understood and contemplated herein that the disclosed methods of treatment may be used to treat any disease or condition that develops uncontrolled cellular proliferation, including but not limited to cancer and metastasis. A representative but non-limiting list of cancers that can be treated using the disclosed methods of potent immune cells is as follows: 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 (head and neck cancer), head and neck squamous cell cancer, 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 cancer of the mouth, throat and lung, cervical cancer (cervical cancer), cervical cancer (cervical carcinoma), breast cancer and epithelial cancer, kidney cancer, genitourinary cancer, lung cancer (pulmony cancer), esophageal cancer, head and neck cancer (head and neck cancer), large intestine cancer, hematopoietic cancer; testicular cancer; colon, rectal, prostate or pancreatic cancer.

Examples of autoimmune diseases that can be treated using the disclosed methods include, but are not limited to: achalasia, acute disseminated encephalomyelitis, acute motor axonopathy, Addison's disease, painful obesity, Adult stele's disease, hypogammaglobulinemia, alopecia areata, Alzheimer's disease, amyloidosis, ankylosing spondylitis, GBM/TBM nephritis, antiphospholipid syndrome, aplastic anemia, autoimmune angioedema, autoimmune 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 polyendocrinopathy syndrome, autoimmune retinopathy, Autoimmune urticaria, axonal and neuronal neuropathy (AMAN), barllosis (Bal disease), Behcet's disease(Behcet's disease), benign mucosal pemphigoid, Behcet's brainstem encephalitis (Bickerstaff's encephalitis), bullous pemphigoid, Castleman's disease (multicentric Castleman disease, CD), celiac disease, Chagas disease (Chagas disease), chronic fatigue syndrome, Chronic Inflammatory Demyelinating Polyneuropathy (CIDP), Chronic Relapsing Multifocal Osteomyelitis (CRMO), Chager-Strauss syndrome (Churg-Strauss syndrome, CSS), Eosinophilic Granulomatosis (EGPA), cicatricial pemphigoid, Cogan's syndrome, colletotrichosis, congenital heart block, Coxseyl viral myocarditis, CREST syndrome, Crohn's disease (Crohn's disease), dermatitis, herpes zoster (Devicer's disease), Graves's disease (Lupus disceus's disease), Graves's disease (Graves's disease), Graves's disease (type 1-type diabetes mellitus), Endometriosis, periappendicitis, eosinophilic esophagitis (EoE), eosinophilic fasciitis, erythema nodosum, primary mixed cryoglobulinemia, Evans syndrome (Evans syndrome), feldy syndrome (Felty syndrome), fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture's syndrome, granulomatous polyangiitis, Graves' disease, Guillain-Barre syndrome (Guillain-Barre syndrome), Hashimoto's encephalopathy (Hashimoto's encephalopathy), Hashimoto's thyroiditis (Hashimoto's thyroiditis), hemolytic anemia, Henoch-Schonlein purpura (HSP ), herpes simplex or herpes gestationis (acne), herpes zoster) (HS suppurative (HS), pymetrorrhagia (HS), and/or herpes zoster) Hypogammaglobulinemia, IgA nephropathy, IgG 4-associated 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 (Lambert-Eaton syndrome), Leucocyte-disrupted vasculitis, lichen planus, lichen sclerosus, xyloid conjunctivitis, Linear IgA disease (LAD), lupus nephritis, lupus vasculitis, chronic Lyme disease, Meniere's disease (Menie disease)re's disease), Microscopic Polyangiitis (MPA), Mixed Connective Tissue Disease (MCTD), Moren's ulcer (Mooren's), Muhaber-Hebmann's disease (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, Alder's thyroiditis (Ord's thyroiditis), recurrent rheumatism (PR), PANDAS, Paraneoplastic Cerebellar Degeneration (PCD), sleep hemoglobinuria (PNH), Parmberg syndrome (Parmberg syndrome), pars plana pars encephalomyelitis (peripheral meningitis), Pannage-Telner syndrome (Parsonnel disease-syndrome), peripheral neuromeningitis, peripheral venous meningitis (peripheral lymphadenitis), peripheral neuroleptitis (peripheral lymphadenitis-syndrome), peripheral venous lymphadenitis (peripheral lymphadenitis), peripheral lymphadenitis (peripheral lymphadenitis) syndrome (peripheral lymphadenitis), neurolept-synephora) and peripheral lymphadenitis (peripheral lymphadenitis) may be-associated with multiple sclerosis (peripheral lymphadenitis) may be-a, Pernicious Anemia (PA), POEMS syndrome, polyarteritis nodosa, type I, type II, type III, polyglandular syndrome, 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, recurrent polychondritis, Restless Leg Syndrome (RLS), retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, rheumatoid vasculitis, sarcoidosis, Schmidt syndrome, Schnitz syndrome, scleritis, scleroderma, Sjogren's syndrome (Schnitz syndrome) ((R))syndrome), sperm and testis autoimmunity, Stiff Person Syndrome (SPS), Subacute Bacterial Endocarditis (SBE), Susac's syndrome, Sydenham's chorea, Sympathetic Ophthalmia (SO), systemic lupus erythematosus, systemic scleroderma, Takayasu's arteritis, temporal arteritis/giant cell arteritis, thrombocytopenic purpura (TTP), torosahent-hunter syndrome (Tolosa-Hunt syndrome,THS), transverse myelitis, type 1 diabetes, Ulcerative Colitis (UC), Undifferentiated Connective Tissue Disease (UCTD), urticaria vasculitis, uveitis, vasculitis, vitiligo, Vogt-Koyanagi-Harada Disease, and Wegener's granulomatosis (or Granulomatous Polyangiitis (GPA)).

