A universal donor selection method for identifying NK cell donors

Abstract

Described herein are compositions comprising universal donor natural killer (NK) cells, populations of such cells, methods for obtaining and preparing such cells, and methods for using such cells and compositions in the medical treatment of cancer and infectious diseases. In one aspect, the disclosure relates to methods for selecting universal donor NK cells for therapeutic administration to a subject in need thereof.

Description

[Technical Field]

[0001] The present disclosure relates generally to methods for donor selection of natural killer (NK) cells, and more particularly to providing methods for selecting universal donor cells for therapeutic administration to a recipient in need thereof. [Background technology]

[0002] Human natural killer (NK) cells express multiple receptors that interact with human leukocyte antigen (HLA) class I molecules. These NK cell receptors belong to one of two major protein superfamilies: the immunoglobulin superfamily or the C-type lectin superfamily. NK cells' ability to distinguish normal tissue from pathological self tissue is largely explained by the inhibitory function of the killer cell immunoglobulin-like receptor (KIR) family, which predominantly recognize classical HLA class I molecules on potential targets. This self-major histocompatibility complex (MFIC) recognition confers the NK cell with the functional capacity to be triggered through its activating receptor, a process called licensing. As a result, licensed NK cells bearing self-MHC-specific receptors are more readily activated than unlicensed NK cells lacking self-MHC-specific receptors. Various KIR family members interact with distinct HLA class I allotypes, resulting in extensive genetic diversity. NK cells also simultaneously express multiple different receptors with different specificities. As a result, any attempt to utilize NK cells for adoptive immunotherapy must address compatibility between the NK cell donor and the recipient. Testing multiple donors to identify a specific donor for a particular patient can be costly and time-consuming. What is needed is a universal source of NK cells that does not suffer from compatibility issues. Summary of the Invention [Means for solving the problem]

[0003] In one aspect, the present disclosure relates to a method of selecting universal donor NK cells for therapeutic administration to a subject in need thereof, the method comprising determining a KIR phenotype of a candidate NK cell from an NK cell donor, wherein the KIR phenotype indicates the presence of one or more variably inherited inhibitory KIRs 2DL1, 2DL2, 2DL3, and 3DL1; and selecting the candidate NK cell as a universal donor NK cell for therapeutic administration when the KIR phenotype indicates the presence of one or more variably inherited inhibitory KIRs 2DL1, 2DL2, 2DL3, and 3DL1.

[0004] In another aspect, the present disclosure relates to a method of selecting universal donor NK cells for therapeutic administration to a recipient subject in need thereof, the method comprising obtaining an HLA genotype of a candidate NK cell from an NK cell donor, the HLA genotype indicating the presence or absence of at least two HLA C1, C2, and Bw4 alleles, and thereby indicating the presence of one or more variably inherited inhibitory KIRs 2DL1, 2DL2, 2DL3, and 3DL1; and selecting the candidate NK cell as a universal donor NK cell for therapeutic administration when the HLA genotype of the candidate NK cell indicates the presence of at least two of the HLA C1, C2, and Bw4 alleles. The method may further include obtaining or having obtained a KIR phenotype of the candidate NK cell, the KIR phenotype indicating the presence or absence of an activating KIR selected from the group consisting of 2DS1 / 2, 2DS3 / 5, 3DS1 and 2DS4; and further selecting the candidate NK cell as being one, wherein a candidate NK cell comprising at least three activating KIRs 2DS1 / 2, 2DS3 / 5, 3DS1 and / or 2DS4 is a pan-NK cell. The method may further include obtaining or having obtained an HLA genotype of a candidate NK cell from an NK cell donor, wherein the 1-ILA genotype indicates the presence or absence of HLA Cl, C2, and Bw4 alleles, and thereby indicates the presence of one or more variably inherited inhibitory KIRs 2DL1, 2DL2, 2DL3, and 3DL1, and further selecting the candidate NK cell as a universal donor NK cell for therapeutic administration when the HLA genotype indicates the presence of at least two HLA alleles HLA Cl, C2, and Bw4.

[0005] The present disclosure also relates to a method of selecting universal donor NK cells for therapeutic administration to a recipient subject in need thereof, the method comprising obtaining or having obtained a KIR genotype of a candidate NK cell, wherein the KIR genotype indicates the presence or absence of an activating KIR selected from the group consisting of 2DS1 / 2, 2DS3 / 5, 3DS1, and 2DS4, and selecting the candidate NK cell as a universal donor NK cell for therapeutic administration when the KIR genotype indicates the presence of at least three activating KIRs 2DS1 / 2, 2DS3 / 5, 3DS1, and / or 2DS4.

[0006] The present disclosure additionally relates to a method of screening a candidate NK cell population from a donor to identify universal NK donor cells within the population in order to provide a source of NK cells for therapeutic administration to a subject in need thereof, the method comprising: (a) obtaining or having obtained an HLA genotype of candidate NK cells from an NK cell donor, the HLA genotype indicating the presence or absence of HLA Cl, C2, and Bw4 alleles, and thereby indicating the presence of one or more variably inherited inhibitory KIRs 2DL1, 2DL2, or 2DL3 and / or 3DL1; wherein candidate NK cells comprising at least two HLA alleles HLA Cl, C2, and Bw4, and thus comprising at least one of variably inherited inhibitory KIRs 2DL1, 2DL2, or 2DL3 and / or 3DL1, are universal donor NK cells. The method may further include obtaining or having obtained a KIR genotype of the candidate NK cells, the KIR genotype indicating the presence or absence of an activating KIR selected from the group consisting of 2DS1 / 2, 2DS3 / 5, 3DS1, and 2DS4; wherein candidate NK cells containing at least three activating KIRs, 2DS1 / 2, 2DS3 / 5, 3DS1, and / or 2DS4, are universal NK cells. In any of these methods, the selected universal donor NK cells may be histologically optimized for at least 50% to 85% of recipient subjects. Any of these methods may also include obtaining or having obtained a CMV-seropositivity status of the candidate NK cells, wherein the NK candidate NK cells are further selected, obtained, or having obtained when the NK cell donor is seropositive for CMV or when the NK cells from the NK cell donor exhibit elevated NKG2C expression compared to a reference level of NKG2C expression. In one embodiment of such a method, the reference level of NKG2C expression is that less than 5% of NK cells express NKG2C. In another embodiment of such a method, high NKG2C expression is that between 5% and about 22% of NK cells express NKG2C.

[0007] In another aspect, the present disclosure provides isolated universal donor NK cells selected or screened by any of the methods discussed herein, wherein the NK cells are NKG2C+. The isolated universal donor NK cells can be activated by incubating the universal donor NK cells in vitro in the presence of IL-21. The IL-21 used for in vitro activation can include soluble IL-21, IL-21-expressing feeder cells (FC21), IL-21 cell membrane particles (PM21), or IL-21 exosomes (EX21).

[0008] In another aspect, the present disclosure provides a method of treating cancer or an infectious disease in a subject, the method comprising administering to the subject donor NK cells selected by any one or more of the methods discussed above or donor NK cells screened by any one or more of the methods discussed above; or isolated pan-NK cells discussed by some or all of the methods discussed above.

[0009] The present disclosure provides a method of treating cancer or an infectious disease in a subject, comprising: (a) obtaining or obtaining an HLA genotype of candidate NK cells from an NK cell donor, wherein the HLA genotype indicates the presence or absence of HLA Cl, C2, and Bw4 alleles, and thereby indicates the presence of one or more variably inherited inhibitory KIRs 2DL1, 2DL2, 2DL3, and 3DL1; (b) obtaining or obtaining a KIR genotype of the candidate NK cells, wherein the KIR genotype indicates the presence or absence of an activating KIR selected from the group consisting of 2DS1 / 2, 2DS3 / 5, 3DS1, and 2DS4; and (c) determining whether or not (i) the HLA genotype indicates the presence of at least two HLA alleles HLA Cl, C2, and Bw4; and (ii) the KIR genotype indicates the presence or absence of at least three activating KIRs. The present invention further relates to a method comprising selecting the candidate NK cells as universal donor NK cells for therapeutic administration when they exhibit the presence of 2DS1 / 2, 2DS3 / 5, 3DS1, and / or 2DS4. In one embodiment, the selected universal donor NK cells can be histologically optimized for at least 50% to 85% of recipient subjects. In another embodiment, the method can further include obtaining or having obtained a CMV-seropositive status of the candidate NK cells, wherein the NK candidate NK cells are further selected, obtained, or having obtained when the NK cell donor is seropositive for CMV or when the NK cells from the NK cell donor exhibit elevated NKG2C expression compared to a reference level of NKG2C expression. The method can further include incubating the selected universal donor NK cells in vitro in the presence of IL-21. The IL-21 used in the in vitro culture may include soluble IL-21, IL-21-expressing feeder cells (FC21), IL-21 cell membrane particles (PM21), and / or IL-21 exosomes (EX21). In this method, the cancer may be selected from blood cancer, lung cancer, esophageal cancer, gastric cancer, pancreatic cancer, liver cancer, biliary tract cancer, colon cancer, rectal cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, kidney cancer, bladder cancer, testicular cancer, prostate cancer, laryngeal cancer, thyroid cancer, brain cancer, or skin cancer.In another aspect of the method, the infectious disease may be caused by a pathogen selected from a virus, a bacterium, or a fungus.

[0010] The present disclosure further relates to a method for preparing a universal donor NK cell population for therapeutic administration to a subject in need thereof, the method comprising: (a) obtaining an initial NK cell population from an NK cell donor, the NK cell donor having a genotype indicating the presence of (i) at least two of variably inherited activating KIRs 2DS1 / 2, 2DS3 / 5, 3DS1, and / or 2DS4; and (ii) at least one of the Cl, C2, and Bw4 alleles; and (b) exposing the initial NK cell population to IL-21 in vitro for a time and under conditions sufficient to expand the initial NK cell population. In one embodiment of the method, the donor genotype may indicate the presence of the Cl, C2, and Bw4 alleles. In another embodiment of the method, step (b) may be performed for a time and under conditions sufficient to achieve at least one population doubling. In another embodiment of the method, a preferred donor may have a CMV seropositivity profile indicating the presence of NKG2C+ NK cells. In another embodiment of the method, exposing the initial NK cell population to IL-21 may comprise contacting the NK cells in vitro with at least one of soluble IL-21, IL-21-expressing feeder cells (FC21), IL-21 cell membrane particles (PM21), and IL-21 exosomes (EX21), or any combination thereof. In another embodiment of the method, the IL-21 present in the feeder cells (FC21), IL-21 cell membrane particles (PM21), and IL-21 exosomes (EX21) may comprise a form of IL-21 selected from (a) an engineered membrane-bound form of IL-21, (b) IL-21 chemically conjugated to the surface of FC21, PM21, or EX21, or (c) IL-21 in a solution that is mixed with and interacts with the NK cells. In another embodiment of the method, any one of FC21, PM21, or EX21 may further comprise (a) an NK stimulatory ligand selected from IL-2, IL-12, IL-18, IL-15, IL-7, ULBP, MICA, OX4OL, an NKG2D agonist, Delta-1, a Notch ligand, an NKp46 agonist, an NKp44 agonist, an NKp30 agonist, other NCR agonists, a CD16 agonist; or (b) membrane-bound TGF-β.In another embodiment of the method, the NK cells may be further exposed to one or more NK stimulatory ligands selected from the group of soluble and / or membrane-bound ligands. In yet another embodiment of the method, a universal donor NK cell population may be prepared.

[0011] In another aspect, the disclosure provides an NK cell population prepared by any one or more of the aforementioned methods, wherein the expanded NK cell population is characterized by an increased ability to produce and secrete the anti-tumor cytokines IFNy or TNFa. In another aspect, the NK cell population prepared by any one or more of the aforementioned methods comprises an expanded NK cell population characterized by increased expression of NKG2D, increased expression of CD16, increased expression of NKp46, and / or increased KIR expression. In one aspect of the method, the IL-21 present in the feeder cells (FC21), IL-21 cell membrane particles (PM21), and IL-21 exosomes (EX21) may comprise a form of IL-21 selected from: (a) a modified membrane-bound form of IL-21; (b) IL-21 chemically conjugated to the surface of FC21, PM21, or EX21; or (c) IL-21 in a solution mixed with the NK cells for interaction. In the method of any of the foregoing embodiments, any one of FC21, PM21, or EX21 can further comprise (a) an NK stimulatory ligand selected from IL-2, IL-12, IL-18, IL-15, IL-7, ULBP, MICA, OX4OL, an NKG2D agonist, Delta-1, a Notch ligand, an NKp46 agonist, an NKp44 agonist, an NKp30 agonist, other NCR agonists, or a CD16 agonist; or (b) membrane-bound TGF-β. In one embodiment, any one of FC21, PM21, or EX21 further comprises a soluble and / or membrane-bound stimulatory ligand.

[0012] The present disclosure additionally relates to modified NK cells or cell lines, wherein the NK cells are transformed to express one or more HLA alleles, including Cl, C2, or Bw4. In the modified NK cells or cell lines of the foregoing embodiments, the NK cells can be transformed to express Cl, C2, and Bw4. In the modified NK cells or cell lines of any of the foregoing embodiments, the NK cells can be further transformed to express one or more variably inherited activating KIRs, including 2DS1 / 2, 2DS3 / 5, 3DS1, or 2DS4. In the modified NK cells or cell lines of any of the foregoing embodiments, the NK cells can be further transformed to express two, three, or more variably inherited activating KIRs, including 2DS1 / 2, 2DS3 / 5, 3DS1, or 2DS4.

[0013] Also disclosed are methods and compositions relating to universal donor NK cells that can be used for therapeutic administration to recipient subjects in need thereof. In one aspect, disclosed herein is a method of selecting universal donor NK cells for therapeutic administration to a recipient subject in need thereof, the method comprising: (a) obtaining or obtaining an HLA genotype of candidate NK cells from an NK cell donor, wherein the HLA genotype indicates the presence or absence of HLA Cl, C2, and Bw4 alleles, and thereby indicates the presence of one or more variably inherited inhibitory KIRs 2DL I, 2DL2, 2DL3, and 3DL1, and / or (b) obtaining or obtaining a KIR genotype of the candidate NK cells, wherein the KIR genotype indicates the presence or absence of an activating KIR selected from the group consisting of 2DS1 / 2, 2DS3 / 5, 3DS1, and 2DS4, and (c) obtaining or obtaining a candidate NK cell when (i) the HLA genotype indicates the presence of at least two HLA alleles HLA Cl, C2, and Bw4, and / or (ii) the KIR genotype indicates the presence or absence of at least three activating KIRs. When the candidate NK cells show the presence of 2DS1 / 2, 2DS3 / 5, 3DS1 and / or 2DS4, they are selected as universal donor NK cells for therapeutic administration.

[0014] Also disclosed herein is a method of screening a candidate NK cell population from a donor to identify universal NK donor cells in the population to provide a source of NK cells for therapeutic administration to a subject in need thereof, the method comprising: (a) obtaining or having obtained an HLA genotype of the candidate NK cells from the NK cell donor, the HLA genotype indicating the presence or absence of HLA Cl, C2, and Bw4 alleles, and thereby indicating the presence of one or more variably inherited inhibitory KIRs 2DL1, 2DL2, or 2DL3 and / or 3DL1; and / or (b) obtaining or having obtained a KIR genotype of the candidate NK cells, the KIR genotype indicating the presence or absence of an activating KIR selected from the group consisting of 2DS1 / 2, 2DS3 / 5, 3DS1, and 2DS4; wherein (i) the KIR genotype comprises at least two HLA alleles HLA Cl, C2, and Bw4, and thereby indicating the presence of one or more variably inherited inhibitory KIRs 2DL1, 2DL2, or 2DL3 and / or 3DL1. Candidate NK cells comprising at least one of 2DL1, 2DL2 or 2DL3 and / or 3DL1, and / or (ii) candidate NK cells comprising at least three activating KIRs 2DS1 / 2, 2DS3 / 5, 3DS1 and / or 2DS4 are pan-NK cells.

[0015] In another aspect, disclosed herein is a method of screening an NK cell population of any of the preceding aspects or a method of selecting universal donor NK cells for therapeutic administration to a recipient subject, wherein the selected universal donor NK cells are histologically optimized for at least 50%-85% of the recipient subject.

[0016] Also disclosed herein is a method of screening an NK cell population of any of the foregoing aspects or a method of selecting universal donor NK cells for therapeutic administration to a recipient subject, further comprising obtaining or having obtained a CMV seropositivity status of the candidate NK cells, wherein the NK candidate NK cells are further selected, obtained or having been obtained when the NK cell donor is seropositive for CMV or when the NK cells from the NK cell donor exhibit elevated NKG2C expression compared to a reference level of NKG2C expression.

[0017] In another aspect, also disclosed are isolated universal donor NK cells selected or screened by the method of any of the preceding aspects. The NK cells of any of the preceding aspects can be NKG2C+. Also disclosed herein are isolated universal NK cells or cells of any of the preceding aspects, wherein one or more NK cells are activated by incubating one or more universal donor NK cells in vitro in the presence of IL-21. In one aspect, the IL-21 used for in vitro activation comprises soluble IL-21, IL-21-expressing feeder cells (FC21), IL-21 cell membrane particles (PM21), IL-21 exosomes (EX21), or any combination thereof.

[0018] In another aspect, disclosed herein is a method of treating, preventing, inhibiting, and / or reducing cancer, metastasis, or infection in a subject in need thereof, comprising administering to the subject donor NK cells selected or screened by the method of any of the preceding aspects; or administering to the subject an isolated pan-NK cell or cells of any of the preceding aspects. For example, in one aspect, provided herein is a method of treating cancer or an infectious disease in a subject, comprising: (a) obtaining or obtaining an HLA genotype of candidate NK cells from an NK cell donor, wherein the HLA genotype indicates the presence or absence of HLA Cl, C2, and Bw4 alleles, and thereby indicates the presence of one or more variably inherited inhibitory KIRs 2DL1, 2DL2, 2DL3, and 3DL1; (b) obtaining or obtaining a KIR genotype of the candidate NK cells, wherein the KIR genotype indicates the presence or absence of an activating KIR selected from the group consisting of 2DS1 / 2, 2DS3 / 5, 3DS1, and 2DS4; and (c) obtaining or obtaining a candidate NK cell when (i) the HLA genotype indicates the presence of at least two HLA alleles HLA Cl, C2, and Bw4; and (ii) the KIR genotype indicates the presence or absence of at least three activating KIRs. Disclosed are methods that include selecting the candidate NK cells as universal donor NK cells for therapeutic administration when they exhibit the presence of 2DS1 / 2, 2DS3 / 5, 3DS1 and / or 2DS4.

[0019] In another aspect, disclosed herein is a method of treating cancer or an infectious disease of any of the preceding aspects, wherein the selected universal donor NK cells are histologically optimized for at least 50%-85% of recipient subjects.

[0020] Also disclosed herein is a method of treating cancer or an infectious disease of any of the foregoing aspects, further comprising obtaining or having obtained a CMV seropositivity status of the candidate NK cells; wherein the candidate NK cells are further selected when the NK cell donor is seropositive for CMV or when the NK cells from the NK cell donor exhibit high NKG2C expression compared to a reference level of NKG2C expression.

[0021] In another aspect, disclosed herein is a method of treating cancer or an infectious disease of any of the preceding aspects, further comprising incubating the selected universal donor NK cells in vitro in the presence of IL-21. In another aspect, the IL-21 used for in vitro culture comprises soluble IL-21, IL-21-expressing feeder cells (FC21), IL-21 cell membrane particles (PM21), or IL-21 exosomes (EX21), or any combination thereof.

[0022] Also disclosed herein are methods of preparing a universal donor NK cell population for therapeutic administration to a subject in need thereof, comprising: (a) obtaining an initial NK cell population from an NK cell donor, the NK cell donor having a genotype indicating the presence of (i) at least two of variably inherited activating KIRs 2DS1 / 2, 2DS3 / 5, 3DS1, and / or 2DS4; and (ii) at least one, two, or all three HLA alleles, including Cl, C2, and Bw4 alleles; and (b) exposing the initial NK cell population to IL-21 in vitro for a time and under conditions sufficient to expand the initial NK cell population.

[0023] In another aspect, disclosed herein is a population of NK cells of any of the preceding aspects, wherein the isolated NK cells are NKG2C+ or CMV-seropositive.A method of preparing an NK cell population, wherein exposing the initial NK cell population to IL-21 comprises contacting the NK cells in vitro with at least one of soluble IL-21, IL-21-expressing feeder cells (FC21), IL-21 cell membrane particles (PM21), and IL-21 exosomes (EX21). For example, disclosed herein are methods for preparing an NK cell population, wherein the IL-21 present in feeder cells (FC21), IL-21 cell membrane particles (PM21), and IL-21 exosomes (EX21) comprises IL-21 in a form selected from (a) a modified membrane-bound form of IL-21, (b) IL-21 chemically conjugated to the surface of FC21, PM21, or EX21, or (c) IL-21 in a solution mixed with or in contact with NK cells. In one embodiment, any one of FC21, PM21, or EX21 further comprises (a) an NK stimulatory ligand selected from IL-2, IL-12, IL-18, IL-15, IL-7, ULBP, MICA, OX4OL, an NKG2D agonist, Delta-1, a Notch ligand, an NKp46 agonist, an NKp44 agonist, an NKp30 agonist, other NCR agonists, a CD16 agonist; or (b) membrane-bound TGF-β.

[0024] In one aspect, disclosed herein is a universal donor NK cell population prepared by the method of any of the preceding aspects. In one aspect, the NK cell population is characterized by an increased ability to produce and secrete the anti-tumor cytokines IFNy or TNFa. In one aspect, the expanded NK cell population is characterized by increased expression of NKG2D, increased expression of CD16, increased expression of NKp46, and increased KIR expression.

[0025] Also disclosed herein are modified NK cells or cell lines, wherein the NK cells are transformed to express one, two or more I ILA alleles, including Cl, C2, or Bw4 (e.g., an NK cell or cell line expressing Cl, C2, and Bw4), and / or are transformed to express one, two, three, four, five, or more variably inherited activating KIRs, including 2DS1 / 2, 2DS3 / 5, 3DS I, or 2DS4.

[0026] One aspect of the present invention includes a method of selecting universal donor NK cells for therapeutic administration, the method comprising: identifying NK donor cells having an HLA genotype comprising at least one of C1, C2, and BW3 alleles as HLA donor cells, thereby indicating the presence of one or more variably inherited inhibitory KIRs comprising at least one of 2DL1, 2DL2, 2DL3, and 3DL1; identifying the number of activating KIRs present on the HLA donor cells; identifying the HLA donor cells as KIR donor cells in response to the number of activating KIRs present on the HLA donor cells exceeding an activation threshold; identifying the NKG2C expression status of the KIR donor cells; and identifying the KIR donor cells as therapeutic donor cells in response to the KIR donor cells being NKG2C positive.

[0027] Another aspect of the present invention includes a method of selecting and modifying universal donor NK cells for therapeutic administration, the method comprising modifying NK donor cells to express an HLA genotype comprising at least one of C1, C2, and BW3 alleles to generate HLA NK cells; obtaining a KIR genotype of the HLA NK cells; transforming the HLA NK cells to express at least three activating KIRs, wherein the three activating KIRs comprise at least one of 2DS1 / 2, 2DS3 / 5, 3DS1, and 2DS4; identifying the cytomegalovirus (CMV) seropositivity status of the NK donor cells; and utilizing the KIR donor cells as therapeutic donor cells in response to the KIR donor cells being CMV seropositive.

[0028] Yet another aspect of the present invention includes a method for selecting, modifying, and preparing universal donor NK cells for therapeutic administration, comprising: determining whether the NK donor cells have an HLA genotype comprising at least one of C1, C2, and BW3 alleles as an HLA donor cell, thereby indicating the presence of one or more variably inherited inhibitory KIRs comprising at least one of 2DL1, 2DL2, 2DL3, and 3DL1; and, in response to the NK donor cells having an HLA genotype comprising at least one of C1, C2, and BW3 alleles, modifying the NK donor cells to an HLA genotype. identifying the HLA donor cells as NK cells; identifying the number of activating KIR present on the HLA donor cells; identifying the HLA donor cells as KIR donor cells in response to the number of activating KIR present on the HLA donor cells exceeding an activation threshold; identifying the NKG2C expression status of the KIR donor cells; identifying the KIR donor cells as therapeutic donor cells in response to the KIR donor cells being NKG2C positive; and stimulating the therapeutic donor cells for a first feeding period with irradiated K562 expressing at least one of membrane-bound IL-21, 4-1BBL, and IL-2.

[0029] Provided herein is a method for preparing an NK cell collection from a donor, comprising: (i) determining from one or more donors (a) an HLA genotype indicating the presence or absence of HLA C1, C2, and Bw4 alleles, thereby indicating the presence of one or more variably inherited inhibitory KIRs 2DL1, 2DL2, 2DL3, and 3DL1; and (b) a KIR genotype indicating the presence or absence of an activating KIR selected from the group consisting of 2DS1 / 2, 2DS3 / 5, 3DS1, and 2DS4; (ii) determining from one or more donors (a) an HLA genotype indicating the presence or absence of at least two HLA alleles HLA C1, C2, and Bw4; and (b) a KIR genotype indicating the presence or absence of at least three activating KIRs selected from the group consisting of 2DS1 / 2, 2DS3 / 5, 3DS1, and 2DS4; and (iii) preparing the NK cell collection from an ex vivo batch of NK cells of the universal donor. In this method, the selection as universal donor NK cells for therapeutic administration may further comprise selecting a donor with a CMV seropositivity profile indicative of the presence of NKG2C+ NK cells.

[0030] In another aspect, disclosed herein is the use of any one or more of donor NK cells selected by the method of any of the preceding aspects, donor NK cells screened by the method of any of the preceding aspects, isolated pan-NK cells of any of the preceding aspects, population of pan-donor NK cells of any of the preceding aspects, modified NK cells or cell lines of any of the preceding aspects in the manufacture of a medicament for treating cancer or an infectious disease in a subject.

[0031] In another aspect, the present specification discloses use of an NK cell population in the manufacture of a medicament for treating cancer or an infectious disease in a subject, wherein the NK cell population comprises: (i) an HLA genotype having at least two HLA alleles selected from HLA C1, C2, and Bw4, indicating the presence of one or more variably inherited inhibitory KIRs selected from 2DL1, 2DL2, 2DL3, and 3DL1; and (ii) a KIR genotype including at least three activating KIRs selected from the group consisting of 2DS1 / 2, 2DS3 / 5, 3DS1, and 2DS4. In the use of any of the aforementioned aspects, the NK cells or NK cell population may be histologically optimized for at least 50% to 85% of recipient subjects. In the use of any of the aforementioned aspects, the donor of the NK cells or NK cell population may be seropositive for CMV, or the NK cells or NK cell population may have elevated NKG2C expression compared to a reference level of NKG2C expression. The use of any of the aforementioned embodiments may include culturing the NK cells or NK cell population in vitro in the presence of IL-21 prior to use in therapy. In the use of any of the aforementioned embodiments, the IL-21 in the in vitro culture may comprise IL-21, IL-21-expressing feeder cells (FC21), IL-21 cell membrane particles (PM21), or IL-21 exosomes. In the use of any of the aforementioned embodiments, the cancer may be selected from blood cancer, lung cancer, esophageal cancer, gastric cancer, pancreatic cancer, liver cancer, biliary tract cancer, colon cancer, rectal cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, kidney cancer, bladder cancer, testicular cancer, prostate cancer, laryngeal cancer, thyroid cancer, brain cancer, or skin cancer. The infectious disease may be caused by a pathogen selected from a virus, bacteria, or fungus. In the use of any of the foregoing embodiments, the NK cell or NK cell population and / or the donor of the NK cell or NK cell population may be selected from a collection comprising two or more cells, populations and / or donors for which the HLA genotype and the KIR genotype have been determined.

