NZ733149A - Methods of treating hematological disorders, solid tumors, or infectious diseases using natural killer cells - Google Patents

Abstract

Provided herein are methods of treating a hematological disorder, a solid tumor, or an infectious disease in a subject in need thereof using natural killer cells in combination with a second agent, or using natural killer cells with genetic modifications for target specificity and/or homing specificity. ity.

Description

METHODS OF TREATING HEMATOLOGICAL ERS, SOLID , OR INFECTIOUS DISEASES USING L KILLER CELLS This application claims benefit of US. Provisional Patent Application No. 62/098,547, filed December 31, 2014, and US. ional Patent Application No. 62/139,952, filed March 30, 2015, the disclosures of each of which are incorporated by nce herein in its entirety. l. FIELD Provided herein are methods of treating a hematological disorder, a solid tumor, or an ious disease in a subject in need thereof using natural killer cells in ation with a second agent, or using natural killer cells with genetic modifications for target city and/or homing specificity. 2. BACKGROUND Natural killer (NK) cells are cytotoxic lymphocytes that constitute a major component of the innate immune system.

NK cells are activated in response to interferons or macrophage-derived cytokines.

NK cells possess two types of surface receptors, labeled “activating receptors” and “inhibitory receptors,” that l the cells’ cytotoxic activity.

Among other activities, NK cells play a role in the host rejection of tumors and have been shown capable of killing virus-infected cells. Natural killer cells can become activated by cells lacking, or displaying reduced levels of, major histocompatibility complex (MHC) proteins.

Activated and expanded NK cells and LAK cells from peripheral blood have been used in both ex vivo therapy and in vivo treatment of patients having advanced cancer, with some success against bone marrow related diseases, such as leukemia; breast cancer; and certain types of lymphoma.

In spite of the advantageous ties ofNK cells in killing tumor cells and virus- infected cells, there s a great need for developing more efficacious NK cells and more efficacious therapeutic regimens that utilize NK cells. 3. SUMMARY OF THE INVENTION The present invention provides methods of treating a disease (e.g., a hematological disorder, a solid tumor, or an ious e) in a subject in need thereof, using natural killer (NK) cells in combination with a second agent that can be used to treat the disease. Also provided herein are methods of ng a disease (e.g., a hematological disorder, a solid tumor, or an infectious disease) in a subject in need thereof, using NK cells with genetic modifications (e.g., NK cells that comprise a chimeric antigen receptor (CAR) and/or a homing or) for target specificity and/or homing city.

In one aspect, provided herein are methods of ng a cancer in a subject in need thereof, comprising: (a) administering to said subject an isolated tion of natural killer (NK) cells or a pharmaceutical composition thereof; and (b) administering to said subject a second agent or a pharmaceutical composition thereof, wherein said second agent can be used to treat said cancer. In a specific embodiment, said cancer is multiple myeloma.

In certain embodiments, the second agent is an antibody or antigen binding fragment thereof that specifically binds to a tumor-associated antigen (TAA). In specific embodiments, the antibody is a monoclonal antibody. In specific embodiments, the TAA is selected from the group consisting of CD123, CLL-l, CD38, CS-l (also ed to as SLAM7, SLAMF7, CD319, and CRACC), CD138, RORl, FAP, MUCl, PSCA, EGFRVIII, EPHAZ, and GD2. In a more specific embodiment, the second agent is an antibody that binds to CS-l. In more c embodiments, the second agent is elotuzumab (HuLuc63, Bristol Myers-Squibb/Abeie humanized anti-CS-l monoclonal antibody).

In certain embodiments, the second agent is an antibody or n binding nt thereof that specifically binds to a tumor microenvironment-associated antigen (TMAA). In specific embodiments, the antibody is a monoclonal antibody. In specific embodiments, the TMAA is selected from the group consisting of , EGF, PDGF, IGF, and bFGF.

In certain embodiments, the second agent is an antibody or antigen binding fragment thereof that specifically binds to and antagonizes the activity of an immune checkpoint protein.

In specific embodiments, the antibody is a monoclonal dy. In specific embodiments, the immune oint protein is selected from the group consisting of CTLA-4, PD-1, PD-Ll, PD- L2, and LAG—3.

In certain embodiments, the second agent is a bispecific killer cell engager (BiKE).