The following examples are included to illustrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Examples of the invention

The assays disclosed herein are directed to testing the efficacy of therapeutic immune cells that will address the problems with current standard methods and meet FDA requirements. To achieve this, Phytohemagglutinin (PHA) is used as a surrogate to induce cytokine production in immune cells. PHA is used in a clinical setting to test the immune response of transplant patients. The use of PHA to induce cytokine production in therapeutic immune cells would remove all of the biological and batch variability from the immune cell potency assay. The assay would eliminate the need for a fully operational research laboratory to test the efficacy of therapeutic immune cells at multiple clinical infusion sites and would provide faster turnaround times for such tests. As part of the FDA-set efficacy test requirements, the assay will provide a method for testing the effector function of therapeutic immune cells.

Testing the efficacy of therapeutic immune cells using PHA as a stimulator

The use of tumor cells to test therapeutic immune cells for effector function has become standard practice. These target cells add biological variability to the assay results. Moreover, this assay setup is more cumbersome and complex due to target conditions, plate setup, and interpersonal technical differences, thereby increasing variability. The therapeutic response of NK cells was compared by PHA cytokine assay with the widely used tumor cell (K562) mediated cytokine assay. The level of cytokines indicates the efficacy of the response from NK cells.

Figure 1 shows the correlation of PHA-induced cytokine assay with the standard K562-induced cytokine assay. The outliers present in figure 1 are the result of variability introduced by using tumor cells. Higher secretion of GM-CSF (normally secreted by tumor cells) was detected in tumor cell mediated assays. This shows a clear advantage of PHA assays due to the lack of variability of tumor cell introduction.

Figure 2 shows a histogram of cytokine levels obtained using a four hour incubation versus a 24 hour incubation (using 100 ten thousand NK cells). Figure 3 shows a histogram of cytokine levels obtained using less than 100 ten thousand cells and more than 100 ten thousand cells. These conditions were tested to normalize the assay. FIG. 4 shows PHA assay results of NK cells expanded with mb-IL-21 and TGF-. beta.s. Figure 5 shows the difference in cytokine profile between donor immune cells and expanded therapeutic NK cell products when induced by PHA.

In determining the optimal number of cells for the assay (10)⁶) Thereafter, multiple donors of fresh peripheral blood NK cells (N ═ 10) and expanded NK cells (N ═ 18) were stimulated with PHA to produce cytokines, and the cytokines in the supernatant were evaluated after 1 hour. The expression of most cytokines is similar between the two types of NK cells, but several cytokines and chemokines related to NK cell function are highly differentially expressed in response to stimulation (fig. 6). Next, the expression of 6 cytokines in fresh and expanded NK cells was measured 1 hour after stimulation with PHA. Compared to expanded NK cells, pentraxin-3 (mean 1,062 vs. 7), IL-8 (mean 821 vs. 11), and chitinase 3-like protein 1 (mean 620 vs. 7) were highly overexpressed in fresh NK cells, and IFN-. gamma. (mean 15 vs. 105), IL-2 (mean 3 vs. 141), and CD30 (mean flat) compared to fresh NK cellsMean 9 vs 156) were highly over-expressed in the expanded NK cells (fig. 7). On the basis of overexpression only, the cut-off values for both IFN- γ and IL-2 expressing 10pg/mL had 94% specificity (16/17) and 89% sensitivity (16/18) in distinguishing expanded NK cells from fresh peripheral blood NK cells (fig. 8). The use of (upregulated) IL-2 together with downregulation of cytokines (pentraxin-3 or chitinase 3-like protein 1) has 100% sensitivity and specificity to distinguish between expanded NK cells and fresh peripheral blood NK cells, where for fresh cells the expression ratio (downregulated: IL-2) is>1, and for expanded cells are<1 (fig. 9). The use of (up-regulated) IFNg together with down-regulated cytokines (pentraxin-3 or chitinase 3-like protein 1) was 95% effective in differentiating between expanded NK cells and fresh peripheral blood NK cells. Combinations of various up-and down-regulating cytokines can be used to increase sensitivity and specificity of defined types (figure 10).

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed invention belongs. The publications cited herein and the materials cited therein are specifically incorporated by reference. It should be understood, however, that any patent, publication, or other disclosure material that is said to be incorporated by reference herein is incorporated in whole or in part only to the extent that the incorporated material does not conflict with existing definitions, statements, 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 by reference. Any material, or portion thereof, that is said to be incorporated by reference herein, but which conflicts with existing definitions, statements, or other disclosure material set forth herein will only be incorporated to the extent that no conflict arises between that incorporated material and the existing disclosure material.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. While the invention has been described with reference to specific examples and embodiments, it will be understood that various changes and further modifications may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention or the inventive concept thereof. In addition, many modifications may be made to adapt a particular situation or apparatus to the teachings of the invention without departing from the essential scope thereof. Such equivalents are intended to be encompassed by the following claims. It is intended that the invention not be limited to the particular embodiments disclosed herein, but that the invention will include all embodiments falling within the scope of the appended claims.