Assays for immune cell recovery

Abstract

Disclosed are compositions and methods for measuring the recovery potential of freshly thawed immune cells. The method involves assaying the level of ADAM-17 cleaved surface receptor expressed on immune cells, and the level of this surface receptor directly correlates with the recovery potential of immune cells (i.e., the greater the increase in ADAM-17 cleaved surface receptor expression level compared to control, the greater the recovery potential). The method can be used to determine whether immune cells have sufficient viability for immunotherapy before use.

Description

Background Art

[0001] This application claims the benefit of U.S. Provisional Application No. 62 / 904,843, filed Sep. 24, 2019, and U.S. Provisional Application No. 62 / 898,170, filed Sep. 10, 2019, which are hereby incorporated by reference in their entirety.

[0002] Cryopreservation of immune cells has been a difficult problem to overcome for immunotherapy. Variability in the viability of cryopreserved immune cells from different lots or different donors is also a significant problem for cell-based immunotherapy. In addition, common assays of viability performed immediately after thawing may have poor predictive values for functional recovery. For example, T cells have more than 90% variability after thawing between groups. Immune cells such as natural killer (NK) cells that are cryopreserved often have poor recovery and function after thawing. NK cells immediately after thawing have a viability of 80-90%, but continue to lose viability over the next 24 hours, and the final survival recovery rate is often as low as 20%. Thus, viability immediately after thawing is not a good indicator of whether cells will survive or be highly functional.

[0003] Interest in NK cell-based immunotherapy has increased again because new protocols for the purification and expansion of a large number of clinical-grade cells have become available. However, since such cells are cryopreserved before use, there is still a need for a reliable method to determine the viability of NK cells after cryopreservation to ensure that the administered cells are dosed according to the number of viable cells.

Summary of the Invention

[0004] Disclosed is a method for measuring cell recovery after a cell membrane damage event based on surrogate measurement of a cell surface receptor (e.g., CD16, etc.) that is cleaved by a metalloprotease (e.g., ADAM17, etc.) activated by the entry of calcium into the cell during the cell membrane damage event.

[0005] The inventors have found that on the day of thawing, the CD16 receptor is lost due to cleavage by ADAM17, but 24 hours later, the surface level of CD16 recovers on live cells. Without being bound by theory, the evidence is consistent with the view that metalloproteases such as ADAM17 have a detrimental effect on NK cell function through CD16 shedding. CD16 is an important protein expressed on NK cells as it is responsible for both antibody-dependent and antibody-independent cytotoxicity of NK cells. The inventors have identified a technique that can predict how well NK cells recover by analyzing the percentage of CD16 present in the live cell population on the day of thawing of cryopreserved samples. This provides an immediate recovery marker on the day of thawing and helps in immunotherapy by ensuring that patients receive only functional live NK cell products for infusion.

[0006] In one aspect, methods are disclosed herein for measuring the likelihood of recovery of immune cells (such as T cells, natural killer (NK) cells, macrophages, dendritic cells, natural killer T (NKT) cells, innate lymphoid cell (ILC), B cells, γδT cells, neutrophils, chimeric antigen receptor (CAR) T cells, and / or CAR NK cells, etc.) after a cell membrane damaging event (including but not limited to freeze-thaw cycles, gene editing, electroporation, magnetofection, detergent permeabilization (such as saponin and / or digitonin, etc.), streptolysin O (SLO) exposure, physical morphological changes (cell squeezing), and / or ethanol exposure (including but not limited to solutions containing ethanol at 30% or less)). The method includes assaying the level of an ADAM-17 cleaved surface receptor (such as CD16, CD62L, or IL-15 receptor (IL-15R), etc.) expressed on the immune cell (including but not limited to assaying via flow cytometry). An increase in the level of the surface receptor (compared to control immune cells or compared to a mixed population of cell membrane damaged immune cells) is directly correlated with the likelihood of immune cell recovery.

[0007] Also disclosed herein is a method for measuring the likelihood of immune cell recovery after a cell membrane damage event of any of the preceding aspects, wherein the level of ADAM-17-cleaved surface receptor expression is assayed within 0 to 24 hours or 12 to 24 hours after the immune cell membrane damage event.

[0008] In one aspect, disclosed herein is a method for measuring the likelihood of immune cell recovery after a cell membrane damage event of any of the preceding aspects, wherein the level of ADAM-17-cleaved surface receptor expressed on an immune cell is represented as a ratio of the level of the surface receptor cleaved by ADAM17 expressed on the cell membrane-damaged immune cell compared to the normal level of the ADAM-17-cleaved surface receptor expressed on the immune cell.

[0009] Also disclosed herein is a method of administering an immunotherapy (e.g., anti-cancer therapy, etc.) to a subject in need thereof, the method comprising: a) obtaining one or more immune cells (e.g., T cells, natural killer (NK) cells, macrophages, dendritic cells, natural killer T (NKT) cells, innate lymphoid cells (ILC), B cells, γδT cells, neutrophils, chimeric antigen receptor (CAR) T cells, and / or CAR NK cells, etc.) previously subject to a cell membrane damage event (including but not limited to freeze-thaw cycles, gene editing, electroporation, magnetofection, detergent permeabilization (e.g., saponin and / or digitonin, etc.), streptolysin O (SLO) exposure, physical morphological changes (cell squeezing), and / or ethanol exposure (including but not limited to solutions containing ethanol at 30% or less)); b) assaying the expression level of an ADAM-17-cleaved surface receptor (e.g., CD16, CD62L, or IL-15 receptor (IL-15R), etc.); and c) administering to the subject a therapeutically effective amount of immune cells that express an increased level of ADAM-17-cleaved surface receptor compared to control immune cells or compared to a mixed population of cell membrane-damaged immune cells.

[0010] In one aspect, provided herein is a method of administering immunotherapy of any of the preceding aspects, wherein the level of ADAM-17-cleaved surface receptor expression is assayed within 0 to 24 hours or 12 to 24 hours after an immunocyte membrane damage event.

[0011] Also provided herein is a method of administering immunotherapy of any of the preceding aspects, wherein the level of ADAM-17-cleaved surface receptor expression is assayed using flow cytometry.

[0012] In one aspect, provided herein is a method of administering immunotherapy of any of the preceding aspects, wherein the level of ADAM-17-cleaved surface receptor expressed on immune cells is represented as a ratio of the level of surface receptor cleaved by ADAM17 expressed on cell membrane-damaged immune cells compared to the normal level of ADAM-17-cleaved surface receptor expressed on immune cells.

[0013] Also provided herein is the use of a therapeutically effective amount of immune cells in immunotherapy as previously subject matter after a cell membrane damage event, wherein the cells express an increased level of ADAM-17-cleaved surface receptor compared to control immune cells or compared to a mixed population of cell membrane-damaged immune cells. In one aspect, the immunotherapy may be, for example, an anti-cancer therapy.

[0014] Also provided herein is a composition for immunotherapy comprising a therapeutically effective amount of immune cells as previously subject matter after a cell membrane damage event, wherein the cells express an increased level of ADAM-17-cleaved surface receptor compared to control immune cells or compared to a mixed population of cell membrane-damaged immune cells. The composition for immunotherapy may further comprise a pharmaceutically acceptable carrier.

[0015] Various aspects of the methods, uses, and compositions for immunotherapy described herein may include methods, uses, and compositions in which the cell membrane damage event may include any one of freeze-thaw cycles, gene editing, electroporation, magnetofection, detergent permeabilization, streptolysin O (SLO) exposure, physical morphological changes (cell squeezing), and / or ethanol exposure. In any of the methods, uses, and compositions described herein, the ADAM-17-cleaved surface receptor expression level may be determined within 0 to 24 hours or within 12 to 24 hours after the cell membrane damage event. In any of the methods, uses, and compositions described herein, the ADAM-17-cleaved surface receptor expressed on immune cells may include CD16, CD62L, or the IL-15 receptor (IL-15R). In any of the methods, uses, and compositions described herein, the immune cells may include T cells, natural killer (NK) cells, chimeric antigen receptor (CAR) T cells, macrophages, dendritic cells, natural killer T (NKT) cells, innate lymphoid cells (ILC), B cells, γδ T cells, neutrophils, and / or CAR NK cells. When the immune cells include natural killer (NK) cells and / or CAR NK cells, the ADAM-17-cleaved surface receptor may include CD16, and / or the cells may include expanded NK or CAR NK cells. When the immune cells include T cells and / or CAR T cells, the ADAM-17-cleaved surface receptor may include CD62L or IL-15R. In any of the methods, uses, and compositions described herein, the level of ADAM-17-cleaved surface receptor expression may be assayed using flow cytometry and / or may be expressed as the ratio of the level of the surface receptor cleaved by ADAM17 expressed on cell membrane-damaged immune cells compared to the normal level of the ADAM-17-cleaved surface receptor expressed on immune cells. Brief Description of the Drawings

[0016] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate some embodiments and, together with the description, exemplify the disclosed compositions and methods.

[0017]

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DETAILED DESCRIPTION OF THE INVENTION

[0018] Before disclosing and describing the compounds, compositions, articles, devices, and / or methods of the present invention, it should be understood that, unless otherwise specified, they are not limited to a particular synthetic method or a particular recombinant biotechnology method, and, unless otherwise specified, they are not limited to particular reagents (since, of course, they can vary). It should also be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0019] A. Definitions As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a pharmaceutical carrier" includes mixtures of two or more such carriers, and the like.

[0020] In this specification, ranges may be expressed as from about a particular value and / or to about another particular value. When such a range is expressed, another embodiment includes from that particular value and / or to that other particular value. Similarly, when a value is expressed as an approximation, by use of the antecedent “about”, it will be understood that the particular value forms another embodiment. Further, it will be understood that each endpoint of a range is significant both in relation to the other endpoint and independently of the other endpoint. It is also understood that some values disclosed herein exist, and each value, in addition to the value itself, is also disclosed herein as “about” that particular value. For example, if the value “10” is disclosed, “about 10” is also disclosed. As will be appropriately understood by those skilled in the art, when it is disclosed that a value is “less than that value”, it will also be understood that the possible ranges between “greater than that value” and that value are also disclosed. For example, if the value “10” is disclosed, “less than 10” as well as “greater than 10” are also disclosed. Also, throughout this application, the data is provided in several different formats, and it is understood that this data represents ranges of endpoints and starting points, as well as any combination of data points. For example, if a particular data point “10” and a particular data point 15 are disclosed, in addition to between 10 and 15, values greater than 10, greater than or equal to 10, less than 10, less than or equal to 10, and equal to 10, greater than 15, greater than or equal to 15, less than 15, less than or equal to 15, and equal to 15 are understood to be 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.

[0021] In this specification and the appended claims, reference is made to several terms defined to have the following meanings.

[0022] “Optional” or “optionally” means that the event or circumstance described hereinafter may or may not occur, and this description includes instances where the event or circumstance occurs and instances where it does not occur.

[0023] "Increase" can refer to any change that results in a greater amount of a symptom, disease, composition, condition, or activity. An increase can be a statistically significant increase in any individual value, median, or mean of a state, symptom, activity, or composition. Thus, an 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%, as long as the increase is statistically significant.

[0024] "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 genetic output of a gene if the genetic output of the gene product containing that substance is less compared to the output of the gene product not containing that substance. Also, for example, a decrease can be a change in the symptoms of a disorder such that the symptoms are less than those previously observed. A decrease can be a statistically significant decrease in any individual value, median, or mean of a state, symptom, activity, or composition. Thus, a 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.

[0025] "Inhibit", "inhibiting", and "inhibition" mean reducing an activity, response, condition, disease, or other biological parameter. This can include, but is not limited to, complete elimination of an activity, response, condition, or disease. This can also include, for example, a 10% reduction in an activity, response, condition, or disease compared to a natural or control level. Thus, a reduction can be a reduction of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount in between, compared to a natural or control level.

[0026] The term "reduce" or other forms of the word, such as "reducing" or "reduction", means a decrease in an event or a characteristic (e.g., tumor growth). This is typically related to some standard or expected value, in other words, it is relative, but it is understood that it is not necessarily required to refer to a standard value or a relative value. For example, "reducing tumor growth" means reducing the growth rate of the tumor as compared to a standard or a control.

[0027] The term "prevent" or other forms of the word, such as "preventing" or "prevention", means stopping a particular event or characteristic, stabilizing or delaying the development or progression of a particular event or characteristic, or minimizing the likelihood of a particular event or characteristic occurring. Prevention is typically more absolute than, for example, reduction, and thus does not require a comparison with a control. As used herein, something can be reduced but may not be able to be prevented, but there may also be cases where something that is reduced can be prevented. Similarly, something can be prevented but may not be able to be reduced, but there may also be cases where something that is prevented can be reduced. It should be understood that when reduction or prevention is used, the use of other words is also explicitly disclosed unless specifically specified otherwise.

[0028] The term "subject" refers to any individual that is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. In one aspect, the subject can be a human, non-human primate, cow, horse, pig, dog, or cat. The subject can also be a guinea pig, rat, hamster, rabbit, mouse, or mole. Thus, the subject can be a human or a veterinary patient. The term "patient" refers to a subject under the treatment of a clinician, for example, a physician.