[0032] The foregoing and other features and advantages of the present disclosure will become apparent to those skilled in the art to which this disclosure pertains upon consideration of the following description of the invention in conjunction with the accompanying drawings, in which like reference numerals refer to like parts throughout the drawings unless otherwise noted. [Brief explanation of the drawings]

[0033] [Figure 1] 1 shows that, according to one embodiment of the present disclosure, increasing the number of activated KIRs is associated with increased lysis of target cells. [Figure 2] 1 shows a table containing representative data showing the population distribution of KIR genotypes, according to one embodiment of the present disclosure. [Figure 3] 1 illustrates a method for selecting universal donor NK cells for therapeutic administration to a recipient subject in need thereof, according to one embodiment of the present disclosure. [Figure 4] 1 shows a method for modifying NK cells to encode and / or express different alleles, KIRs and / or receptors according to one embodiment of the present disclosure. [Figure 5] 1 illustrates a method for harvesting and preparing universal donor NK cells for therapeutic administration to a recipient subject in need thereof, according to one embodiment of the present disclosure. [Figure 5A] A schematic representation of the assessment of the presence (gray) or absence (black) of KIR genes (bottom) by KIR typing of donors across the HLA-C1, C2, Bw4 spectrum (top) is shown. [Figure 5B] Analysis of PBMCs and donor-matched NK cells by flow cytometry to determine KIR expression on NK cells. Expression of 2DL2 / 3, 2DL1, and 3DL1 was assessed using KIR-specific antibodies REA147 / CH-L, 143211, and DX9, respectively. The percentage of NK cells expressing each KIR for individual donors is shown. [Figure 6] 1 illustrates a method for harvesting and preparing universal donor NK cells for therapeutic administration to a recipient subject having a first disease type in need thereof, according to one embodiment of the present disclosure. [Figure 7]1 illustrates a method of identifying a recipient with a first disease type and providing treatment using universal donor NK cells, according to one embodiment of the present disclosure. [Figure 8] Using flow cytometry, we show that all CMV+ donors have NK cells expressing NKG2C and that NKG2C expression increases after expansion. [Figure 9] Using mRNA level measurements, we show that NKG2C expression increases after expansion. DETAILED DESCRIPTION OF THE INVENTION

[0034] Those skilled in the art will appreciate that elements in the figures are illustrated for simplicity and clarity and have not necessarily been drawn to scale. For example, the dimensions of some elements in the figures may be exaggerated relative to other elements to facilitate an improved understanding of embodiments of the present disclosure.

[0035] Components of the apparatus and methods are represented in the drawings by conventional symbols, where appropriate, and only those specific details relevant to an understanding of the embodiments of the present disclosure are shown, so as not to obscure the present disclosure with details that will be readily apparent to those skilled in the art having the benefit of the description herein.

[0036] Reference will now be made to the drawings, in which like numbered features shown therein refer to like elements unless otherwise noted. The present disclosure relates generally to methods for donor selection of natural killer (NK) cells, and more specifically to providing methods for selecting universal donor cells for therapeutic administration to a recipient in need thereof.

[0037] In this disclosure, reference will be made to a number of terms that shall be defined to have the following meanings. "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that both the occurrence and non-occurrence of the event or circumstance are encompassed by the description. A "primer" is a portion of a probe capable of supporting a certain enzymatic manipulation and capable of hybridizing to a target nucleic acid in such a way that the enzymatic manipulation can occur. Primers can be made of any combination of nucleotides or nucleotide derivatives or analogs available in the art that do not interfere with the enzymatic manipulation.

[0038] A "probe" is a molecule capable of interacting with a target nucleic acid, typically in a sequence-specific manner, e.g., through hybridization. Nucleic acid hybridization is well understood in the art and is discussed herein. Typically, a probe can be made of any combination of nucleotides or nucleotide derivatives or analogs available in the art.

[0039] The terms "peptide," "polypeptide," and "protein" are used interchangeably to refer to a polymer of amino acid residues. The term "sequence identity," as used herein, refers to a quantitative measure of the degree of identity between two sequences of substantially equal length. The percent identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between the two aligned sequences, divided by the length of the shorter sequence, multiplied by 100.

[0040] Approximate alignment of nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics, Vol. 2, pp. 482-489 (1981). This algorithm may be applied to amino acid sequences using a scoring matrix developed by Dayhoff, "Atlas of Protein Sequences and Structure," M. O. Dayhoff, ed., 5th Supplement, Vol. 3, pp. 353-358, National Biomedical Research Foundation, Washington, DC, USA, and standardized by Gribskov, Nucl. Acids Res., Vol. 14, No. 6, pp. 6745-6763, 1986. An exemplary implementation of this algorithm for determining percent sequence identity is provided in the "BestFit" utility application by the Genetics Computer Group (Madison, Wis.). Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters.For example, BLASTN and BLASTP can be used with the following default parameters: Genetic Code - Standard; Filter - None; Strand Both; Cutoff = 60; Expectation = 10; Matrix = BLOSUM62; Description = 50 sequences; Sort Order = HIGHSCORE; Database = Non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS Translations + Swiss Protein + Sp Update + P1R. More information about these programs can be found on the GenBank website. Generally, substitutions are conservative amino acid substitutions, limited to exchanges within members of Group 1: glycine, alanine, valine, leucine, and isoleucine; Group 2: serine, cysteine, threonine, and methionine; Group 3: proline; Group 4: phenylalanine, tyrosine, and tryptophan; and Group 5: aspartic acid, glutamic acid, asparagine, and glutamine.

[0041] Techniques for determining nucleic acid and amino acid sequence identity are known in the art. Typically, such techniques involve determining the nucleotide sequence of a gene's mRNA and / or determining the amino acid sequence encoded thereby and comparing that sequence to a second nucleotide or amino acid sequence. Genomic sequences can also be determined and compared in this manner. Generally, identity refers to the exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotide or polypeptide sequences, respectively. Two or more sequences (polynucleotide or amino acid) can be compared by determining their percent identity.

[0042] Because various modifications may be made to the cells and methods described above without departing from the scope of the invention, it is intended that all matter contained in the above description and in the examples provided below be interpreted as illustrative and not limiting.

[0043] "Increase" can refer to any change that results in a greater degree of a symptom, disease, composition, condition, or activity. An increase can be any individual, median, or mean increase in a condition, symptom, activity, or composition by a statistically significant degree. Thus, an increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% increase, so long as the increase is statistically significant.

[0044] "Reduction" can refer to any change that results in a lesser degree of symptoms, disease, composition, condition, or activity. A substance is also understood to reduce the genetic output of a gene when the genetic output of the gene product in the presence of the substance is less than the output of the gene product in the absence of the substance. For example, a reduction can also be a change in the symptoms of a disorder such that the symptoms are less severe than those previously observed. A reduction can be any individual, median, or mean decrease in a condition, symptom, activity, or composition that is statistically significant. Thus, a reduction can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% reduction, so long as the reduction is statistically significant.

[0045] "Inhibit," "inhibiting," and "inhibition" refer to a decrease in an activity, response, condition, disease, or other biological parameter. This can include, but is not limited to, the complete elimination of the activity, response, condition, or disease. It can also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to native or control levels. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any intermediate degree of reduction as compared to native or control levels.

[0046] "Reduce" or other forms of this word, such as "reducing" or "reduction," means to lower an event or characteristic (e.g., tumor growth). This is typically understood to be relative to some standard or expected value; in other words, it is relative, but not always with reference to a standard or relative value. For example, "reducing tumor growth" means reducing the rate of tumor growth compared to a standard or control.

[0047] "Prevent" or other forms of this word, such as "preventing" or "prevention," mean to stop a particular event or characteristic, stabilize or slow the development or progression of a particular event or characteristic, or minimize the likelihood of a particular event or characteristic occurring. Prevent is typically more absolute than, for example, reduce, and does not necessarily require comparison to a control. As used herein, something may be reduced but not prevented, but something that is reduced may be prevented. Similarly, something may be prevented but not reduced, but something that is prevented may be reduced. Where reduce or prevent is used, unless specifically indicated otherwise, it is understood that the use of other words is expressly disclosed.

[0048] The term "subject" refers to any individual who is the target of administration or treatment. The subject may be a vertebrate, e.g., a mammal. In one embodiment, the subject may be a human, a non-human primate, a bovine, an equine, a porcine, a canine, or a feline. The subject may also be a guinea pig, a rat, a hamster, a rabbit, a mouse, or a mole. Thus, the subject may be a human or an animal patient. The term "patient" refers to a subject under the care of a clinician, e.g., a physician.

[0049] The term "therapeutically effective" refers to the amount of composition used being sufficient to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration need only result in a reduction or alteration, not necessarily elimination.

[0050] The term "treatment" refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, condition, or disorder. The term includes active treatment, i.e., treatment specifically directed toward reversing a disease, condition, or disorder, and also includes causal treatment, i.e., treatment directed toward eliminating the cause of the associated disease, condition, or disorder. In addition, the term includes palliative treatment, i.e., treatment designed to alleviate symptoms rather than cure the disease, condition, or disorder; preventative treatment, i.e., treatment directed toward minimizing or partially or completely suppressing the occurrence of the associated disease, condition, or disorder; and supportive treatment, i.e., treatment used to supplement another specific therapy directed toward reversing the associated disease, condition, or disorder.

[0051] "Administration" to a subject includes any route of introducing or delivering an agent to a subject. Administration can be by any suitable route, including oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-articular, parenteral, intraarteriolar, intradermal, intracerebroventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation, via an implanted reservoir, parenteral (e.g., subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intraperitoneal, intrahepatic, intralesional, and intracranial injection or infusion), and the like. "Concurrent administration," "administration in combination," "simultaneous administration," or "administered simultaneously," as used herein, means that compounds are administered at the same time in time or essentially immediately after each other. In the latter case, the two compounds are administered at times sufficiently close together that results are observed that are indistinguishable from those achieved when the compounds are administered at the same time in time. "Systemic administration" refers to the introduction or delivery of an agent to a subject via a route that introduces or delivers the agent to a wide area of ​​the subject's body (e.g., greater than 50% of the body), for example, via an entry point into the circulatory or lymphatic system. In contrast, "local administration" refers to the introduction or delivery of an agent to a subject via a route that introduces or delivers the agent to an area of ​​or immediately adjacent to the site of administration, but does not introduce the agent systemically in therapeutically significant amounts. For example, a locally administered agent may be readily detectable in the local vicinity of the site of administration, but may be undetectable or detectable only in negligible amounts in distant parts of the subject's body. Administration includes self-administration and administration by another.

[0052] "Treate," "treating," "treatment," and grammatical variations thereof, as used herein, include administration of a composition intended or intended to partially or completely prevent, delay, eradicate, cure, ameliorate, alleviate, alter, repair, improve, reverse, stabilize, palliate, and / or reduce the intensity or frequency of one or more diseases or conditions, symptoms of a disease or condition, or the underlying causes of a disease or condition. Treatment according to the present invention may be applied defensively, prophylactically, palliatively, or remedially. Prophylactic treatment is administered to a subject prior to onset (e.g., before overt signs of cancer appear), at early onset (e.g., at the onset of early signs and symptoms of cancer), or after the development of cancer has been confirmed. Prophylactic administration may occur from one (or several) days before symptoms of disease or infection appear, to several years.

[0053] I. Selecting a Universal Donor NK cells are licensed (gain enhanced killing ability) when they express inhibitory killer immunoglobulin receptors (KIRs) for self-HLA class I molecules. This allows NK cells to recognize "self" and spare self-cells from killing. Therefore, targets lacking self-HLA class I molecules are more likely to elicit recognition by licensed NK cells. Inhibitory KIR genes known to be associated with NK alloreactivity are (i) 2DL1, which binds to HLA-C group 2 alleles; (ii) 2DL2 and 2DL3, which bind to HLA-C group 1 alleles; and (iii) 3DL1, which binds to HLA-B Bw4 alleles. According to the ligand-deficiency model, each NK cell expressing an inhibitory KIR gene will be alloreactive only if the corresponding ligand is present in the donor and not in the recipient—for example, a donor carrying a group C1 allele will be alloreactive to any individual lacking the group C1 allele. Thus, a donor carrying HLA from the CI, C2 and Bw4 families would be predicted, according to this model, to be alloreactive to any recipient lacking CI, or C2, or Bw4.

[0054] While inhibitory KIRs prevent alloreactivity, activating KIRs recognize activating ligands that promote NK cell lysis. There is considerable variability in the inheritance of activating KIRs—any individual can have between 0 and 7 aKIRs. Data from stem cell transplant patients suggest that patients receiving allografts from donors with a higher number of activating KIRs have better outcomes than those receiving allografts from donors with fewer activating KIRs. Other studies have demonstrated a protective benefit against leukemia in individuals who inherit a higher number of activating KIRs. Our laboratory has shown that NK cells with a higher number of activating KIRs are more potent at inducing target cell lysis (Figure 1). Additionally, multivariate analysis indicates that activating KIRs 2DS1 and 3DS1 are associated with disease-free survival.

[0055] Finally, NKG2C is an activating receptor expressed late in NK cell development and recognizes HLA-E rather than HLA-B or -C. NKG2C expression is induced in patients with CMV infection and correlates with an adaptive NK cell phenotype and improved leukemia-free survival.

[0056] Thus, a "universal" donor is one with an HLA genotype carrying the C1, C2, and Bw4 alleles, a KIR genotype that possesses inhibitory KIRs (2DL I, 2DL2, or 3 and 3DL1) that bind to Cl, C2, and Bw4 (leading to maximal licensing) and a high proportion of activating KIRs (more than three variably inherited activating genes, including 2DS I and 3DS1), and a history of CMV exposure resulting in high NKG2C expression.

[0057] Considering available data for Caucasian donors, the CI / C2 / Bw4 allele is found in 32% of the population. 25.3% of the 23 KIR genotypes, representing 80% of the population, meet all of these criteria (Figure 2)—approximately 90% of adults have been exposed to CMV. Therefore, an "ideal" NK cell donor can be identified in approximately 1 in 16 healthy individuals. It is understood, and contemplated herein, that by screening for and / or selecting donor NK cells from this 1 in 16 healthy individuals, one can obtain "universal" donor NK cells that are histologically optimized for at least 50% to 85% of recipient subjects.

[0058] Thus, in one aspect, the present disclosure relates to a method for selecting universal donor NK cells for therapeutic administration to a subject in need thereof, the method comprising: determining the KIR phenotype of candidate NK cells from an NK cell donor, wherein the KIR phenotype indicates the presence of one or more variably inherited inhibitory KIRs 2DL1, 2DL2, 2DL3, and 3DL1; and selecting the candidate NK cells as universal donor NK cells for therapeutic administration when the KIR phenotype indicates the presence of one or more variably inherited inhibitory KIRs 2DL1, 2DL2, 2DL3, and 3DL1. In this method, the KIR phenotype can be determined using an image-based method, such as magnetic resonance imaging, which can facilitate high-throughput phenotypic imaging. Micro-computed tomography scanning technology can provide high-precision imaging suitable for supporting phenotypic analysis. Genome-scale RNAi screening can also be applied.

[0059] In one embodiment, the present disclosure encompasses a method 300 of selecting universal donor NK cells for therapeutic administration to a recipient subject in need thereof, as shown in FIG. 3. At 302, it is determined whether the donor cells have HLA Cl, C2, and Bw4 alleles. In one embodiment, the presence of HLA Cl, C2, and Bw4 alleles is determined by obtaining or having obtained an HLA genotype of the candidate NK cells from the NK cell donor, where the HLA genotype indicates the presence or absence of the HLA Cl, C2, and Bw4 alleles, and thereby the presence or absence of each of one or more variably inherited inhibitory KIRs 2DL1, 2DL2, 2DL3, and 3DL1. At 304, in response to the donor cells lacking at least one of the HLA Cl, C2, and Bw4 alleles, the donor cells are marked as sub-optimal. At 306, in response to the donor cells having at least one of the HLA Cl, C2, and Bw4 alleles, it is determined whether the donor cells have a number of activating KIRs equal to or greater than an activation threshold, the activation threshold being the minimum number of activating KIRs present. As a non-limiting example, in one embodiment, the threshold may be at least one activating KIR, where the presence of one or more activating KIRs meets the activation threshold. In an alternative embodiment, the activation threshold is 2, 3, 4, 5, 6, or 7 activating KIRs, where the presence of at least one of 2, 3, 4, 5, 6, or 7 activating KIRs, respectively, is met. In one embodiment, the presence of activating KIRs is determined by obtaining or having obtained a KIR genotype of the candidate NK cell, where the KIR genotype indicates the presence or absence of activating KIRs. At 308, in response to the donor cells lacking a number of activating KIRs above the activation threshold, the donor is identified as a non-universal donor.

[0060] At 310, in response to the donor cell having a number of activating KIRs above an activation threshold, it is determined whether the activating KIRs are selected from the group consisting of 2DS1 / 2, 2DS3 / 5, 3DS1, and 2DS4. At 312, in response to the donor cell lacking a KIR selected from the group consisting of 2DS1 / 2, 2DS3 / 5, 3DS1, and 2DS4, the donor cell is identified as a non-universal donor cell.

[0061] In one exemplary embodiment, the KIR genotype indicates the presence or absence of each of activating KIRs selected from the group consisting of 2DS1 / 2, 2DS3 / 5, 3DS1, and 2DS4. At 314, in response to the donor being tested for CMV seropositivity status, it is determined whether the donor is CMV+. At 316, in response to the donor testing CMV seronegative, the donor cells are identified as non-universal donor cells.

[0062] At 318, in response to the donor cell having NKG2C activating receptor expression, the donor cell is identified as a universal donor cell. At 320, in response to the donor lacking the NKG2C activating receptor expression phenotype, the donor cell is identified as a non-universal donor cell. At 322, in response to the donor cell meeting the criteria in at least one, two, three, four, or five of steps 302, 306, 310, 314, and / or 318, the donor cell is identified as a universal donor cell.

[0063] In some embodiments, donor cells identified as universal are selected as universal donor NK cells for therapeutic administration to a subject in need thereof. As described above, NKG2C is an activating receptor expressed late in NK cell development and recognizes HLA-E rather than HLA-B or -C. NKG2C expression is induced in patients with CMV infection and correlates with an adaptive NK cell phenotype and improved leukemia-free survival. Therefore, identifying candidate donor cells from individuals with elevated NKG2C or CMV-seropositive individuals may further increase the efficacy of donor NK cells. Accordingly, also disclosed herein are methods for screening NK cell populations or selecting universal donor NK cells for therapeutic administration to a recipient subject, which methods further include obtaining or having obtained the CMV-seropositive status of the candidate NK cells; and when the NK cell donor is seropositive for CMV or when the NK cells from the NK cell donor exhibit elevated NKG2C expression compared to a reference level of NKG2C expression, the candidate NK cells are further selected. The reference level may be, for example, a predetermined reference value for NKG2C expression obtained from a control donor or the mean value of NKG2C expression levels obtained from a set of CMV-seronegative control donors. Those skilled in the art will understand that the presence or absence of one of the elements described in 302, 306, 310, 314, and / or 318 does not prevent a donor from ultimately being considered a universal donor.

[0064] In another embodiment, a donor is marked as optimal when (i) the HLA genotype indicates the presence of at least two I ILA alleles I ILA Cl, C2 and Bw4, and / or (ii) the KIR genotype indicates the presence of at least three activating KIRs 2DS1 / 2, 2DS3 / 5, 3DS1 and / or 2DS4.

[0065] Also disclosed herein is a method for screening a candidate NK cell population from a donor to identify universal NK donor cells in the population to provide a source of NK cells for therapeutic administration to a subject in need thereof. This method is substantially the same as method 300, except that the candidate NK cell population is screened. The method for screening a candidate NK cell population includes method steps 302-318.

[0066] In another embodiment, the method of screening a candidate NK cell population comprises (a) obtaining or obtaining an HLA genotype of candidate NK cells from an NK cell donor, the HLA genotype indicating the presence or absence of HLA Cl, C2, and Bw4 alleles, and thereby indicating the presence of one or more variably inherited inhibitory KIRs 2DL1, 2DL2, or 2DL3 and / or 3DL I, and / or (b) obtaining or obtaining a KIR genotype of candidate NK cells, the KIR genotype indicating the presence or absence of an activating KIR selected from the group consisting of 2DS1 / 2, 2DS3 / 5, 3DS1, and 2DS4; wherein (i) candidate NK cells comprise at least two HLA alleles HLA Cl, C2, and Bw4, and thus comprise at least one of variably inherited inhibitory KIRs 2DL1, 2DL2, or 2DL3 and / or 3DL1, and / or (ii) at least three activating KIRs Candidate NK cells containing 2DS1 / 2, 2DS3 / 5, 3DS1 and / or 2DS4 are pan-NK cells.

[0067] In one aspect, disclosed herein is a method of screening an NK cell population of any of the preceding aspects or a method of selecting universal donor NK cells for therapeutic administration to a recipient subject, wherein the selected universal donor NK cells are histologically optimized for at least 50%-85% of the recipient subject.

[0068] It is understood, and contemplated herein, that the screening and selection methods of the present disclosure ultimately result in isolated universal donor NK cells. Accordingly, disclosed herein are isolated universal donor NK cells, wherein the isolated universal donor NK cells comprise at least two I ILA alleles I ILA Cl, C2, and Bw4; and / or at least three activating KIRs 2DS1 / 2, 2DS3 / 5, 3DS1, and / or 2DS4. In one embodiment, the isolated universal donor NK cells are NKG2C+ or are derived from a CMV-seropositive donor source.

[0069] It is further understood that, rather than selecting or screening candidate donor NK cells from a donor source to obtain universal donor NK cells with the correct genotypic characteristics, as depicted in method 400 shown in Figure 4, NK cells or cell lines can be modified to encode and / or express various alleles, KIRs, and / or receptors. Accordingly, disclosed herein at 402 are modified NK cells or cell lines, wherein the NK cells have been transformed to express one, two, or more HLA alleles, including Cl, C2, or Bw4 (e.g., an NK cell or cell line expressing Cl, C2, and Bw4). At 404, the NK cells or cell line are modified, wherein the NK cells are transformed to express HLA alleles indicative of the presence of one or more variably inherited inhibitory KIRs 2DL1, 2DL2, 2DL3, and / or 3DL1. At 406, the NK cells or cell line are modified to encode and / or express activating KIRs 2DS1 / 2, 2DS3 / 5, 3DS1, and / or 2DS4, and / or are transformed to express one, two, three, four, five, or more variably inherited activating KIRs, including 2DS1 / 2, 2DS3 / 5, 3DS1, or 2DS4. At 408, the NK cells or cell line are modified to activate NKG2C (e.g., by exposing the cell line to CMV-seropositive conditions). Method steps 402-408 can be selectively completed depending on the underlying gene expression or cell activation present in the NK cell line being utilized; additionally, these steps can be performed on donor cells marked as suboptimal (e.g., steps 304, 308, 312, 306 of method 300) and / or donor cells marked as optimal (e.g., step 318 of method 300).

[0070] It is understood, and contemplated herein, that isolated universal donor NK cells and engineered universal donor NK cells or cell lines can be activated and / or expanded in the presence of one or more NK cell effector agents (e.g., stimulatory peptides, cytokines, and / or adhesion molecules) to overcome many of the challenges associated with cytokine toxicity. Examples of NK cell activators and stimulatory peptides include, but are not limited to, IL-21, 41BBL, IL-2, IL-12, IL-15, IL-18, IL-7, ULBP, MICA, LFA-1, 2B4, BCM / SLAMF2, CCR7, OX4OL, NKG2D agonists, Delta-I, Notch ligands, NKp46 agonists, NKp44 agonists, NKp30 agonists, other NCR agonists, CD16 agonists; and / or TGF-β and / or other homing-inducing signaling molecules. Examples of cytokines include, but are not limited to, IL-2, IL-12, IL-21, and IL-18.

[0071] Examples of adhesion molecules include, but are not limited to, LFA-1, MICA, and BCM / SLAMF2. These NK cell effector agents may be soluble in solution or present as membrane-bound agents on the surface of PM particles, exosomes (EX), or feeder cells (FC). PM particles, EX exosomes, and / or FC cells can be engineered to express membrane-type NK cell activators and stimulatory peptides. Alternatively, the NK cell activators and stimulatory peptides can be chemically conjugated to the surface of PM particles, EX exosomes, or FC feeder cells. For example, PM particles, feeder cells (FC), or exosomes (EX) (FC21 cells, PM21 particles, and EX21 exosomes, respectively) prepared from feeder cells expressing membrane-bound IL-21. Thus, in one aspect, disclosed herein is an isolated universal donor NK cell or cell line, wherein the universal donor NK cell or cell line is activated and / or expanded by incubating the universal donor NK cells in vitro in the presence of one or more activating agents, stimulatory peptides, cytokines and / or adhesion molecules, including, but not limited to, 41BBL, IL-2, IL-12, IL-15, IL-18, IL-7, ULBP, MICA, LFA-1, 2B4, BCM / SLAMF2, CCR7, OX4OL, NKG2D agonist, Delta-1, Notch ligand, NKp46 agonist, NKp44 agonist, NKp30 agonist, other NCR agonist, CD16 agonist; and / or TGF-β (e.g., IL-21). In one embodiment, the IL-21 used for in vitro activation comprises soluble IL-21, IL-21-expressing feeder cells (FC21), IL-21 cell membrane particles (PM21) or IL-21 exosomes (EX21).It is understood, and is contemplated herein, that FC21 cells, PM21 particles, and EX21 exosomes expressing membrane-bound IL-21 may further comprise one or more additional activators, stimulatory peptides, cytokines, and / or adhesion molecules, including, but not limited to, 41BBL, IL-2, IL-12, IL-15, IL-18, IL-7, ULBP, MICA, LFA-1, 2B4, BCM / SLAMF2, CCR7, OX4OL, NKG2D agonists, Delta-1, Notch ligands, NKp46 agonists, NKp44 agonists, NKp30 agonists, other NCR agonists, CD16 agonists; and / or TGF-β (e.g., PM21 particles, EX21 exosomes, or FC cells expressing 41BBL and membrane-bound interleukin-21). NK cells can additionally be exposed to additional ligands, both soluble and membrane-bound.