In specific embodiments, the BiKE comprises a first single chain variable fragment (scFv) that specifically binds to a TAA. In further specific embodiments, the TAA is selected from the group consisting of CD123, CLL-l, CD38, CS-l, CD138, RORl, FAP, MUCl, PSCA, EGFRvIII, EPHAZ, and GDZ. In specific embodiments, the BiKE comprises a second scFv that specifically binds to CD16.

In certain embodiments, the second agent is an anti-inflammatory agent.

In certain embodiments, the second agent is an modulatory agent. In specific embodiments, the second agent is lenalidomide or pomalidomide.

In certain embodiments, the second agent is a cytotoxic agent.

In certain embodiments, the second agent is a cancer vaccine.

In certain embodiments, the second agent is a chemotherapeutic.

In n embodiments, the second agent is an HDAC inhibitor. In other specific ments, the second agent is romidepsin (ISTODAX®, Celgene).

In certain embodiments, the second agent is an siRNA.

In some embodiments, the isolated tion ofNK cells or a pharmaceutical composition thereof is stered before the second agent or a pharmaceutical composition thereof. In some embodiments, the isolated population ofNK cells or a pharmaceutical composition thereof is administered after the second agent or a pharmaceutical composition thereof. In other embodiments, the isolated population ofNK cells or a ceutical composition thereof is administered at the same time as the second agent or a pharmaceutical composition thereof.

In specific ments, the step of administering to said subject an isolated population ofNK cells or a pharmaceutical composition thereof is by injection, infusion, intravenous (IV) administration, intrafemoral administration, or intratumor administration. In specific embodiments, the step of administering to said subject an isolated population ofNK cells or a pharmaceutical composition f is performed with a devise, a matrix, or a scaffold.

In specific embodiments, the step of administering to said subject an ed population ofNK cells or a pharmaceutical composition thereof is by injection. In specific embodiments, the injection ofNK cells is local injection. In more specific ments, the local injection is directly into a solid tumor (e.g., a sarcoma). In specific ments, stration ofNK cells is by injection by syringe. In specific embodiments, administration ofNK cells by injection is aided by laparoscopy, endoscopy, ultrasound, computed tomography, magnetic resonance, or radiology.

In specific embodiments, the step of administering to said subject a second agent or a ceutical composition thereof is by injection, infusion, intravenous (IV) stration, intrafemoral administration, or intratumor administration. In specific embodiments, the step of administering to said subject a second agent or a pharmaceutical composition thereof is performed with a devise, a matrix, or a scaffold.

In various embodiments, the NK cells are fucosylated on the cell surface.

In some ments, the isolated population ofNK cells or a pharmaceutical composition f is administered in a single dose. In other embodiments, the isolated population ofNK cells or a pharmaceutical composition thereof is administered in multiple doses.

In some embodiments, the second agent or a pharmaceutical composition thereof is administered in a single dose. In other embodiments, the second agent or a ceutical composition thereof is administered in le doses.

In another aspect, provided herein are methods of treating a cancer in a t in need f, comprising administering to said subject an isolated population ofNK cells or a pharmaceutical composition thereof, wherein the NK cells comprise a chimeric antigen receptor (CAR), wherein said CAR comprises an extracellular domain, a transmembrane domain, an intracellular stimulatory , and optionally a co-stimulatory domain. Also provided herein are methods of treating a cancer in a subject in need thereof, comprising stering to said subject an isolated population ofNK cells or a pharmaceutical composition thereof, wherein the NK cells comprise a homing receptor, and methods of treating a cancer in a subject in need thereof, comprising administering to said subject an ed population of Natural Killer (NK) cells or a pharmaceutical composition thereof, wherein the NK cells comprise a chimeric antigen or (CAR) and a homing receptor, wherein said CAR comprises an extracellular , a transmembrane domain, an intracellular stimulatory , and optionally a co-stimulatory domain. In various ments, the CAR comprises an extracellular domain, a transmembrane domain, an intracellular stimulatory domain, and a co-stimulatory domain.

In specific embodiments, the NK cells comprising the CAR and/or the homing receptor are derived from CD34+ hematopoietic stem cells (HSCs) that are engineered to express the CAR and/or the homing receptor.