[0029] The term "therapeutically effective" refers to an amount of the composition being used that is sufficient to reduce one or more causes or symptoms of a disease or disorder. Such reduction only requires reduction or modification and does not need to be elimination.

[0030] The term "treatment" refers to the medical management of a patient with the intention of curing, alleviating, stabilizing, or preventing a disease, pathological condition, or disorder. This term includes active treatment, i.e., treatment specifically directed towards the improvement of a disease, pathological condition, or disorder, and causal treatment, i.e., treatment directed towards the elimination of the cause of the related disease, pathological condition, or disorder. In addition, this term includes palliative treatment, i.e., treatment designed for the relief of symptoms rather than the cure of a disease, pathological condition, or disorder, preventive treatment, i.e., treatment directed towards minimizing or partially or completely inhibiting the onset of a related disease, pathological condition, or disorder, and adjuvant treatment, i.e., treatment used to supplement another specific therapy directed towards the improvement of a related disease, pathological condition, or disorder.

[0031] "Biocompatibility" generally refers to materials and any metabolites or their degradation products that are generally non-toxic to the recipient and do not cause significant side effects in the subject.

[0032] The term "comprising" is intended to mean that compositions and methods, etc., include the recited elements but do not exclude other elements. When used to define compositions and methods, "consisting essentially of" shall mean including the recited elements but excluding any other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements defined herein does not exclude trace contaminants and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, etc., from the isolation and purification methods. "Consisting of" shall mean excluding more than trace amounts of other components and substantial method steps for administering the compositions provided and / or claimed in this disclosure. Each of these transitional terms defines an embodiment that is within the scope of this disclosure.

[0033] A "control" is an alternative object or sample used in an experiment for comparison purposes. The control may be "positive" or "negative".

[0034] An "effective amount" of a drug refers to an amount of the drug sufficient to provide the desired effect. The amount of a drug that is "effective" will vary from subject to subject depending on many factors such as the age and general condition of the subject, the particular drug(s), etc. Thus, it is not always possible to specify a quantified "effective amount". However, the appropriate "effective amount" for any given subject may be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, unless otherwise specified, an "effective amount" of a drug can also refer to an amount that covers both a therapeutically effective amount and a prophylactically effective amount. The "effective amount" of a drug required to achieve a therapeutic effect can vary according to factors such as the age, sex, and weight of the subject. The dosing schedule can be adjusted to provide an optimal therapeutic response. For example, the dose may be administered in several divided doses per day, or the dose may be proportionally reduced as indicated by the urgency of the therapeutic situation.

[0035] A "pharmaceutically acceptable" component is a component that is not biological or otherwise undesirable, i.e., a component that can be incorporated into the pharmaceutical formulations provided by the present disclosure and administered to a subject as described herein without causing a significantly undesirable biological effect or interacting in a harmful manner with any of the other components of the formulation in which it is included. When used in connection with administration to humans, the term generally means that the component meets the required criteria of toxicity and manufacturing tests or is included in the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration.

[0036] "Pharmaceutically acceptable carrier" (which may be referred to as "carrier") generally means a carrier or excipient useful in the preparation of a generally safe and non-toxic pharmaceutical or therapeutic composition, including carriers acceptable for veterinary and / or human pharmaceutical or therapeutic use. The term "carrier" or "pharmaceutically acceptable carrier" may include, but is not limited to, phosphate buffered saline, water, emulsions (such as oil / water or water / oil emulsions), and / or various types of wetting agents. As used herein, the term "carrier" encompasses, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or material well known in the art for use in pharmaceutical formulations, as well as materials further described herein.

[0037] "Pharmacological activity" (or simply "activity") in a "pharmacological activity" derivative or analog refers to a derivative or analog (e.g., salt, ester, amide, complex, metabolite, isomer, fragment, etc.) having the same type of pharmacological activity as the parent compound and to an extent that is approximately equivalent.

[0038] "Therapeutic agent" refers to any composition having a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., the treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., the prevention of a disorder or other undesirable physiological condition (e.g., non-immunogenic cancer). These terms also include, but are not limited to, pharmaceutically acceptable pharmacologically active derivatives of the beneficial agents specifically mentioned herein, such as salts, esters, amides, prodrugs, active metabolites, isomers, fragments, analogs, etc. When the term "therapeutic agent" is used, or when a specific agent is specifically identified, it should be understood that this term includes the agent itself, as well as pharmaceutically acceptable pharmacologically active salts, esters, amides, prodrugs, complexes, active metabolites, isomers, fragments, analogs, etc.

[0039] A "therapeutically effective amount" or "therapeutically effective dosage" of a composition (e.g., a composition comprising an agent) refers to an amount effective to obtain a desired therapeutic result. In some embodiments, the desired therapeutic result is the control of type I diabetes. In some embodiments, the desired therapeutic result is the control of obesity. The therapeutically effective amount of a given therapeutic agent will typically vary with factors such as the type and severity of the disorder or disease being treated, as well as the age, sex, and weight of the subject. The term can also refer to the amount of a therapeutic agent, or the rate of delivery of a therapeutic agent (e.g., amount over time), effective to promote a desired therapeutic effect such as pain relief. The exact desired therapeutic effect will vary according to the condition being treated, the tolerance of the subject, the agent and / or pharmaceutical formulation being administered (e.g., the potency of the therapeutic agent, the concentration of the agent in the formulation, etc.), and various other factors understood by those of skill in the art. In some cases, the desired biological or medical response is achieved after multiple administrations of the composition over several days, weeks, or years.

[0040] "Immune cell(s)" refers to any immune cell, e.g., T cells, natural killer (NK) cells, macrophages, dendritic cells, γδ T cells, natural killer T (NKT) cells, innate lymphoid cells (ILC), B cells, neutrophils, chimeric antigen receptor (CAR) T cells, and / or CAR NK cells, or any combination thereof.

[0041] It is to be understood that "ADAM-17-cleaved surface receptor" encompasses receptors that can also be the subject of shedding or loss mediated by other proteases.

[0042] Throughout this application, various publications are referenced. The disclosures of these publications are hereby incorporated by reference in their entirety into this application to more fully describe the state of the art relevant to this application. The disclosed references are also discussed in the context of the articles relying on the references, and the materials contained therein are hereby incorporated by reference individually and specifically into this specification.

[0043] B. Methods for Measuring the Potential for Cell Recovery The present invention provides a method for measuring the potential for recovery of immune cells after a cell membrane damage event (such as freeze-thaw cycles that occur during cryopreservation, gene editing, electroporation, magnetofection, detergent permeabilization (such as saponin and / or digitonin, etc.), streptolysin O (SLO) exposure, physical morphological changes (cell squeezing), and / or ethanol (including but not limited to solutions containing 30% or less ethanol) exposure, etc.). This method includes measuring the level of ADAM-17-cleaved surface receptors expressed on immune cells as a surrogate for damage-inducing ADAM-17 activity, and an increase in the level of this surface receptor compared to non-damaged cells (i.e., the level of the ADAM-17-cleaved surface receptor is maintained or has less loss of the ADAM-17-cleaved surface receptor) is directly correlated with less damage and a higher potential for immune cell recovery. That is, the less loss of the ADAM-17-cleaved surface receptor, the higher the potential for immune cell recovery. In one aspect, the present invention provides a method for measuring the potential for recovery of immune cells after a cell membrane damage event (such as freeze-thaw cycles that occur during cryopreservation, gene editing, electroporation, magnetofection, detergent permeabilization (such as saponin and / or digitonin, etc.), streptolysin O (SLO) exposure, physical morphological changes (cell squeezing), and / or ethanol (including but not limited to solutions containing 30% or less ethanol) exposure, etc.), and this method includes assaying the level of ADAM-17-cleaved surface receptors (such as CD16, CD62L, or IL-15 receptor (IL-15R), etc.) expressed on immune cells, and an increase in the level of the surface receptor is directly correlated with the potential for immune cell recovery. That is, the greater the surface expression of the ADAM-17-cleaved surface receptor on immune cells (i.e., the smaller the loss of the surface receptor due to ADAM-17 cleavage and the closer the surface receptor level is to that of an undamaged control), the higher the potential for immune cells to recover.Thus, disclosed herein is a method of measuring the likelihood of immune cell recovery after a cell membrane damage event, including assaying the level of ADAM-17-cleaved surface receptors expressed on immune cells, wherein an increase in the level of surface receptors is directly correlated with the likelihood of immune cell recovery.

[0044] The methods disclosed herein enable the assessment of the likelihood of recovery from a cell membrane damage event. Immune cell recovery refers to the restoration of function by immune cells. Living (viable) cells are recovered cells, although some living cells exhibit some loss of normal function. Cell viability can be determined, for example, but not limited to, using viable cell counts performed using trypan blue exclusion. Alternatively, any of many methods known in the art for determining cell viability can be used. Preferably, the recovered cells contain all or at least most of the typical activities seen for a particular type of immune cell. Cell function can be evaluated using a variety of assays known to those of skill in the art.

[0045] It is understood and contemplated herein that a "cell membrane damage event" can refer to any event or manipulation of a cell that results in deformation or permeabilization of the cell membrane (e.g., permanent, reversible, or transient permeabilization, etc.). Such events include, but are not limited to, freeze-thaw cycles (such as those resulting from cryopreservation), gene editing, electroporation, magnetofection, detergent permeabilization (such as permeabilization with saponin and / or digitonin), streptolysin O (SLO) exposure, physical morphological changes (cell squeezing), and / or ethanol exposure (including but not limited to solutions containing up to 30% ethanol).

[0046] For example, cryopreservation of immune cells can be performed using freezing and thawing processes (and equipment), in which the cells are frozen at a rate of about 1°C per minute until reaching a liquid nitrogen temperature of -80°C or about -200°C, can be stored at this temperature indefinitely, and then must be thawed very rapidly. Immune cells can be frozen after isolation or in a blood sample. Immune cells can be cryopreserved using a variety of different devices, such as Mr. Frosty (registered trademark), a polycarbonate freezing container provided by Nalgene (registered trademark), shock freezing, freezing in styrofoam insulation, or using a temperature-controlled freezer. A storage temperature of -80°C or about -80°C is preferred. In many cases, dimethyl sulfoxide (DMSO) or another cryopreservative (e.g., glycerol) is also used to help protect the cells. Typically, about 5% to about 15% DMSO, and / or glycerol, ethylene glycol, dextran, hydroxyethyl starch, or a sugar-based cryoprotectant such as trehalose is used. Once frozen, the immune cells will have a relatively long storage period and can then be thawed when needed. When immune cells are needed, they can be thawed. Thawing refers to the process of defrosting the cells, i.e., raising the temperature above the freezing point. The cells may be further warmed to a normal cell culture temperature such as about 37°C. The present invention provides a method for measuring the potential for recovery of freshly thawed immune cells.

[0047] Immune cells may be thawed using a variety of methods known to those of skill in the art. For example, immune cells can be thawed using a water bath or a controlled heat transfer device. As used herein, a water bath is a laboratory water bath. Water baths are available in various volumes and include temperature control to heat the water in the bath to a desired temperature. Water baths often include various means for providing a uniform temperature within the water bath, including means for moving the water, such as circulation and agitation. For example, various suitable laboratory water baths can be obtained from Thermo Scientific, Benchmark Scientific, or Sheldon Manufacturing, Inc. Preferably, the water bath contains water at a temperature of 37°C, such that the temperature of the immune cells rises to body temperature after thawing is complete. A controlled heat transfer device, such as the VIAThaw device from Asymptote, Ltd. (a division of GE Healthcare Life Sciences, now Cytiva), achieves the same process without using water as the heat transfer medium. Generally, frozen immune cells should be thawed immediately after removal from frozen storage. The immune cells should then be thawed rapidly (i.e., within minutes) by immersing the container holding the cells in a water bath until only small chips of ice remain in the container containing the cells. The cells are then transferred to a pre-warmed growth medium suitable for the cells (e.g., RPMI-1640 medium). Additional steps, such as centrifuging the immune cells and resuspending them in fresh growth medium, can also be performed.

[0048] As disclosed herein, the potential for recovery of immune cells from cell membrane damage directly correlates with ADAM-17 activation, which in turn can be evaluated by assaying the level of ADAM17-cleaved surface receptors expressed on immune cells. ADAM metallopeptidase domain 17 (ADAM17), also known as tumor necrosis factor α-converting enzyme (TACE), is a 70 kD enzyme belonging to the ADAM protein family of disintegrins and metalloproteases. ADAM17 is understood to be involved in the release of a wide variety of membrane-anchored cytokines, cell adhesion molecules, receptors, ligands, and enzymes in a process known as "shedding." ADAM17 can cleave the portion of a transmembrane receptor that is already bound to an agonist (e.g., CD16, CD62L, and / or IL-15R), enabling the agonist to go to and stimulate a receptor on another cell. The ADAM-17-cleaved surface receptors expressed on immune cells can vary depending on the type of immune cell. For example, in some embodiments, the immune cell is a natural killer (NK) cell and the ADAM-17-cleaved surface receptor is CD16. In other aspects, the immune cell is a T cell and the ADAM-17-cleaved surface receptor is CD62L or IL-15R.