[0072] As discussed above, additional activation and / or expansion of universal donor NK cells increases the efficacy of the cells when administered to a recipient. Accordingly, in one aspect, disclosed herein is a method of preparing a universal donor NK cell population for therapeutic administration to a subject in need thereof, the method comprising: (a) obtaining an initial NK cell population from an NK cell donor, the NK cell donor having a genotype indicating the presence of (i) at least two of variably inherited activating KIRs 2DS1 / 2, 2DS3 / 5, 3DS1, and / or 2DS4; and (ii) at least one, two, or all three HLA alleles, including Cl, C2, and Bw4 alleles; and (b) activating HLA alleles that include 11-21, 41BBL, IL-2, IL-12, IL-15, IL-18, IL-7, ULBP, MICA, LFA-1, 2B4, BCM / SLAMF2, CCR7, OX4OL, NKG2D agonists, Del, and / or IL-18. The method includes exposing the initial NK cell population in vitro to one or more activating agents, stimulatory peptides, cytokines, and / or adhesion molecules, including, but not limited to, NKG2C-1, Notch ligands, NKp46 agonists, NKp44 agonists, NKp30 agonists, other NCR agonists, CD16 agonists; and / or TGF-β (e.g., IL-21), for a time and under conditions sufficient to expand the initial NK cell population. It is understood, and contemplated herein, that exposure to one or more activating agents can be for a time and under conditions sufficient to achieve at least one population doubling. In one exemplary embodiment, expansion increases NKG2C-high expression in NK cells expressing NKG2C from 5% to about 22% to 11% to about 30%.

[0073] In one embodiment, the isolated universal donor NK cells or cell line or NK cell population is characterized by an increased ability to produce and secrete the anti-tumor cytokines IFNy or TNFa. In one embodiment, the expanded NK cell population is characterized by increased expression of NKG2D, increased expression of CD16, increased expression of NKp46, and increased KIR expression.

[0074] II. Donor Selection In one aspect, donors are screened in a stepwise manner, eliminating donors who do not meet the criteria from further testing (see FIG. 3). Initially, KIR genotyping of NK cell donors can be performed using reverse sequence-specific oligonucleotide (SSO) methodology (e.g., One Lambda), including differentiation between functional and deletion mutants of KIR2DL4. In another exemplary embodiment, activating KIR content is determined by scoring the total number of activating KIR genes. DS-designated KIRs and functional KIR2DL4s were all considered active. In one aspect, donors with a common activating KIR (KIR2DS4 and functional versions of KIR2DL4) and a high number of five variably inherited activating KIRs are selected. In another aspect, donors are selected based on the number of inherited B-KIR segments (e.g., three or four centromeric and telomeric B alleles). In one exemplary embodiment, a high number is three, four, or five variably inherited activating KIRs. In another exemplary embodiment, the high number is 4 variably inherited activating KIRs. In yet another exemplary embodiment, the high number is 1 or more variably inherited activating KIRs.

[0075] In one embodiment, NK cell donors are HLA typed at a moderate or high resolution level for alleles at the HLA-B and -C loci by SSO-PCR (amplification and oligonucleotide sequencing) using commercially available kits. In another embodiment, KIR-ligand classes are predicted using the KIR Ligand Calculator maintained by the European Bioinformatics Institute at the European Molecular Biology Lab (EMBL-EBI). Individuals with all three Cl, C2, and Bw4 classes are selected.

[0076] In one embodiment, the donor is finally tested for CMV. Testing of CMV+ donors can confirm the presence of NKG2C+ NK cells. Alternatively, donors are screened for the presence of NKG2C+ NK cells above an expected threshold (e.g., about 20%) prior to CMV exposure.

[0077] In one aspect, disclosed herein is a method 500 for screening optimal universal donor NK cell donors to prepare optimal universal donor NK cells for use in treating various diseases, as shown in Figure 5. For example, in step 502, optimal cell donors (as defined by method 300 of Figure 3) are screened for communicable diseases. In this exemplary embodiment, optimal universal donor NK cell donors (donors) will undergo infectious disease testing and screening as required for HCT / P donors at BTMB institutions in accordance with Title 21 Code of Federal Regulations Part 1271, the FDA guidance document "Eligibility Determination for Donors of Human Cells, Tissues, and Cellular and Tissue-Based Products (HCT / Ps)," and any previously issued supplemental guidance documents. Testing will be performed in a CLIA-certified and FDA-registered laboratory under contract with BTMB in accordance with BTMB guidelines for HCT / P donors and a separate donor protocol utilizing FDA-approved tests. Donors will be tested for infectious disease markers (IDMs) within seven days before or after collection using the specimen / test methodology in Table 1. IDMs include Hepatitis B Virus, Hepatitis C Virus, HTLV-I and II, HIV-1, -2, and -O, Syphilis, Trypanosoma cruzi (Chagas Disease), West Nile Virus, and CMV.

[0078] [Table 1]

[0079] III. Manufacturing and Vial Filling Estimation In one embodiment, expanded donor NK cell preparations are manufactured prior to or upon the occurrence of patient need. In one embodiment, the donor undergoes standard infectious disease screening and other donor screening (as required by 21 CFR 1271C) within seven days of collection. In step 504 of method 500, peripheral blood mononuclear cells (MNC / PBMC) are collected from the donor in response to the donor's lack of IDM. In another exemplary embodiment, source peripheral blood mononuclear cells (PBMC) are collected and NK cells are expanded by standard methods. In 506, depleted MNCs are formed by CD3+ immunodepletion of the collected MNCs. In one exemplary embodiment, the MNCs / PBMCs are depleted of CD3+ T cells using MACS colloidal superparamagnetic CD3 microbeads.

[0080] At 508, the depleted MNCs are stimulated with feeder cells for a first feeding period and a first feeding interval to expand and activate NK cells. In one exemplary embodiment, the feeder cells are irradiated feeder cells (IFCs). In another exemplary embodiment, the depleted MNCs are expanded by repeated weekly stimulation with irradiated CSTX002 feeder cells (cryopreserved or fresh). CSTX002 is treated with 100 Gy (10,000 rad) gamma irradiation either (i) prior to cryopreservation or (ii) prior to its addition to fresh MNC or NK cell cultures. Validation of irradiation demonstrated that 25 Gy eliminated detectable proliferation, and co-culture with NK cells resulted in an additional 99.9% effective IFC elimination. In one exemplary embodiment, IFCs are added to a medium containing RPMI-1640, 10% FBS, 2 mM Glutamax, and 100 IU / mL recombinant human IL-2 (Proleukin, Prometheus) at a TNC to IFC ratio of about 1:2. In this exemplary embodiment, the first feeding period is 10-15 days, and the first feeding interval is 1-5 days. In another exemplary embodiment, the first feeding period is 14 days, and the first feeding interval is 1-3 days. In one exemplary embodiment, MNC or NK cells are restimulated with IFCs at a TNC to IFC ratio of about 1:1 and cultured for 7 days (e.g., on days 8-14). In this embodiment, a first feeding interval is utilized, in which cultures are monitored for cell number at 1-3 day intervals during expansion days 8-14, and fresh IL-2 is added at 100 IU / mL and TGF-β at 10 ng / mL. To prevent overgrowth and maximize yield, NK cell cultures are maintained at 5-10 x 10 6 cells / cm 2 Depending on the culture vessel, fresh medium is also provided by replacing at least half of the medium as needed.

[0081] At 510, CD3+ depletion is determined. In response to CD3+ depletion exceeding a threshold, step 506 is repeated. In one example, CD3+ depletion is determined one day before the end of day 6 of feeder cell stimulation. In this embodiment, samples for cell counting, immunophenotyping, and viability determination are obtained from the MNC and / or NK cell culture (e.g., under feeder cell stimulation). In one exemplary embodiment, the threshold for CD3+ depletion is the presence of more than 5% CD3+ cells. Here, in one exemplary embodiment, repeating step 506 includes performing a second CD3+ depletion cycle over the first feeding period on day 7. After depletion, samples for cell counting, immunophenotyping, and viability determination will be obtained from the CD3-negative NK cell fraction.

[0082] At 512, in response to CD3+ depletion falling below a threshold, the MNC and / or NK cells are cultured with interleukin-2 (IL-2) and / or transforming growth factor β (TGFβ) for a second feeding period at a second feeding interval. In one exemplary embodiment, the second feeding period is about 5-8 days, and the second feeding interval is about 1-5 days. In another exemplary embodiment, the second feeding period is 7 days, and the second feeding interval is about 1-3 days. In this exemplary embodiment, fresh IL-2 is added at 100 IU / mL, and 10 ng / mL of TGF-β is added at the second feeding interval during the first 7 days of the first feeding period.

[0083] At 514, immunophenotyping and viability determination is performed on the cultured natural killer cells. In one example, on day 13 of the first feeding period, a sample for cell count, immunophenotyping, and viability determination is obtained from the NK cell culture. In response to the presence of less than 0.33% CD3+ cells, the test can be repeated immediately or before harvest on day 14 of the first feeding period. In response to CD3+ depletion exceeding a second threshold (e.g., 0.33%), further depletion as described in step 506 is performed immediately on day 13 or after harvest on day 14. A sample for cell count, immunophenotyping, and viability determination is obtained from the CD3-depleted NK cell fraction, and the remainder will again be cultured overnight with IL-2 and TGF-β. In one exemplary embodiment, in response to not performing CD3+ depletion, then immunophenotyping on day 7 will not be performed.

[0084] At 516, the cultured NK cells are concentrated to a dose concentration. In one example, the dose concentration is 2×10 6 NC / mL ~ 2 × 10 8 NC / mL. At 518, the dose concentration of cultured NK cells is cryopreserved. In one exemplary embodiment, the NK cells are cryopreserved in NK freezing medium. In an exemplary embodiment, the NK freezing medium comprises 10% DMSO, 12.5% ​​(w / v) human serum albumin (HSA), USP, and / or in Plasma-Lyte A (USP). Method 500 describes how to treat a particular patient starting at 520, details of which follow.

[0085] In one exemplary embodiment, such as when treating a herpes simplex virus (HSV) patient with HSV, the HSV patient (i.e., a person diagnosed with HSV) receives 5.0 x 10 7Patients will receive a single daily dose of banked NK cells administered at cells / kg / dose for up to five consecutive days. In this exemplary embodiment, HSV patients with a history of transfusion or infusion reaction will be premedicated with diphenhydramine 1 mg / kg (maximum 50 mg) IV and acetaminophen 10 mg / kg (maximum 650 mg) PO. HSV patients will undergo repeat eligibility assessments over the following days (D1-D4) to determine if they are eligible for repeat dosing. Doses will be given to HSV patients once daily for five consecutive days.

[0086] In another exemplary embodiment, such as when treating COVID patients (e.g., those with COVID-19 infection or SARS-COV-2), NK cells are provided as a treatment. In one exemplary embodiment, a patient with a history of transfusion or infusion reaction is premedicated with diphenhydramine 1 mg / kg (maximum 50 mg) IV and acetaminophen 10 mg / kg (maximum 650 mg). In another exemplary embodiment, a patient receives their first NK cell dose within 48 hours of hospitalization with COVID. In yet another exemplary embodiment, allogeneic expanded NK cells are titrated by patient weight to quantitatively and qualitatively restore innate immune function against COVID. In this exemplary embodiment, a COVID patient is provided with a dose of 10 NK cells / kg patient weight (e.g., a dose that would replenish the total NK cell content in the peripheral blood of an average patient). In another exemplary embodiment, a COVID patient will receive a maximum of two doses.

[0087] In another embodiment, source peripheral blood mononuclear cells (PBMCs) are collected and NK cells are expanded by standard methods. In yet another embodiment, PBMCs are depleted of CD3+ T cells using MACS colloidal superparamagnetic CD3 microbeads. The resulting cells are co-cultured with irradiated feeder cells and / or membrane particles in medium supplemented with fetal bovine serum and IL-2. On day 7, the cultures are restimulated. In one embodiment, the NK cell preparation is lot-release tested and cryopreserved for subsequent infusion on day 14. NK cells are cryopreserved in single-dose aliquots (e.g., 50 mL containing 10 NK cells / mL). Assuming an initial donor blood volume equivalent to one unit (450 mL), a median content of 1.26 x 10 NK cells / mL, and a median expansion of 2,800-fold over two weeks, each donor can generate enough NK cells for 31 unit-dose bags. Assuming an initial donor apheresis containing a median of 3 x 10 NK cells after CD3 depletion (MD Anderson experiment), each donor can produce an average of 168 unit dose bags. One bag is sufficient for one dose of 10 NK cells / kg for a 50 kg individual. For adult patients, a dose of 10 NK cells / kg may require up to 2-3 bags per patient per dose. One exemplary assumption is that the freezing medium contains 10% DMSO, and the DMSO administered for a 10 NK cell / kg dose may be 0.1 ml / kg.

[0088] VI. Genotyping, Sequencing and Polymerase Chain Reaction (PCR) Immunoassays and Fluorescent Dyes The steps of a variety of useful immunodetection methods are described in the scientific literature. In its simplest and most straightforward sense, an immunoassay is a binding assay involving the binding between an antibody and an antigen. Many types and formats of immunoassays are known, any of which 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 blotting, dot blotting, gel shift assay, flow cytometry, protein array, multiplexed bead array, magnetic capture, in vivo imaging, fluorescence resonance energy transfer (FRET), and fluorescence recovery after photobleaching / localization (FRAP / FLAP).

[0089] In general, immunoassays involve contacting a sample suspected of containing a molecule of interest (such as a disclosed biomarker) with an antibody to the molecule of interest, or contacting an antibody to the molecule of interest (such as an antibody to a disclosed biomarker) with a molecule capable of being bound by that antibody, optionally under conditions effective to form an immune complex. Contacting a sample with an antibody to a molecule of interest, or a molecule capable of being bound by an antibody to a molecule of interest, under conditions effective and for a period of time sufficient to form immune complexes (primary immune complexes) is generally simply a matter of bringing the molecule or antibody into contact with the sample and incubating the mixture for a period of time long enough for the antibody to form an immune complex with, i.e., bind to, any molecules present (e.g., antigens) to which the antibody can bind. In many forms of immunoassays, the sample-antibody composition, such as a tissue section, ELISA plate, dot blot, or Western blot, can then be washed to remove any nonspecifically bound antibody species, leaving only the specifically bound antibody present in the primary immune complexes to be detected.

[0090] In practicing the methods of the present invention, the expression level of a nucleic acid molecule can be determined by any method, including, but not limited to, Southern analysis, Northern analysis, polymerase chain reaction (PCR) (see, e.g., PCR Protocols: A Guide to Methods and Applications, Innis et al., eds., 1990, Academic Press, NY), reverse transcriptase PCR (RT-PCT), anchored PCR, competitive PCR (see, e.g., U.S. Pat. No. 5,747,251), rapid amplification of cDNA ends (RACE) (see, e.g., Gene Cloning and Analysis: Current Innovations, 1997, pp. 99-115); ligase chain reaction (LCR) (see, e.g., European Patent No. 01001014). 320308), one-sided PCR (Ohara et al., Proc. Natl. Acad. Sci. 1989, vol. 86, pp. 5673-5677), in situ hybridization, Taqman-based assays (Holland et al., Proc. Natl. Acad. Sci. 1991, vol. 88, pp. 7276-7280), differential display (e.g., Liang et al., Proc. Natl. Acad. Sci. 1991, vol. 88, pp. 7276-7280), and the like. (Liang et al., Nucl. Acid. Res., 1993, 21, 3269-3275) and other RNA fingerprinting methods, nucleic acid sequence-based amplification (NASBA) and other transcription-based amplification systems, Qbeta replicase, strand displacement amplification (SDA), repair chain reaction (RCR), nuclease protection assays, subtraction-based methods, Rapid-Scan™, and the like.

[0091] Hybridization techniques can use nucleic acid probes to detect polynucleotides encoding specific characteristics of NK cells. These techniques generally involve contacting and incubating nucleic acid molecules in a biological sample obtained from a subject with a nucleic acid probe under conditions that allow specific hybridization between the nucleic acid probe and a complementary sequence of the nucleic acid molecule. After incubation, unhybridized nucleic acids are removed, and the presence and amount of nucleic acids hybridized to the probe are detected and quantified. Genotyping is performed by PCR, hybridization probes, and / or direct DNA sequencing.

[0092] Immunoassays can include methods for detecting or quantifying the amount of a molecule of interest (such as a disclosed biomarker or an antibody thereof) in a sample, which generally involve the detection or quantification of any immune complexes formed during the binding process. In general, the detection of immune complex formation is well known in the art and can be achieved through the application of numerous techniques. These methods are generally based on the detection of a label or marker, such as any radioactive, fluorescent, biological, or enzymatic tag, or any other known label.

[0093] As used herein, labels include fluorescent dyes, members of binding pairs such as biotin / streptavidin, metals (e.g., gold), and / or epitope tags that specifically interact with molecules that can be detected, such as by generating colored substrates or fluorescence. Suitable substances for detectably labeling proteins include fluorescent dyes (also referred to herein as fluorescent dyes and fluorophores) and enzymes that react with colorimetric substrates (e.g., horseradish peroxidase). Because fluorescent dyes are detectable even in very low amounts, their use is generally preferred in the practice of the present invention. Furthermore, when reacting multiple antigens with a single array, each antigen is labeled with a different fluorescent compound for simultaneous detection. The labeled spots on the array are detected using a fluorimeter, and the presence of a signal indicates the antigen bound to the specific antibody.

[0094] A fluorophore is a compound or molecule that emits light. Typically, a fluorophore absorbs electromagnetic energy at one wavelength and emits electromagnetic energy at a second wavelength. Representative fluorophores include, but are not limited to, 1,5 IAEDANS; 1,8-ANS; 4-methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein; 5-carboxyfluorescein (5-FAM); 5-carboxynaphthofluorescein; 5-carboxytetramethylrhodamine (5-TAMRA); 5-hydroxytryptamine (5-HAT); 5-ROX (carboxy-X-rhodamine); 6-carboxyrhodamine 6G; 6-CR 6G; 6-JOE; 7-amino-4-methylcoumarin; 7-aminoactinomycin D (7-AAD); 7-hydroxy-4-methylcoumarin; 9-amino-6-chloro-2-methoxyacridine (ACMA); ABQ; acid fuchsin; acridine orange; acridine red; acridine yellow; acriflavine; acriflavine feuilletine SITSA; aequorin (photoprotein); AFP - autofluorescent protein - (Quantum Biotechnologies), see sgGFP, sgBFP; Alexa Fluor 350™; Alexa Fluor 430™; Alexa Fluor 488™; Alexa Fluor 532™; Alexa Fluor 546™ 546(Trademark); Alexa Fluor 568(Trademark); Alexa Fluor 594(Trademark); Alexa Fluor 633(Trademark); Alexa Fluor 647(Trademark); Alexa Fluor 660(Trademark); Alexa Fluor 680(Trademark); Alizarin Complexone; Alizarin Red; Allophycocyanin (APC); AMC, AMCA-S; Aminomethylcoumarin (AMCA); AMCA-X; Aminoactinomycin D; Aminocoumarin;Aniline blue; anthrosyl stearate; APC-Cy7; APTRA-BTC; APTS; Astrazon Brilliant Red 4G; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7 GLL; atabrine; ATTO-TAG™ CBQCA; ATTO-TAG™ FQ; auramine; aurophosphine G; aurophosphine; BAO 9 (bisaminophenyloxadiazole); BCECF (high pH); BCECF (low pH); berberine sulfate; β-lactamase; BFP blue-shifted GFP (Y66H); blue fluorescent protein; BFP / GFP FRET; bimane; bisbenzimide; bisbenzimide (Hoechst); bis-BTC; Blancophor FFG; Blancophor SV; B0130™-1; BOBO™-3; Bodipy 492 / 515 (Bodipy 492 / 515); Bodipy 493 / 503 (Bodipy 493 / 503); Bodipy 500 / 510 (Bodipy 500 / 510); Bodipy 505 / 515; Bodipy 530 / 550 (Bodipy 530 / 550); Bodipy 542 / 563 (Bodipy 542 / 563); Bodipy 558 / 568 (Bodipy 558 / 568); Bodipy 564 / 570 (Bodipy 564 / 570; Bodipy 576 / 589 (Bodipy 576 / 589); Bodipy 581 / 591 (Bodipy 581 / 591); Bodipy 630 / 650-X (Bodipy 630 / 650-X); Bodipy 650 / 665-X (Bodipy 650 / 665-X); Bodipy 665 / 676 (Bodipy 665 / 676); Bodipy Fl (Bodipy Fl); Bodipy FL ATP (Bodipy FL ATP); Bodipy F1-ceramide (Bodipy F1-ceramide); Bodipy R6G SE (Bodipy R6G SE); Bodipy TMR (Bodipy TMR); Bodipy TMR-X (Bodipy TMR-X) conjugate; Bodipy TMR-X, SE (Bodipy TMR-X, SE); Bodipy TR (Bodipy TR); Bodipy TR ATP(Bodipy TR ATP);Bodipy TR-X SE; BO-PRO™-1; BO-PRO™-3; Brilliant Sulfoflavin FF; BTC; BTC-5N; calcein; calcein blue; calcium crimson; calcium green; calcium green-1 Ca2+ dye; calcium green-2 Ca2+; calcium green-5N Ca2+; calcium green-C18 Ca2+; calcium orange; calcofluor white; carboxy-X-rhodamine (5-ROX); Cascade Blue™; Cascade yellow; catecholamines; CCF2 (GeneBlazer); CFDA; CFP (cyan fluorescent protein); CFP / YFP FRET; chlorophyll; chromomycin A; chromomycin A; CL-NERF; CMFDA; coelenterazine; coelenterazine cp; coelenterazine f; coelenterazine fcp; coelenterazine h; coelenterazine hcp; coelenterazine ip; coelenterazine n; coelenterazine 0; coumarin phalloidin; C-phycocyanin; CPM I methylcoumarin; CTC; CTC formazan; Cy2™; Cy3.1 8; Cy3.5™; Cy3™; Cy5.1 8; Cy5.5™; Cy5™; Cy7™; Cyan GFP; Cyclic AMP Fluorosensor (FiCRhR); Dabsyl; Dansyl; Dansylamine; Dansylcadaverine; Dansyl chloride; DansylDHPE; Dansyl fluoride; DAPI; Dapoxil; Dapoxil 2; Dapoxil 3'DCFDA; DCFH (Dichlorodihydrofluorescein diacetate); DDAO; DHR (Dihydrorhodamine 123); Di-4-ANEPPS; Di-8-ANEPPS (non-ratio); DiA(4-di16-ASP); dichlorodihydrofluorescein diacetate (DCFH); DiD-lipophilic tracer; DiD(Di1C18(5)); DIDS; dihydrorhodamine 123 (DHR); Dil(DilC18(3)); Idinitrophenol; DiO(DiOC18(3)); DiR; DiR(Di1C18(7)); DM-NERF (high pH); DNP; dopamine; DsRed; DTAF; DY-630-NHS; DY-635-NHS; EBFP; ECFP; EGFP;ELF 97; Eosin; Erythrosine; Erythrosine ITC; Ethidium Bromide; Ethidium Homodimer-1 (EthD-1); Euchrysin; EukoLight; Europium(111) Chloride; EYFP; Fast Blue; FDA; Feulgen (pararosaniline); FIF (formaldehyde-induced fluorescence); FITC; Furazo Orange; Fluo-3; Fluo-4; Fluorescein (FITC); Fluorescein diacetate; Fluoroemerald; Fluorogold (hydroxystilbamidine); Fluor-Ruby; FluorX; FM 1-43™; FM 4-46; Fura Red™ (high pH); Fura Red™ / Fluo-3; Fura-2; Fura-2 / BCECF; Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF; Genacryl Pink 3G; Genacryl Yellow 5GF; GeneBlazer; (CCF2); GFP (S65T); GFP red-shifted (rsGFP); GFP wild-type non-UV-excitable (wtGFP); GFP wild-type, UV-excitable (wtGFP); GFPuv; GI oxalate; Granular Blue; Hematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; I IPTS; Hydroxycoumarin; Hydroxystilbamidine (Fluor Gold); Hydroxytryptamine; Indo-1 High Calcium; Indo-1 Low Calcium; Indodicarbocyanine (DiD); Indotricarbocyanine (DiR); Intrawhite Cf; JC-1; JO J0-1; JO-PRO-1; LaserPro; Laurodan; LDS 751 (DNA); LDS 751 (RNA); Leucophor PAF; Leucophor SF;Leucophor WS; Lissamine rhodamine; Lissamine rhodamine B; Calcein / ethidium homodimer; LOLO-1; LO-PRO-1; Lucifer Yellow; LysoTracker Blue; LysoTracker Blue-White; LysoTracker Green; LysoTracker Red; LysoTracker Yellow; LysoSensor Blue; LysoSensor Green; LysoSensor Yellow / Blue; Mag Green; Magdala Red (Phloxine B); Magfla Red; Magfla 2; Magfla 5; Magind 1; Magnesium Green; Magnesium Orange; Malachite Green; Marina Blue; Maxilon Brilliant Flavin 10 GFF; Maxilon Brilliant Flavin 8 GFF; Merocyanine; Methoxycoumarin; Mitotracker Green FM; Mitotracker Orange; Mitotracker Red; Mithramycin; Monobromobimane; Monobromobimane (mBBr-GSH); Monochlorobimane; MPS (Methyl Green Pyronine Stilbene); NBD; NBD-amine; Nile Red; Nitrobenzoxadiazole; Noradrenaline; Nuclear Fast Red; Nuclear Yellow; Nylosan Brilliant Flavin E8G; Oregon Green™; Oregon Green™ 488; Oregon Green™ Green™ 500; Oregon Green™ 514; Pacific Blue; Pararosaniline (Feulgen); PBFI; PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5; PE-Texas Red (Red 613); Phloxine B (Magdala Red); Phorwite AR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R;Photoresist; Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26 (Sigma); PKH67; PMIA; Pontochrome Blue Black; POPO-1; POPO-3; PO-PRO-1; PO-I PRO-3; Primulin; Procion Yellow; Propidium iodide (; PI); PyMPO; Pyrene; Pyronine; Pyronine B; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Resorufin; RH 414; Rhodamine 2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5 GLD; Rhodamine 6G; Rhodamine B; Rhodamine B 200; Rhodamine B Extra; Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine phallicidin; Rhodamine phalloidin; Rhodamine Red; Rhodamine WT; Rose Bengal; R-phycocyanin; R-phycoerythrin (PE); rsGFP; S65A; S65C; S65L; S65T; Sapphire GFP; SBFI; Serotonin; Sevron Brilliant Red 2B; Sevron Brilliant Red 4G; Sevron Brilliant Red B; Sevron Orange; Sevron Yellow L; sgBFP (TM) (SuperGlow BFP); sgGFP™ (SuperGlow GFP); SITS (primulin; stilbene isothiosulfonic acid); SNAFL calcein; SNAFL-1; SNAFL-2; SNARF calcein; SNARFI; sodium green; SpectrumAqua; Spectrum Green; Spectrum Orange; Spectrum Red; SPQ (6-methoxy-N-(3-sulfopropyl)quinolinium); stilbene; sulforhodamine B and C; sulforhodamine Extra; SYTO 11;SYTO 12;SYTO 13;SYTO 14;SYTO 15;SYTO 16;SYTO 17;SYTO 18;SYTO 20;SYTO 21;SYTO 22;SYTO 23;SYTO 24;SYTO 25;SYTO 40;SYTO 41;SYTO 42;SYTO 43;SYTO 44;SYTO 45;SYTO 59;SYTO 60;SYTO 61;SYTO 62;SYTO 63;SYTO 64;SYTO 80;SYTO 81;SYTO 82;SYTO 83;SYTO 84;SYTO 85;SYTOX blue;SYTOX green;SYTOX orange;tetracycline;Tetramethylrhodamine (TRITC); Texas Red™; Texas Red-X™ conjugate; Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazole Orange; Thioflavin 5; Thioflavin S; Thioflavin TON; Thiolite; Thiozole Orange; Tinopol CBS (Calcofluor White); TIER; TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITC isothiocyanate tetramethylrhodamine; True Blue; Tru Red; Ultralite; Uranine B; Uvitex SFC; wt GFP; WW 781; X-rhodamine; XRITC; xylene orange; Y66F; Y66H; Y66W; yellow GFP; YFP; YO-PRO-1; YO-PRO 3; YOY0-1; YOY0-3; Sybr Green; thiazole orange (an intercalating dye); semiconductor nanoparticles such as quantum dots; or caged fluorophores (which can be activated by light or other electromagnetic energy sources), or combinations thereof;

[0095] In one aspect, a modifier unit such as a radionuclide is 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, and fluorine-18. In another aspect, the radionuclide is attached to a linking group or attached via a chelating group, which is then attached to the compound directly or using a linker. Examples of radionuclides useful in this embodiment 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. Radiolabeling techniques such as these are routinely used in the radiopharmaceutical industry.