In various ments, the extracellular domain of the CAR is an antigen binding domain. In specific embodiments, the antigen binding domain is an scFv domain. In certain embodiments, the antigen binding domain cally binds to a TAA. In specific embodiments, the TAA is selected from the group consisting of CD123, CLL-l, CD38, CD20, and CS-l. In more specific embodiments, the antigen-binding domain comprises a single—chain Fv (scFv) or antigen-binding fragment derived from an antibody that binds CS-l. In more specific embodiments, the antigen-binding domain comprises a single-chain version of elotuzumab and/or an antigen-binding fragment of elotuzumab, In specific embodiments, the n- binding domain comprises a -chain Fv (scFv) or antigen-binding fragment derived from an antibody that binds CD20 In various embodiments, the intracellular stimulatory domain of the CAR is a CD3 zeta ing domain.

In various embodiments, the co-stimulatory domain of the CAR comprises the ellular domain of CD28, 4-1BB, PD-l, 0X40, CTLA-4, NKp46, NKp44, NKp30, DAP10 or DAPl2.

In various embodiments, the homing receptor is a actic receptor. In specific embodiments, the chemotactic receptor is selected from the group ting of CXCR4, VEGFR2, and CCR7.

In one embodiment, provided herein is a method of treating an individual having multiple myeloma, comprising administering to the individual (1) lenalidomide or pomalidomide and (2) NK cells that comprise a CAR (“CAR NK cells”), wherein said CAR NK cells are effective to treat multiple myeloma in said dual. In specific embodiments of the method of treating an individual with multiple myeloma, said CAR NK cells comprise a CAR extracellular domain, which extracellular domain is a CS-l binding domain. In specific embodiments, the CS-l g domain comprises an scFv or antigen-binding fragment of an dy that binds CS-l. In certain specific embodiments, the CS-l binding domain comprises a single-chain version of elotuzumab and/or an antigen-binding nt of umab.

In another embodiment, provided herein is a method of treating an individual having multiple myeloma, comprising administering to the individual (1) domide or pomalidomide; (2) elotuzumab; and (3) CAR NK cells, wherein said CAR NK cells are effective to treat multiple myeloma in said individual. In certain specific embodiments of the method of treating an dual with multiple myeloma, said CAR NK cells comprise a CAR extracellular domain, which extracellular domain is a CS-l binding domain. In specific embodiments, the CS-l binding domain comprises an scFv or antigen-binding fragment of an antibody that binds CS-l.

In r embodiment, provided herein is a method of treating an individual having a blood cancer (e.g., Burkitt’s lymphoma), comprising administering to the individual (1) romidepsin and (2) CAR NK cells, wherein said CAR NK cells are effective to treat the blood cancer (e.g., Burkitt’s lymphoma) in said individual. In certain specific embodiments of the method of treating an individual with blood cancer (e.g., Burkitt’s lymphoma), said CAR NK cells comprise a CAR extracellular , which extracellular domain is a CD20 binding domain. In specific ments, the CD20 binding domain comprises an scFv or antigen- binding fragment of an antibody that binds CD20.

In specific embodiments, the step of administering to said subject an isolated tion ofNK cells or a pharmaceutical composition thereof is by injection, infusion, intravenous (IV) administration, intrafemoral administration, or intratumor administration. In ic embodiments, the step of administering to said subject an isolated population ofNK cells or a ceutical ition f is performed with a devise, a matrix, or a ld, In specific embodiments, the step of administering to said subject an isolated population ofNK cells or a pharmaceutical composition thereof is by injection. In specific embodiments, the injection ofNK cells is local injection. In more specific embodiments, the local injection is directly into a solid tumor (e.g., a sarcoma). In specific embodiments, administration ofNK cells is by injection by syringe. In specific embodiments, administration ofNK cells by injection is aided by laparoscopy, endoscopy, ound, computed tomography, magnetic nce, or radiology. [003 6] In various embodiments, the NK cells are fucosylated on the cell surface. [003 7] In some embodiments, the isolated population ofNK cells or a pharmaceutical composition thereof is administered in a single dose. In other embodiments, the ed population ofNK cells or a pharmaceutical composition thereof is administered in multiple doses.

In another aspect, ed herein are methods of treating a viral infection in a subject in need thereof, comprising: (a) stering to said subject an isolated tion of natural killer (NK) cells or a pharmaceutical composition thereof, and (b) administering to said subject a second agent or a pharmaceutical composition thereof, wherein said second agent can be used to treat said viral infection.

In certain ments, the second agent is an antibody or antigen binding fragment thereof that specifically binds to and antagonizes the activity of an immune checkpoint protein.