[0049] An immune cell, as defined herein, is any cell of the immune system that produces cytokines (i.e., cytokine-producing immune cells). Examples of cytokine-producing immune cells include lymphocytes, neutrophils, macrophages, and natural killer cells. Lymphocytes include B cells, T cells, and NK cells. In some embodiments, the immune cell is a T cell, a natural killer (NK) cell, a macrophage, a dendritic cell, a natural killer T (NKT) cell, an innate lymphoid cell (ILC), a B cell, a γδ T cell, a neutrophil, a chimeric antigen receptor (CAR) T cell, and / or a CAR NK cell. Immune cells can be obtained from cell cultures or from a subject.

[0050] In some embodiments, the immune cells are T cells. 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 their cell surface. Examples of T cells include T helper cells (TH cells), cytotoxic T cells (TC cells), effector T cells, memory T cells, regulatory or "suppressor" T cells, γδ T cells, and natural killer T cells (NKT cells, distinguished from NK cells and recognizing glycolipid antigens rather than peptides presented by MHC molecules). Different types of T cells differ from each other in their cytokine production patterns.

[0051] In some embodiments, the immune cells are NK cells. Natural killer cells are a type of cytotoxic lymphocyte of the immune system. NK cells provide a rapid response to virus-infected cells and respond to transformed cells. Typically, immune cells detect peptides from pathogens presented by major histocompatibility complex (MHC) molecules on the surface of infected cells, which triggers cytokine release and causes lysis or apoptosis. NK cells are unique in that they have the ability to recognize stressed cells regardless of whether pathogen-derived peptides are present on MHC molecules. These cells were named "natural killer" because of the initial belief that they did not require prior activation to kill targets. NK cells are large granular lymphocytes (LGLs) and are known to differentiate and mature in the bone marrow and then enter the circulation from there.

[0052] In some embodiments, the immune cells are immune cells for immunotherapy. Immune cells for immunotherapy are useful for the treatment of diseases such as cancer. Immune cells for immunotherapy include tumor-infiltrating lymphocytes (TILs), T cells, and / or NK cells that have been described for use in the treatment of cancer. Immune cells for immunotherapy are also cells engineered to contain chimeric antigen receptors (CARs), including but not limited to CAR T cells and CAR NK cells, which are also used in the treatment of cancer.

[0053] Since it is beneficial to be able to administer a large number of immune cells in immunotherapy, in some embodiments, the immune cells are expanded immune cells. The expanded immune cells are immune cells that grow ex vivo to grow a large number of immune cells. The expanded immune cells can be expanded from simultaneous immune cells or from other cell types. For example, NK cells can be expanded from peripheral blood mononuclear cells or hematopoietic stem cells. In some embodiments, the expanded immune cells are autologous cells that can be easily administered to a subject without inducing an immune response. However, in some embodiments, the expanded immune cells are allogeneic immune cells, and their inherent alloreactivity can be beneficial. In further embodiments, the expanded immune cells are genetically engineered to contain chimeric antigen receptors to help the immune cells target diseased tissue. The preparation of expanded immune cells includes activating and expanding the immune cells. Some 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 NK cells ex vivo. For example, in some embodiments, the NK cells to be evaluated are NK cells expanded with IL-21. NK cells expanded with IL-21 include NK cells stimulated by soluble IL-21, feeder cells composed of membrane-bound IL-21 (mbIL-21), cell membrane particles containing mbIL-21, exosomes containing mbIL-21, and solid supports having mbIl-21.

[0054] Measuring the possibility of recovery of immune cells after a cell membrane damage event (such as freeze-thaw cycles, gene editing, electroporation, magnetofection, detergent permeabilization (such as saponin and / or digitonin, etc.), streptolysin O (SLO) exposure, physical morphological changes (cell squeezing), and / or ethanol (including but not limited to solutions containing 30% or less ethanol) exposure, etc.) may occur at any time after the cell membrane damage event. The freshly damaged immune cells are cells damaged within the past 48 hours. In some embodiments, the freshly damaged immune cells are those damaged within 0 to 24 hours, while in other embodiments, the freshly damaged immune cells are those damaged within 0 to 12 hours. In further embodiments, the freshly damaged immune cells are those damaged within 12 to 24 hours. For example, the immune cells can be assayed within 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 90, 105, 120, 150, 180 minutes, 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, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 hours after the immune cell membrane damage event.

[0055] In a method for measuring the likelihood of recovery of immune cells after a cell membrane damage event (including, but not limited to, freeze-thaw cycles, gene editing, electroporation, magnetofection, detergent permeabilization (e.g., saponin and / or digitonin, etc.), streptolysin O (SLO) exposure, physical morphological changes (cell squeezing), and / or ethanol exposure (including but not limited to solutions containing up to 30% ethanol)), an increase in the level of ADAM-17-cleaved surface receptors correlates directly with the likelihood of immune cell recovery. That is, the greater the increase in ADAM-17-cleaved surface receptor expression (i.e., the fewer surface receptors cleaved by ADAM17 and the closer to the control where the expression level is not damaged), the higher the likelihood of immune cell recovery. Similarly, the greater the decrease in ADAM-17-cleaved surface receptor expression, the lower the likelihood of cell recovery. The increase in the level of ADAM-17-cleaved surface receptors can be determined by comparison with a control value. The control value can be a previously identified control value or a control value measured simultaneously with the determination of the ADAM-17-cleaved surface receptor level. The control can be a positive control such as non-damaged cells, in which case the comparison can be made with respect to the amount of loss, and cells with a higher likelihood of recovery are cells with less loss of ADAM-17-cleaved surface receptors, and more loss of ADAM-17-cleaved surface receptors has a lower likelihood of recovery. Alternatively, the control can be a negative control with damaged or non-damaged cell surfaces with low or no ADAM-17-cleaved surface receptor expression, and cells with a higher likelihood of recovery have increased ADAM-17-cleaved surface receptor expression compared to that control. Since the recovery ability is directly correlated, more recovery is seen with an increase, and the increase or decrease can also be with respect to other immune cells in the population, and cells expressing increased ADAM-17-cleaved surface receptors have a higher likelihood of recovery than cells from the same sample with fewer ADAM-17-cleaved surface receptors.

[0056] In some embodiments, the level of the ADAM-17-cleaved surface receptor expressed on immune cells is expressed as the ratio of the level of the surface receptor cleaved by ADAM17 expressed on damaged immune cells compared to the normal level of the ADAM-17-cleaved surface receptor expressed on immune cells. Thus, in one aspect, the present disclosure provides a method for measuring the likelihood of recovery of immune cells after a cell membrane damage event, wherein the level of the ADAM-17-cleaved surface receptor expressed on immune cells is expressed as the ratio of the level of the surface receptor cleaved by ADAM17 expressed on cell membrane-damaged immune cells compared to the normal level of the ADAM-17-cleaved surface receptor expressed on immune cells.

[0057] The amount of the ADAM-17-cleaved surface receptor can be assayed using any method capable of detecting the amount of protein on the cell surface. For example, the amount of the ADAM-17-cleaved surface receptor can be detected using an immunoassay or a cell sorting method. There are many different formats and variants of immunoassays. The immunoassay may be performed in multiple steps involving adding reagents and washing or separating at different points in the assay. Immunoassays include heterogeneous immunoassays that include multiple steps, and homogeneous immunoassays that simply mix a reagent and a sample and involve physical measurements. Types of immunoassays include competitive homogeneous immunoassays, competitive heterogeneous immunoassays, one-site non-competitive immunoassays, and two-site non-competitive immunoassays. Immunoassays also include enzyme-linked immunosorbent assay (ELISA), lateral flow immunoassay, enzyme-linked immunosorbent spot (ELIspot) assay, antibody array assay and bead-based assay, magnetic immunoassay, Western blot, and radioimmunoassay.

[0058] In some embodiments, the level of ADAM-17 cleavage surface receptor expression is assayed using flow cytometry. Flow cytometry is a cell sorting method that labels cells with fluorescent markers and then passes the cells through a flow cytometer that uses light scattering to characterize the cells. Similarly, mass cytometry (CyTOF) may be used in the assay.

[0059] The steps of various useful immunoassay methods are described in scientific literature such as, for example, Maggio et al., Enzyme-Immunoassay, (1987), and Nakamura, et al., Enzyme Immunoassays: Heterogeneous and Homogeneous Systems, Handbook of Experimental Immunology, Vol. 1: Immunochemistry, 27.1-27.20 (1986), each of which is hereby incorporated by reference in its entirety, particularly for the teachings regarding the immunoassay method. The simplest and most direct meaning of an immunoassay is a binding assay involving the binding between an antibody and an antigen. Many types and formats of immunoassays are known, and all are suitable for detecting the disclosed biomarkers. Examples of immunoassays are enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunoprecipitation assay (RIPA), immunobead capture assay, Western blot, dot blot, gel shift assay, flow cytometry, protein array, multiplex bead array, magnetic capture, in vivo imaging, fluorescence resonance energy transfer (FRET), and fluorescence recovery after photobleaching / localization (FRAP / FLAP).

[0060] Generally, an immunoassay involves contacting a sample suspected of containing a molecule of interest (such as a disclosed biomarker) with an antibody to the molecule of interest, or an antibody to the molecule of interest (such as an antibody to a disclosed biomarker) with a molecule to which the antibody can bind, under conditions effective to form an immune complex. Contacting the sample with an antibody to the molecule of interest, or a molecule to which the antibody can bind, under conditions effective and for a period of time sufficient to form an immune complex (a primary immune complex) generally simply involves contacting the molecule or antibody with the sample and incubating the mixture for a period of time long enough for the antibody to form an immune complex with any molecule present to which the antibody can bind, i.e., bind to that any molecule. In many forms of immunoassay, the sample-antibody composition, such as a tissue section, ELISA plate, dot blot or Western blot, is then washed to remove any non-specifically bound antibody species and enable detection of only the specifically bound antibody in the primary immune complex.

[0061] An immunoassay can include methods for detecting or quantifying the amount of a molecule of interest (such as a disclosed biomarker or an antibody thereto) in a sample, and these methods generally involve detection or quantification of any immune complex formed during the binding process. Generally, detection of immune complex formation is well known in the art and can be accomplished by applying a number of approaches. These methods generally are based on detection of a label or marker, such as any radioactive, fluorescent, biological, or enzymatic tag, or any other known label.

[0062] As used herein, a label can include a fluorescent dye, a member of a binding pair such as biotin / streptavidin, a metal (e.g., gold), or an epitope tag that can specifically interact with a molecule that can be detected, for example, by generating a colored substrate or fluorescence. Substances suitable for detectably labeling a protein include fluorescent dyes (also known herein as fluorescent dyes and fluorophores), and enzymes that react with a colorimetric substrate (e.g., horseradish peroxidase). The use of fluorescent dyes is generally preferred in the practice of the present invention because fluorescent dyes can be detected in very small amounts. Further, when multiple antigens are reacted in a single array, each antigen can be labeled with a different fluorescent compound for simultaneous detection. A fluorometer is used to detect the labeled spots on the array, which is the presence of a signal indicating the antigen bound to a specific antibody.

[0063] Modifying units such as radionuclides can be incorporated into or directly attached to any of the compounds described herein by halogenation. Examples of radionuclides useful in this embodiment include, but are not limited to, tritium, iodine-125, iodine-131, iodine-123, iodine-124, astatine-210, carbon-11, carbon-14, nitrogen-13, fluorine-18. In another aspect, the radionuclide may be attached to a linking group or bound by a chelating group, which in turn binds directly or via a linker to the compound. Examples of radionuclides useful in this aspect include, but are not limited to, Tc-99m, Re-186, Ga-68, Re-188, Y-90, Sm-153, Bi-212, Cu-67, Cu-64, and Cu-62. Such radiolabeling techniques are routinely used in the radiopharmaceutical industry.

[0064] Radioactive labeled compounds are useful as imaging agents for diagnosing neurological diseases (e.g., neurodegenerative diseases) or mental states, or for tracking the progression or treatment of such diseases or states in mammals (e.g., humans). The radioactive labeled compounds described herein can be conveniently used in combination with imaging techniques such as positron emission tomography (PET) or single photon emission computed tomography (SPECT).

[0065] The label may be either direct or indirect. In direct labeling, the detection antibody (an antibody for the molecule of interest) or the detection molecule (a molecule that can be bound by an antibody against the molecule of interest) contains the label. Detection of the label indicates the presence of the detection antibody or detection molecule, which in turn indicates, respectively, the presence of the molecule of interest or the presence of an antibody against the molecule of interest. In indirect labeling, an additional molecule or moiety is contacted with the immune complex or generated at the site of the immune complex. For example, a signal generating molecule or moiety such as an enzyme can bind to or associate with the detection antibody or detection molecule. The signal generating molecule can then generate a detectable signal at the site of the immune complex. For example, an enzyme can generate a visible or detectable product at the site of the immune complex when supplied with an appropriate substrate. ELISA uses this type of indirect labeling.