[0096] Radiolabeled compounds are useful as imaging agents for diagnosing neurological diseases (e.g., neurodegenerative diseases) or psychiatric conditions in mammals (e.g., humans) or monitoring the progression or treatment of such diseases or conditions. The radiolabels described herein can be advantageously used in conjunction with imaging techniques such as positron emission tomography (PET) or single photon emission computed tomography (SPECT).

[0097] Labels can be either direct or indirect. In direct labeling, the detection antibody (antibody against the molecule of interest) or the detection molecule (molecule capable of being bound by the antibody against the molecule of interest) contains a label. Detection of the label indicates the presence of the detection antibody or detection molecule, which in turn indicates the presence of the molecule of interest or the antibody against the molecule of interest, respectively. 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 be attached to or linked to 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, when provided with a suitable substrate, generates a visible or detectable product at the site of the immune complex. ELISA uses this type of indirect labeling.

[0098] As another example of indirect labeling, an additional molecule (which may be referred to as a binder) capable of binding to either the molecule of interest or an antibody to the molecule of interest (the primary antibody), such as a secondary antibody directed against the primary antibody, may be contacted with the immune complex. This additional molecule may carry a label or a signal-generating molecule or moiety. The additional molecule may be an antibody, which may therefore be referred to as a secondary antibody. When the secondary antibody binds to the primary antibody, it may form a so-called sandwich with the first (or primary) antibody and the molecule of interest. The immune complex may be contacted with a labeled secondary antibody under conditions effective and for a period of time sufficient to allow the formation of secondary immune complexes. The secondary immune complexes may then generally be washed to remove any nonspecifically bound labeled secondary antibody, and the remaining label in the secondary immune complexes may be detected. The additional molecule may also be or include one of a pair of molecules or moieties capable of binding to each other, such as a biotin / avidin pair. In this format, the detection antibody or detection molecule must include the other member of the pair.

[0099] Other indirect labeling methods include detecting primary immune complexes by a two-step approach: for example, a molecule (e.g., a first binding agent) such as an antibody that exhibits binding affinity for the molecule of interest or a corresponding antibody can be used to form secondary immune complexes as described above.

[0100] After washing, the secondary immune complexes can be contacted with another molecule (which can be referred to as a second binding agent) that exhibits binding affinity for the first binding agent, again under conditions effective and for a period of time sufficient to allow immune complex formation (thus forming tertiary immune complexes). The second binding agent can be linked to a detectable label or signal-generating molecule or moiety, allowing detection of the tertiary immune complexes thus formed. This system can provide signal amplification.

[0101] Immunoassays involving the detection of a substance itself, such as a protein or an antibody to a specific protein, include label-free assays, protein separation methods (e.g., electrophoresis), solid support capture assays, or in vivo detection. Label-free assays are generally diagnostic tools that determine the presence or absence of a specific protein or an antibody to a specific protein in a sample. Protein separation methods are additionally useful for assessing the physical properties of proteins, such as size or net charge. Capture assays are generally more useful for quantitatively assessing the concentration of a specific protein or an antibody to a specific protein in a sample. Finally, in vivo detection is useful for assessing the spatial expression pattern of a substance, for example, where the substance can be found in a subject, tissue, or cell.

[0102] At sufficient concentrations, the molecular complex ([Ab-Ag]n) generated by antibody-antigen interaction is visible to the naked eye, but even small amounts can be detected and measured due to its ability to scatter light. Complex formation indicates the presence of both reactants, and in immunoprecipitation assays, a fixed concentration of reagent antibody is used to measure the specific antigen ([Ab-Ag]n) and the reagent antigen is used to detect the specific antibody ([Ab-Ag]n). When the reagent species is pre-coated onto cells (as in hemagglutination assays) or very small particles (as in latex agglutination assays), the "agglomeration" of the coated particles becomes visible even at much lower concentrations. Various assays based on this basic principle are commonly used, including Ouchterlony immunodiffusion assays, rocket immunoelectrophoresis, and immunoturbidimetric and nephelometric assays. The main limitation of such assays is their limited sensitivity (low detection limit) compared to assays that utilize labels, and in some cases, the procedure is further complicated by the fact that very high concentrations of analyte actually inhibit complex formation, necessitating countermeasures. Some of these Group 1 assays date back immediately to the discovery of antibodies, and none of them have an actual "label" (e.g., Ag-enz). Other types of label-free immunoassays rely on immunosensors, and a variety of instruments are now commercially available that can directly detect antibody-antigen interactions. Most rely on generating an evanescent wave at a sensor surface with immobilized ligand, which allows continuous monitoring of binding to the ligand. Immunosensors allow for easy investigation of kinetic interactions and may find wide application in immunoanalysis in the future, given the emergence of low-cost, specialized instruments.

[0103] Detection of specific proteins using immunoassays can involve separation of proteins by electrophoresis. Electrophoresis is the migration of charged molecules in solution in response to an electric field. The migration rate depends on the strength of the electric field; the net charge, size, and shape of the molecule, and also on the ionic strength, viscosity, and temperature of the medium through which the molecules move. As an analytical tool, electrophoresis is simple, rapid, and sensitive. It is used analytically to examine the properties of singly charged species and as a separation technique.

[0104] Typically, samples are run on a support matrix, such as paper, cellulose acetate, starch gel, agarose, or polyacrylamide gel. The matrix suppresses convective mixing caused by heating and provides a record of the electrophoresis run; at the end of the run, the matrix can be stained and used for scanning, autoradiography, or storage. In addition, the most commonly used support matrices—agarose and polyacrylamide—provide a means of separating molecules by size in that they are porous gels. Porous gels can act as sieves by slowing or, in some cases, completely blocking the migration of larger macromolecules while allowing smaller molecules to migrate freely. Dilute agarose gels are generally more rigid and easier to handle than polyacrylamide at the same concentration, so agarose is used to separate large macromolecules, such as nucleic acids, large proteins, and protein complexes. Polyacrylamide, which is easy to handle and to increase in concentration, is used to separate many proteins and small oligonucleotides, which require small gel pore sizes for retardation.

[0105] Proteins are amphoteric compounds; therefore, their net charge is determined by the pI of the medium in which they are suspended. In a solution with a pH above their isoelectric point, proteins have a net negative charge and migrate in an electric field toward the anode. Below their isoelectric point, proteins are positively charged and migrate toward the cathode. In addition, regardless of their size, the net charge carried by a protein—i.e., the charge carried per unit mass (or length, for given proteins and nucleic acids, being linear macromolecules) of the molecule—varys from protein to protein. Therefore, at a given pH and under non-denaturing conditions, electrophoretic separation of proteins depends on both the size and charge of the molecule.

[0106] Sodium dodecyl sulfate (SDS) is an anionic detergent that denatures proteins by "wrapping" around the polypeptide backbone—and SDS binds to proteins with high specificity at a mass ratio of 1.4:1. SDS then imparts a negative charge to the polypeptide, proportional to its length. Furthermore, protein disulfide bridges must usually be reduced (denatured) before the protein can adopt the random coil structure necessary for size separation; this is accomplished with 2-mercaptoethanol or dithiothreitol (DTI). Thus, in denaturing SDS-PAGE separations, migration is determined by molecular weight, not by the intrinsic charge of the polypeptide.

[0107] Molecular weight determinations are performed by subjecting proteins of known molecular weight to SDS-PAGE along with the protein to be characterized. A linear relationship exists 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 relative molecular weight (Mr) by electrophoresis is to plot a standard curve of the logl OMW of known samples versus the distance migrated and read the logMr of the sample after measuring the distance migrated on the same gel.

[0108] In two-dimensional electrophoresis, proteins are first fractionated based on one physical property and then fractionated in a second step based on another. For example, the first dimension can be isoelectric focusing, conveniently performed in a tube gel, and the second dimension can be SDS electrophoresis in a slab gel. The leading ion in the Laemmli buffer system is chloride, and the trailing ion is glycine. Thus, the resolving gel and stacking gel are constructed in Tris-HCl buffers (of various concentrations and pH), while the running tank buffer is Tris-glycine. All buffers contain 0.1% SDS.

[0109] One example of an electrophoretic immunoassay contemplated by the present method is Western blot analysis. Western blotting, or immunoblotting, allows for the determination of the molecular mass of proteins present in various samples and the measurement of the relative amounts of proteins. Detection methods include chemiluminescent and chromogenic detection methods.

[0110] Typically, proteins are separated by gel electrophoresis, usually SDS-PAGE. The proteins are transferred to a piece of special blotting paper, such as nitrocellulose, although other types of paper or membranes can also be used. The proteins retain the same separation pattern as when they were placed on the gel. The blot is incubated with a generic protein (such as milk protein), which binds to any remaining sticky spots on the nitrocellulose. An antibody is then added to the solution, which is capable of binding to its specific protein.

[0111] Attachment of specific antibodies to specific immobilized antigens can be easily visualized by indirect enzyme immunoassay techniques, which typically use chromogenic (e.g., alkaline phosphatase or horseradish peroxidase) or chemiluminescent substrates. Other probing possibilities include the use of fluorescent or radioisotope labels (e.g., fluorescein, 1251). Probes for detection of antibody binding can be conjugated anti-immunoglobulin, conjugated Staphylococcus aureus protein A (which binds IgG), or probes for biotinylated primary antibodies (e.g., conjugated avidin / streptavidin).

[0112] The power of this technique lies in the simultaneous detection of specific proteins by their antigenicity and their molecular mass. Proteins are first separated by mass on SDS-PAGE and then specifically detected in an immunoassay step. Therefore, protein standards (ladders) can be run simultaneously to approximate the molecular mass of proteins of interest in heterogeneous samples.

[0113] Gel shift assays or electrophoretic mobility shift assays (EMSAs) can be used to detect interactions between DNA binding proteins and their cognate DNA recognition sequences in both a qualitative and quantitative manner.

[0114] In a typical gel shift assay, purified proteins or crude cell extracts are incubated with a labeled (e.g., 32P-radiolabeled) DNA or RNA probe, followed by separation of the complex from free probe in a non-denaturing polyacrylamide gel. The complex migrates more slowly through the gel than the unbound probe. Depending on the activity of the binding protein, the labeled probe can be either double-stranded or single-stranded. For detection of DNA-binding proteins, such as transcription factors, either purified or partially purified proteins or nuclear cell extracts can be used. For detection of RNA-binding proteins, either purified or partially purified proteins 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 DNA or RNA fragments or oligonucleotides or other unrelated sequences containing the binding site of the protein of interest. Specific interactions can be identified from differences in the nature and strength of complexes formed in the presence of specific and nonspecific competitors. Gel shift methods can include, for example, detecting proteins in gels, such as polyacrylamide electrophoresis gels, using a colloidal form of COOMASSIE (Imperial Chemical Industries, Ltd.) Blue stain. In addition to the conventional protein assay methods referenced above, a combined cleaning and protein staining composition is described in U.S. Pat. No. 5,424,000 (incorporated herein by reference in its entirety for its teachings regarding gel shift methods). Solutions can include phosphoric, sulfuric, and nitric acids and acid violet dye.

[0115] Radioimmunoprecipitation assay (RIPA) is a highly sensitive assay that uses radiolabeled antigens to detect specific antibodies in serum. The antigen is allowed to react with serum and then precipitated using special reagents, such as protein A sepharose beads. The bound radiolabeled immunoprecipitate is then analyzed, typically by gel electrophoresis. Radioimmunoprecipitation assay (RIPA) is often used as a confirmatory test to diagnose the presence of HIV antibodies. RIPA is also referred to in the art as a pharmacoassay, precipitation assay, radioimmunoprecipitation assay; radioimmunoprecipitation analysis; radioimmunoprecipitation analysis, and radioimmunoprecipitation analysis.

[0116] While the immunoassays described above that use electrophoresis to separate and detect specific proteins of interest allow for the assessment of protein size, such assays are not very sensitive for assessing protein concentration. However, immunoassays are also contemplated in which proteins or protein-specific antibodies are bound to a solid support (e.g., a tube, well, bead, and / or cell) in combination with a method for detecting the protein or protein-specific antibody on the support to capture the antibody or protein of interest, respectively, from a sample. Examples of such immunoassays include radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA), flow cytometry, protein arrays, multiplex bead assays, and / or magnetic capture.

[0117] Radioimmunoassays (RIAs) are classical quantitative assays that measure the binding of unlabeled substances to specific antibodies or other receptor systems by detecting antigen-antibody reactions using radioactively labeled substances (radioligands) either directly or indirectly. Radioimmunoassays are used, for example, to test hormone levels in blood without the need for bioassays. Non-immunogenic substances (e.g., haptens) can also be measured when coupled to large carrier proteins (e.g., bovine γ-globulin or human serum albumin) that are capable of inducing antibody formation. RIA involves mixing a radioactive antigen (often radioisotopes 1251 or 1311 are used because iodine atoms can easily be incorporated into tyrosine residues in proteins) with an antibody against that antigen. The antibody is typically linked to a solid support, such as a tube or beads. A known amount of unlabeled or "cold" antigen is then added, and the amount of displaced labeled antigen is measured. Initially, the radioactive antigen binds to the antibody. When cold antigen is added, the two compete for antibody binding sites - and as the concentration of cold antigen increases, it binds more to the antibody, displacing the radioactive variant. Bound antigen is separated from unbound antigen in solution, and a binding curve is plotted using the radioactivity of each. This technique is both highly sensitive and specific.

[0118] Enzyme-linked immunosorbent assays (ELISAs), or more generally, enzyme immunoassays (EIAs), are immunoassays capable of detecting protein-specific antibodies. In such assays, the detectable label attached to either the antibody- or antigen-binding reagent is an enzyme. This enzyme, when exposed to its substrate, reacts to produce a chemical moiety that can be detected, for example, by spectrophotometric, fluorometric, 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.

[0119] Variations of the ELISA method are known to those skilled in the art. In one variation, an antibody that binds to a protein is immobilized on a selected surface that exhibits protein affinity, such as the wells of a polystyrene microtiter plate. A test composition suspected of containing a marker antigen is then added to the wells. After binding and washing away non-specifically bound immune complexes, the bound antigen can be detected.

[0120] Detection can be achieved by adding a second antibody specific to the target protein and linked to a detectable label. This type of ELISA is a simple "sandwich ELISA." Detection can also be achieved by adding the second antibody, followed by a third antibody that exhibits binding affinity to the second antibody and is linked to a detectable label.

[0121] Another variation is the competitive ELISA. In a 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 before or during incubation with the coated wells. The presence of reactive species in the sample serves to reduce the amount of labeled species available for binding to the wells, thus reducing the final signal. Regardless of the format used, ELISAs share certain common features, such as coating, incubation, or binding, washing to remove nonspecifically bound species, and detecting bound immune complexes. The antigen or antibody can be linked to a solid support, such as a plate, bead, dipstick, membrane, or column matrix, and the sample to be analyzed can be applied to this immobilized antigen or antibody. When coating a plate with either the antigen or antibody, the wells of the plate are generally incubated with a solution of the antigen or antibody, either overnight or for a specified period of time. The wells of the plate can then be washed to remove incompletely adsorbed material. Any remaining available surfaces of the wells can then be "coated" with a nonspecific protein that is antigenically neutral with respect to the test antisera. Such nonspecific proteins include bovine serum albumin (BSA), casein, and milk powder solution. Coating allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera to the surface.

[0122] Rather than a direct procedure, ELISAs can also employ secondary or tertiary detection means. Thus, after binding the protein or antibody to a well, reducing background by coating with a non-reactive substance, and removing unbound material by washing, the immobilizing surface is contacted with the clinical or biological control sample under test under conditions effective to allow immune complex (antigen / antibody) formation. Detection of the immune complex then requires either a labeled secondary binding agent or a secondary binding agent in conjunction with a labeled third binding agent.

[0123] An enzyme-linked immunospot assay (ELISPOT) is an immunoassay capable of detecting antibodies specific for a protein or antigen. In such assays, the detectable label attached to either the antibody- or antigen-binding reagent is an enzyme. This enzyme, when exposed to its substrate, reacts to produce a chemical moiety that can be detected by, for example, spectrophotometric, fluorometric, 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 the antigen. The test sample is exposed to the antigen and then reacted in the same manner as in an ELISA assay. Detection differs from traditional ELISA in that it is determined by counting spots on a 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 antigen-specific cells in the sample.

[0124] By "under conditions effective to permit immune complex (antigen / antibody) formation" it is meant 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.

[0125] Suitable conditions also refer to incubation at a temperature and for a period of time sufficient to allow effective binding. Incubation steps can typically be from about 1 minute to 12 hours, at a temperature of about 20°C to 30°C, or can be incubated overnight at about 0°C to about 10°C. After all incubation steps in an ELISA, the contacted surface can be washed to remove uncomplexed material. The washing procedure can include washing with a solution such as PBS / Tween® or borate buffer. Following the formation of specific immune complexes between the test sample and the originally bound material and subsequent washing, the appearance of even minute amounts of immune complexes can be determined.

[0126] To provide a detection method, as described above, the second or third antibody can have an associated label that allows for detection. This can be an enzyme that can generate color development when incubated with an appropriate chromogenic substrate. Thus, for example, the first or second immune complex can be contacted with the labeled antibody and incubated for a time and under conditions that favor the occurrence of further immune complex formation (e.g., incubation in a PBS-containing solution, such as PBS-Tween®, at room temperature for 2 hours).

[0127] After incubation with the labeled antibody and subsequent removal of unbound material by washing, in the case of peroxidase as the enzyme label, the amount of label can be quantified by incubation with a colorimetric substrate, such as urea and bromocresol purple or 2,2'-azido-di-(3-ethyl-benzthiazoline-6-sulfonic acid [ABTS] and 1-1202. Quantitation can then be achieved by measuring the degree of color development, for example, using a visible spectrum spectrophotometer. Protein arrays are proteins immobilized on surfaces, including glass, membranes, microtiter wells, mass spectrometer plates, and beads or other particles. Solid-phase ligand-binding assay systems using proteins. These assays are highly parallel (multiplexed) and often miniaturized (microarrays, protein chips). Advantages include rapid automation, high sensitivity, reagent savings, and the provision of extensive data in a single experiment. Bioinformatics support is crucial; data processing requires sophisticated software and data comparison and analysis. However, software, as well as hardware and many of the detection systems, can be adapted from those used for DNA arrays.

[0128] One major format is the capture array, in which ligand-binding reagents (usually antibodies, but also 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 allow multiple immunoassays to be performed in parallel, both individually testing several analytes in, for example, serum, and simultaneously testing many serum samples. In proteomics, capture arrays are used to quantify and compare protein levels in various samples in health and disease, e.g., protein expression profiling. Array formats for in vitro functional interaction screening use proteins other than specific ligand binders, such as protein-protein, protein-DNA, protein-drug, receptor-ligand, and enzyme-substrate. The capture reagents themselves are selected and screened against many proteins, which can also be done in a multiplex array format against multiple protein targets.

[0129] Protein sources for constructing arrays include cell-based expression systems for recombinant proteins, purification from natural sources, in vitro production using cell-free translation systems, and synthetic methods for peptides. Many of these methods can be automated for high-throughput production. For capture arrays and protein function analysis, it is important that proteins be correctly folded and functional; 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 antibodies for cross-reactivity, identifying autoantibodies, and selecting ligand-binding proteins.

[0130] Protein arrays are designed as miniaturized versions of well-known immunoassay methods, such as ELISA and dot blotting, which often utilize fluorescent readouts. Robotics and high-throughput detection systems allow multiple assays to be performed in parallel. Common physical supports include glass slides, silicon, microwells, nitrocellulose or PVDF membranes, and magnetic and other microbeads. While the most well-known format involves delivery of protein microdroplets to a planar surface, alternative configurations include CD centrifuge devices based on developments in microfluidics (Gyros, Monmouth Junction, NJ) and specialized chip designs, such as engineered microchannels in plates (e.g., The Living Chip™, Biotrove, Woburn, MA) and microscopic 3D posts on silicon surfaces (Zyomyx, Hayward, CA).Particles in suspension can also be used as the basis for arrays if they are coded for identification; systems include color-coding microbeads (Luminex, Austin, TX; Bio-Rad Laboratories) and semiconductor nanocrystals (e.g., QDot™, Quantum Dot, Hayward, CA) and barcoding beads (UltraPlex™, SmartBead Technologies Ltd, Babraham, Cambridge, UK) and multimetallic microrods (e.g., Nanobarcode™ particles, Nanoplex Technologies, Mountain View, CA). Beads can also be assembled into planar arrays on semiconductor chips (LEAPS technology, BioArray Solutions, Warren, NJ).

[0131] Protein immobilization involves both the coupling reagent and the properties of the surface to be coupled. A good protein array support surface is chemically stable before and after the coupling procedure, allows good spot morphology, exhibits minimal nonspecific binding, does not contribute to detection system background, and is compatible with a variety of detection systems. The immobilization method used is reproducible, applicable to proteins of various properties (e.g., size, hydrophilicity, hydrophobicity, etc.), amenable to high throughput and automation, and compatible with preserving full functional protein activity. The orientation of surface-bound proteins is recognized as a critical factor for their active presentation to ligands or substrates; for capture arrays, the most efficient binding results are achieved when the capture reagent is properly oriented, which generally requires site-specific labeling of the protein.

[0132] Both covalent and noncovalent protein immobilization methods are used and offer various advantages and disadvantages. Passive adsorption to surfaces is methodologically simple but offers little quantification or orientation control; it may or may not alter the functional properties of the protein, and reproducibility and efficiency vary. Covalent coupling methods provide stable attachment, are applicable to a variety of proteins, and have good reproducibility; however, orientation can vary, chemical derivatization can alter protein function, and a stable interaction surface is required. Biological capture methods that utilize tags on proteins provide stable attachment and bind to proteins specifically and in a reproducible orientation, but the biological reagent must first be properly immobilized, may require special processing on the array, and have variable stability.

[0133] Several immobilization chemistries and tags have been described for the creation of protein arrays. Substrates for covalent attachment include glass slides coated with amino- or aldehyde-containing silane reagents. The Versalinx™ system (Prolinx, Bothell, WA) achieves reversible covalent coupling through the interaction between proteins derivatized with phenyldiboronic acid and salicylhydroxamic acid immobilized on the support surface. This results in low background binding and intrinsic fluorescence, and allows the immobilized proteins to retain their functionality. Noncovalent attachment of unmodified proteins occurs within porous structures such as three-dimensional polyacrylamide gel-based HydroGel™ (PerkinElmer, Wellesley, MA); this substrate has been reported to provide particularly low background on glass microarrays, along with high capacity and retention of protein function. Widely used biological coupling methods rely on biotin / streptavidin or hexahistidine / Ni interactions to appropriately modify proteins. Biotin can be conjugated to a poly-lysine backbone immobilized on surfaces such as titanium dioxide (Zyomyx) or tantalum pentoxide (Zeptosens, Witterswil, Switzerland).

[0134] Array fabrication methods include robotic contact printing, inkjet printing, piezoelectric spotting, and photolithography. Several commercially available arrayers (e.g., manufactured and sold by Packard Biosciences) and manual instruments (e.g., manufactured and sold by V&P Scientific) are available. Bacterial colonies can be robotically gridded onto PVDF membranes to induce in situ protein expression. Nanoarrays with spots on the nanometer spatial scale are at the limit of spot size and density, enabling thousands of reactions to be performed on a single chip less than 1 square millimeter. BioForce Laboratories has developed a nanoarray with 1,521 protein spots in 85 square microns, equivalent to 25 million spots per square centimeter, the limit of optical detection; readout methods are fluorescence and atomic force microscopy (AFM).

[0135] Fluorescent labeling and detection methods are widely used. The same instrumentation used to read DNA microarrays can also be applied to protein arrays. For differential display methods, capture (e.g., antibody) arrays can be probed with fluorescently labeled proteins from two different cell states, where cell lysates are directly conjugated with different fluorophores (e.g., Cy-3, Cy-5) and mixed, with color serving as a readout for changes in target abundance. The sensitivity of the fluorescent readout can be amplified 10- to 100-fold by tyramide signal amplification (TSA) (PerkinElmer Lifesciences). Planar waveguide technology (Zeptosens) allows for ultrasensitive fluorescence detection, with the added advantage of no intervening washing steps. High sensitivity can also be achieved with suspension beads and particles using phycoerythrin as a label (Luminex®) or the properties of semiconductor nanocrystals (Quantum Dot).Several novel alternative readout methods are being developed, particularly in the commercial biotechnology industry.These include the application of surface plasmon resonance (e.g., manufactured and sold by FITS Biosystems, Intrinsic Bioprobes, Tempe, AZ), rolling circle DNA amplification (e.g., manufactured and sold by Molecular Staging, New Haven, CT), mass spectrometry (e.g., manufactured and sold by Intrinsic Bioprobes; Ciphergen, Fremont, CA), resonant light scattering (e.g., manufactured and sold by Genicon Sciences, San Diego, CA), and atomic force microscopy (e.g., manufactured and sold by BioForce Laboratories).