In specific embodiments, the antibody is a monoclonal antibody. In specific embodiments, the immune checkpoint protein is selected from the group ting of CTLA-4, PD-l, PD-Ll, PD- L2, and LAG—3.

In certain embodiments, the second agent is a bispeciflc killer cell engager (BiKE).

In some embodiments, the isolated population ofNK cells or a pharmaceutical composition thereof is administered before the second agent or a pharmaceutical composition thereof. In some embodiments, the isolated population ofNK cells or a pharmaceutical composition thereof is administered after the second agent or a pharmaceutical composition thereof. In other embodiments, the isolated population ofNK cells or a ceutical composition thereof is administered at the same time as the second agent or a ceutical ition thereof.

In specific embodiments, the step of administering to said subject an isolated population ofNK cells or a pharmaceutical composition thereof is by injection, infusion, intravenous (IV) administration, intrafemoral administration, or intratumor administration. In ic embodiments, the step of administering to said subject an isolated tion ofNK cells or a pharmaceutical ition thereof is med with a devise, a matrix, or a scaffold.

In specific embodiments, the step of administering to said subject an isolated population ofNK cells or a pharmaceutical composition thereof is by injection. In specific embodiments, the injection ofNK cells is local injection. In more specific embodiments, the local injection is directly into a solid tumor (e.g., a sarcoma). In specific embodiments, administration ofNK cells is by injection by e. In specific embodiments, administration ofNK cells by injection is aided by laparoscopy, endoscopy, ultrasound, computed aphy, magnetic resonance, or radiology.

In specific embodiments, the step of administering to said subject a second agent or a pharmaceutical composition thereof is by injection, on, intravenous (IV) administration, intrafemoral stration, or intratumor administration. In specific embodiments, the step of administering to said subject a second agent or a pharmaceutical composition thereof is WO 09668 performed with a devise, a matrix, or a scaffold.

In various embodiments, the NK cells are fucosylated on the cell surface.

In some embodiments, the isolated population ofNK cells or a pharmaceutical composition thereof is administered in a single dose. In other ments, the ed population ofNK cells or a pharmaceutical composition thereof is administered in multiple doses.

In some embodiments, the second agent or a pharmaceutical ition thereof is administered in a single dose. In other ments, the second agent or a pharmaceutical composition thereof is administered in multiple doses.

In another aspect, provided herein are methods of treating a viral infection in a subject in need f, comprising administering to said t an isolated population ofNK cells or a pharmaceutical composition f, wherein the NK cells comprise a chimeric antigen receptor (CAR), wherein said CAR ses an extracellular domain, a transmembrane domain, an intracellular stimulatory domain, and optionally a co-stimulatory domain. Also provided herein are methods of treating a viral infection in a subject in need thereof, comprising administering to said subject an isolated population ofNK cells or a pharmaceutical composition thereof, wherein the NK cells comprise a homing receptor, and methods of treating a viral ion in a subject in need thereof, comprising administering to said subject an isolated population of Natural Killer (NK) cells or a pharmaceutical composition thereof, wherein the NK cells comprise a chimeric antigen receptor (CAR) and a homing receptor, wherein said CAR comprises an extracellular domain, a transmembrane domain, an intracellular stimulatory , and optionally a co-stimulatory domain. In various embodiments, the CAR ses an extracellular domain, a transmembrane , an ellular stimulatory domain, and a co- stimulatory domain.

In specific embodiments, the NK cells comprising the CAR and/or the homing receptor are derived from CD34+ hematopoietic stem cells (HSCs) that are engineered to express the CAR and/or the homing receptor.

In various embodiments, the extracellular domain of the CAR is an antigen binding domain. In specific embodiments, the antigen binding domain is an scFv domain.

In various embodiments, the intracellular stimulatory domain of the CAR is a CD3 zeta signaling domain.

In various embodiments, the co-stimulatory domain of the CAR comprises the intracellular domain of CD28, 4-1BB, PD-l, 0X40, , NKp46, NKp44, NKp30, DAPIO or DAP12.

In various embodiments, the homing receptor is a actic receptor. In specific embodiments, the chemotactic receptor is selected from the group consisting of CXCR4, VEGFR2, and CCR7.

In specific embodiments, the step of administering to said subject an isolated tion ofNK cells or a pharmaceutical composition thereof is by injection, infusion, intravenous (IV) administration, intrafemoral administration, or umor administration. In specific embodiments, the step of administering to said subject an isolated population ofNK cells or a pharmaceutical composition thereof is performed with a devise, a matrix, or a scaffold.