[0066] As another example of indirect labeling, an additional molecule (which may be referred to as a binder) that can bind to either the molecule of interest or an antibody (primary antibody) against the molecule of interest, for example, a second antibody against the primary antibody, can be contacted with the immune complex. The additional molecule may have a label or signal generating molecule or moiety. Since the additional molecule can be an antibody, it may be referred to as a secondary antibody. Binding of the secondary antibody to the primary antibody can form a so-called sandwich with the first (or primary) antibody and the molecule of interest. The immune complex can be contacted with the labeled secondary antibody for a sufficient period under conditions effective to form a secondary immune complex. Then, generally, after washing the secondary immune complex to remove any non-specifically bound labeled secondary antibody, the remaining label in the secondary immune complex can be detected. The additional molecule can also be or include one of a pair of molecules or moieties that can bind to each other, such as the biotin / avidin pair. In this manner, the detection antibody or molecule should include the other member of the pair.

[0067] Other modes of indirect labeling include the detection of primary immune complexes by a two-step approach. For example, a molecule such as an antibody (which may be referred to as a first binder) having binding affinity for the molecule of interest or the corresponding antibody can be used to form a secondary immune complex as described above. After washing, the secondary immune complex can be contacted with another molecule (which may be referred to as a second binder) having binding affinity for the first binder for a sufficient period under conditions effective to form an immune complex here too (thus forming a tertiary immune complex). The second binder can be linked to a detectable label or signal generating molecule or moiety to enable detection of the tertiary immune complex thus formed. This system can provide signal amplification.

[0068] Immunoassays involving detection as a substance such as a protein or an antibody against a specific protein include label-free assays, protein separation methods (i.e., electrophoresis), solid support capture assays, or in vivo detection. Label-free assays are generally diagnostic means for determining the presence or absence of a specific protein in a sample or an antibody against a specific protein. Protein separation methods are further useful for evaluating the physical properties of proteins such as size or net charge. Capture assays are generally more useful for quantitatively evaluating the concentration of a specific protein in a sample or an antibody against a specific protein. Finally, in vivo detection is useful for evaluating the spatial expression pattern of a substance, i.e., where the substance can be found in a subject, tissue, or cell.

[0069] If the concentration is sufficient, the molecular complex ([Ab-Ag]n) generated by the antibody-antigen interaction is visible to the naked eye, but even smaller amounts can be detected and measured due to their ability to scatter light beams. The formation of the complex indicates the presence of both reactants, and in immunoprecipitation assays, a specific antigen ([Ab-Ag]n) is measured using a reagent antibody at a certain concentration, and a specific antibody ([Ab-Ag]n) is detected using a reagent antigen. If the reagent species has been previously coated on cells (in the case of hemagglutination reaction assays) or very small particles (in the case of latex agglutination assays), the "clamping" of the coated particles is visible at much lower concentrations. Various assays based on these basic principles are commonly used, including the Ouchterlony immunodiffusion assay, rocket immunoelectrophoresis, and immunoturbidimetry and nephelometry assays. The main limitation of such assays is that, compared to assays using labels, their sensitivity is limited (the detection limit is low), and in some cases, very high concentrations of the analyte can actually inhibit complex formation and require precautions to make the procedure more complex. Some of these Group 1 assays date back to the discovery of antibodies, and none of them actually have a "label" (e.g., Ag-enz). Other types of label-free immunoassays rely on immunosensors, and various instrumentation capable of directly detecting antibody-antigen interactions is currently commercially available. Most rely on generating an evanescent wave on a sensor surface with an immobilized ligand, which allows for continuous monitoring of binding to the ligand. Immunosensors enable easy investigation of kinetic interactions and, with the emergence of low-cost specialized instrumentation, may be widely applied to immunoanalysis in the future.

[0070] The use of an immunoassay to detect a specific protein may involve the separation of proteins by electrophoresis. Electrophoresis is the movement of charged molecules in a solution in response to an electric field. Their migration rates depend on the strength of the electric field, the net charge, size, and shape of the molecules, as well as the ionic strength, viscosity, and temperature of the medium through which the molecules migrate. As an analytical tool, electrophoresis is simple, rapid, and sensitive. It is used analytically to study the properties of single charged species and as a separation technique.

[0071] The sample is electrophoresed in a support matrix such as paper, cellulose acetate, starch gel, agarose, or polyacrylamide gel. The matrix suppresses mixing due to convection caused by heating and provides a record of the electrophoresis migration. At the end of the electrophoresis, the matrix can be stained and used for scanning, autoradiography, or storage. In addition, agarose and polyacrylamide, the most commonly used support matrices, provide a means of separating molecules by size since they are porous gels. The porous gel can function as a sieve by allowing smaller molecules to move freely while retarding or, in some cases, completely preventing the movement of larger macromolecules. Dilute agarose gels are generally more rigid and easier to handle than polyacrylamide gels of the same concentration, so agarose is used to separate larger macromolecules such as nucleic acids, large proteins, and protein complexes. Polyacrylamide, which is easier to handle and prepare at higher concentrations, is used to separate most proteins and small oligonucleotides that require small gel pore sizes for retardation.

[0072] Proteins are amphoteric compounds, and thus their net charge is determined by the pH of the medium in which they are suspended. In a solution where the pH is higher than its isoelectric point, the protein has a net negative charge and migrates towards the anode in an electric field. When below its isoelectric point, the protein is positively charged and migrates towards the cathode. The net charge carried by a protein is, in addition, independent of its size, i.e., the charge carried per unit mass of the molecule (or, considering proteins and nucleic acids as linear macromolecules, the length) varies from protein to protein. Thus, at a given pH and under non-denaturing conditions, the electrophoretic separation of proteins is determined by both the size and charge of the molecule.

[0073] Sodium dodecyl sulfate (SDS) is an anionic detergent that denatures proteins by "wrapping" the polypeptide backbone, and SDS binds quite specifically to proteins at a mass ratio of 1.4:1. By doing so, SDS imparts a negative charge to the polypeptide in proportion to its length. Furthermore, it is usually necessary to reduce (denature) the disulfide bridges in the protein before it takes on the random coil conformation necessary for size separation, which is done using 2-mercaptoethanol or dithiothreitol (DTT). Thus, in the denaturing SDS-PAGE separation, migration is determined by the molecular weight rather than the internal charge of the polypeptide.

[0074] The determination of molecular weight is carried out by SDS-PAGE of proteins of known molecular weight together with the protein to be characterized. There is a linear relationship between the logarithm of the molecular weight of an SDS-denatured polypeptide or native nucleic acid and its Rf. Rf is calculated as the ratio of the distance migrated by the molecule to the distance migrated by the marker dye front. A simple method for determining the relative molecular weight (Mr) by electrophoresis is to plot a standard curve of migration distance versus log10MW for known samples and read the logMr of the sample after measuring its migration distance on the same gel.

[0075] In two-dimensional electrophoresis, proteins are fractionated first based on one physical property and then in a second step based on another physical property. For example, isoelectric focusing can be used in the first dimension and can be conveniently carried out in a tube gel, and SDS electrophoresis in a slab gel can be used in the second dimension. An example of the procedure is that of O’Farrell, P.H., High Resolution Two-dimensional Electrophoresis of Proteins, J. Biol. Chem. 250:4007-4021 (1975), which is hereby incorporated by reference in its entirety for its teaching regarding two-dimensional electrophoresis. Other examples include, but are not limited to, those found in Anderson, L and Anderson, NG, High resolution two-dimensional electrophoresis of human plasma proteins, Proc. Natl. Acad. Sci. 74:5421-5425 (1977), Ornstein, L., Disc electrophoresis, L. Ann. N.Y. Acad. Sci. 121:321349 (1964), each of which is hereby incorporated by reference in its entirety for its teaching regarding electrophoresis. Laemmli, U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227:680 (1970), which is hereby incorporated by reference in its entirety for its teaching regarding electrophoresis, discloses a discontinuous system for separating proteins denatured by SDS. The leading ion of the Laemmli buffer system is chloride and the trailing ion is glycine. Thus, the separating gel and the stacking gel are composed of Tris-HCl buffers (with different concentrations and pHs), and the tank buffer is Tris-glycine. All buffers contain 0.1% SDS.

[0076] An example of an immunoassay using electrophoresis contemplated by the present method is Western blot analysis. The Western blot method or immunoblot method enables the determination of the molecular mass of proteins and the measurement of the relative amounts of proteins present in different samples. Detection methods include chemiluminescence and chromogenic detection. Standard methods for Western blot analysis can be found, for example, in D.M. Bollag et al., Protein Methods (2d edition 1996) and E. Harlow & D. Lane, Antibodies, a Laboratory Manual (1988), U.S. Patent No. 4,452,901, each of which is hereby incorporated by reference in its entirety for teachings regarding the Western blot method. Generally, proteins are separated by gel electrophoresis, usually SDS-PAGE. The proteins are transferred to a special blotting paper, such as a sheet of nitrocellulose, although other types of paper or membranes can also be used. The proteins retain the same separation pattern as they had on the gel. The blot is incubated with a general protein (such as milk protein) to bind to any remaining sticky spots on the nitrocellulose. Next, an antibody is added to a solution that can bind to that specific protein.

[0077] The attachment of a specific antibody to a specific immobilized antigen can usually be easily visualized by indirect enzyme immunoassay techniques using a chromogenic substrate (e.g., alkaline phosphatase or horseradish peroxidase) or a chemiluminescent substrate. Other possibilities for the probe include the use of fluorescence or radioisotope labels (e.g., fluorescein, 125 I). The probe for the detection of antibody binding can be a conjugated anti-immunoglobulin, a conjugated staphylococcal protein A (which binds to IgG), or a probe to a biotinylated primary antibody (e.g., conjugated avidin / streptavidin).

[0078] The ability of this technique lies in simultaneously detecting specific proteins based on their antigenicity and their molecular mass. First, the proteins are separated by mass in SDS-PAGE and then specifically detected in an immunoassay step. In this way, protein standards (ladders) can be run simultaneously to approximate the molecular mass of the protein of interest in a heterogeneous sample.

[0079] The gel shift assay or electrophoretic mobility shift assay (EMSA) can be used to detect the interaction between DNA-binding proteins and their cognate DNA recognition sequences in both qualitative and quantitative manners. Exemplary techniques are described in Ornstein L., Disc electrophoresis-I: Background and theory, Ann. NY Acad. Sci. 121:321-349 (1964), and Matsudiara, PT and DR Burgess, SDS microslab linear gradient polyacrylamide gel electrophoresis, Anal. Biochem. 87:386-396 (1987), each of which is hereby incorporated by reference in its entirety for the teachings regarding the gel shift assay.

[0080] In a typical gel shift assay, a purified protein or crude cell extract is incubated with a label (e.g., 32After incubation with a DNA or RNA probe labeled with [[P radioactive label]], the complex can be separated from the free probe through a non-denaturing polyacrylamide gel. The complex migrates through the gel more slowly than the unbound probe. Depending on the activity of the binding protein, the labeled probe can be either double-stranded or single-stranded. For the detection of DNA-binding proteins such as transcription factors, either purified or partially purified protein, or nuclear cell extracts can be used. For the detection of RNA-binding proteins, either purified or partially purified protein, or nuclear or cytoplasmic cell extracts can be used. The specificity of a DNA or RNA-binding protein for a putative binding site is established by competition experiments using a DNA or RNA fragment, or an oligonucleotide containing the binding site for the protein of interest, or other unrelated sequences. The difference in the nature and strength of the complexes formed in the presence of specific and non-specific competitors allows the identification of specific interactions. See Promega, Gel Shift Assay FAQ available at <http: / / www.promega.com / faq / gelshfaq.html> (last accessed March 25, 2005). This is hereby incorporated by reference in its entirety for teachings regarding the gel shift assay.

[0081] The gel shift method can include, for example, detecting proteins in a gel such as a polyacrylamide electrophoresis gel using a colloidal form of a COOMASSIE (Imperial Chemicals Industries, Ltd) blue dye. Such methods are described, for example, in Neuhoff et al., Electrophoresis 6:427-448 (1985), and Neuhoff et al., Electrophoresis 9:255-262 (1988), each of which is hereby incorporated by reference in its entirety for teachings regarding the gel shift method. In addition to the conventional protein assay methods referenced above, a combined wash and protein staining composition is described in U.S. Patent No. 5,424,000, which is hereby incorporated by reference in its entirety for teachings regarding the gel shift method. The solution can include phosphoric acid, sulfuric acid, and nitric acid, as well as an acid violet dye.

[0082] Radioimmunoprecipitation assay (RIPA) is a sensitive assay that uses a radioactively labeled antigen to detect specific antibodies in serum. After reacting the antigen with the serum, it is precipitated using a special reagent such as protein A Sepharose beads, for example. Next, the bound radioactively labeled immunoprecipitate is analyzed as usual by gel electrophoresis. Radioimmunoprecipitation assay (RIPA) is often used as a confirmatory test for diagnosing the presence of HIV antibodies. RIPA is also sometimes referred to in the art as the Farr assay, precipitin assay, radioimmunoprecipitin assay, radioimmunoprecipitation analysis, radioimmunoprecipitation analysis, and radioimmunoprecipitation analysis.