[0136] Capture arrays form the basis of diagnostic chips and arrays for expression profiling. Capture arrays utilize high-affinity capture reagents, such as conventional antibodies, single domains, engineered scaffolds, peptides, or nucleic acid aptamers, to bind and detect specific target ligands in a high-throughput manner. Antibody arrays have the required specificity characteristics and acceptable background, and some are commercially available (e.g., as manufactured and sold by BD Biosciences, San Jose, CA; Clontech, Mountain View, CA; and / or BioRad; Sigma, St. Louis, MO). Antibodies for capture arrays are either produced by conventional immunization (polyclonal sera and hybridomas) or as recombinant fragments, usually expressed in E. coli, after selection from phage or ribosome display libraries (e.g., manufactured and sold by Cambridge Antibody Technology, Cambridge, UK; BioInvent, Lund, Sweden; Affitech, Walnut Creek, CA; and / or Biosite, San Diego, CA). In addition to conventional antibodies, Fab, and scFv fragments, single V-domains from camelids or engineered human equivalents (e.g., manufactured and sold by Domantis, Waltham, MA) can also be useful in arrays.

[0137] The term "scaffold" refers to the ligand-binding domain of a protein, which is engineered to produce multiple variants capable of binding diverse target molecules with antibody-like specificity and affinity properties. These variants can be generated in a genetic library format and selected against individual targets by phage, bacterial, or ribosome display. Such ligand-binding scaffolds or frameworks include "Affibodies®" based on Staph. aureus protein A (e.g., manufactured and sold by Affibody, Bromma, Sweden), "Trinectins" based on fibronectin (e.g., manufactured and sold by Phylos, Lexington, MA), and "Anticalins®" based on lipocalin structures (e.g., manufactured and sold by Pieris Proteolab, Freising-Gweihenstephan, Germany). These can be used in capture arrays in the same way as antibodies, and may offer advantages such as robustness and ease of manufacture.

[0138] Non-protein capture molecules, particularly single-stranded nucleic acid aptamers, that bind protein ligands with high specificity and affinity are also used in arrays (e.g., manufactured and sold by SomaLogic, Boulder, CO). Aptamers are selected from libraries of oligonucleotides by the Selex™ procedure, and their interaction with proteins can be covalently enhanced through the incorporation of brominated deoxyuridine and UV-activated crosslinks (photoaptamers). Photocrosslinking to the ligand reduces the cross-reactivity of the aptamer due to specific steric requirements.

[0139] Aptamers offer the advantages of ease of production by automated oligonucleotide synthesis and the stability and robustness of DNA; on photoaptamer arrays, binding can be detected using universal fluorescent protein stains.

[0140] Protein analytes bound to antibody arrays can be detected directly or via secondary antibodies in sandwich assays. Direct labeling is used to compare different samples with different colors. When pairs of antibodies directed against the same protein ligand are available, sandwich immunoassays offer high specificity and sensitivity, making them the method of choice for low-abundance proteins 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 alteration. Optimal sensitivity and specificity are required for all methods, where low background provides high signal-to-noise. To cover a wide range of analyte concentrations, sensitivity must be appropriately adjusted accordingly; serial dilutions of samples or the use of antibodies with different affinities are solutions to this problem. Proteins of interest are often present in low concentrations in biological fluids and extracts, such as cytokines or low-expression products within cells, requiring detection in the pg range or below.

[0141] An alternative to arrays of capture molecules is one created through "molecular imprinting" technology, where peptides (e.g., from the C-terminal region of a protein) are used as templates to generate structurally complementary, sequence-specific voids in a polymerizable matrix; these voids can then specifically capture (denatured) proteins with the appropriate primary amino acid sequence (e.g., manufactured and sold as ProteinPrint™ by Aspira Biosystems, Burlingame, CA).

[0142] Another methodology that can be used diagnostically and in expression profiling is the ProteinChip® array (e.g., manufactured and sold by Ciphergen, Fremont, CA), in which a solid-phase chromatographic surface binds proteins with similar charge or hydrophobic characteristics from a mixture such as plasma or tumor extract, and the retained proteins are detected using SELDI-TOF mass spectrometry. By immobilizing large numbers of purified proteins, large-scale functional chips have been constructed and used to assay a wide range of biochemical functions, such as protein interactions with other proteins, drug-target interactions, and enzyme-substrate interactions. Generally, this requires an expression library cloned into E. coli, yeast, etc., from which the expressed proteins are then purified and immobilized, e.g., via His tags. Cell-free protein transcription / translation is a viable option for synthesizing proteins that are poorly expressed in bacteria or other in vivo systems.

[0143] For the detection of protein-protein interactions, protein arrays can be useful as an in vitro alternative when the cell-based yeast two-hybrid system is insufficient, such as for interactions involving secreted proteins or proteins with disulfide bridges. High-throughput analysis of biochemical activity on arrays has been described for yeast protein kinases and for various functions of the yeast proteome (protein-protein and protein-lipid interactions), in which a large proportion of the entire yeast open reading frame was expressed and immobilized on a microarray. Large-scale "proteome chips" promise to be extremely useful in identifying functional interactions, drug screening, etc. (e.g., manufactured and sold by Proteometrix, Branford, CT).

[0144] Phage or ribosome display libraries can be screened using protein arrays as two-dimensional displays of individual elements to select specific binding partners, including antibodies, synthetic scaffolds, peptides, and aptamers. In this way, "library-versus-library" screening can be performed. Combining the screening of drug candidates with chemical libraries against protein target arrays identified from genome projects is another application of this approach.

[0145] Multiplexed bead assays, such as BD™ cytometric bead arrays, are a series of discrete spectrally distinct particles that can be used to capture and quantify soluble analytes. The analytes are then measured by fluorescence-based emission detection and flow cytometry analysis. Multiplexed bead assays generate data comparable to ELISA-based assays, but in a "multiplexed" or simultaneous format. For cytometric bead arrays, as with any sandwich-format assay, unknown concentrations are calculated by plotting unknowns against a standard curve, for example, using known standards. Furthermore, multiplexed bead assays enable quantification of soluble analytes in samples that was previously unthinkable due to sample volume limitations. In addition to quantitative data, powerful visual images can be generated, revealing unique profiles or signatures that provide additional information to the user at a glance.

[0146] V. Methods of Using the Composition In one aspect, disclosed herein is a method of treating, preventing, inhibiting and / or reducing cancer, metastasis, or infection in a subject, comprising administering to the subject any of the isolated or modified universal donor NK cells or cell lines disclosed herein, or any universal donor NK cells or cell lines selected or screened by methods 300, 400, or prepared by any of the methods disclosed herein, or modified universal donor NK cells or cell lines.

[0147] For example, in one aspect, the present specification discloses a method for treating cancer or an infectious disease in a subject, the method comprising identifying and / or obtaining universal donor cells as described in method 300 of FIG. 3 and / or modifying universal donor cells as described in method 400 of FIG. 4. In another aspect, a method of treating cancer or an infectious disease in a subject comprising identifying and / or obtaining universal donor cells comprises (a) obtaining or obtaining an HLA genotype of candidate NK cells from an NK cell donor, wherein the HLA genotype indicates the presence or absence of HLA CI, C2, and Bw4 alleles, and thereby indicates the presence of one or more variably inherited inhibitory KIRs 2DL1, 2DL2, 2DL3, and 3DL1; (b) obtaining or obtaining a KIR genotype of the candidate NK cells, wherein the KIR genotype indicates the presence or absence of an activating KIR selected from the group consisting of 2DS1 / 2, 2DS3 / 5, 3DS1, and 2DS4; and (c) obtaining or obtaining a universal donor cell when (i) the HLA genotype indicates the presence of at least two I ILA alleles HLA CI, C2, and Bw4; and (ii) the KIR genotype indicates the presence or absence of at least three activating KIRs 2DS1 / 2, 2DS3 / 5, 3DS When the candidate NK cells show the presence of I and / or 2DS4, they are selected as universal donor NK cells for therapeutic administration.

[0148] In one aspect, disclosed herein is a method of treating cancer or an infectious disease, wherein the selected universal donor NK cells are histologically optimized with at least 50%-85% of recipient subjects. In one aspect, the method of treating cancer or an infectious disease of any of the preceding aspects further comprises obtaining or having obtained a CMV-seropositive status of the candidate NK cells; wherein the candidate NK cells are further selected when the NK cell donor is seropositive for CMV or when the NK cells from the NK cell donor exhibit elevated NKG2C expression compared to a reference level of NKG2C expression.

[0149] Method 500, illustrated in FIG. 5 and continuing from above, begins at 520 and describes a method for treating a particular patient. In step 520, NK cells are generated at a concentration within a percentage of the patient / recipient's assigned dose level. In one exemplary embodiment, the concentration of TGF-βi NK cells / kg is within 20% of the patient's assigned dose level. For each patient to be treated with NK cells, a platelet-reactive antibody test is performed to allow for exclusion of TGF-βi NK cell preparations from donors with the patient's alloimmunized HLA type. The patient's weight is used to calculate the TGF-βi NK dose, the patient's assigned dose level, and the planned infusion date. In one exemplary embodiment, pooled TGF-βi NK cells are prepared from the remaining donors (e.g., donors not excluded) for distribution into doses. These doses are verified for NK cell, T cell, and endotoxin doses.

[0150] In step 522, it is determined that the CD3+ cells present in the NK cells are below the T cell threshold for the assigned dose level. If it is determined that the CD3+ cells present in the NK cells are above the T cell threshold for the assigned dose level, the dose is excluded. In one exemplary embodiment, the T cell threshold is less than or equal to the cumulative T cell dose maximum (see Table 2 below) for the patient's assigned dose level.

[0151] In step 524, the endotoxin dose of the non-excluded donor cells is determined to be below the endotoxin threshold and identified as donor-eligible cells. In one exemplary embodiment, the endotoxin threshold is below 5 EU / kg. In step 526, doses of NK cells are provided to the patient over a threshold dose cycle. In one exemplary embodiment, the threshold dose cycle is six 21-day cycles consisting of irinotecan, temozolomide, dinutuximab, and sargramostim and universal donor TGF-βi ex vivo expanded NK cells (e.g., donor-eligible cells). Universal donor-expanded TGF-βi NK cells are administered at a dose of 1×10 on day 8 of the 21-day cycle. 8The therapy is administered IV at a dose of NK cells / kg patient weight. In one exemplary embodiment, there is dose escalation. In another exemplary embodiment, there is no dose escalation.

[0152] It is understood, and contemplated herein, that activating and / or expanding universal donor NK cells prior to therapeutic administration to a subject can help overcome many of the difficulties associated with cytokine toxicity. In one embodiment, the method of treating cancer or infectious disease of any of the foregoing embodiments further comprises incubating the selected universal donor NK cells in vitro in the presence of one or more NK cell effector agents (e.g., stimulatory peptides, cytokines, and / or adhesion molecules) (e.g., IL-21). Examples of NK cell activators and stimulatory peptides include, but are not limited to, IL-21, 41BBL, IL-2, IL-12, IL-15, IL-18, IL-7, ULBP, MICA, LFA-1, 2B4, BCM / SLAMF2, CCR7, OX4OL, NKG2D agonists, Delta-1, Notch ligands, NKp46 agonists, NKp44 agonists, NKp30 agonists, other NCR agonists, CD16 agonists; and / or TGF-β and / or other homing-inducing signaling molecules. Examples of cytokines include, but are not limited to, IL-2, IL-12, IL-21, and IL-18. Examples of adhesion molecules include, but are not limited to, LFA-1, MICA, and BCM / SLAMF2.

[0153] These NK cell effector agents are either soluble in solution or present as membrane-bound agents on the surface of PM particles, exosomes (EX), or feeder cells (FC). PM particles, EX exosomes, and / or FC cells can be engineered to express membrane-type NK cell activators and stimulatory peptides. Alternatively, the NK cell activators and stimulatory peptides can be chemically conjugated to the surface of PM particles, EX exosomes, or FC feeder cells. For example, PM particles, feeder cells (FC), or exosomes (EX) prepared from feeder cells expressing membrane-bound IL-21 (FC21 cells, PM21 particles, and EX21 exosomes, respectively). It is understood, and is contemplated herein, that membrane-bound IL-21-expressing FC21 cells, PM21 particles, and EX21 exosomes (e.g., PM21 particles, EX21 exosomes, or FC cells expressing 41BBL and membrane-bound interleukin-21) can further comprise one or more additional activators, stimulatory peptides, cytokines, and / or adhesion molecules, including, but not limited to, 41BBL, IL-2, IL-12, IL-15, IL-18, IL-7, ULBP, MICA, LEA-I, 2B4, BCM / SLAMF2, CCR7, OX4OL, NKG2D agonists, Delta-1, Notch ligands, NKp46 agonists, NKp44 agonists, NKp30 agonists, other NCR agonists, CD16 agonists; and / or TGF-β.

[0154] It is understood that the pathogen can be a virus. Thus, in one embodiment, the pathogen is herpes simplex virus type 1, herpes simplex virus type 2, varicella zoster virus, Epstein-Barr virus, cytomegalovirus, human herpes virus type 6, smallpox virus, vesicular stomatitis virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, or the like. C) viruses, hepatitis D virus, hepatitis E virus, rhinovirus, coronavirus, influenza A virus, influenza B virus, measles virus, polyomavirus, human papillomavirus, respiratory syncytial virus, adenovirus, coxsackievirus, dengue virus, mumps virus, poliovirus, rabies virus, Rous sarcoma virus, reovirus, yellow fever virus, Ebola virus, Marburg virus, Lassa fever virus, Eastern equine encephalitis virus, Japanese encephalitis virus, St. Louis encephalitis virus, Murray Valley fever virus, West Nile virus, Rift Valley fever virus, rotavirus A, rotavirus B, rotavirus C, Sindbis virus, simian immunodeficiency virus, human T-cell leukemia virus type 1, hantavirus, rubella virus, simian immunodeficiency virus, human immunodeficiency virus type 1 and / or human immunodeficiency virus type 2.

[0155] Also disclosed are methods in which the pathogen is a bacteria, such as Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium bovis BCG strains, BCG substrains, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium africanum, Mycobacterium kansasii, Mycobacterium marinum, Mycobacterium ulcerans, Mycobacterium avium subspecies paratuberculosis, Nocardia asteroides, or the like. asteroides, other Nocardia species, Legionella pneumophila, other Legionella species, Acinetobacter baumannii, Salmonella typhi, Salmonella enterica, other Salmonella species, Shigella boydii, Shigella dysenteriae, Shigella sonnei, Shigella flexneri, other Shigella species, Yersinia pestis, Pasteurella haemolytica, Pasteurella multocida multocida, other Pasteurella species, Actinobacillus pleuropneumoniae, Listeria monocytogenes, Listeria ivanoviiivanovii, Brucella abortus, other Brucella species, Cowdria ruminantium, Borrelia burgdorferi, Bordetella avium, Bordetella pertussis, Bordetella bronchiseptica, Bordetella trematum, Bordetella hinzii, Bordetella petrii, Bordetella parapertussis, Bordetella ansorpii, other Bordetella species, Burkholderia mallei, Burkholderia pseudomallei Pseudomonas pseudomallei, Burkholderia cepacia, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydia psittaci, Coxiella burnetii, Rickettsia species, Ehrlichia species, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Escherichia coli, Vibrio cholerae cholerae, Campylobacter species, Neisseria meningitidis, Neisseria gonorrhea, Pseudomonas aeruginosaaeruginosa, other Pseudomonas species, Haemophilus influenzae, Haemophilus ducreyi, other Hemophilus species, Clostridium tetani, other Clostridium species, Yersinia enterocolitica, and / or other Yersinia species, and / or Mycoplasma species. In one embodiment, the bacterium is not Bacillus anthracis.

[0156] Also disclosed is a method of treating an infection, wherein the pathogen is a fungus selected from the group of fungi consisting of Candida albicans, Cryptococcus neoformans, Histoplasma capsulatum, Aspergillus fumigatus, Coccidioides immitis, Paracoccidioides brasiliensis, Blastomyces dermatitidis, Pneumocystis carinii, Penicillium marneffei, and / or Alternaria alternata.

[0157] The pathogens include Toxoplasma gondii, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, other Plasmodium species, Entamoeba histolytica, Naegleria fowleri, Rhinosporidium seeberi, Giardia lamblia, Enterobius vermicularis, Enterobius gregorii, Ascaris lumbricoides, Ancylostoma duodenale, and Necator americanus, Cryptosporidium spp., Trypanosoma brucei, Trypanosoma cruzi, Leishmania major, other Leishmania species, Diphyllobothrium latum, Hymenolepis nana, Hymenolepis diminuta, Echinococcus granulosus, Echinococcus multilocularis, Echinococcus vogeli, Echinococcus oligarthrus, Diphyllobothrium latum, Clonorchis sinensis;Clonorchis viverrini, Fasciola hepatica, Fasciola gigantica, Dicrocoelium dendriticum, Fasciolopsis buski, Metagonimus yokogawai, Opisthorchis viverrini), Opisthorchis felineus, Clonorchis sinensis, Trichomonas vaginalis, Acanthamoeba species, Schistosoma intercalatum, Schistosoma haematobium, Schistosoma japonica Also disclosed are methods of treating infections in which the parasite is selected from the group of parasites consisting of Schistosoma japonicum, Schistosoma mansoni, other Schistosoma species, Trichobilharzia regenti, Trichinella spiralis, Trichinella britovi, Trichinella nelsoni, Trichinella nativa, and / or Entamoeba histolytica;

[0158] The disclosed compositions can be used to treat any disease in which uncontrolled cell proliferation occurs, such as cancer. A representative, but non-limiting list of cancers for which the disclosed compositions can be used to treat is as follows: lymphoma, B-cell lymphoma, T-cell lymphoma, mycosis fungoides, Hodgkin's disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, head and neck squamous cell carcinoma, lung cancer, including small cell lung cancer and non-small cell lung cancer, neuroblastoma / glioblastoma, ovarian cancer, skin cancer, liver cancer, thyroid cancer, melanoma, squamous cell carcinoma of the oral cavity, pharynx, larynx, and lung, cervical cancer, cervical carcinoma, breast cancer and epithelial cancer, renal cancer, genitourinary tract cancer, lung cancer, esophageal cancer, head and neck cancer, colon cancer, hematopoietic cancer; testicular cancer; colon cancer, rectal cancer, stomach cancer, prostate cancer, and / or pancreatic cancer.

[0159] In the exemplary embodiment shown in Figure 6, NK cells are utilized in a therapeutic formulation method 600 for treating cancer, such as neuroblastoma. In step 602, donor eligibility as a suitable donor is confirmed. In one exemplary embodiment, a suitable donor is confirmed as described above in Figure 3, and / or suitable donor cells are modified as in Figure 4. Suitable donors are therefore those with an HLA genotype bearing the C1, C2, and Bw4 alleles, those with inhibitory KIRs (2DL1, 2DL2, or 3 and 3DL1) that bind to C1, C2, and Bw4 (leading to maximal licensing) and a KIR genotype with a high proportion of activating KIRs (three or more variably inherited activating genes including 2DS1 and 3DS1), and those who have been exposed to CMV, resulting in high NKG2C expression.

[0160] In step 604, CD3+ immunodepletion of the MNCs of the optimal cell donor is performed. In one exemplary embodiment, the CD3+ immunodepletion is the same as in step 506 of method 500. In step 606, the depleted optimal donor cells are expanded for a blastoma duration. In one exemplary embodiment, the blastoma duration is 10-18 days. In another exemplary embodiment, the blastoma duration is 14 days. In another exemplary embodiment, the blastoma interval (e.g., when expansion-inducing elements are added) is 1-3 days. During expansion, in step 608, the depleted optimal donor cells are stimulated with irradiated K562 feeder cells expressing membrane-bound interleukin (II) 11-21, 11-2, and / or 4-1BBL. In one exemplary embodiment, NK cells are generated during stimulation with irradiated K562 feeder cells (e.g., at a concentration of 100 IU / mL) expressing membrane-bound IL-21 and 4-1BBL and IL-2. Irradiated feeder cells (IFC) are added at a TNC to IFC ratio of approximately 1:2 for the first 7 days of the blastoma period, and at a 1:1 ratio for the second 7 days of the blastoma period. In one exemplary embodiment, fresh IL-2 is added every blastoma interval.

[0161] At 612, TGF-βi NK cells are generated by applying or imprinting transforming growth factor β (TGF-β) to the confirmed donor-eligible cells. In one exemplary embodiment, donor-eligible cells are chronically stimulated with TGF-β (e.g., at a concentration of 10 ng / mL). In another exemplary embodiment, fresh TGF-β is added at blastoma intervals during the blastoma period. Addition of TGF-β during expansion does not impair the fold expansion (465-3200-fold expansion) or viability (>96%) of the final NK cell preparation after expansion. When cultured with tumor targets, TGF-βi NK cells exhibit a pro-inflammatory phenotype with hypersecretion of interferon-γ and tumor necrosis factor-α, which increases both the proportion of cytokine-producing NK cells in the culture and the amount of cytokines produced by each of these cells, thereby increasing anti-tumor cytokine secretion, compared to typically expanded NK cells. These cells exhibit phenotypic and transcriptional changes that confer resistance to inhibition by TGF-β.

[0162] At 614, the cultured NK cells are concentrated to a dose concentration. In one example, the dose concentration is 2×10 6 NC / mL ~ 2 × 10 8 The total volume of NK cells is NC / mL. At 616, the expanded and transformed NK cells are cryopreserved at a dose concentration. In one embodiment, the NK cells are cryopreserved in NK freezing medium. In another exemplary embodiment, the NK freezing medium comprises 10% DMSO, 12.5% ​​(w / v) human serum albumin (HSA), USP, and / or in Plasma-Lyte A, USP.

[0163] 7 describes recipient / patient eligibility and treatment with NK cells in a recipient eligibility and treatment method 700 for treating one or more cancers, such as neuroblastoma. At 702, it is determined whether the recipient has a histologically confirmed, recurrent, non-metastatic, supratentorial World Health Organization (WHO) grade III / IV malignant brain tumor. In one exemplary embodiment, the brain tumor includes anaplastic ependymoma, embryonal tumor, anaplastic neuroectodermal tumor, AT / RT, anaplastic astrocytoma, anaplastic oligoastrocytoma, anaplastic oligodendroglioma, anaplastic xanthoastrocytoma multiforme, glioblastoma multiforme, gliosarcoma, and / or malignant glioma NOS.

[0164] At 704, in response to the recipient not having a histologically confirmed recurrent non-metastatic supratentorial WHO grade III / IV malignant brain tumor, the recipient is marked as sub-optimal (e.g., not a candidate for receiving NK donor cells). At 706, in response to the recipient having a histologically confirmed recurrent non-metastatic supratentorial WHO grade III / IV malignant brain tumor, it is determined whether the recipient is considered a candidate for resection / open biopsy of the recurrent tumor (resection candidate) and / or placement of an intracavitary / intratumorally placed Ommaya reservoir (Ommaya candidate). At 708, in response to the recipient not being considered a resection candidate or an Ommaya candidate, the recipient is marked as sub-optimal.

[0165] At 710, in response to the recipient being considered a resection and / or Ommaya candidate, it is determined whether the recipient has a Lansky score of 50 or greater if the recipient is 16 years of age or younger (optimal Lansky score) or a Karnofsky score of 50 or greater if the recipient is over 16 years of age (optimal Karnofsky score). In one exemplary embodiment, optimal candidates are 3 years of age or older and under 25 years of age at the time of enrollment in the study. At 712, in response to the recipient not being considered to have an optimal Lansky score or optimal Karnofsky score for their age, the recipient is marked as suboptimal.

[0166] At 714, in response to the recipient being deemed to have an optimal Lansky score or optimal Karnofsky score for their age, it is determined whether the recipient has organ function above a functional threshold. In one exemplary embodiment, the functional threshold is having sufficient bone marrow function without transfusions or growth factors during 21 days of NK cell administration. In another exemplary embodiment, sufficient bone marrow function is defined as a white blood cell (WBC) count of 2.5 x 103 / microliter or greater, a hemoglobin (Hgb) of 9 gm / dL or greater, an absolute neutrophil count (ANC) of 1,000 cells / microliter or greater, and a platelet count of 75,000 cells / microliter or greater. In one exemplary embodiment, the functional threshold is having sufficient liver function and / or sufficient renal function. In one exemplary embodiment, sufficient liver function is defined as ALT, AST, and alkaline phosphatase being less than 2 times the ULN and bilirubin being less than 1.5 times the ULN, and sufficient renal function is defined as BUN or creatinine being less than 1.5 times the ULN. At 716, in response to the recipient being deemed not to have organ function above the organ function threshold, the recipient is marked as suboptimal.

[0167] At 718, in response to the recipient being deemed to have organ function above the organ function threshold, it is determined whether the recipient received toxic therapy within the treatment period. In one exemplary embodiment, an optimal recipient will have completed first-line treatment with radiation therapy and / or chemotherapy before receiving universal donor NK cell therapy. In one exemplary embodiment, the treatment period is at least 12 weeks from the completion of initial radiation therapy. In another exemplary embodiment, the treatment period is at least 6 weeks from the completion of any cytotoxic chemotherapy regimen. In yet another exemplary embodiment, the treatment period is a minimum of 2 weeks from the last dose of any toxic agent. In this exemplary embodiment, the recipient is deemed to have recovered from any toxicity of the toxic agent prior to universal NK donor cell treatment. In one exemplary embodiment, the treatment period is between the diagnosis of cancer and the present time. In another exemplary embodiment, the toxic therapy is systemic steroids (excluding replacement therapy), and the treatment period is at least 3 days prior to NK cell infusion. In another exemplary embodiment, the toxic therapy is bevacizumab, and the treatment period is at least 6 weeks prior to the start of NK cell infusion. At 720, in response to the recipient being deemed to have received a toxic therapy within the treatment period, the recipient is marked as suboptimal. At 722, in response to the recipient being deemed to have received a toxic therapy outside the treatment period, the recipient is marked as optimal to receive universal donor NK cell therapy.

[0168] At 724, NK cells (created using method 600 of FIG. 6) are created having an NK cell concentration within a percentage of the assigned dose level (e.g., as set forth in Table 2 below). In one exemplary embodiment, therapy duration is 3 months and / or until disease progression, the occurrence of a comorbidity that prevents further treatment administration, the occurrence of one or more unacceptable adverse events, the patient electing to discontinue, significant patient noncompliance with the protocol, or a general or specific change in the patient's condition that, as determined by the clinician, renders the patient unable to tolerate further treatment. At 726, a dose of NK cells is provided for use in the optimal recipient over a threshold dose cycle (see, e.g., Table 2 below). In one example, the dose of NK cells is provided using methods such as intravenous, intramuscular, etc.

[0169] At 728, a dose of NK cells is provided for use in the Ommaya reservoir over a threshold dose cycle (see, e.g., Table 2 below). The patient proceeds to surgery for tumor resection and Ommaya placement. In one exemplary embodiment, a first dose of TGFβi NK cells is administered at least 14 days after Ommaya reservoir placement. TGFβi NK cell infusions from the Ommaya reservoir are administered once a week for three weeks, followed by one week of rest, for a total of three (four-week) cycles. If the patient shows stable or improving disease, then the patient continues with therapy for a total of 12 cycles. In one exemplary embodiment, an optimal recipient receives three cycles of TGFβi NK cell infusions, each cycle being four weeks in duration. During the first three weeks, TGFβi NK cells are infused weekly. The fourth week is a rest week. TGFβi NK cell infusions should be delivered at least 3 days apart (e.g., Friday of week 1 and Monday of week 2). Dose titration is based on recipient body surface area (BSA).