In specific embodiments, the step of administering to said subject an isolated population ofNK cells or a pharmaceutical composition thereof is by injection. In c embodiments, the injection ofNK cells is local ion. In more specific embodiments, the local injection is directly into a solid tumor (e.g, a sarcoma). In specific embodiments, administration ofNK cells is by injection by syringe. In specific embodiments, administration ofNK cells by injection is aided by laparoscopy, endoscopy, ultrasound, computed tomography, magnetic resonance, or ogy.

In various embodiments, the NK cells are fucosylated on the cell surface. [005 5] In some embodiments, the isolated population ofNK cells or a ceutical composition thereof is administered in a single dose. In other embodiments, the isolated population ofNK cells or a pharmaceutical composition thereof is administered in multiple doses.

The present ion also provides kits for treating a disease (e.g, a hematological disorder, a solid tumor, or an infectious disease) in a subject in need thereof, which comprise an ed population ofNK cells and a second agent that can be used to treat the disease.

In one aspect, provided herein are kits for treating a cancer in a subject in need thereof, comprising: (a) an isolated population ofNK cells or a ceutical composition thereof; and (b) a second agent or a pharmaceutical composition thereof, wherein said second agent can be used to treat said . The second agent can be any that may be used in the methods of treating a cancer as provided above. [005 8] In another , ed herein are kits for treating a viral infection in a subject in need thereof, comprising: (a) an isolated population ofNK cells or a pharmaceutical composition thereof; and (b) a second agent or a pharmaceutical composition thereof, wherein said second agent can be used to treat said viral infection. The second agent can be any that may be used in the methods of treating a viral ion as provided above.

In various embodiments of the methods or kits provided herein, the NK cells are placental intermediate natural killer (PiNK) cells. In certain embodiments, the PiNK cells are derived from placental cells. In specific embodiments, the placental cells are obtained from tal perfusate. In specific embodiments, the placental cells are obtained from placental tissue that has been mechanically and/or enzymatically disrupted.

In various embodiments of the methods or kits provided herein, the NK cells are ted NK cells. In certain embodiments, the activated NK cells are produced by a process comprising: (a) seeding a tion of hematopoietic stem or progenitor cells in a first medium comprising interleukin-15 (IL-15) and, optionally, one or more of stem cell factor (SCF) and interleukin-7 (IL-7), wherein said IL-15 and optional SCF and IL-7 are not sed within an undefined component of said medium, such that the population expands, and a plurality of hematopoietic stem or itor cells within said population of hematopoietic stem or progenitor cells differentiate into NK cells during said expanding; and (b) expanding the cells from the step (a) in a second medium comprising interleukin-2 (IL-2), to produce a population of activated NK cells. In certain embodiments, the activated NK cells are ed by a process comprising: expanding a population of hematopoietic stem or progenitor cells in a first medium comprising one or more of stem cell factor (SCF), interleukin-7 (IL-7) and interleukin-15 (IL- ), and wherein said SCF, IL-7 and IL-15 are not comprised within an undefined component of said medium, and wherein a plurality of hematopoietic stem or progenitor cells within said population of hematopoietic stem or progenitor cells differentiate into NK cells during said expanding, and wherein a second step of said method comprises expanding the cells from the first step in a second medium comprising interleukin-2 (IL-2), to e activated NK cells.

In specific embodiments, the first medium further comprises one or more of Fms- yrosine kinase 3 ligand (Flt3 -L), thrombopoietin (Tpo), interleukin-2 (IL-2), or heparin. In further specific embodiments, the first medium further comprises fetal bovine serum or human serum. In r specific embodiments, the SCF is t at a concentration of about 1 to about 150 ng/mL in the first medium. In further specific embodiments, the Flt3 -L is present at a tration of about 1 to about 150 ng/mL in the first medium. In further specific embodiments, the IL-2 is present at a concentration of about 50 to about 1500 IU/mL in the first medium. In further specific embodiments, the IL-7 is present at a concentration of about 1 to about 150 ng/mL in the first medium. In further specific embodiments, the IL-15 is t at a concentration 1 to about 150 ng/mL in the first medium. In further specific embodiments, the Tpo is present at a tration of about 1 to about 150 ng/mL in the first medium. In further specific embodiments, the heparin is present at a concentration of about 0.1 to about 30 U/mL in the first medium.