[0083] The above-described immunoassays that utilize electrophoresis to separate and detect a specific protein of interest enable the evaluation of protein size, but they are not very sensitive for the evaluation of protein concentration. However, immunoassays that bind a protein or an antibody specific to the protein to a solid support (e.g., a tube, well, bead, or cell) to capture the antibody or the protein of interest from a sample, respectively, are also contemplated in combination with a method for detecting the protein or the antibody specific to the protein on the support. Examples of such immunoassays include radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), flow cytometry, protein arrays, multiplex bead assays, and magnetic capture.

[0084] Radioimmunoassay (RIA) is a classical quantitative assay for detecting antigen-antibody reactions by using a radioactively labeled substance (radioactive ligand), either directly or indirectly, to measure the binding of an unlabeled substance to a specific antibody or other receptor system. Radioimmunoassay is used, for example, to test hormone levels in blood without the need to use a bioassay. Non-immunogenic substances (e.g., haptens) can also be measured if they are coupled to a larger carrier protein (e.g., bovine gamma-globulin or human serum albumin) that can induce antibody formation. In RIA, radioisotopes 125 I or 131Often, the use of I) involves mixing it with an antibody against that antigen. The antibody is generally linked to a solid support such as a tube or beads. Next, an unlabeled antigen or “cold” antigen is added in a known amount, and the displacement amount of the labeled antigen is measured. First, the radioactive antigen is bound to the antibody. When the cold antigen is added, these two compete for the antibody-binding site, and when the concentration of the cold antigen is high, more of it binds to the antibody and displaces the radioactive variant. The bound antigen is separated from the unbound antigen in the solution, and the binding curve is plotted using the radioactivity of each. This technique is extremely sensitive and specific.

[0085] Enzyme-linked immunosorbent assay (ELISA), or EIA (enzyme immunoassay) in general, is an immunoassay that can detect antibodies specific to a protein. In such an assay, the detectable label conjugated to either an antibody-binding or an antigen-binding reagent is an enzyme. When exposed to a substrate, this enzyme reacts to produce a chemical moiety that can be detected, for example, by spectrophotometry, fluorometry, or visual means. Enzymes that can be used to detectably label reagents useful for detection include, but are not limited to, horseradish peroxidase, alkaline phosphatase, glucose oxidase, β-galactosidase, ribonuclease, urease, catalase, malate dehydrogenase, staphylococcal nuclease, asparaginase, yeast alcohol dehydrogenase, α-glycerophosphate dehydrogenase, triosephosphate isomerase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase.

[0086] Variations of the ELISA technique are known in the art. In one variation, an antibody capable of binding to a protein can be immobilized on a selected surface exhibiting protein affinity, such as in a well of a polystyrene microtiter plate. Next, a test composition suspected of containing a marker antigen can be added to the well. After binding and washing to remove non-specifically bound immune complexes, the bound antigen can be detected. Detection can be achieved by adding a second antibody specific for the target protein conjugated to a detectable label. This type of ELISA is a simple "sandwich ELISA". Detection can also be achieved by adding a second antibody followed by a third antibody having binding affinity for the second antibody, which third antibody is conjugated to a detectable label.

[0087] Another variation is competitive ELISA. In competitive ELISA, a test sample competes for binding with a known amount of labeled antigen or antibody. The amount of reactive species in the sample can be determined by mixing the sample with a known labeled species either before or during incubation using the coated well. The presence of reactive species in the sample acts to reduce the amount of labeled species capable of binding to the well, so that the final signal is reduced.

[0088] Regardless of the format used, ELISA has certain common features, such as coating, incubation or binding, washing to remove non-specifically bound species, and detection of the bound immune complex. Antigens or antibodies can be linked to a solid support, such as in the form of plates, beads, test strips, membranes or column matrices, and the sample to be analyzed is applied to the immobilized antigen or antibody. When coating a plate with either an antigen or an antibody, generally the wells of the plate are incubated with a solution of the antigen or antibody overnight or for a specific time. The wells of the plate can then be washed to remove incompletely adsorbed material. Subsequently, any remaining available surface of the wells can be "coated" with a non-specific protein that is antigenically neutral with respect to the test antiserum. These include solutions of bovine serum albumin (BSA), casein, and skim milk. Coating enables the blocking of non-specific adsorption sites on the immobilized surface, thus reducing the background caused by non-specific binding of the antiserum to the surface.

[0089] In ELISA, secondary or tertiary detection means can also be used instead of a direct procedure. Therefore, after binding of the protein or antibody to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilized surface is contacted with a control clinical or biological sample that is tested under conditions effective to allow immune complex (antigen / antibody) formation. Detection of the immune complex subsequently requires a labeled second binder, or a second binder combined with a labeled third binder.

[0090] Enzyme-linked immunosorbent spot assay (ELISPOT) is an immunoassay that can detect antibodies specific to a protein or antigen. In such an assay, the detectable label conjugated to either an antibody-binding or antigen-binding reagent is an enzyme. When exposed to a substrate, this enzyme reacts to produce a chemical moiety that can be detected, for example, by spectrophotometry, fluorometry, or visual means. Enzymes that can be used to detectably label reagents useful for detection include, but are not limited to, horseradish peroxidase, alkaline phosphatase, glucose oxidase, β-galactosidase, ribonuclease, urease, catalase, malate dehydrogenase, staphylococcal nuclease, asparaginase, yeast alcohol dehydrogenase, α-glycerophosphate dehydrogenase, triosephosphate isomerase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase. In this assay, a nitrocellulose microtiter plate is coated with an antigen. After exposing a test sample to the antigen, it is reacted in the same manner as in an ELISA assay. Detection is different from conventional ELISA in that it is determined by enumeration of spots on the nitrocellulose plate. The presence of spots indicates that the sample has reacted with the antigen. The spots can be counted to determine the number of cells in the sample that are specific to the antigen.

[0091] "Under conditions effective to form an immune complex (antigen / antibody)" means that the conditions include diluting the antigen and antibody in solutions such as BSA, bovine gamma globulin (BGG), and phosphate buffered saline (PBS) / Tween to reduce non-specific binding and promote a reasonable signal-to-noise ratio.

[0092] Suitable conditions also mean that the incubation is at a temperature and for a period sufficient to allow effective binding. The incubation step typically may be at a temperature of about 1 minute to 12 hours, about 20°C to 30°C, or may be incubated overnight at about 0°C to about 10°C.

[0093] After all incubation steps in ELISA, the contacted surface can be washed to remove uncomplexed materials. The washing procedure can include washing with a solution such as PBS / Tween or borate buffer. After a specific immune complex is formed between the test sample and the original binding substance and subsequent washing is performed, the occurrence of the immune complex can be determined even in trace amounts.

[0094] To provide a detection means, the second or third antibody can have an associated label for enabling detection as described above. This can be an enzyme that can generate color when incubated with an appropriate chromogenic substrate. For this purpose, for example, the first or second immune complex can be contacted and incubated with the labeled antibody for a certain period of time, under conditions and for a period favorable for the development of further immune complex formation (e.g., incubation at room temperature for 2 hours in a PBS-containing solution such as PBS-Tween).

[0095] After incubation with the labeled antibody, following washing to remove unbound substances, the amount of the label can be quantified, for example, in the case of peroxidase as an enzyme label, by incubation with a chromogenic substrate such as urea and bromocresol purple, or 2,2'-azino-di-(3-ethyl-benzothiazoline-6-sulfonic acid [ABTS] and H2O2). Then, quantification can be achieved, for example, by measuring the degree of color generation using a visible spectrum spectrophotometer.

[0096] A protein array is a solid-phase ligand-binding assay system that uses proteins immobilized on surfaces including glass, membranes, microtiter wells, mass spectrometry plates, and beads or other particles. The assay is highly parallel (multiplexed) and often miniaturized (microarray, protein chip). These advantages include being rapid and automatable, being able to be highly sensitive, being economical in terms of reagents, and providing abundant data in a single experiment. Bioinformatics support is important. Advanced software and data comparative analysis are required for data processing. However, the software can be adapted from that used for DNA arrays, as can many hardware and detection systems.

[0097] One of the main formats is the capture array, in which ligand-binding reagents, usually antibodies but which can also be alternative protein scaffolds, peptides, or nucleic acid aptamers, are used to detect target molecules in mixtures such as plasma or tissue extracts. In diagnostics, capture arrays can be used to perform multiple immunoassays in parallel, for example both tests for several analytes in individual sera and simultaneous tests on many serum samples. In proteomics, capture arrays are used to quantify and compare protein levels in different samples in healthy and diseased states, i.e., for protein expression profiling. Proteins other than specific ligand-binding agents are used in array formats for in vitro functional interaction screens such as protein–protein, protein–DNA, protein–drug, receptor–ligand, enzyme–substrate, etc. The capture reagents themselves are selected and screened against many proteins, and this can also be done in a multiplex array format against multiple protein targets.

[0098] For array construction, protein sources include cell-based expression systems for recombinant proteins, purification from natural sources, in vitro production by cell-free translation systems, and methods for peptide synthesis. Many of these methods can be automated for high-throughput production. For capture arrays and protein functional analysis, it is important that the proteins are correctly folded and functional, but this is not always the case, for example, when recombinant proteins are extracted from bacteria under denaturing conditions. Nevertheless, arrays of denatured proteins are useful for screening antibody cross-reactivity, identifying autoantibodies, and selecting ligand-binding proteins.

[0099] Protein arrays are designed as miniaturizations of familiar immunoassay methods such as ELISA and dot blotting, which are often facilitated by robotics and high-throughput detection systems that utilize fluorescence readouts and enable the performance of multiple assays in parallel. Commonly used physical supports include glass slides, silicon, microwells, nitrocellulose or PVDF membranes, as well as magnetic and other microbeads. The most familiar format is microdroplets of proteins delivered to a planar surface, although alternative architectures include CD centrifugal devices (Gyros, Monmouth Junction, NJ) based on developments in microfluidics, and special chip designs such as engineered microchannels in a plate (e.g., The Living Chip™, Biotrove, Woburn, MA), and tiny 3D posts on a silicon surface (Zyomyx, Hayward CA). Particles in suspension can also be used as the basis of an array, provided they are coded for identification, and systems include color coding for microbeads (Luminex, Austin, TX; Bio-Rad Laboratories) and semiconductor nanocrystals (e.g., QDots™, Quantum Dot, Hayward, CA), as well as barcoding for beads (UltraPlex™, SmartBead Technologies Ltd, Babraham, Cambridge, UK) and polymetallic microrods (e.g., Nanobarcodes™ particles, Nanoplex Technologies, Mountain View, CA). Beads can also be assembled into planar arrays on semiconductor chips (LEAPS technology, BioArray Solutions, Warren, NJ).

[0100] Protein immobilization involves both a coupling reagent and the nature of the surface to which it is coupled. A good protein array support surface is chemically stable before and after the coupling procedure, allows for good spot morphology, exhibits minimal non-specific binding, does not contribute to the background in the detection system, and is compatible with different detection systems. The immobilization method used is reproducible, applicable to proteins of different characteristics (size, hydrophilicity, hydrophobicity), suitable for high-throughput and automation, and compatible with the retention of fully functional protein activity. The orientation of surface-bound proteins is recognized as an important factor in presenting it in an active state to ligands or substrates, and for capture arrays, the most efficient binding results are obtained by using oriented capture reagents that generally require site-specific labeling of the protein.

[0101] Both covalent and non-covalent methods of protein immobilization are used, with various advantages and problems. Passive adsorption to the surface is methodologically simple but offers little quantitative or orientational control, which can either modify or not modify the functional properties of the protein, and reproducibility and efficiency vary. Covalent coupling methods provide stable linkages, can be applied to a variety of proteins, and have good reproducibility, but the orientation can vary, chemical derivatization can modify the function of the protein, and a stable interaction surface is required. Biological capture methods that utilize tags on the protein provide stable linkages and bind specifically and reproducibly to the protein in an oriented manner, but the biological reagents must be fully immobilized first, special handling is required for the array, and stability varies.

[0102] Several immobilization chemistries and tags have been described for the manufacture of protein arrays. Substrates for covalent bonding include slide glasses coated with amino or aldehyde-containing silane reagents. In the Versalinx™ system (Prolinx, Bothell, WA), reversible covalent bonds are achieved by the interaction between a protein derivatized with phenyl diboronic acid and salicylhydroxamic acid immobilized on a support surface. This also allows for low background binding, low autofluorescence, and retention of the function of the immobilized protein. Non-covalent binding of non-modified proteins occurs within a porous structure such as HydroGel™ (PerkinElmer, Wellesley, MA) based on a three-dimensional polyacrylamide gel, and this substrate has been reported to confer particularly low background on glass microarrays with high protein functional capacity and retention. Widely used biological coupling methods are those via appropriately modified biotin / streptavidin or hexahistidine / Ni interactions of proteins. Biotin may be conjugated to a polylysine backbone immobilized on a surface such as titanium dioxide (Zyomyx) or tantalum pentoxide (Zeptosens, Witterswil, Switzerland).