[0170] [Table 2]

[0171] It is also contemplated herein that the disclosed methods of treating, preventing, inhibiting, or reducing cancer or metastasis in a subject may further include the administration of any anti-cancer agent (e.g., gemcitabine, etc.) that would further aid in the reduction, inhibition, treatment, and / or elimination of cancer or metastasis. Anti-cancer agents that can be used in the disclosed bioresponsive hydrogels for the disclosed methods of reducing, inhibiting, treating, and / or elimination of cancer or metastasis in a subject, or as additional therapeutic agents in addition to the disclosed pharmaceutical compositions, modified particles, and / or bioresponsive hydrogels (including bioresponsive hydrogels in which modified particles are encapsulated), include, but are not limited to, abemaciclib, abiraterone acetate, abitrexate (methotrexate), ... Abraxane (paclitaxel-albumin-stabilized nanoparticles), ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris (brentuximab vedotin), ADE, Ado-trastuzumab emtansine, Adriamycin (doxorubicin hydrochloride), afatinib dimaleate, Afinitor (everolimus), Akynzeo (netupitant and palonosetron hydrochloride), Aldara (A ldara (imiquimod), aldesleukin, Alecensa (alectinib), alectinib, alemtuzumab, Alimta (pemetrexed disodium), Aliqopa (copanlisib hydrochloride), Alkeran injection (melphalan hydrochloride), Alkeran tablets (melphalan), Aloxi (palonosetron hydrochloride), Alunbrig (brigatinib), Ambox Ambochlorin (chlorambucil), Amifostine, Aminolevulinic acid, Anastrozole, Aprepitant, Aredia (Pamidronate disodium), Arimidex (Anastrozole), Aromasin (Exemestane), Arranon (Nelarabine), Arsenic trioxide, Arzerra (Ofatumumab),Asparaginase, Erwinia chrysanthemi, Atezolizumab, Avastin (bevacizumab), Avelumab, Axitinib, Azacitidine, Bavencio (avelumab), BEACOPP, Becenum (carmustine), Beleoodaq (belinostat), belinamustine hydrochloride, BEP, Besponsa (inotuzumab ozogamicin), Bevacizumab, Bexarotene, Bexxar (tositumomab and iodine I1 31 Tositumomab), bicalutamide, BiCNU (carmustine), bleomycin, blinatumomab, Blincyto (blinatumomab), bortezomib, Bosulif (bosutinib), bosutinib, brentuximab vedotin, brigatinib, BuMel, busulfan, Busulfex (busulfan), cabazitaxel, Cabometyx (cabozantinib-S-malate), cabozantinib-S-malate, CAF, Campas (C ampath (alemtuzumab), Camptosar (irinotecan hydrochloride), capecitabine, CAPDX, Carac (topical fluorouracil), carboplatin, carboplatin-Taxol, carfilzomib, Carmubris (carmustine), carmustine, carmustine implant, Casodex (bicalutamide), CEM, ceritinib, Cerubidine (daunorubicin hydrochloride), Cervarix Cervarix (recombinant HPV bivalent vaccine), cetuximab, CEV, chlorambucil, chlorambucil-prednisone, CHOP, cisplatin, cladribine, Clafen (cyclophosphamide), clofarabine, Clofarex (clofarabine), chlorar (clofarabine), CMF, cobimetinib, Cometriq (cabozantinib-S-malate), copanlisib hydrochloride, COPDAC, COPP, COPP-ABV,Cosmegen (dactinomycin), Cotellic (cobimetinib), crizotinib, CVP, cyclophosphamide, Cyfos (ifosfamide), Cyramza (ramucirumab), cytarabine, cytarabine liposome, Cytosar-U (cytarabine), Cytoxan (cyclophosphamide), dabrafenib, dacarbazine, Dacogen (decitabine), dactinomycin cin, daratumumab, Darzalex (daratumumab), dasatinib, daunorubicin hydrochloride, daunorubicin hydrochloride and cytarabine liposome, decitabine, defibrotide sodium, Defitelio (defibrotide sodium), degarelix, denileukin diftitox, denosumab, DepoCyt (cytarabine liposome), dexamethasone, dexrazoxane hydrochloride, dinutuximab, docetaxel, doxycycline Doxil (doxorubicin hydrochloride liposome), doxorubicin hydrochloride, doxorubicin hydrochloride liposome, Dox-SL (doxorubicin hydrochloride liposome), DTIC-Dome (dacarbazine), durvalumab, Efudex (fluorouracil-topical), Elitek (rasburicase), Ellence (epirubicin hydrochloride), elotuzumab, Eloxatin (oxaliplatin) tin), eltrombopag olamine, Emend (aprepitant), Empliciti (elotuzumab), enasidenib mesylate, enzalutamide, epirubicin hydrochloride, EPOCH, Erbitux (cetuximab), eribulin mesylate, Erivedge (vismodegib), erlotinib hydrochloride, Erwinaze (asparaginase Erwinia chrysanthemi), Ethyol (amifostine), Etopophos (etoposide phosphate), etoposide, etoposide phosphate, Evacet (doxorubicin hydrochloride liposomal), everolimus,Evista (raloxifene hydrochloride), Evomela (melphalan hydrochloride), exemestane, 5-FU (fluorouracil injection), 5-FU (fluorouracil - topical), Fareston (toremifene), Farydak (panobinostat), Faslodex (fulvestrant), FEC, Femara (letrozole), filgrastim, Fludara (fludarabine phosphate), fludarabine phosphate, Fluoroplex (fluorouracil - topical), fluorouracil injection, fluorouracil - topical, flutamide, Folex (methotrexate), Folex PFS PFS) (methotrexate), FOLFIRI, FOLFIRI-bevacizumab, FOLFIRI-cetuximab, FOLFIRI NOX, FOLFOX, Folotyn (pralatrexate), FU-LV, fulvestrant, Gardasil (recombinant HPV quadrivalent vaccine), Gardasil 9 9) (recombinant HPV nonavalent vaccine), Gazyva (obinutuzumab), gefitinib, gemcitabine hydrochloride, gemcitabine-cisplatin, gemcitabine-oxaliplatin, gemtuzumab ozogamicin, Gemzar (gemcitabine hydrochloride), Gilotrif (afatinib dimaleate), Gleevec (imatinib mesylate), Gliadel (carmustine implant), Gliadel wafer wafer) (carmustine implant), glucarpidase, goserelin acetate, Halaven (eribulin mesylate), Hemangeol (propranolol hydrochloride), Herceptin (trastuzumab), HPV bivalent vaccine, recombinant, HPV nonavalent vaccine, recombinant, HPV quadrivalent vaccine, recombinant, Hycamtin (topotecan hydrochloride), Hydrea (hydroxyurea),Hydroxyurea, Hyper-CVAD, Ibrance (palbociclib), ibritumomab tiuxetan, ibrutinib, ICE, Iclusig (ponatinib hydrochloride), Idamycin (idarubicin hydrochloride), idarubicin hydrochloride, idelalisib, Idhifa (enasidenib mesylate), Ifex (ifosfamide), ifosfamide, Ifosfamidam (ifosfamide), IL-2 (aldesleukin-11), ), imatinib mesylate, Imbruvica (ibrutinib), Imfinzi (durvalumab), imiquimod, Imlygic (talimogene laherparepvec), Inlyta (axitinib), inotuzumab ozogamicin, interferon alpha-2b, recombinant, interleukin-2 (aldesleukin), Intron A (recombinant interferon alpha-2b), iodine I131 tositumomab and tositumomab, ipilimumab, Iressa ( Gefitinib), Irinotecan Hydrochloride, Irinotecan Hydrochloride Liposomal, Istodax (Romidepsin), Ixabepilone, Ixazomib Citrate, Ixempra (Ixabepilone), Jakafi (Ruxolitinib Citrate), JEB, Jevtana (Cabazitaxel), Kadcyla (Ado-Trastuzumab Emtansine), Keoxifene (Raloxifene Hydrochloride), Kepivance (Palifermin), Keytruda Keytruda (pembrolizumab), Kisqali (ribociclib), Kymriah (tisagenlecleucel), Kyprolis (carfilzomib), lanreotide acetate, lapatinib ditosylate, Lartruvo (olaratumab), lenalidomide, lenvatinib mesylate, Lenvima (lenvatinib mesylate), letrozole, leucovorin calcium, Leukeran (chlorambucil), leuprolide acetate,Leustatin (cladribine), Levulan (aminolevulinic acid), Linfolizin (chlorambucil), LipoDox (doxorubicin hydrochloride liposomal), lomustine, Lonsurf (trifluridine and tipiracil hydrochloride), Lupron (leuprolide acetate), Lupron, Lupron Depot (leuprolide acetate), Lupron Depot - Pediatric (leuprolide acetate), Lynparza (olaparib), Marqibo (vincristine sulfate liposomal), Matulane (procarbazine hydrochloride), mechlorethamine hydrochloride, megestrol acetate, Mekinist (trametinib), melphalan, melphalan hydrochloride, mercaptopurine, mesna, Mesnex (mesna), metazolastone azolastone (temozolomide), methotrexate, methotrexate LPF (methotrexate), methylnaltrexone bromide, Mexate (methotrexate), Mexate-AQ (methotrexate), midostaurin, mitomycin C, mitoxantrone hydrochloride, Mitozytrex (mitomycin C), MOPP, Mozobil (plerixafor), Mustargen (mechlorethamine hydrochloride), mutamycin (Mu tamycin (mitomycin C), Myleran (busulfan), Mylosar (azacitidine), Mylotarg (gemtuzumab ozogamicin), nanoparticle paclitaxel (paclitaxel-albumin-stabilized nanoparticles), Navelbine (vinorelbine tartrate), necitumumab, nelarabine, Neosar (cyclophosphamide), neratinib maleate, Nerlynx (neratinib maleate), netupitant, and and palonosetron hydrochloride, Neulasta (pegfilgrastim), Neupogen (filgrastim), Nexavar (sorafenib tosylate), Nilandron (nilutamide), nilotinib, nilutamide, Ninlaro (ixazomib citrate), niraparibut tosylate monohydrate, nivolumab, Nolvadex (tamoxifen citrate), Nplate (romiplostim), obinutuzumab,Odomzo (sonidegib), OEPA, ofatumumab, OFF, olaparib, olaratumab, omacetaxine mepesuccinate, Oncaspar (peguaspargase), ondansetron hydrochloride, Onivyde (irinotecan hydrochloride liposomal), Ontak (denileukin diftitox), Opdivo (nivolumab), OPPA, osimertinib, oxaliplatin, paclitaxel, paclitaxel albumin-stabilized nanoparticle formulation, PAD, palbociclib, Palifermin, palonosetron hydrochloride, palonosetron hydrochloride and netupitant, pamidronate disodium, panitumumab, panobinostat, Paraplat (carboplatin), Paraplatin (carboplatin), pazopanib hydrochloride, PCV, PEB, pegaspargase, pegfilgrastim, peginterferon alfa-2b, PEG-Intron (peginterferon alfa-2b), pembrolizumab, pemetrexed disodium, Perjeta ta) (pertuzumab), pertuzumab, Platinol (cisplatin), Platinol-AQ (cisplatin), plerixafor, pomalidomide, Pomalyst (pomalidomide), ponatinib hydrochloride, Portrazza (necitumumab), pralatrexate, prednisone, procarbazine hydrochloride, Proleukin (aldesleukin), Prolia (denosumab), Promacta (eltrombopag) amine), propranolol hydrochloride, Provenge (sipuleucel-T), Purinethol (mercaptopurine), Purixan (mercaptopurine), radium-223 dichloride, raloxifene hydrochloride, ramucirumab, rasburicase, R-CHOP, R-CVP, recombinant human papillomavirus (l-IPV) bivalent vaccine, recombinant human papillomavirus (HPV) nonavalent vaccine, recombinant human papillomavirus (HPV) quadrivalent vaccine, recombinant interferon alpha-2b,Regorafenib, Relistor (methylnaltrexone bromide), R-EPOCH, Revlimid (lenalidomide), Rheumatrex (methotrexate), ribociclib, R-ICE, Rituxan (rituximab), Rituxan Hycera Hycela (Rituximab and Hyaluronidase Human), Rituximab, Rituximab and Hyaluronidase Human, Rolapitant Hydrochloride, Romidepsin, Romiplostim, Rubidomycin (Daunorubicin Hydrochloride), Rubraca (Rucaparib Camsylate), Rucaparib Camsylate, Ruxolitinib Phosphate, Rydapt (Midostaurin), Sclerosol Intrapleural Aerosol (Talc), Siltuximab, Sipuleucel-T, Somatuline Depot (Lanreotide Acetate), Sonidegib, Sorafenib Tosylate, Sprycel (Dasatinib), STANFORD V), Sterile Talc Powder (Talc), Steritalc (Talc), Stivarga (Regorafenib), Sunitinib Malate, Sutent (Sunitinib Malate), Sylatron (Peginterferon alfa-2b), Sylvant (Siltuximab), Synribo (Omacetaxine Mepesuccinate), Tabloid (Thioguanine), TAC, Tafinlar (Dabrafenib), Tagrisso (Osimertinib), Talc, Talimogene Laherparepvec, Tamoxifen Citrate, Tarabine PFS PFS) (cytarabine), Tarceva (erlotinib hydrochloride), Targretin (bexarotene), Tasigna (nilotinib), Taxol (paclitaxel), Taxotere (docetaxel), Tecentriq (atezolizumab),Temodar (temozolomide), temozolomide, temsirolimus, thalidomide, Thalomid (thalidomide), thioguanine, thiotepa, tisagenlecleucel, Tolak (topical fluorouracil), topotecan hydrochloride, toremifene, Torisel (temsirolimus), tositumomab and iodine I131 tositumomab, Totect (dexrazoxane hydrochloride), TPF, trabectedin, trametinib, trastuzumab, Treanda (bendamustine hydrochloride), trifluridine and tipiracil hydrochloride, Trisenox (arsenic trioxide), Tykerb (lapatinib ditosylate) ), Unituxin (dinutuximab), uridine triacetate, VAC, vandetanib, VAMP, Varubi (rolapitant hydrochloride), Vectibix (panitumumab), VeIP, Velban (vinblastine sulfate), Velcade (bortezomib), Velsar (vinblastine sulfate), vemurafenib, Venclexta (venetoclax), venetoclax, Verzenio (abemaciclib), Viadur (leuprolide acetate), Vidaza (azacitidine), vinblastine sulfate, Vincasar PFS PFS) (vincristine sulfate), vincristine sulfate, vincristine sulfate liposome, vinorelbine tartrate, VIP, vismodegib, Vistogard (uridine triacetate), Voraxaze (glucarpidase), vorinostat, Votrient (pazopanib hydrochloride), Vyxeos (daunorubicin hydrochloride and cytarabine liposome), Wellcovorin (leucovorin calcium), Xalkori (crizotinib), Xeloda (capecitabine), XELIRI, XELOX, Xgeva (denosumab), Xofigo (radium-223 dichloride),Xtandi (enzalutamide), Yervoy (ipilimumab), Yondelis (trabectedin), Zaltrap (Ziv-aflibercept), Zarxio (filgrastim), Zejula (niraparibut tosylate monohydrate), Zelboraf (vemurafenib), Zevalin (ibritumomab tiuxetan), Zinecard (dexrazoxane hydrochloride), Ziv- The anti-cancer agent may include any anti-cancer agent known in the art, including aflibercept, Zofran (ondansetron hydrochloride), Zoladex (goserelin acetate), zoledronic acid, Zolinza (vorinostat), Zometa (zoledronic acid), Zydelig (idelalisib), Zykadia (ceritinib) and / or Zytiga (abiraterone acetate). Checkpoint inhibitors include, but are not limited to, antibodies that block PD-1 (nivolumab (BMS-936558 or MDX1106), CT-011, MK-3475), PD-LI (MDX-1105 (BMS-936559), MPDL3280A, MSB0010718C), PD-L2 (rHIgM12B7), CTLA-4 (ipilimumab (MDX-010), tremelimumab (CP-675,206)), IDO, B7-H3 (MGA271), B7-H4, TIM3, and LAG-3 (BMS-986016).

[0172] References Almalte Z, Samarani S, Iannello A et al., "Novel associations between activating killer-cell immunoglobulin-like receptor genes and childhood leukemia," Blood, 2011, Vol. 118, No. 5, pp. 1323-1328.

[0173] Braud VM, Allan DS, O'Callaghan CA et al., "HLA-E binds to natural killer cell receptors CD94 / NKG2A, B and C," Nature, 1998, Vol. 391, No. 6669, pp. 795-799.

[0174] Cichocki F, Cooley S, Davis Z et al., "CD56dimCD57+NKG2C+ NK cell expansion is associated with reduced leukemia relapse after reduced-intensity HCT," Leukemia, 2016, Vol. 30, No. 2, pp. 456-463.

[0175] Foley B, Cooley S, Verneris MR et al., "Cytomegalovirus reactivation after allogeneic transplantation promotes a lasting increase in educated NKG2C+ natural killer cells with potent function," Blood, 2012, Vol. 119, No. 11, pp. 2665-2674.

[0176] Foley B, Cooley S, Verneris MR et al., "Human cytomegalovirus (CMV)-induced memory-like NKG2C(+) NK cells are transplantable and expand in vivo in response to recipient CMV antigen," J Immunol., 2012, Vol. 189, No. 10, pp. 5082-5088.

[0177] Mancusi A, Ruggeri L, Urbani E et al., "Haploidentical hematopoietic transplantation from KIR ligand-mismatched donors with activating KIRs reduces nonrelapse mortality," Blood, 2015, Vol. 125, No. 20, pp. 3173-3182.

[0178] Pittari G, Fregni G, Roguet L, et al., "Early evaluation of natural killer activity in post-transplant acute myeloid leukemia patients," Bone Marrow Transplant., 2010, Vol. 45, No. 5, pp. 862-871.

[0179] Ruggeri L, Mancusi A, Perruccio K, Burchielli E, Martelli MF, Velardi A, "Natural killer cell alloreactivity for leukemia therapy," J Immunother., 2005, Vol. 28, No. 3, pp. 175-182.

[0180] Stringaris K, Adams S, Uribe M et al., "Donor KIR Genes 2DL5A, 2DS1, and 3DS1 are associated with a reduced rate of leukemia relapse after HLA-identical sibling stem cell transplantation for acute myeloid leukemia but not other hematologic malignancies," Biol Blood Marrow Transplant., 2010, Vol. 16, No. 9, pp. 1257-1264.

[0181] In the foregoing specification, specific embodiments have been described. However, those skilled in the art will recognize that various modifications and changes can be made without departing from the scope of the present disclosure, as defined in the following claims. Accordingly, the specification and figures should be interpreted in an illustrative rather than a restrictive sense, and all such modifications are intended to be included within the scope of the present teachings.

[0182] Any element or elements that may suggest or further emphasize a benefit, advantage, solution to a problem, and any benefit, advantage, or solution should not be construed as a critical, required, or essential feature or element of any claim. The present disclosure is defined solely by the appended claims, including any amendments made during the pendency of this application and all equivalents of such claims as issued.

[0183] Furthermore, as used herein, relationship terms such as first and second, above and below, etc., may be used only to distinguish one entity or operation from another and do not necessarily require or imply any actual relationship or order between such entities or operations. The terms "comprise," "comprising," "have," "having," "including," "including," "containing," "containing," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises, has, includes, or contains a list of elements not only includes those elements, but may also include other elements not expressly listed or inherent to such process, method, article, or apparatus. An element preceded by "including," "having," "including," or "containing" does not, without further constraints, exclude the presence of additional identical elements in the process, method, article, or apparatus that comprises, has, includes, or contains the element. The terms "a" and "an" are defined as one or more, unless otherwise specified herein. The terms "substantially," "essentially," "approximately," "about," or any other variation thereof, are defined as close, as understood by one of ordinary skill in the art. In one non-limiting embodiment, these terms are defined as, for example, within 10%, within 5% in another possible embodiment, within 1% in another possible embodiment, and within 0.5% in another possible embodiment. The term "coupled," as used herein, is defined as connected or in contact, either temporarily or permanently, although not necessarily directly, and not necessarily mechanically. A device or structure that is "configured" in a certain way is configured in at least that way, but may also be configured in ways not listed.

[0184] Before the present compounds, compositions, articles, devices, and / or methods are disclosed and described, it is to be understood that, unless otherwise specified, they are not limited to particular synthetic methods or to particular recombinant biotechnology methods, or to particular reagents, unless otherwise specified, as such can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. The references disclosed are also individually and specifically incorporated by reference herein for the material contained therein that is discussed in the sentence in which the reference is relied upon.

[0185] To the extent that material is not specified for any one of the foregoing embodiments or components thereof, it should be understood that one of ordinary skill in the art would recognize suitable material for the intended purpose. Any and all items, texts, patents, patent publications, and patent application numbers referenced herein are hereby incorporated by reference in their entirety for all purposes.

[0186] Ranges may be expressed herein as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are both significant relative to the other endpoint, and independently of the other endpoint. It is understood that there are a number of values ​​disclosed herein, and that each value is also herein disclosed as "about" that particular value in addition to the value itself. For example, if the value "10" is disclosed, then "about 10" is also disclosed. When a value is disclosed, it is understood that "less than or equal to" that value, "greater than or equal to" that value, and possible ranges between those values ​​are also disclosed, as appropriately understood by one of ordinary skill in the art. For example, when the value "10" is disclosed, "less than or equal to 10" as well as "greater than or equal to 10" is also disclosed. It is also understood that throughout this application, data is provided in a number of different formats and that this data represents endpoints and starting points and ranges for any combination of those data points.

[0187] For example, if a specific data point "20" and a specific data point 25 are disclosed, it is understood that a disclosure of greater than, greater than or equal to, less than, less than, less than, and equal to 20 and 25 is considered the same as a disclosure of 20 to 25. It is also understood that each unit between the two specified units is also disclosed. For example, if 20 and 25 are disclosed, then 21, 22, 23, and 24 are also disclosed.

[0188] This Abstract of the Disclosure is provided to enable the reader to quickly ascertain the nature of the technical disclosure. It is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims. Additionally, in the foregoing Detailed Description, various features may be seen grouped together in various embodiments for the purpose of streamlining the disclosure. This method of disclosure is not to be interpreted as reflecting an intention that the claimed embodiments require more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive subject matter lies in less than all features of a single disclosed embodiment. Accordingly, the following claims are hereby incorporated into the Detailed Description, with each claim standing on its own as separately claimed subject matter. [Example]

[0189] In order that the present invention may be more fully understood, the following examples are set forth. The examples described herein are provided to illustrate the methods and compositions provided herein and should not be construed in any way as limiting the scope thereof.

[0190] Example 1: Selecting the "ideal" donor to create a consistent and potent "off-the-shelf" NK cell therapy preparation NK cells are licensed (gain enhanced killing ability) when they express inhibitory killer immunoglobulin receptors (KIRs) for self-HLA class I molecules. This allows NK cells to recognize "self" and spare self-cells from killing. Therefore, targets lacking self-HLA class I molecules are more likely to elicit recognition by licensed NK cells. Inhibitory KIR genes known to be associated with NK alloreactivity are (i) 2DL1, which binds to HLA-C group 2 alleles; (ii) 2DL2 and 2DL3, which bind to HLA-C group 1 alleles; and (iii) 3DL1, which binds to HLA-B Bw4 alleles. According to the ligand-deficiency model, NK cells expressing inhibitory KIR genes will be alloreactive only if the corresponding ligand is present in the donor and not in the recipient. For example, a donor carrying a group C1 allele will be alloreactive to any individual lacking the group C1 allele. Thus, a donor carrying HLA from the C1, C2, and Bw4 families would be predicted, according to this model, to be alloreactive to any recipient lacking C1, or C2, or Bw4.

[0191] While inhibitory KIRs prevent alloreactivity, activating KIRs recognize activating ligands that promote NK cell lysis. There is considerable variability in the inheritance of activating KIRs, with any individual having between 0 and 7 aKIRs. Data from patients undergoing stem cell transplantation suggest that patients receiving allografts from donors with a higher number of activating KIRs have better outcomes than those receiving allografts from donors with fewer activating KIRs. Other studies have demonstrated a protective benefit against leukemia in individuals who inherit a higher number of activating KIRs. Our laboratory has shown that NK cells with a higher number of activating KIRs more potently induce target cell lysis (Figure 1). Additionally, multivariate analysis has shown that activating KIRs 2DS1 and 3DS1 are associated with disease-free survival.

[0192] Finally, NKG2C is an activating receptor expressed late in NK cell development and recognizes HLA-E rather than HLA-B or -C. NKG2C expression is induced in patients with CMV infection and correlates with an adaptive NK cell phenotype and improved leukemia-free survival.

[0193] Therefore, an "optimal" donor would be one with an HLA genotype carrying the C1, C2, and Bw4 alleles, a KIR genotype that possesses inhibitory KIRs (2DL1, 2DL2, or 3 and 3DL1) that bind to C1, C2, and Bw4 (leading to maximal licensing) and a high proportion of activating KIRs (three or more variably inherited activating genes including 2DS1 and 3DS1), and a history of exposure to CMV, resulting in high NKG2C expression.

[0194] Considering the available data for Caucasian donors, the C1 / C2 / Bw4 allele is found in 32% of the population. 25.3% of the 23 KIR genotypes, representing 80% of the population, meet these criteria. Approximately 90% of adults have been exposed to CMV. As shown in Figure 8 by flow cytometry, all CMV+ donors had NK cells expressing NKG2C, which increased after expansion, such as described in 606 of method 600 illustrated in Figure 6. As shown in Figure 9 by mRNA level measurements, NKG2C expression increased after expansion, such as described in 606 of method 600 illustrated in Figure 6.

[0195] Therefore, an "ideal" NK cell donor can be identified in approximately 1 in 16 healthy individuals. Donor Selection: For maximum cost savings and time efficiency, donors are screened using a tiered algorithm, and donors who do not meet the criteria are excluded from further testing.

[0196] Donor selection involves HLA and KIR gene typing, KIR phenotyping, and NK generation (Figure 5A, top). Donors can be KIR-typed to assess the presence (gray) or absence (black) of KIR genes (Figure 5A, bottom). In one example, PBMCs and donor-matched NK cells were analyzed by flow cytometry to determine KIR expression on NK cells. Expression of 2DL2 / 3, 2DL1, and 3DL1 was determined using KIR-specific antibodies REA147 / CH-L, 143211, and DX9, respectively. The percentage of NK cells expressing each KIR for each donor was determined (Figure 5B).

[0197] For NK cell donors, KIR genotyping using reverse sequence-specific oligonucleotide (SSO) methodology (e.g., One Lambda) may allow for differentiation between functional and deletion mutants of KIR2DL4. KIR-B content can be determined using the B Content Calculator maintained by EMBL-EBI (www.ebi.ac.uk / ipd / kir / donor_b_content.html). Active KIR content will be determined by scoring the total number of active KIR genes. DS-designated KIRs and functional KIR2DL4s were all considered active. Donors with a common active KIR (KIR2DS4 and a functional version of KIR2DL4) and at least three of the five variably inherited active KIRs will be selected.