[0103] Array manufacturing methods include robotic contact printing, inkjet processing, piezoelectric spotting, and photolithography. Several commercially available arrays (e.g., Packard Biosciences) and manual devices (V&P Scientific) are available. Bacterial colonies can be robotically gridded onto PVDF membranes for in situ induction of protein expression.

[0104] The limits of spot size and density are such that the spots are on the nanometer spatial scale and are nanoarrays capable of performing thousands of reactions on a single chip less than 1 mm square. BioForce Laboratories has developed a nanoarray of 1521 protein spots in 85 square microns at the optical detection limit, which corresponds to 25 million spots per square cm. The readout methods are fluorescence and atomic force microscopy (AFM).

[0105] Fluorescent labeling and detection methods are widely used. The same instrumentation used to read DNA microarrays is applicable to protein arrays. For differential display, capture (e.g., antibody) arrays can be probed with fluorescently labeled proteins from two different cell states, where the cell lysates are directly conjugated and mixed with different fluorophores (e.g., Cy-3, Cy-5) such that the colors function as a readout of changes in target abundance. Fluorescent readout sensitivity can be amplified 10 - 100 fold by tyramide signal amplification (TSA) (PerkinElmer Lifesciences). Planar waveguide technology (Zeptosens) enables ultra-high sensitivity fluorescence detection and has the added advantage of not requiring a washing procedure. High sensitivity can also be achieved with suspension beads and particles using phycoerythrin as a label (Luminex), or by using the properties of semiconductor nanocrystals (Quantum Dot). In particular, in the field of commercial biotechnology, several novel alternative readouts have been developed. These include the adaptation of surface plasmon resonance (HTS Biosystems, Intrinsic Bioprobes, Tempe, AZ), rolling circle DNA amplification (Molecular Staging, New Haven CT), mass spectrometry (Intrinsic Bioprobes; Ciphergen, Fremont, CA), resonance light scattering (Genicon Sciences, San Diego, CA), and atomic force microscopy (BioForce Laboratories).

[0106] Capture arrays form the basis of diagnostic chips and arrays for expression profiling. These use high-affinity capture reagents such as conventional antibodies, single domains, engineered scaffolds, peptides, or nucleic acid aptamers to bind to and detect specific target ligands in a high-throughput format.

[0107] Antibody arrays have the required properties of specificity and acceptable background, and some are commercially available (BD Biosciences, San Jose, CA; Clontech, Mountain View, CA; BioRad; Sigma, St. Louis, MO). Antibodies for capture arrays are made either by conventional immunization (polyclonal sera and hybridomas) or, after selection from phage or ribosome display libraries, usually as recombinant fragments expressed in Escherichia coli (Cambridge Antibody Technology, Cambridge, UK; BioInvent, Lund, Sweden; Affitech, Walnut Creek, CA; Biosite, San Diego, CA). In addition to conventional antibodies, Fab and scFv fragments, single V domains from camels, or engineered human equivalents (Domantis, Waltham, MA) may also be useful in arrays.

[0108] The term "scaffold" refers to the ligand-binding domain of a protein that has been engineered into a plurality of variants capable of binding to diverse target molecules with antibody-like properties of specificity and affinity. The variants are produced in the form of a gene library and can be selected against individual targets by phage, bacterial, or ribosome display. Such ligand-binding scaffolds or frameworks include "Affibody" (Affibody, Bromma, Sweden) based on the Staph. aureus protein A, "Trinectin" (Phylos, Lexington, MA) based on fibronectin, and "Anticalin" (Pieris Proteolab, Freising-Weihenstephan, Germany) based on the lipocalin structure. These can be used in capture arrays, similar to antibodies, and can have the advantages of robustness and ease of manufacture.

[0109] Single-stranded nucleic acid aptamers, non-protein capture molecules that bind to protein ligands with particularly high specificity and affinity, are also used in arrays (SomaLogic, Boulder, CO). Aptamers are selected from a library of oligonucleotides by the Selex™ procedure, and their interaction with those proteins can be enhanced covalently through the incorporation of bromodeoxyuridine and UV activation cross-linking (photoaptamers). Photo-cross-linking to the ligand reduces the cross-reactivity of the aptamer due to specific steric requirements. Aptamers have the advantages of ease of generation by automated oligonucleotide synthesis, as well as the stability and robustness of DNA. In photoaptamer arrays, binding can be detected using universal fluorescent protein staining.

[0110] Protein analytes that bind to the antibody array can be detected directly or via a secondary antibody in a sandwich assay. Direct labeling is used for the comparison of different samples of different colors. When pairs of antibodies targeting the same protein ligand are available, the sandwich immunoassay provides high specificity and sensitivity and is thus a preferred method for proteins present in low abundance such as cytokines. They also offer the possibility of detecting protein modifications. Label-free detection methods, including mass spectrometry, surface plasmon resonance, and atomic force microscopy, avoid ligand modification. Optimal sensitivity and specificity, with low background to give a high signal-to-noise ratio, are required from any method. Since analyte concentrations span a wide range, sensitivity needs to be appropriately adjusted. Serial dilution of the sample or use of antibodies with different affinities are solutions to this problem. The protein of interest is often a low-concentration protein in body fluids and extracts that requires detection in the pg range or below, such as cytokines or low-expressing products in cells.

[0111] An alternative to the array of capture molecules is made through "molecular imprinting" technology, where a peptide (e.g., from the C-terminal region of a protein) is used as a template to generate structurally complementary sequence-specific cavities in a polymeric matrix, and this cavity can then specifically capture (denature) a (denatured) protein with the appropriate primary amino acid sequence (ProteinPrint™, Aspira Biosystems, Burlingame, CA).

[0112] Another methodology that can be used for diagnostics and expression profiling is the ProteinChip® array (Ciphergen, Fremont, CA), where the solid-phase chromatography surface binds proteins with similar charge or hydrophobicity characteristics from a mixture such as plasma or tumor extract, and SELDI-TOF mass spectrometry is used to detect the retained proteins.

[0113] Large-scale functional chips are constructed by immobilizing a large number of purified proteins and are used to assay a wide range of biochemical functions such as protein-protein interactions, drug-target interactions, and enzyme substrates. Generally, these require an expression library, which is cloned into Escherichia coli, yeast, or the like, and then the expressed protein is purified and immobilized therefrom, for example, via a His tag. Cell-free protein transcription / translation is a viable alternative for the synthesis of proteins that do not express well in bacterial or other in vivo systems.

[0114] To detect protein-protein interactions, protein arrays can be an in vitro alternative to cell-based yeast two-hybrid systems and may be useful when the latter are lacking, such as for interactions involving secreted proteins or proteins with disulfide bridges. High-throughput analysis of biochemical activities on arrays has been described for yeast protein kinases and for various functions of the yeast proteome (protein-protein and protein-lipid interactions), where most of the entire yeast open reading frames are expressed and immobilized on microarrays. Large-scale "proteome chips" are promised to be very useful in the identification of functional interactions, drug screening, etc. (Proteometrix, Branford, CT).

[0115] As a two-dimensional display of individual elements, protein arrays can be used to screen phage or ribosome display libraries to select specific binding partners including antibodies, synthetic scaffolds, peptides, and aptamers. In this way, "library-to-library" screening can be performed. Screening of drug candidates in combinatorial chemical libraries against arrays of protein targets identified from genomic projects is another application of this approach.

[0116] For example, multiplex bead assays such as BD (trademark) Cytometric Bead Array are a series of spectra that are separate particles and can be used to capture and quantify soluble analytes. The analyte is then measured by fluorescence-based emission and detection by flow cytometry analysis. Multiplex bead assays are equivalent to ELISA-based assays but generate data in a "multiplexed" or simultaneous fashion. Unknown concentrations are calculated for cytometric bead arrays in the same manner as for any sandwich format assay, i.e., using known standards and plotting the unknown against a standard curve. Furthermore, multiplex bead assays enable quantification of soluble analytes in samples that have not been previously considered due to sample volume limitations. In addition to quantitative data, powerful visual images can be generated that reveal unique profiles or signatures that provide additional information to the user at a glance.

[0117] C. Methods of Immunotherapy The present invention also provides a method of immunotherapy comprising administering a therapeutically effective amount of immune cells to a subject in need thereof, wherein the immune cells have just been damaged and their viability has been determined prior to administration using the methods of recovery possibility described herein. Thus, in one aspect, a method of administering immunotherapy (e.g., anti-cancer treatment, etc.) to a subject in need thereof is disclosed herein, the method comprising: a) one or more immune cells (e.g., T cells, natural killer (NK) cells, macrophages, dendritic cells, neutrophils, γδ T cells, natural killer T (NKT) cells, innate lymphoid cells (ILC), B cells, chimeric antigen receptor (CAR) T cells, and / or CAR NK cells, etc.) that have been previously subjected to a cell membrane damage event (including but not limited to freeze-thaw cycles, gene editing, electroporation, magnetofection, detergent permeabilization (e.g., saponin and / or digitonin, etc.), streptolysin O (SLO) exposure, physical morphological changes (cell squeezing), and / or ethanol exposure (including but not limited to solutions containing 30% or less ethanol)) to obtain; b) assaying the expression level of an ADAM-17-cleaved surface receptor (e.g., CD16, CD62L, or IL-15 receptor (IL-15R), etc.); c) administering to the subject a therapeutically effective amount of immune cells that express an increased level of the ADAM-17-cleaved surface receptor compared to control immune cells or compared to a mixed population of cell membrane-damaged immune cells.

[0118] As described above, the method of the present immunotherapy includes administering a therapeutically effective amount of immune cells to a subject in need thereof, and the immune cells subjected to a cell membrane damage event (including, but not limited to, freeze-thaw cycles, gene editing, electroporation, magnetofection, detergent permeabilization (such as saponin and / or digitonin, etc.), streptolysin O (SLO) exposure, physical morphological changes (cell squeezing), and / or ethanol exposure (including, but not limited to, solutions containing ethanol at 30% or less)) are determined to have survived and been recovered prior to administration by determining that they express increased levels of ADAM-17-cleaved surface receptors. The recovered cells are likely to provide effective immunotherapy and are likely to survive the administration process (thus increasing the uptake of the transferred cells), and as a result, are useful for enabling the evaluation of the recovery of immune cells prior to administration for immunotherapy. In one aspect, by measuring the recovery of immune cells (by assaying the expression level of ADAM-17-cleaved surface receptors), the clinician can select the transferred cells that have recovered (i.e., express increased levels of ADAM-17-cleaved surface receptors), thereby increasing the proportion of cells that survive transfer to the subject and are likely to be an immunotherapy for the subject, as understood and contemplated herein.

[0119] Immunotherapy, as used herein, refers to cell-based immunotherapy in which immune cells such as lymphocytes (including but not limited to tumor-infiltrating lymphocytes (TIL)), macrophages, dendritic cells, natural killer (NK) cells, natural killer T (NKT) cells, innate lymphoid cells (ILC), B cells, γδ T cells, neutrophils, cytotoxic T lymphocytes, chimeric antigen receptor (CAR) T cells, and / or CAR NK cells are administered to a subject to achieve a therapeutic effect. Cell-based immunotherapy is most frequently used as an anti-cancer treatment for subjects diagnosed with cancer. Cell-based immunotherapy includes adoptive cell transfer in which immune cells are extracted from a patient or another individual and then administered to improve immune function. For example, in autologous cancer immunotherapy, T cells or NK cells are extracted from a patient, optionally genetically modified, cultured in vitro, and then returned to the same patient. Alternatively, allogeneic cell-based immunotherapy involves cells isolated and expanded from a donor other than the patient receiving the immune cells. In some embodiments, the methods of immunotherapy include the use of T cells or natural killer (NK) cells that have experienced cell membrane damage events (including but not limited to freeze-thaw cycles, gene editing, electroporation, magnetofection, detergent permeabilization (such as saponin and / or digitonin), streptolysin O (SLO) exposure, physical morphological changes (cell squeezing), and / or ethanol (including but not limited to solutions containing 30% or less ethanol)). Further embodiments of the methods of the present immunotherapy include the use of NK cells such as expanded NK cells.

[0120] Since there is a delay between the time the cells are obtained and the time the cells are needed for immunotherapy, it is common to freeze the cells so that they can be stored until needed. Methods for freezing and thawing immune cells are described herein and are known to those of skill in the art. In some embodiments, the immune cells are thawed using a water bath. In other aspects, the cells may be manipulated to provide more effective treatment, such as by insertion of a therapeutic vector or peptide, gene editing, or construction of a chimeric antigen receptor. These manipulations often involve permeabilization of the cell membrane via electroporation, magnetofection, detergent permeabilization (e.g., saponin and / or digitonin, etc.), streptolysin O (SLO) exposure, physical morphological changes (cell squeezing), and / or ethanol exposure (including but not limited to solutions containing up to 30% ethanol). It is understood and contemplated herein that these manipulations or cryopreservation efforts (i.e., freeze-thaw cycles) result in cell membrane damage.

[0121] Measuring the potential for recovery of immune cells after a cell membrane damage event (such as freeze-thaw cycles, gene editing, electroporation, magnetofection, cell squeezing, detergent permeabilization (such as saponin and / or digitonin), streptolysin O (SLO) exposure, physical morphological changes (cell squeezing), and / or ethanol exposure (including but not limited to solutions containing up to 30% ethanol) for use in the disclosed methods of immunotherapy) may occur at any time after the cell membrane damage event. The freshly damaged immune cells are cells that have been damaged within the past 48 hours. In some embodiments, the freshly damaged immune cells are those damaged within 0 to 24 hours, while in other embodiments, the freshly damaged immune cells are those damaged within 0 to 12 hours. In further embodiments, the freshly damaged immune cells are those damaged within 12 to 24 hours. For example, the immune cells can be assayed 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 90, 105, 120, 150, 180 minutes, 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, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 hours after the cell membrane damage event of the immune cells.

[0122] Measuring the potential for recovery of immune cells after a cell membrane damage event can be done using any method capable of detecting the amount of protein on the cell surface. For example, the amount of ADAM-17 cleaved surface receptor can be detected using an immunoassay or a cell sorting method. There are many different formats and variants of immunoassays. An immunoassay may be performed in multiple steps of adding reagents and washing or separating at different points in the assay. Immunoassays include heterogeneous immunoassays that include multiple steps, and homogeneous immunoassays that simply mix a reagent and a sample and involve making a physical measurement. Types of immunoassays include competitive homogeneous immunoassays, competitive heterogeneous immunoassays, one-site non-competitive immunoassays, and two-site non-competitive immunoassays. Immunoassays also include enzyme-linked immunosorbent assay (ELISA), lateral flow immunoassay, enzyme-linked immunosorbent spot (ELIspot) assay, antibody array assay and bead-based assay, magnetic immunoassay, and radioimmunoassay.

[0123] In a method of immunotherapy using immune cells previously subjected to a cell membrane damage event, an increase in the level of ADAM-17 cleaved surface receptor directly correlates with the potential for immune cell recovery. That is, the greater the increase in ADAM-17 cleaved surface receptor expression, the higher the potential for immune cell recovery. In some embodiments, a method of performing immunotherapy is disclosed herein, wherein the level of ADAM-17 cleaved surface receptor expressed on immune cells is represented as a ratio of the level of surface receptor cleaved by ADAM17 expressed on cell membrane damaged immune cells compared to the normal level of ADAM-17 cleaved surface receptor expressed on immune cells.

[0124] 1. Pharmaceutical Carrier / Drug Delivery As described above, the composition can also be administered in vivo in a pharmaceutically acceptable carrier. "Pharmaceutically acceptable" means a material that is not biologically or otherwise undesirable, i.e., a material that can be administered to a subject, together with a nucleic acid or vector, without causing any undesirable biological effects or interacting in a harmful manner with any of the other components of the pharmaceutical composition. The carrier can be necessarily selected, as is well known to those skilled in the art, to minimize any degradation of the active ingredient and to minimize any harmful side effects in the subject.

[0125] The composition can be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, ex vivo, topically (including topical intranasal administration or administration by inhalant). As used herein, "topical intranasal administration" means delivering the composition to the nose and nasal passages through one or both nostrils and can include delivery by a spray or droplet mechanism or by aerosolization of the nucleic acid or vector. Administration of the composition by inhalant can be effected through the nose or mouth via delivery by a spray or droplet mechanism. Delivery can also be directly to any region of the respiratory system (e.g., the lungs) via an endotracheal tube. The exact amount of the composition required will vary from subject to subject depending on the species, age, weight, and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its method of administration, etc. Accordingly, it is not possible to specify an exact amount for all compositions. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation in light of the teachings herein.

[0126] Parenteral administration of the composition, when used, generally features injection. Injectables can be prepared in conventional forms as either liquid solutions or suspensions, solid forms suitable for solution of the suspension in a liquid prior to injection, or emulsions. Recently revised approaches to parenteral administration involve the use of sustained or controlled release such that a constant dosage is maintained. See, for example, U.S. Patent No. 3,610,795 (incorporated herein by reference).

[0127] The materials may be in solution, suspension (e.g., incorporated into microparticles, liposomes, or cells). These may target specific cell types via antibodies, receptors, or receptor ligands. The following references are examples of the use of this technology for targeting specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem., 2:447-451, (1991), Bagshawe, K.D., Br. J. Cancer, 60:275-281, (1989), Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988), Senter, et al., Bioconjugate Chem., 4:3-9, (1993), Battelli, et al., Cancer Immunol. Immunother., 35:421-425, (1992), Pietersz and McKenzie, Immunolog. Reviews, 129:57-80, (1992), and Roffler, et al., Biochem. Pharmacol, 42:2062-2065, (1991)). Vehicles such as "stealth" and other antibody-conjugated liposomes (including lipid-mediated drugs targeting colon cancer), receptor-mediated targeting of DNA via cell-specific ligands, lymphocyte-specific tumor targeting, and highly specific therapeutic retroviral targeting of mouse glioma cells in vivo. The following references are examples of the use of this technology for targeting specific proteins to tumor tissue (Hughes et al., Cancer Research, 49:6214-6220, (1989), and Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187, (1992)). Generally, receptors are involved in either constitutive or ligand-induced endocytosis pathways. These receptors cluster within clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through acidified endosomes where the receptors are sorted, and then are either recycled to the cell surface, stored intracellularly, or degraded in lysosomes.Internal migration pathways serve various functions such as nutrient uptake, removal of activating proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligands, and regulation of receptor levels. Many receptors follow two or more intracellular pathways depending on cell type, receptor concentration, type of ligand, ligand valence, and ligand concentration. The molecular and cellular mechanisms of receptor-mediated endocytosis have been outlined (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)).

[0128] a) A pharmaceutically acceptable carrier The present composition containing the antibody can be used for treatment in combination with a pharmaceutically acceptable carrier.

[0129] Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of a pharmaceutically acceptable salt is used in the formulation to make it isotonic. Examples of pharmaceutically acceptable carriers include, but are not limited to, physiological saline, Ringer's solution, and dextrose solution. The pH of the solution is preferably from about 5 to about 8, more preferably from about 7 to about 7.5. Further carriers include sustained-release preparations such as a semipermeable matrix of a solid hydrophobic polymer containing the antibody, and this matrix is in the form of a shaped article, for example, a film, liposome, or microparticle. It will be apparent to those skilled in the art that certain carriers may be more preferred depending, for example, on the route of administration and the concentration of the composition being administered.

[0130] Pharmaceutical carriers are known to those skilled in the art. These are typically standard carriers for drug administration to humans, including solutions such as sterile water, physiological saline, and buffer solutions at physiological pH. These compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.

[0131] In addition to the selected molecule, the pharmaceutical composition may include a carrier, thickening agent, diluent, buffer, preservative, surfactant, etc. The pharmaceutical composition may also include one or more active ingredients, such as antibacterial agents, anti-inflammatory agents, anesthetics, etc.

[0132] The present pharmaceutical composition can be administered in several ways depending on whether local or systemic treatment is desired and the area to be treated. Administration may be local (including ocular, vaginal, rectal, intranasal), oral, inhalation, or parenteral, such as by intravenous drip, subcutaneous, intraperitoneal, or intramuscular injection. The disclosed antibodies can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.

[0133] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are vegetable oils such as propylene glycol, polyethylene glycol, olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including physiological saline and buffer media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution, or fixed oils. Intravenous vehicles include fluids and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), etc. Preservatives and other additives, such as antibacterial agents, antioxidants, chelating agents, and inert gases, may also be present.

[0134] Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, solutions, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners, etc. may be necessary or desirable.

[0135] Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersion aids, or binders may be desirable.

[0136] Some of the present compositions may be administered as pharmaceutically acceptable acid or base addition salts formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl, and aryl amines, and substituted ethanolamines.

[0137] b) Therapeutic use Effective dosages and schedules for administering the compositions can be determined empirically, and making such determinations is within the skill of the art. The dosage ranges for administration of the compositions are large enough to produce the desired effect that affects the symptoms of the disorder. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, etc. Generally, the dosage will vary depending on the patient's age, condition, gender, and degree of the disease, the route of administration, or whether other drugs are included in the regimen, which can be determined by one of ordinary skill in the art. The dosage can be adjusted by the individual physician in any case of contraindication. The dosage can vary and can be administered in one or more doses per day over one day or several days. Guidelines for appropriate dosages for a given class of pharmaceuticals can be found in the literature. For example, guidelines for selecting an appropriate dosage for an antibody can be found in the literature regarding the therapeutic use of antibodies, such as Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, N.J., (1985) ch. 22 and pp. 303-357, Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York (1977) pp. 365-389. A typical daily dosage of an antibody used alone can range from about 1 μg to up to 100 mg or more per kg of body weight per day, depending on the factors described above.

[0138] In one aspect, a method of administering immunotherapy is disclosed herein, wherein the immunotherapy is an anti-cancer treatment for a subject diagnosed with cancer. Using the disclosed method of immunotherapy, any disease in which uncontrolled cell growth occurs, such as cancer, can be treated, inhibited, reduced, decreased, alleviated, and / or prevented. A representative but non-limiting list of cancers that can be treated using the disclosed compositions 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, squamous cell carcinoma of the head and neck, lung cancer such as small cell lung cancer and non-small cell lung cancer, neuroblastoma / glioblastoma, ovarian cancer, skin cancer, liver cancer, melanoma, oral, pharyngeal, laryngeal cancer, and squamous cell carcinoma of the lung, cervical cancer, cervical carcinoma, breast cancer, as well as epithelial cancer, kidney cancer, genitourinary cancer, lung cancer, esophageal cancer, head and neck cancer, colorectal cancer, hematopoietic cancer, testicular cancer, colon cancer, rectal cancer, prostate cancer, or pancreatic cancer. Thus, in one aspect, a method of treating, inhibiting, reducing, decreasing, alleviating, and / or preventing cancer and / or metastasis in a subject is disclosed herein, the method comprising: a) obtaining one or more immune cells previously subject to a cell membrane damage event; b) assaying the expression level of the ADAM-17-cleaved surface receptor; and c) administering to the subject a therapeutically effective amount of immune cells that express an increased level of the ADAM-17-cleaved surface receptor compared to control immune cells.

[0139] D. Examples The following examples are set forth to provide those of ordinary skill in the art with a complete disclosure and description of how to make and evaluate the compounds, compositions, articles, devices, and / or methods claimed herein, and are intended to be purely exemplary and not to limit the disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperatures, etc.), but some errors and deviations should be accounted for. Unless otherwise specified, parts are parts by weight, temperature is in °C, or ambient temperature, and pressure is at or near atmospheric pressure.

Example

[0140] 1. Example 1: Percentage of CD16 and total recovery rate Natural killer cells were expanded ex vivo for 14 days using K562 mbIL21.41bbL feeder cells. NK cells were frozen on day 14 in nine different cryopreservation media and frozen at a rate of 1 °C / min using a Mr.Frosty Device. NK cells were thawed in a 37 °C water bath using a standard protocol, counted, and evaluated for flow cytometry analysis immediately after thawing. All cells were left standing overnight in a T25 flask containing the same number of viable cells under each condition. NK cells were counted on day 1 after thawing. The total recovery rate from day 0 and day 1 was calculated and shown in Figures 3 and 4.

[0141] The method used to generate these results will be described in more detail here. On day 14 of cell expansion, viable cell counting was completed using trypan blue cell exclusion. Next, as described above, cells were frozen using different cryopreservation media. On day 0 of thawing, the cells were thawed in a 37 °C water bath with gentle stirring until only one small piece of ice remained in the vial. Next, medium was added to the thawed cell suspension, and the thawed cells in the medium were spun at 400×g for 5 minutes to separate the cells. Thereafter, the cells were resuspended in complete medium and viable cell counting was performed using trypan blue exclusion. Subsequently, an aliquot of NK cells was stained for CD16 and a viability dye using a general flow cytometry staining protocol. Subsequently, using the initial cell suspension, a specified number of viable cells were left standing overnight in complete medium containing IL-2.

[0142] On day 1 after thawing, viable cell counting was performed using trypan blue exclusion. The cell recovery rate was calculated from the overnight standing.

[0143] As shown in Fig. 5, NK cells were thawed, the cryopreservation medium was washed away, and the cells were resuspended in culture medium and allowed to stand for 24 hours of recovery. CD16 expression on NK cells was determined by flow cytometry within 60 minutes after thawing. Loss of CD16 was regulated by ADAM17, and NK cells lacking ADAM17 showed enhanced recovery of CD16 expression (Fig. 6).