[0198] NK cell donors can be HLA typed at a high resolution for alleles at the HLA-B and C loci by SSO-PCR (amplification and oligonucleotide sequencing) using commercially available kits. KIR ligand classes can be predicted using the KIR Ligand Calculator (www.ebi.ac.uk / ipd / kir / ligand.html) maintained by the European Bioinformatics Institute at the European Molecular Biology Lab (EMBL-EBI). Individuals carrying all three C1, C2, and Bw4 classes must be selected.

[0199] Donors are also tested for CMV, and if testing reveals a CMV+ donor, the presence of NKG2C+ NK cells is confirmed. OTS NK cell preparation manufacturing and vial filling estimate: Expanded donor NK cell products are manufactured prior to subject enrollment. All donors undergo standard infectious disease screening and other donor screening (as required by 21 CFR 1271.C) within seven days of collection. Source PBMCs are collected and NK cells expanded as outlined in the CMC section of the FDA IND application. Briefly, PBMCs are depleted of CD3+ T cells using MACS colloidal superparamagnetic CD3 microbeads. Resulting cells are co-cultured with irradiated feeder cells and / or membrane particles in medium supplemented with fetal bovine serum and IL-2. On day 7, cultures are restimulated. NK cell products undergo lot release testing and cryopreservation for subsequent infusions on day 14. Sterility testing is partially completed at the time of cryopreservation, and all testing is completed prior to product release. NK cells are stored for 10 days. 8 Cryopreserved in 50 mL single-dose aliquots containing 1.26 x 10 NK cells / mL. Primary donor blood draw equivalent to 1 unit (450 mL), 1.26 x 10 5Assuming a median content of 10 NK cells / mL and a median expansion of 2,800-fold in 2 weeks, each donor will generate enough NK cells for 31 unit dose bags. After CD3 depletion, a median of 3 x 10 8 Assuming an initial donor apheresis containing NK cells, each donor may be able to produce an average of 168 unit dose bags. One bag represents 10 units for a 50 kg individual. 8 One dose of 10 NK cells / kg is sufficient. For adult patients, 8 For a dose of 1000 / kg, up to 2-3 bags may be required per patient per dose. Assuming the freezing medium contains 10% DMSO, 10 8 The DMSO administered for a dose / kg can be 0.1 ml / kg.

[0200] Example 2: Phase I / II clinical trial testing the safety and feasibility of IL-21-expanded natural killer cells for remission induction in relapsed / refractory acute myeloid leukemia 1.0 Background and Rationale 1.1 Relapsed AML and hematopoietic stem cell transplantation (HSCT) Hematopoietic stem cell transplantation (HSCT) is an effective treatment for AML. HSCT has a long-term disease-free survival rate of approximately 60% for patients transplanted in initial remission. After relapse, this rate drops to approximately 40% if the patient was in remission at the time of HSCT. Patients with relapsed AML who do not undergo HSCT have a long-term disease-free survival rate of 5–10%. Many relapsed patients have refractory, chemotherapy-resistant disease, never achieve a remission that would qualify them for potentially curative HSCT, or develop significant complicating comorbidities during the prolonged, intensive reinduction of their disease. Therefore, improved strategies for achieving remission before transplantation in relapsed patients are critical to improving the survival of these patients.

[0201] 1.2 Reinduction chemotherapy for AML Reinduction chemotherapy in relapsed AML results in highly variable remission rates, due in part to the heterogeneity of this population. A meta-review of 31 trials over the past 20 years revealed no single superior regimen. In one study, the mean second complete remission (CR2) rate for high-risk patients (those with a first complete remission (CR1) of less than 1 year) was 27.6% ± 15.5 (weighted mean ± SD), whereas for low-risk patients (those with a CR1 of 1 year or more), the CR2 rate was 56.1% ± 25.9. In another study, patients with high-risk disease (primarily refractory disease or a CR1 of less than 6 months) had a CR2 rate of only 10%, compared with over 50% for patients with low-risk disease (a CR1 of 18 months or more), and patients with favorable karyotypes achieve a second or third remission more frequently than patients with unfavorable karyotypes.

[0202] The importance of high-dose cytosine arabinoside (cytarabine, Ara-C) as an integrated agent in first-line and salvage regimens for the treatment of AML is well established. Fludarabine is widely used to deplete patients' lymphocytes before lymphocyte infusion, and fludarabine-containing regimens, usually combined with cytarabine with or without an anthracycline, are used for reinduction of primary refractory or relapsed AML. Fludarabine has been demonstrated to enhance the increased intracellular accumulation of Ara-CTP, the active metabolite of cytarabine, in AML blasts. This led to the development of the highly active FLAG (fludarabine, cytarabine, G-CSF) regimen for AML.

[0203] As originally described, FLAG chemotherapy has shown excess toxicity in patients over 60 years of age, but in clinical trials in this age group, fludarabine and cytarabine have been safely delivered at reduced doses from 5 to 4 days.

[0204] 1.3 Use of colony-stimulating factors in the treatment of AML Granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) enhance neutrophil recovery after high-dose chemotherapy. The use of G-CSF during induction therapy for AML results in excellent event-free survival rates. Additionally, they have been used to enhance the sensitivity of myeloid leukemia stem cells to cytarabine by increasing the accumulation of Ara-CTP, thus enhancing the anti-leukemic effect of combination chemotherapy, such as FLAG. Furthermore, GM-CSF has been shown to enhance NK cell activity against AML blasts in vitro and in the autologous transplant setting.

[0205] 1.4 Human NK cells as mediators of antitumor therapy Human NK cells are a subset of peripheral blood lymphocytes typically defined by expression of CD56 or CD16 and the absence of the T cell receptor CD3. Several studies suggest that NK cells have a role in tumor surveillance. Cell lines susceptible to NK lysis are referred to as "NK-sensitive" targets. The prototypical NK-sensitive target is the leukemia cell line K562. Activation of NK cells by cytokines, specifically IL-2, endows them with the ability to lyse tumor targets that are normally insensitive to NK lysis (NK-resistant targets).

[0206] NK cells are regulated by KIR receptor-ligand interactions and exhibit cytotoxicity against specific HLA class I-mismatched targets. Alloreactive HLA-haploidentical NK cells in the SCT setting have been reported to enhance engraftment, reduce GvHD, and prevent leukemia relapse. The infusion of human haploidentical NK cells without hematopoietic cell transplantation has been studied in AML patients. These cells were administered after cytoreductive chemotherapy to induce lymphopenia and support homeostatic expansion of NK cells after infusion. NK cells were obtained by donor leukapheresis, with or without secondary positive selection of CD56+ cells, followed by depletion of CD3+ T cells, and then activated overnight with IL-2.

[0207] max 2×107 Infusion of NK cells / kg was well tolerated and induced remission in 5 of 15 patients with refractory AML. Graft-versus-host disease and persistent pancytopenia did not occur. Donor cells were detectable for up to 4 weeks.

[0208] The poor antitumor efficacy of autologous NK cells in previous studies may be due to several factors, including the resistant nature of tumors, factors released by tumors, and killer immunoglobulin receptors (KIRs). Although the effectors mediating graft-versus-host disease (GvHD) and graft-versus-tumor (GVT) remain uncertain, some mouse models suggest that in vivo GVT activity strongly correlates with in vitro NK activity. Using an allogeneic transplant model for the A20 leukemia cell line, allogeneic NK infusion prevented leukemia relapse and had no adverse effects on leukocyte engraftment. In vitro cytotoxicity assays demonstrated superior lytic activity of allogeneic IL2-activated NK cells compared with syngeneic or autologous NK cells. Transplantation with syngeneic or autologous lymphodepleted infusions without NK cell infusion resulted in disease-free survival rates of 10% and 15%, respectively. However, adoptive transfer of IL2-activated syngeneic NK cells improved survival rates to 50%. In contrast, treatment with allogeneic NK cells resulted in an even more potent antitumor effect, with 85% of animals surviving disease-free. The hypothesis that inhibition of class I-induced NK cell lysis is important in antitumor therapy is strongly supported by these in vivo mouse experiments, which show that allogeneic NK cells exhibit greater in vivo antitumor activity than autologous NK cells.

[0209] 1.5 Selection of KIR-mismatched donors and recipients NK cells recognize "self" on autologous targets through HLA class I-associated KIRs. This process inhibits NK cell lysis of the target. Four inhibitory KIR genes have been shown to be associated with NK alloreactivity and have known HLA specificity: 2DL1 binds to HLA-C group 2 alleles, 2DL2 and 2DL3 bind to HLA-C group 1 alleles, and 3DL1 binds to HLA-B Bw4 alleles. According to the ligand-deficiency model for each KIR gene present, alloreactivity occurs only if the corresponding ligand is absent in the patient but present in the donor. Exemplary data for Caucasian donors are presented in Table 3, which summarizes the analysis of HLA Bw and C group loci and KIR expression for donor GVL alloreactivity. The C1 / C2 / Bw4 allele is found in 32% of the population. Of the 23 KIR genotypes representing 80% of the population, 25.3% meet these criteria. Approximately 90% of adults have been exposed to CMV, so an "ideal" NK cell donor could be identified in approximately 1 in 16 healthy individuals.

[0210] [Table 3]

[0211] In the indicated combinations, alloreactivity is expected to occur in the GVL direction. In this study, this model was used to select donors with the highest likelihood of KIR reactivity in the GVL direction. Donors with C1, C2, and Bw4 HLA with the greatest mismatches will provide GVL to the most recipients.

[0212] 1.7 Ex vivo expansion of NK cells A major obstacle to adoptive NK cell immunotherapy is obtaining sufficient cell numbers, due to the fact that these cells represent a small fraction of peripheral leukocytes, have poor ex vivo proliferation, and have a limited in vivo lifespan. Common gamma chain cytokines are important in the activation, maturation, and proliferation of NK cells. Others have described improved ex vivo expansion using soluble cytokines, artificial antigen-presenting cells (aAPCs), and aAPCs engineered with costimulatory molecules and / or membrane-bound IL-15 (mIL-15). Our group created a membrane-bound IL-21 fusion protein (mIL21) and found that ex vivo expansion of NK cells was superior when stimulated with K562 aAPCs genetically modified to express mIL21 and the costimulatory molecules CD86 and CD137L. Freshly isolated peripheral blood mononuclear cells (PBMCs) were co-cultured with irradiated K562 aAPCs at a ratio of 2:1 (aAPCs:PBMCs) in the presence of 50 IU / ml rhIL-2, and then restimulated every 7 days with aAPCs at a ratio of 1:1.

[0213] K562-mIL21 aAPCs were able to promote a mean 37,200-fold expansion of NK cells by day 21, with 85% of donors achieving at least a 5,000-fold expansion (see also Example 1). Expanded cells expressed extremely high levels of CD16 and NCR and retained their pre-expansion KIR repertoire. These cells demonstrated high cytotoxicity and ADCC engagement against tumor targets.

[0214] Thus, the use of mIL21-expressing aAPCs allows for clinically significant NK cell expansion from small peripheral blood samples. 1.8 Clinical Trial Objectives Relapsed AML requires remission before allogeneic HSCT to optimize survival, but chemotherapy response rates are low. HLA haploidentical NK-enriched peripheral blood cell infusion has been shown to be safe in patients with poor-prognosis AML. While not powered for such evaluation, this study showed a promising, but not statistically significant, trend in remission rates. NK cell therapy for AML, particularly relapsed AML, is limited by the low number of NK cells achievable through leukapheresis. However, as described herein, the ability to expand large numbers of NK cells ex vivo from small blood draws would alleviate the need for donor leukapheresis.

[0215] The objective of this study is to determine the safety, feasibility, and maximum tolerated dose of mIL21-expanded haploidentical NK cells in conjunction with FLAG chemotherapy in patients with relapsed / refractory AML.

[0216] 2.0 Eligibility 2.1 Patient Inclusion Criteria 1. Patients with relapsed or primary refractory AML. Patients with AML who have relapsed after allogeneic stem cell transplantation, including those who have received donor lymphocyte infusions, are eligible as long as they do not have active GvHD and are not receiving immunosuppressive medication.

[0217] 2. Have a haploidentical family peripheral blood donor selected for the best possible KIR reactivity. 3. Patient age 18 years or older.

[0218] 4. Performance status: Karnofsky or Lansky Performance Scale (PS) 70 or above. 5. Renal function: Serum creatinine less than 2 mg / dl or creatinine clearance 40 cc / min or more. For pediatric patients, creatinine less than 2 mg / dl or less than twice the upper limit of normal for age (whichever is less).

[0219] 6. Pulmonary Function: Hemoglobin-corrected values ​​of FEV1, FVC, and DLCO >50% of predicted. For pediatric patients, if pulmonary function testing is not available (most children under 7 years of age), pulse oximetry >92% on room air.

[0220] 7. Liver function: Total bilirubin less than 2 mg / dl or less than 2.5 times the ULN for age (excluding Gilbert's syndrome) and SGPT (ALT) less than 2.5 times the ULN for age. 8. Cardiac function: Left ventricular ejection fraction >40%. No uncontrolled arrhythmias or uncontrolled symptomatic heart disease.

[0221] 9. Negative serological test to exclude pregnancy within 2 weeks prior to enrollment in women of childbearing potential (non-childbearing potential defined as premenarchal, postmenopausal for more than 1 year, or surgically sterilized).

[0222] 10. Sexually active men and women of childbearing potential must agree to use a form of contraception deemed effective by the investigator and medically acceptable. 11. Human immunodeficiency virus (HIV)-negative serum.

[0223] 2.2 Patient exclusion criteria 1. Investigational treatment within 4 weeks prior to initiating treatment on this protocol. 2.Congestive heart failure within 6 months prior to screening.

[0224] 3. Unstable angina within 6 months prior to screening. 4. Myocardial infarction within 6 months prior to screening. 5. Uncontrolled infection, defined as an infection that does not resolve spontaneously or shows no evidence of significant resolution after initiation of appropriate therapy, excluding chronic asymptomatic viral infections (e.g., HPV, BK virus, HCV, etc.).

[0225] 2.3 Donor Eligibility Criteria and Pre-Donation Assessment 1. Donors must be 16 years of age or older and weigh at least 50 kg (110 lbs).

[0226] 2. Donors should be selected for optimal NK alloreactivity (defined as having KIR genes present on donor NK cells for which the relevant HLA haplotype (KIR ligand) is present in the donor but not in the recipient) or be HLA haploidentical relatives selected on the basis of activating KIR gene content.

[0227] 3. Donors must meet standard institutional eligibility and donor qualification criteria for therapeutic cell product donation. 4. For women of childbearing potential (not of childbearing potential defined as premenarche, surgically sterilized, or >12 months postmenopausal), not pregnant as defined by a negative serum pregnancy test (βHCG).

[0228] 5.Evaluation: Medical history and physical examination Laboratory tests: hematology, electrolytes, chemistry Infectious disease screening and serology HLA and KIR typing 3.0 Treatment Planning In this study, the first NK cell infusion is referred to as day 0 (D0), and treatment plan activities before or after D0 are expressed as minus days (D-) or plus days (D+).

[0229] 3.1 Donor peripheral blood NK One unit of peripheral blood (approximately 500 mL) will be collected from the donor to initiate NK cell expansion for aAPCs over a 14-day period.

[0230] 3.2 FLAG Treatment Administration According to Standard Treatment Practice After donor peripheral blood collection for NK cell expansion, the recipient may begin FLAG chemotherapy as soon as deemed appropriate by the treating physician. G-CSF will be administered daily, starting one day before the first dose of fludarabine / cytarabine, and continued until the post-nadir absolute neutrophil count (ANC) is ≥ 1000. G-CSF may be maintained at the physician's discretion to maintain a high peripheral blast count for patient safety. Fludarabine is administered at a dose of 30 mg / m 2 / day for 5 days, this dose is based on the actual BSA calculated from actual weight and height. Approximately 4 hours later, cytarabine was administered at 2 g / m 2 / day for 5 days. Patients over 60 years of age will undergo a dose modification by receiving fludarabine and cytarabine for only 4 days.

[0231] A rest period of 2 to 14 days before NK cell infusion. 3.3 NK Cell Infusions on Days 0-14 for a Total of 6 Doses in a Dose Escalation Schema NK cell infusions may begin as soon as the release criteria for expanded cells are met, beginning no earlier than 2 days and no later than 15 days after the last dose of fludarabine / cytarabine. NK cells will be delivered three times per week for at least a 4-day period (e.g., MWF (Monday, Wednesday, Friday), MTuTh (Monday, Tuesday, Thursday), TuThF (Tuesday, Thursday, Friday), etc.). NK cells will be infused according to the Stem Cell Transplantation and Cellular Therapy (SCTCT) Department's standard operating procedures (SOPs) for therapeutic cell infusions.

[0232] Anaphylactic Medication: Have the following medications immediately available prior to NK cell infusion. If anaphylaxis occurs, notify and call your physician (MD).

[0233] Epinephrine (1:1000) 0.5 mL subcutaneously Diphenhydramine 50 mg intravenously In cases of anaphylaxis, a physician (MD) should be consulted before administering corticosteroids.

[0234] Follow the MD Anderson Cancer Center (MDACC) Hypersensitivity Reaction (HSR) algorithm for additional supportive care measures. Premedication: Before NK cell infusion, diphenhydramine 25 mg was administered intravenously.

[0235] Because expanded NK cells may exhibit increased toxicity due to their activated phenotype, the initial NK cell cohort will be administered at a dose well below the currently established safe dose for apheresis-derived NK cells and will follow a dose-escalation scheme to avoid accumulating patients at suboptimal doses.

[0236] [Table 4]

[0237] This study will use the principle of rapid dose escalation to allow rapid progression to currently safe doses of NK cells. To be eligible to receive one or more NK infusions, patients must meet the following requirements: 1. Not taking corticosteroids within the previous 72 hours.

[0238] 2. No need for ventilator support or supplemental oxygen. 3. Karnofsky or Lansky performance status of 70% or higher. NK cell doses will be based on total nucleated cell (TNC) counts and CD56+CD3- percentages determined by flow cytometry. The maximum volume of infused cell preparation is 100 ml. Infused cells will be delivered on a NK cell / kg recipient body weight basis. Total CD3+ T cells will be 1 x 10 for all cohorts. 5 The number of NK cells transfused in the current cohort must be less than 10 5If more than 1 x 10 CD3+ cells / kg recipient body weight are to be delivered, the NK cell infusion dose should be 1 x 10 CD3+ cells / kg recipient body weight. 5 The NK cell infusion dose will be reduced to the highest cohort dose that is less than 1 / kg recipient body weight. Some donor NK cell expansions may not produce enough cells to reach the planned NK cell dose. If it is not possible to deliver the target NK cells / kg recipient body weight, then the NK cell infusion dose will be reduced to the highest achievable cohort dose. For statistical analysis, the patient data will be included in that cohort, and additional subjects will be enrolled at the current dose level.

[0239] 4.0 Drug Information 4.1 Cytosine arabinoside (cytarabine, Ara-C) Cytarabine is an antimetabolite. Injectable cytarabine is commercially available as a solution. Handling, reconstitution, and administration should follow institutional guidelines. Cytarabine can cause cardiac hypertrophy, coma, and neurotoxicity (dose dependent; cerebellar toxicity occurs with high-dose cytarabine [36–48 g / m ]). 2 / cycle or more; incidence may be as high as 55% in patients with renal impairment), personality changes, somnolence, alopecia (total), desquamation, rash (severe), gastrointestinal ulcers, peritonitis, pneumatosis cystoides intestinalis, hyperbilirubinemia, liver abscess, liver damage, necrotizing colitis, peripheral neuropathy (motor and sensory), corneal toxicity, hemorrhagic conjunctivitis, pulmonary edema, acute respiratory distress syndrome, and sepsis.

[0240] Formulation: As a solution for intravenous use in 100, 500, 1000 or 2000 mg vials Commercially available products Storage: Room temperature Stability: 28 days at room temperature Administration: Cytarabine is further diluted in 5% dextrose or 0.9% sodium chloride.

[0241] 4.2 Fludarabine Fludarabine is an antimetabolite. Fludarabine injection is commercially available as a lyophilized cake that is reconstituted with sterile water. Institutional guidelines should be followed for handling, reconstitution, and administration. Fludarabine, at doses higher than those administered in this study, can cause blood count reductions, immune system suppression, nausea and vomiting, fever, hypersensitivity reactions, tumor lysis, transient elevations in serum transaminases, hemolysis, and neurotoxicity.

[0242] Formulation: Commercially available as a white lyophilized cake in 50 mg vials for intravenous use. Storage: Room temperature Mix: Add 2 mL sterile water to the vial to give a final concentration of 25 mg / mL.

[0243] Stability: The intravenous solution must be used within 8 hours of mixing. Administration: Fludarabine is further diluted in 100 mL of 5% dextrose or 0.9% sodium chloride.

[0244] 4.3 Filgrastim (G-CSF; Granulocyte Colony-Stimulating Factor) Filgrastim stimulates the production, maturation, and activation of neutrophils. It also activates neutrophils, increasing both their migration and cytotoxicity. Filgrastim is used in chemotherapy-induced neutropenia (non-myeloid malignancies, acute myeloid leukemia, and bone marrow transplantation); severe chronic neutropenia (SCN); and patients undergoing peripheral blood progenitor cell (PBPC) harvesting.

[0245] Filgrastim has been associated with: Allergic reactions: Rash, hives, wheezing, dyspnea, tachycardia, and / or hypotension have occurred with the first or subsequent doses. Reactions tend to occur more frequently with intravenous administration and within 30 minutes of administration.

[0246] Respiratory distress syndrome: Rare cases of adult respiratory distress syndrome have been reported; patients should be instructed to report respiratory distress. Splenic rupture: Rare cases of splenic rupture have been reported; patients should be instructed to report left upper quadrant or shoulder pain.

[0247] Pharmacodynamics / kinetics Onset of effect: 24 hours; plateaus in 3-5 days Duration: ANC decreases by 50% within 2 days after discontinuation of G-CSF; white blood cell count returns to normal range in 4-7 days; peak plasma levels can be maintained for up to 12 hours Absorption: Subcutaneous: 100% Distribution: 150 mL / kg; no evidence of drug accumulation over the 11-20 day period Metabolism: Degraded systemically Elimination half-life: 1.8-3.5 hours. Time to peak serum: 2-6 hours. Dosage: Subcutaneous: ≤5 mcg / kg / day, starting 24-72 hours after chemotherapy; continue until absolute neutrophil count goal is achieved. Pediatric patients must receive specially calculated doses. Adult doses should be rounded to the nearest vial size (300 mcg or 480 mcg). Autologous stem cell harvest: 5 mcg / kg subcutaneously every 12 hours for 5 days (10 doses total) Dosage Formulation: Injection, solution: 300mcg / mL (1mL, 1.6mL) Injection, solution [prefilled syringe]: 300 mcg / 0.5 mL 5.0 Research Evaluation 5.1 Before starting FLAG standard of care (baseline): 5.1.1 Medical History and Physical Examination 5.1.2 CBC with differential 5.2 Before each NK infusion: 6.2.1 Medical History and Physical Examination 6.2.2 CBC with Differential 6.2.3 Pulse oximetry 5.3 After the last infusion: CBC with differential twice weekly while patient is neutropenic 5.4 After neutrophil recovery: CBC with differential weekly from 1st NK infusion until D+56.

[0248] 5.5 Disease Assessment: After neutrophil recovery and / or approximately D+28, whichever occurs first: 1. Unilateral bone marrow biopsy and aspirate for cytology, flow cytometry, MRD, chimerism (STR or FISH), cytogenetics, and FISH (for known tumor markers) 2. If recovery has not occurred by day +28, then a second bone marrow will be obtained at the time of neutrophil recovery or about day +56, whichever comes first.

[0249] 5.6 Peripheral blood samples to address secondary research objectives will be sent to Dr. Lee's laboratory (MOD1.020) 1.Before starting FLAG standard treatment (baseline).

[0250] 2. Before each NK infusion and 2 hours (± 1 hour) after the infusion is completed. 3. D+14 (±3 days), +16 (±3 days), +18 (±3 days), and +21 (±3 days), then weekly until D+56, as long as infused NK cells can be reliably detected. Samples may be obtained ±3 days before and ±5 days after the target date of D+28. For each sample, draw up to 40 mL (maximum 0.5 mL / kg) into a green-top Na-heparin tube, and draw up to 10 mL of serum (one red-top tube).

[0251] 6.0 Adverse Events 6.1 Assessment of Adverse Event Attributes The study component of the treatment plan for this study is NK cell infusion. FLAG chemotherapy and GCSF are considered standard of care, and their associated adverse events are well known. Therefore, for the purposes of this study, in the presence of an adverse event suspected to be directly related to NK cell infusion, the event will be attributed to the NK cell infusion.

[0252] Events known to be caused by and direct consequences of FLAG chemotherapy, as well as events known to be associated with drugs used in the treatment of GvHD, infections, and supportive care, will be scored as unrelated to NK cell infusion.

[0253] The principal investigator will be the final adjudicator in determining event attribution. 6.2 Grading of Adverse Event Severity From the start of the first NK cell infusion until D+56, the severity of adverse events (AEs) will be graded according to the Common Terminology Criteria for Advanced Treatment of Encephalopathy (CTCAE) version 4.0.

[0254] Events not listed in the CTCAE tables will be scored as follows: Common grading: Grade 1: Mild: Painful, but not interfering with daily activities, and no treatment beyond preventative measures is required.

[0255] Grade 2: Moderate: Pain that interferes with some daily activities and requires treatment. Grade 3: Severe: Not responding to first-line treatment, causing pain that interferes with normal daily activities.

[0256] Grade 4: Life-threatening: Suffering that is immediately life-threatening. 6.3 Potential adverse events associated with allogeneic NK cell infusion: 1. Acute adverse events: Events lasting less than 24 hours: Grade I chills Grade I cough Grade I or II angioedema Grade I or II dyspnea Grade I or II hypotension Grade I or II tachycardia Grade I or II headache Events lasting less than 48 hours: Grade I or II fatigue Grade I or II neuropathic pain Grade I or II vomiting Grade I or II SGPT change Grade I or II hypoalbuminemia Grade I or II hypocalcemia Grade I or II fever Grade I or II pruritus Grade I rash Grade I or II lymphopenia Grade I or II neutropenia Grade I or II leukopenia Grade I or II cytokine release / acute infusion reaction 2. Events lasting less than 72 hours: Grade I or II nausea Tumor lysis syndrome 3. Cytopenia 2-3 weeks after the first NK cell infusion Fludarabine and cytarabine are expected to cause transient myelosuppression lasting 2–3 weeks. However, hematologic toxicity due to allogeneic NK cells can occur later, thus determining hematologic recovery beyond the expected chemotherapy-induced nadir. For example, 10–15% of patients who receive donor lymphocyte infusion therapy after allogeneic HSCT experience myelosuppression.

[0257] Cytopenias in this setting are usually due to T-cell suppression of host hematopoietic cells. Although this situation is unlikely after T-cell-depleted NK cell infusion, the possibility of NK-mediated myelosuppression cannot be ruled out a priori. In addition, the time it takes to restore normal hematopoiesis is highly dependent on the presence of a normal bone marrow reserve, which is nearly nonexistent in the setting of intensively treated patients with multiple relapses.

[0258] 4. Acute graft-versus-host disease GvHD is associated with allogeneic T cells. Because the infused cells are subject to T cell depletion, GvHD is not expected and has generally not occurred in prior studies using allogeneic NK cell therapy. However, small numbers of T cells can be infused, or NK cells can engraft and cause GvHD syndrome.

[0259] GvH of overall grade >2 is not expected to occur. Adverse events considered serious 1. Refractory GvHD 2. Infection during neutropenia requiring hospitalization 3. Any expected or unexpected event believed to be related to the NK cell product resulting in an irreversible condition and / or leading to death.

[0260] Anticipated adverse events known to be associated with FLAG chemotherapy The known toxicities of the combination of fludarabine, cytarabine, and G-CSF (FLAG) are well described in published Phase 1 and Phase 2 studies. Expected toxicities, as well as myelosuppression, cytopenias, and infections, first observed after initiation of FLAG and before administration of NK cells will not be attributed to NK cells for purposes of determining DLTs.

[0261] Adverse events related to FLAG (% Grade III and IV): 1. Liver: ALT (25%), bilirubin (7%), AST (7%), alkaline phosphatase (5%).

[0262] 2.Gastrointestinal tract: ALT (25%), mucositis (5%), nausea / vomiting (30%), diarrhea (6%), and constipation (4%).

[0263] 3.Other: Bleeding (5%), rash (5%), BUN (4%), drug fever (3%), headache (3%), and vision changes (1%).

[0264] 4. Median time to recovery from bone marrow suppression and associated cytopenias from day 0 of chemotherapy (95% CI): neutrophils 32 (27-35) days, platelets 41 (35-47) days. 5. During the cytopenic period, patients are at risk for infection.

[0265] Adverse event data collection From D0 to D+56, adverse event documentation will reflect the dates of onset and resolution and the highest grade. Intermittent events should be labeled as such and tracked until resolution.

[0266] If a patient leaves the study while an event is still ongoing, it will be followed until resolution unless another therapy is initiated. Pre-existing medical conditions will only be recorded if an exacerbation occurs during the active treatment period. Comorbid events will not be scored separately.

[0267] Adverse events will be documented based on progress notes in the electronic (Clinic Station) patient record, including flow sheets. PDMS / CORe will be used as the electronic case report form for this protocol, and all protocol-specific data will be entered into PDMS / CORe.

[0268] Combination drug therapy Patients treated with this protocol will require supportive care treatment (concomitant medications). These medications are considered standard of care and do not contribute scientifically to the protocol, therefore no data are captured regarding the various medications required or their side effects.

[0269] 7.0 Statistical considerations The primary objective of this study is to evaluate the safety and feasibility of an expanded haploidentical donor NK cell product following a FLAG conditioning regimen for the treatment of relapsed / refractory acute myeloid leukemia and to define the maximum tolerated dose (MTD). The maximum tolerated dose endpoint for NK cell infusion is described herein. The safety and feasibility endpoint is defined as the ability to generate NK cells and infuse the maximum tolerated cell dose without undue toxicity limitations in at least 7 of 10 subjects. Secondary endpoints include determining the activation status and persistence of haploidentical NK cells, the immunophenotype and function of haploidentical NK cells, AML disease remission rates, transplant feasibility rates for patients receiving this regimen, and time to transplant for patients with available donors.

[0270] Cytokine-mediated activation of NK cells will be determined by a flow-based activation assay that determines CD107a expression on NK cells in response to standardized targets. NK cell function will be assessed by cytolysis of standardized targets. Remission will be defined as bone marrow recovery with less than 5% blasts in the bone marrow. Clinical response will be correlated with in vivo NK cell expansion, cytokine levels, expression of activation markers, and expression of NK cell ligands in the patient's AML blasts. At indicated times, additional study samples will be collected for laboratory evaluation of in vivo activation of expanded NK cells to study the effects of this therapy on the immune system. The occurrence of toxicity and adverse events will be monitored.

[0271] 7.1 Dose Escalation Dose-limiting toxicity (DLT) is defined as: 1. Grade 3 or higher infusion allergic reaction related to NK cell infusion.

[0272] 2. Acute combined GvHD of grade 3 or higher that does not resolve to grade 1 or lower with treatment within 1 week 3. Unexpected toxicity of greater than Grade 3 that is suspected, probably, or definitely related to NK cell infusion. Grade 3 toxicity that resolves within 72 hours will not be considered a DLT.

[0273] Because NK cells delivered at doses corresponding to dose levels 1-4 have been shown to be safe in other Phase I trials, we will utilize rapid dose escalation through those dose levels. We will use a standard 3+3 design for dose levels 5-6. Once the 3+3 portion of the study is performed, we will limit co-enrollment at any dose level to the minimum number of subjects required to declare the MTD exceeded (e.g., a dose level may begin with co-enrollment of two subjects, but culminate in the enrollment of a third subject, and at least one of the first two subjects must be observed free of DLT by Day +28).

[0274] For dose levels 1 to 4, one patient will be treated at each dose level 1 (10 6 / kg / dose, 3 times per week x 6 doses). If this patient does not exceed the toxicity limits defined for the rapid titration phase (see first bullet point below), the next patient will be treated at the next dose level. If any related toxicity of grade 2 or higher as described is observed at any time in dose levels 1-4, a standard 3+3 will be initiated immediately, and two additional patients will be enrolled at the current dose level. If 3+3 is not initiated through the first four doses, the standard 3+3 design will continue until dose level 5 (10 8 / kg / dose). Three patients will be treated and evaluated for toxicity. If zero of three patients experience a DLT, three patients in the next cohort will be treated at the next higher dose level. If one of three patients treated at a dose level experiences a DLT, three more patients will be treated at the same dose level. If the incidence of DLT among these six patients is 1 in 6, the next cohort will be treated at the next higher dose level. If three or more of six patients treated at a dose level experience a DLT, the MTD is considered exceeded. Unless six patients have already been treated at that dose, three more patients will be treated at the next lower dose as described above. The MTD is defined as the highest dose studied at which six patients are treated and at most two patients experience a DLT. If two of six patients experience a DLT, that dose level will be discontinued and declared the MTD.

[0275] The cohort defined as the MTD may be expanded to up to 10 patients for further evaluation of toxicity and correlative data. If more than one-third of patients experience DLT at any time during the expansion, the expansion cohort will be terminated. If the MTD expansion cohort is terminated due to excessive toxicity, the next lower dose may be expanded to 10 patients and explored. All patients treated at the MTD will be included in the expansion analysis and monitoring.

[0276] During the rapid titration phase, more stringent criteria for toxicity will be utilized to ensure patient safety: Occurrence of NK cell-related Grade 2 toxicity by any one patient within 21 days of initiating NK cell infusion, excluding Grade 2 fever, chills / chills, fatigue, vomiting / nausea, pruritus / itching, electrolyte imbalance, hypoalbuminemia, and lymphopenia: Current and subsequent (if any) cohorts will be expanded to include up to 3 patients.

[0277] If the MTD is not established at dose level 6, this dose level will be expanded to 10 patients to further evaluate the safety and anti-tumor therapeutic response of the expanded NK cells.

[0278] If a stopping rule is applied at any time during cohort expansion, patient enrollment into that expansion cohort will be suspended. After the last patient in the cohort completes treatment, clinical and safety data will be analyzed and dose escalation will follow the dose escalation rules defined above.

[0279] MTD - Maximum Tolerated Dose is defined as the highest dose level at which no more than two patients experience DLT on treatment in a cohort of six patients. If two out of six patients experience DLT, that dose level will be discontinued and declared as the MTD.

[0280] 7.2 Test Size Adequacy The dose-escalation phase of this study may enroll up to six patients per cohort. After determining the maximum tolerated dose of NK cells, we will enroll subjects until there are 10 subjects in the study who have successfully received NK cell infusion at the MTD level or the highest dose level. We expect to accrue these patients over two years. Patients who do not meet the criteria for receiving NK cell infusion will not be included in determining the primary feasibility objective. For each enrolled patient who does not receive NK cell infusion at the planned dose level, an additional patient will be enrolled. We anticipate that up to six patients may not be able to receive NK cells at the MTD or highest dose level due to toxicity of the FLAG regimen. Therefore, this study may complete dose level 6 with as few as 17 subjects or may enroll up to 46 subjects.

[0281] A secondary objective of this study will be determination of complete remission (CR) at 56 days after NK cell infusion. For efficacy, we will measure outcomes based on patient risk. The remission rate in patients with relapsed AML across multiple regimens is 56.1% for low-risk patients and 27.6% for high-risk patients.

[0282] 7.3 Research Termination Rules Adverse events will be defined according to the criteria of the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTC AE) version 4.0.

[0283] If three or more subjects experience adverse events > Grade 4 or severe (> Grade 4) infections involving the cardiopulmonary, hepatic (excluding albumin), nervous, or renal systems that are suspected, probable, or definitely caused by the infused NK cell product, we will temporarily close the study to new patient enrollment in order to review whether the safety criteria and / or consent form may need to be modified.

[0284] If any death suspected, probably, or definitely attributable to the infused NK cells occurs in a study participant within 30 days of NK cell infusion, we will temporarily close the study to new patient enrollment to review safety criteria and / or consent forms for potential modifications. If a death occurs more than 30 days after NK cell infusion, the study will be temporarily stopped and reviewed only if the cause of the death is definitely attributable to NK cell therapy.

[0285] 7.4 Analysis of Secondary Study Endpoints 7.4.1 Analysis of NK Cell Numerical Expansion In Vivo: Peripheral blood will be obtained before therapy, during NK cell therapy, and after NK cell therapy. The studies may include flow cytometric analysis and sorting as well as molecular studies. Donor NK cell expansion will be defined as an increase in the absolute number of circulating donor-derived NK cells above post-infusion levels. The following chimerism methods will be used to determine the origin and number of circulating NK cells: 7.4.2 Chimerism Research: Chimerism can be determined by flow cytometry using haplotype-specific antibodies.

[0286] Chimerism can be determined by STR polymorphisms. When there is gender mismatch between donor and recipient, assays based on determining sex chromosome frequencies may be used. Testing may be modified by the Principal Investigator or designee.

[0287] 7.5 Clinical Outcomes We will summarize the demographic and clinical characteristics of patients in this study using descriptive statistics. We will estimate complete remission (CR) and time to transplant (TTT) rates with the Kaplan-Meier estimator and tabulate them with 95% confidence intervals. We will estimate CR and TTP with 95% confidence intervals. We will estimate the proportion of patients with successful in vivo NK cell expansion with 95% confidence intervals. We will model CR and TTT as a function of NK cell dose using Cox proportional hazards regression.

[0288] 7.6 Accumulation Rate Estimation The inventors expect to enroll a minimum of 15 eligible patients per year. The protocol may take 3 years to complete.

[0289] 8.0 Research Criteria 8.1 Recovery: Defined as the first day of sustained ANC ≥ 1000 / uL. 8.2 Prolonged Neutropenia: Failure to achieve recovery within 28 days after NK cell infusion.

[0290] 8.3 Disease Progression: Detection of persistent or progressive underlying disease by bone marrow and / or peripheral blood examination. 8.4 Study Withdrawal: 8.4.1 Unable to infuse NK cell products due to product contamination or insufficient cell dose.

[0291] 8.4.2 Graft failure requiring further treatment. 8.4.3 Disease progression requiring further treatment. 8.4.4 The patient responds to treatment and then begins another therapy (e.g., stem cell transplant).

[0292] 8.4.5 Unexpected patterns of toxicity. 8.4.6 Patient Withdrawal of Informed Consent. 8.4.7 Patient non-compliance with treatment schema.

[0293] 8.4.8 After completion of treatment, D+56. Example 3: Cytotoxicity of natural killer cells expanded from PBMCs from universal donors NK cells are prepared by expansion from PBMCs obtained from a universal donor identified by the method described in Figure 3. Expansion is performed in the presence of membrane-bound IL-21 in the form of irradiated feeder cells bearing membrane-bound IL-21, IL-21-bearing cell membrane particles, or IL-21-bearing exosomes. PBMCs are initially isolated from buffy coats and grown in cell culture medium supplemented with 10% FBS and maintained at 37°C in a humidified atmosphere with 5% CO2. From day 5 onwards, the medium is changed every other day by replacing half of the medium with fresh medium supplemented with 100 U of IL-2. Cells are counted every other day, and culture contents are checked regularly from day 7 onwards. NK cells are expanded for at least 7–14 days. A cytotoxicity assay is performed as follows: the ovarian carcinoma-derived target cell line SKOV3 transfected with green fluorescent protein (GFP) is used to measure the antitumor cytotoxicity of effector NK cells expanded from universal donor PBMCs. Target cells are cultured alone (control wells) or co-cultured with NK cells for 45 minutes at 37°C in a 5% CO atmosphere. Cells are then centrifuged, resuspended in antibody-containing labeling buffer, incubated, and analyzed by flow cytometry. Cytotoxicity is determined based on the absolute amount of viable target cells (GFP+ / antibody-) remaining in each well compared to the average VTCs in the "target alone" control wells.

[0294] Cytotoxicity E:T(%)=(VTCE:T / Average VTCT control)×100 The cytotoxicity of NK cells expanded from PBMCs obtained from universal donors is shown to exhibit increased cytotoxicity against SKOV3 cells compared to NK cells expanded from PBMCs obtained from control donors who do not meet the universal donor criteria provided herein.

[0295] Example 4: Treatment with NK cells expanded from PBMCs from a universal donor At least 15 AML patients will be selected as described in Example 2 and treated for approximately 3 years using NK cells derived from universal donors and expanded according to Example 3, according to the clinical trial protocol detailed in Example 2 (Section 3). Peripheral blood will be obtained from each patient before therapy, during NK cell therapy, and after NK cell therapy. Flow cytometry analysis and selection, as well as molecular studies, will be performed during treatment. Complete remission rates (CR) and time to transplant (TTT) will be determined using the Kaplan-Meier estimator and tabulated with 95% confidence intervals. CR and TTP will be determined with 95% confidence intervals. The proportion of patients with successful in vivo NK cell expansion will be determined with 95% confidence intervals. Cox proportional hazards regression will be used to model CR and TTT as a function of NK cell dose. Recovery will be defined as the first day of a sustained ANC of 1000 / uL or greater. Prolonged neutropenia will be defined as failure to achieve recovery within 28 days after NK cell infusion. Disease progression is determined when bone marrow and / or peripheral blood tests detect persistent or progressive underlying disease. The majority of patients with AML have a good outcome.

Claims

1. A method for selecting universal donor NK cells, the method comprising selecting candidate NK cells having (i) an HLA genotype carrying C1, C2 and Bw4 alleles and (ii) a KIR genotype or phenotype carrying an inhibitory KIR including 2DL1, 2DL2 or 2DL3, and 3DL1 as universal donor NK cells.

2. 2. The method of claim 1, further comprising selecting candidate NK cells having a KIR genotype carrying at least three activating KIRs, including 2DS1 and 3DS1, as universal donor NK cells.

3. The method according to claim 1, a) assessing the expression of 2DL1, 2DL2 or 2DL3, and 3DL1 in said candidate NK cells; b) obtaining the HLA genotype of said candidate NK cells; and / or c) obtaining the KIR genotype of said candidate NK cells; The method further comprises:

4. 10. The method of claim 1, further comprising selecting a donor with a CMV seropositive profile as a source of said candidate NK cells.

5. The method of claim 4, wherein the selected universal donor NK cells are NKG2C+.

6. The method of any one of claims 1 to 5, wherein the selected universal donor NK cells are activated by incubating the universal donor NK cells in vitro in the presence of IL-21.

7. 7. The method of claim 6, wherein the IL-21 comprises at least one of soluble IL-21, IL-21-expressing feeder cells (FC21), IL-21 cell membrane particles (PM21), and IL-21 exosomes (EX21).

8. The IL-21 present in FC21, PM21, and EX21 comprises a form of IL-21 selected from (a) a modified membrane-bound form for IL-21, (b) IL-21 chemically conjugated to the surface of FC21, PM21, or EX21, or (c) IL-21 in a solution mixed to contact with the NK cells, and any one of FC21, PM21, or EX21 is selected from (a) 41BBL, IL-2, IL-12, IL-18, IL- 15, IL-7, ULBP, MICA, LFA-1, 2B4, BCM / SLAMF2, CCR7, OX4OL, NKG2D agonist, Delta-1, Notch ligand, NKp46 agonist, NKp44 agonist, NKp30 agonist, other NCR agonist, and CD16 agonist; or (b) membrane-bound TGF-β.

9. 9. The method of claim 8, wherein any one of FC21, PM21, or EX21 further comprises 41BBL.

10. 1. A method for preparing a universal donor NK cell population, said method comprising: (a) selecting an NK cell donor with (i) an HLA genotype having C1, C2, and Bw4 alleles and (ii) a KIR genotype or phenotype carrying an inhibitory KIR including 2DL1, 2DL2, or 2DL3, and 3DL1; and (b) exposing said initial NK cell population to IL-21 in vitro for a time and under conditions sufficient to expand said initial NK cell population from said NK cell donor to obtain a population of universal donor NK cells; A method comprising:

11. 11. The method of claim 10, wherein the NK cell donor is further selected by having a KIR genotype that possesses at least three activating KIRs, including 2DS1 and 3DS1.

12. The method according to claim 10, i) assessing the expression of 2DL1, 2DL2 or 2DL3, and 3DL1 in said NK cell donor; ii) obtaining the HLA genotype of said NK cell donor; and / or iii) obtaining the KIR genotype of said NK cell donor; The method further comprises:

13. 11. The method of claim 10, wherein step (b) is carried out for a time and under conditions to achieve at least one population doubling.

14. 11. The method of claim 10, wherein the NK cell donor is further selected by having a CMV seropositivity profile indicative of the presence of NKG2C+ NK cells.

15. 11. The method of claim 10, wherein exposing the primary NK cell population to IL-21 comprises contacting the primary NK cell population in vitro with at least one of soluble IL-21, IL-21-expressing feeder cells (FC21), IL-21 cell membrane particles (PM21), and IL-21 exosomes (EX21).

16. The IL-21 present in FC21, PM21, and EX21 comprises a form of IL-21 selected from (a) a modified membrane-bound form of IL-21, (b) IL-21 chemically conjugated to the surface of FC21, PM21, or EX21, or (c) IL-21 in a solution mixed with the NK cells so as to be in contact with each other, and any one of FC21, PM21, or EX21 is selected from (a) 41BBL, IL-2, IL-12, IL-18, IL-19, IL-21, IL-22, IL-19, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, IL-39, IL-40, IL-41, IL-42, IL-43, IL-44, IL-45, IL-46, IL-47, IL-48, IL-49, IL-50, IL-51, IL-52, IL-53, IL-54, IL-55, IL-56, IL-57, IL-58, IL-59 ...

16. The method of claim 15, further comprising one or more activating agents, stimulatory peptides, cytokines and / or adhesion molecules selected from: IL-15, IL-7, ULBP, MICA, LFA-1, 2B4, BCM / SLAMF2, CCR7, OX4OL, NKG2D agonists, Delta-1, Notch ligands, NKp46 agonists, NKp44 agonists, NKp30 agonists, other NCR agonists and CD16 agonists; or (b) membrane-bound TGF-β.

17. 17. The method of claim 16, wherein any one of FC21, PM21, or EX21 further comprises 41BBL.

18. 18. The method of any one of claims 10 to 17, wherein the universal donor NK cell population has an increased ability to produce and secrete the anti-tumor cytokines IFNγ or TNFα, increased expression of NKG2D, increased expression of CD16, increased expression of NKp46, and / or increased expression of KIR compared to the initial NK cell population.

19. A modified NK cell or cell line derived from a universal donor NK cell selected by the method of claim 1, wherein the modified NK cell or cell line has been transformed to express HLA C1, C2 and / or Bw4 alleles, and the modified NK cell or cell line has been further transformed to express one or more inhibitory KIRs including 2DL1, 2DL2, 2DL3, and / or 3DL1.

20. 20. The modified NK cell or cell line of claim 19, wherein the modified NK cell or cell line is transformed to express HLA C1, C2 and Bw4 alleles.

21. 20. The modified NK cell or cell line of claim 19, wherein the modified NK cell or cell line is further transformed to express an inhibitory KIR comprising 2DL1, 2DL2 or 2DL3, and 3DL1.

22. 20. The modified NK cell or cell line of claim 19, wherein the modified NK cell or cell line is further transformed to express one or more activating KIRs, including 2DS1 / 2, 2DS3 / 5, 3DS1 and / or 2DS4.

23. 23. The modified NK cell or cell line of claim 22, wherein the modified NK cell or cell line is further transformed to express three or more activating KIRs, including 2DS1 and 3DS1.

24. 20. The modified NK cell or cell line of claim 19, wherein the modified NK cell or cell line is further modified to activate NKG2C.

25. 25. The modified NK cell or cell line of any one of claims 19 to 24, wherein the modified NK cell or cell line is activated in vitro by incubating the modified NK cell or cell line in the presence of IL-21.

26. 26. The modified NK cell or cell line of claim 25, wherein the IL-21 comprises at least one of soluble IL-21, IL-21-expressing feeder cells (FC21), IL-21 cell membrane particles (PM21), and IL-21 exosomes (EX21).

27. The IL-21 present in FC21, PM21, and EX21 comprises a form of IL-21 selected from (a) a modified membrane-bound form of IL-21, (b) IL-21 chemically conjugated to the surface of FC21, PM21, or EX21, or (c) IL-21 in a solution mixed to contact with the NK cells, and any one of FC21, PM21, or EX21 is selected from (a) 41BBL, IL-2, IL-12, IL-18, IL-15, IL-7 , ULBP, MICA, LFA-1, 2B4, BCM / SLAMF2, CCR7, OX4OL, NKG2D agonist, Delta-1, Notch ligand, NKp46 agonist, NKp44 agonist, NKp30 agonist, other NCR agonist, and CD16 agonist; or (b) membrane-bound TGF-β.

28. 28. The modified NK cell or cell line of claim 27, wherein any one of FC21, PM21, or EX21 further comprises 41BBL.

29. 1. A population of NK cells for use in treating cancer or an infectious disease in a subject, said population of NK cells comprising: (i) an HLA genotype carrying the C1, C2, and Bw4 alleles; and (ii) a KIR genotype or phenotype possessing an inhibitory KIR comprising 2DL1, 2DL2 or 2DL3, and 3DL1; Including, The NK cell population further comprising culturing said NK cell population in vitro in the presence of IL-21 prior to said use.

30. 30. The NK cell population of claim 29, wherein the NK cell population further comprises a KIR genotype possessing at least three activating KIRs, including 2DS1 and 3DS1.

31. 30. The NK cell population of claim 29, wherein the NK cell population is derived from an NK cell donor with a CMV seropositive profile.

32. The NK cell population of claim 29, wherein the IL-21 comprises IL-21, IL-21-expressing feeder cells (FC21), IL-21 cell membrane particles (PM21), or IL-21 exosomes (EX21).

33. The IL-21 present in FC21, PM21, and EX21 comprises a form of IL-21 selected from (a) a modified membrane-bound form of IL-21, (b) IL-21 chemically conjugated to the surface of FC21, PM21, or EX21, or (c) IL-21 in a solution mixed with the NK cells so as to be in contact with each other, and any one of FC21, PM21, or EX21 is selected from (a) 41BBL, IL-2, IL-12, IL-18, IL-1 33. The NK cell population of claim 32, further comprising one or more activating agents, stimulatory peptides, cytokines and / or adhesion molecules selected from: IL-5, IL-7, ULBP, MICA, LFA-1, 2B4, BCM / SLAMF2, CCR7, OX4OL, NKG2D agonists, Delta-1, Notch ligands, NKp46 agonists, NKp44 agonists, NKp30 agonists, other NCR agonists, and CD16 agonists; or (b) membrane-bound TGF-β.

34. 34. The NK cell population of claim 33, wherein any one of FC21, PM21, or EX21 further comprises 41BBL.

35. The NK cell population according to any one of claims 29 to 34, wherein the cancer is selected from blood cancer, lung cancer, esophageal cancer, gastric cancer, pancreatic cancer, liver cancer, biliary tract cancer, colon cancer, rectal cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, kidney cancer, bladder cancer, testicular cancer, prostate cancer, laryngeal cancer, thyroid cancer, brain cancer, or skin cancer, and the infectious disease is caused by a pathogen selected from a virus, a bacterium, or a fungus.

36. 1. A method for preparing a NK cell collection from a donor, said method comprising: (i) selecting from one or more donors a universal donor having (i) an HLA genotype carrying C1, C2, and Bw4 alleles and (ii) a KIR genotype or phenotype carrying an inhibitory KIR including 2DL1, 2DL2, or 2DL3, and 3DL1; and (ii) preparing said NK cell collection from an ex vivo batch of said universal donor NK cells; A method comprising:

37. 37. The method of claim 36, wherein the universal donor is further selected by having a KIR genotype that possesses at least three activated KIRs, including 2DS1 and 3DS1.

38. The method of claim 36, a) assessing the expression of 2DL1, 2DL2 or 2DL3, and 3DL1 in said one or more donors; b) obtaining the HLA genotype of said one or more donors; and / or c) obtaining the KIR genotype of said one or more donors; The method further comprises:

39. 37. The method of claim 36, wherein the universal donor is further selected by having a CMV seropositivity profile indicative of the presence of NKG2C+ NK cells.

40. 37. The method of claim 36, further comprising activating said NK cell collection by incubating said NK cell collection in vitro in the presence of IL-21.

41. 41. The method of claim 40, wherein the IL-21 comprises IL-21, IL-21-expressing feeder cells (FC21), IL-21 cell membrane particles (PM21), or IL-21 exosomes (EX21).

42. The IL-21 present in FC21, PM21, and EX21 comprises a form of IL-21 selected from (a) a modified membrane-bound form of IL-21, (b) IL-21 chemically conjugated to the surface of FC21, PM21, or EX21, or (c) IL-21 in a solution mixed with the NK cells so as to be in contact with each other, and any one of FC21, PM21, or EX21 is selected from (a) 41BBL, IL-2, IL-12, IL-18, IL-19, IL-21, IL-22, IL-19, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, IL-39, IL-40, IL-41, IL-42, IL-43, IL-44, IL-45, IL-46, IL-47, IL-48, IL-49, IL-50, IL-51, IL-52, IL-53, IL-54, IL-55, IL-56, IL-57, IL-58, IL-59 ...

42. The method of claim 41, further comprising one or more activating agents, stimulatory peptides, cytokines and / or adhesion molecules selected from: IL-15, IL-7, ULBP, MICA, LFA-1, 2B4, BCM / SLAMF2, CCR7, OX4OL, NKG2D agonists, Delta-1, Notch ligands, NKp46 agonists, NKp44 agonists, NKp30 agonists, other NCR agonists, and CD16 agonists; or (b) membrane-bound TGF-β.

43. 43. The method of claim 42, wherein any one of FC21, PM21, or EX21 further comprises 41BBL.

Images