[0144] Although the present invention has been specifically shown and described with reference to its preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined in the appended claims. All patents, publications, and references cited in the foregoing specification are hereby incorporated by reference in their entirety into this specification. Various embodiments of the present invention are shown below. 1. A method for measuring the possibility of recovery of immune cells after a cell membrane damage event, comprising assaying the level of the ADAM-17-cleaved surface receptor expressed on the immune cells, wherein an increase in the level of the surface receptor is directly correlated with the possibility of immune cell recovery. 2. The method for measuring the possibility of recovery of immune cells after a cell membrane damage event according to 1 above, wherein the membrane damage event includes freeze-thaw cycles, gene editing, electroporation, magnetofection, detergent permeabilization, streptolysin O (SLO) exposure, physical morphological changes (cell squeezing), and / or ethanol exposure. 3. The method for measuring the possibility of recovery of immune cells after a cell membrane damage event according to 2 above, wherein the membrane damage event includes a freeze-thaw cycle. 4. The method for measuring the possibility of recovery of immune cells after a cell membrane damage event according to 3 above, wherein the immune cells are thawed using a water bath. 5. The method for measuring the possibility of recovery of immune cells after a cell membrane damage event according to any one of 1 to 4 above, wherein the level of the ADAM-17-cleaved surface receptor expression is assayed within 0 to 24 hours after the immune cell membrane damage event. 6. The method for measuring the possibility of recovery of immune cells after a cell membrane damage event according to any one of 1 to 5 above, wherein the level of the ADAM-17-cleaved surface receptor expression is assayed within 12 to 24 hours after the immune cell membrane damage event. 7. The method for measuring the possibility of recovery of immune cells after a cell membrane damage event according to any one of 1 to 6 above, wherein the ADAM-17-cleaved surface receptor expressed on the immune cells includes CD16, CD62L, or IL-15 receptor (IL-15R). 8. The method for measuring the possibility of recovery of immune cells after a cell membrane damage event according to any one of 1 to 7 above, wherein the immune cells include T cells, natural killer (NK) cells, chimeric antigen receptor (CAR) T cells, CAR NK cells, macrophages, dendritic cells, natural killer T (NKT) cells, innate lymphoid cells (ILC), B cells, γδ T cells, or neutrophils. 9. The method for measuring the possibility of recovery of immune cells after a cell membrane damage event according to any one of 1 to 8 above, wherein the immune cells are natural killer (NK) cells or CAR NK cells, and the ADAM-17-cleaved surface receptor is CD16. 10. The method for measuring the possibility of recovery of immune cells after the cell membrane damage event according to item 9 above, wherein the NK cells are expanded NK cells. 11. The method for measuring the possibility of recovery of immune cells after the cell membrane damage event according to any one of items 1 to 8 above, wherein the immune cells are T cells or CAR T cells, and the ADAM-17-cleaved surface receptor includes CD62L or IL-15R. 12. The method for measuring the possibility of recovery of immune cells after the cell membrane damage event according to any one of items 1 to 11 above, wherein the level of ADAM-17-cleaved surface receptor expression is assayed using flow cytometry. 13. The method for measuring the possibility of recovery of immune cells after the cell membrane damage event according to any one of items 1 to 12 above, wherein the level of the ADAM-17-cleaved surface receptor expressed on the immune cells is represented as the ratio of the level of the surface receptor cleaved by ADAM17 expressed on the cell membrane-damaged immune cells compared to the normal level of the ADAM-17-cleaved surface receptor expressed on the immune cells. 14. A method of administering immunotherapy to a subject in need thereof, comprising: a) obtaining one or more immune cells previously subject to a cell membrane damage event; b) assaying the immune cells to determine the expression level of the ADAM-17-cleaved surface receptor; c) administering to the subject a therapeutically effective amount of immune cells that express an increased level of the ADAM-17-cleaved surface receptor compared to control immune cells. 15. The method of administering immunotherapy according to item 14 above, further comprising using the assay result of the expression level of the ADAM-17-cleaved surface receptor for screening or selection of a manufacturing lot of an immunotherapy drug. 16. The method of administering immunotherapy according to item 14 or 15 above, wherein the immunotherapy is an anti-cancer treatment for a subject diagnosed with cancer. 17. The method of administering immunotherapy according to any one of items 14 to 16 above, wherein the cell membrane damage event includes freeze-thaw cycles, gene editing, electroporation, magnetofection, detergent permeabilization, streptolysin O (SLO) exposure, physical morphological changes (cell squeezing), and / or ethanol exposure. 18. The method of administering immunotherapy according to item 17 above, wherein the cell membrane damage event includes freeze-thaw cycles. 19. The method of administering immunotherapy according to item 18 above, wherein the immune cells are thawed using a water bath. 20. A method of administering the immunotherapy according to any one of 14 to 19 above, wherein the level of expression of the ADAM-17-cleaved surface receptor is assayed within 0 to 24 hours after the cell membrane damage event. 21. A method of administering the immunotherapy according to any one of 14 to 19 above, wherein the level of expression of the ADAM-17-cleaved surface receptor is assayed within 12 to 24 hours after the cell membrane damage event. 22. A method of administering the immunotherapy according to any one of 14 to 21 above, wherein the ADAM-17-cleaved surface receptor expressed on the immune cells comprises CD16, CD62L, or interleukin-15 receptor (IL-15R). 23. A method of administering the immunotherapy according to any one of 14 to 22 above, wherein the immune cells comprise T cells, natural killer (NK) cells, chimeric antigen receptor (CAR) T cells, CAR NK cells, macrophages, dendritic cells, natural killer T (NKT) cells, innate lymphoid cells (ILC), B cells, γδ T cells, or neutrophils. 24. A method of administering the immunotherapy according to any one of 14 to 23 above, wherein the immune cells are natural killer (NK) cells or CAR NK cells, and the ADAM-17-cleaved surface receptor is CD16. 25. A method of administering the immunotherapy according to 24 above, wherein the NK cells are expanded NK cells. 26. A method of administering the immunotherapy according to any one of 14 to 25 above, wherein the immune cells are T cells or CAR T cells, and the ADAM-17-cleaved surface receptor comprises CD62L or IL-15R. 27. A method of administering the immunotherapy according to any one of 14 to 26 above, wherein the level of expression of the ADAM-17-cleaved surface receptor is assayed using flow cytometry. 28. A method of administering the immunotherapy according to any one of 14 to 27 above, wherein the level of the ADAM-17-cleaved surface receptor expressed on the immune cells is represented as a ratio of the level of the surface receptor cleaved by ADAM17 expressed on the cell membrane-damaged immune cells to the normal level of the ADAM-17-cleaved surface receptor expressed on the immune cells. 29. Use in an immunotherapy of a therapeutically effective amount of immune cells previously subject to a cell membrane damage event, wherein the cells express an increased level of ADAM-17-cleaved surface receptor compared to control immune cells. 30. The use according to 29 above, wherein the immunotherapy is anti-cancer therapy. 31. Use according to any one of claims 29 or 30, wherein the cell membrane damage event comprises a freeze-thaw cycle, gene editing, electroporation, magnetofection, detergent permeabilization, streptolysin O (SLO) exposure, physical morphological changes (cell squeezing), and / or ethanol exposure. 32. Use according to any one of claims 29 or 30, wherein the cell membrane damage event comprises a freeze-thaw cycle. 33. Use according to claim 32, wherein the immune cells are thawed using a water bath. 34. Use according to any one of claims 29 to 33, wherein the level of ADAM-17-cleaved surface receptor expression is determined within 0 to 24 hours after the cell membrane damage event. 35. Use according to any one of claims 29 to 33, wherein the level of ADAM-17-cleaved surface receptor expression is assayed within 12 to 24 hours after the cell membrane damage event. 36. Use according to any one of claims 29 to 35, wherein the ADAM-17-cleaved surface receptor expressed on the immune cells comprises CD16, CD62L, or interleukin-15 receptor (IL-15R). 37. Use according to any one of claims 29 to 36, wherein the immune cells comprise T cells, natural killer (NK) cells, chimeric antigen receptor (CAR) T cells, CAR NK cells, macrophages, dendritic cells, natural killer T (NKT) cells, innate lymphoid cells (ILC), B cells, γδ T cells, or neutrophils. 38. Use according to any one of claims 29 to 37, wherein the immune cells comprise natural killer (NK) cells and / or CAR NK cells, and the ADAM-17-cleaved surface receptor is CD16. 39. Use according to claim 38, wherein the NK cells and / or CAR NK cells comprise expanded NK cells. 40. Use according to any one of claims 29 to 36, wherein the immune cells comprise T cells and / or CAR T cells, and the ADAM-17-cleaved surface receptor comprises CD62L or IL-15R. 41. Use according to any one of claims 29 to 40, wherein the level of ADAM-17-cleaved surface receptor expression is assayed using flow cytometry. 42. Use according to any one of 29 to 41 above, wherein the level of the ADAM-17-cleaved surface receptor expressed on the immune cell is represented as the ratio of the level of the surface receptor cleaved by ADAM17 expressed on the cell membrane-damaged immune cell compared to the normal level of the ADAM-17-cleaved surface receptor expressed on the immune cell. 43. An immunotherapy composition comprising a therapeutically effective amount of immune cells previously subject to a cell membrane damage event and expressing an increased level of ADAM-17-cleaved surface receptor compared to control immune cells. 44. The immunotherapy composition according to 43 above, wherein the cell membrane damage event comprises freeze-thaw cycles, gene editing, electroporation, magnetofection, detergent permeabilization, streptolysin O (SLO) exposure, physical morphological changes (cell squeezing), and / or ethanol exposure. 45. The immunotherapy composition according to 43 or 44 above, wherein the membrane damage event comprises freeze-thaw cycles. 46. The immunotherapy composition according to 45 above, wherein the immune cells are thawed using a water bath. 47. The immunotherapy composition according to any one of 43 to 46 above, wherein the level of ADAM-17-cleaved surface receptor expression is determined within 0 to 24 hours after the cell membrane damage event. 48. The immunotherapy composition according to any one of 43 to 46 above, wherein the level of ADAM-17-cleaved surface receptor expression is assayed within 12 to 24 hours after the cell membrane damage event. 49. The immunotherapy composition according to any one of 43 to 48 above, wherein the ADAM-17-cleaved surface receptor expressed on the immune cell comprises CD16, CD62L, or IL-15 receptor (IL-15R). 50. The immunotherapy composition according to any one of 43 to 49 above, wherein the immune cells comprise T cells, natural killer (NK) cells, chimeric antigen receptor (CAR) T cells, CAR NK cells, macrophages, dendritic cells, natural killer T (NKT) cells, innate lymphoid cells (ILC), B cells, γδT cells, or neutrophils. 51. The immunotherapy composition according to 50 above, wherein the immune cells comprise natural killer (NK) cells and / or CAR NK cells, and the ADAM-17-cleaved surface receptor is CD16. 52. The immunotherapy composition according to 51 above, wherein the NK cells and / or CAR NK cells comprise expanded NK cells. 53. The immunotherapy composition according to item 50 above, wherein the immune cells include T cells and / or CAR T cells, and the ADAM-17-cleaved surface receptor includes CD62L or IL-15R. 54. The immunotherapy composition according to any one of items 43 to 53 above, wherein the level of the ADAM-17-cleaved surface receptor expression is assayed using flow cytometry. 55. The immunotherapy composition according to any one of items 43 to 54 above, wherein the level of the ADAM-17-cleaved surface receptor expressed on the immune cells is represented as the ratio of the level of the surface receptor cleaved by ADAM17 expressed on the cell membrane-damaged immune cells to the normal level of the ADAM-17-cleaved surface receptor expressed on the immune cells. 56. The immunotherapy composition according to any one of items 43 to 55 above, further comprising a pharmaceutically acceptable carrier.

[0145] References Becker et al., Cancer Immunol. Immunother 65, 477 - 484 (2016). Bethune et al., Curr Opin Biotechnol., 48:142 - 152 (2017). Fang et al., Semin Immunol., 31:37 - 54 (2017). Koepsell et al., Transfusion, 53(2):404 - 10(2013). Luo et al., Cryobiology, 79:65 - 70(2017). Mandelboim et al., Proceedings of the National Academy of Sciences of the United States of America, 96(10):5640 - 5644(1999). Rezvani et al., Front Immunol., 6, 578(2015). Worrell et al., Diabetes Metab Res Rev, 27(8):737 - 45(2011). Xin et al., Proc Natl Acad Sci U S A. 114(4):740 - 745(2017).

Claims

**Claim 1** A method for measuring the recovery of natural killer (NK) cells after a freeze-thaw cycle of cell membrane damage, comprising assaying the level of the ADAM-17-cleaved surface receptor CD16 expressed on the NK cells, wherein an increase in the level of the ADAM-17-cleaved surface receptor CD16 expression compared to the expression of the ADAM-17-cleaved surface receptor CD16 on negative control NK cells with damaged cell surfaces is directly correlated with the NK cell recovery, and the level of the ADAM-17-cleaved surface receptor CD16 expression is assayed within 12 to 24 hours after the NK cell thawing. **Claim 2** The method according to claim 1, wherein the level of the ADAM-17-cleaved surface receptor CD16 expression is assayed using flow cytometry. **Claim 3** The method according to claim 1, wherein the NK cells are thawed using a water bath. **Claim 4** The method according to claim 1, wherein the NK cells include chimeric antigen receptor NK cells.

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