In specific embodiments, said IL-2 in the second step above is present at a concentration 50 to about 1500 IU/mL in the second medium.

In specific embodiments, said second medium additionally comprises one or more of fetal calf serum (FCS), transferrin, insulin, ethanolamine, oleic acid, linoleic acid, ic acid, bovine serum albumin (B SA) and emagglutinin.

In specific embodiments, the hematopoietic stem or progenitor cells are CD34+.

In specific embodiments, the hematopoietic stem or progenitor cells comprise hematopoietic stem or progenitor cells from human placental perfusate and hematopoietic stem or progenitor cells from umbilical cord, wherein said placental perfusate and said umbilical cord blood are from the same placenta.

In specific embodiments, the feeder cells in step (b) above se mitomycin C- treated peripheral blood mononuclear cells (PBMC), K562 cells or tissue culture-adherent stem cells.

In specific embodiments, the NK cells are CD3‘CD56+CD16_, In a further specific ment, the I\K cells are additionally CD94+CD117+. In another further specific embodiment, the I\K cells are additionally CD161‘. In another further specific embodiment, the NK cells are additionally NKG2D+. In another further specific ment, the NK cells are additionally NKp46+. In another further specific embodiment, the NK cells are additionally CD226+.

In various embodiments of the s or kits provided herein, the NK cells are Three—Step s NK (TSPNK) cells. In specific embodiments, the TSPNK cells are NK progenitor cells. In n embodiments, the TSPNK cells are ed by a process comprising: (a) culturing hematopoietic stem cells or progenitor cells in a first medium comprising Flt3L, TPO, SCF, IL-7, G—CSF, IL—6 and GM-CSF; (b) subsequently culturing said cells in a second medium comprising Flt3L, SCF, IL-15, and IL-7, IL-17 and IL-15, G—CSF, IL-6 and GM —CSF; and (c) subsequently culturing said cells in a third medium comprising SCF, IL- , IL—7, IL-2, G—CSF, IL-6 and GM-CSF.

In specific embodiments, the duration of culturing step (a) is 7-9 days, the duration of ing step (b) is 5—7 days, and the duration of culturing step (c) is 5-9 days. In c embodiments, the duration of ing step (a) is 7-9 days, the duration of culturing step (b) is 5- 7 days, and the duration of culturing step (c) is 21-35 days.

In specific ments, the hematopoietic stem or progenitor cells used in the process are CD34+.

In specific embodiments, the hematopoietic stem or itor cells comprise hematopoietic stem or progenitor cells from human placental perfusate and hematopoietic stem or progenitor cells from umbilical cord, wherein said placental perfusate and said umbilical cord blood are from the same placenta.

In specific embodiments, CD34- cells comprise more than 80% of the TSPNK cells at the end of step (a) of the process of producing TSPNK cells above.

In specific embodiments, the TSPNK cells comprise no more than 40% CD3- CD56+ cells.

In specific embodiments, the TSPNK cells comprise cells which are CD52+ CD1 17+.

In various embodiments of the methods or kits bed herein, the NK cells are produced by a process comprising: (a) culturing hematopoietic stem or progenitor cells in a first medium comprising a stem cell mobilizing agent and thrombopoietin (Tpo) to produce a first tion of cells; (b) culturing the first population of cells in a second medium comprising a stem cell mobilizing agent and interleukin-15 (IL-15), and lacking Tpo, to produce a second population of cells; and (c) ing the second tion of cells in a third medium comprising IL-2 and IL-15, and lacking a stem cell mobilizing agent and LMWH, to produce a third population of cells; wherein the third population of cells comprises natural killer cells that are CD56+, CD3-, CD16— or CDl6+, and CD94+ or CD94-, and wherein at least 80% of the natural killer cells are Viable.

The cancer in any one of the methods or kits provided herein can be a hematological cancer or a solid tumor.

In preferred embodiment of any one of the methods or kits provided herein, the t is a human. 3.1. Terminology As used herein, “natural killer cell” or “NK cells” without r modification, includes natural killer cells derived from any tissue , and include mature l killer cells as well as natural killer progenitor cells. In some embodiments, NK cells are placental intermediate l killer (PiNK) cells as described in Section 5.1.1. In some embodiments, NK cells are ted NK cells as described in Section 5.1.2. In some embodiments, NK cells are Three—Step Process NK (TSPNK) cells as described in Section 513 Natural killer cells can be derived from any tissue source, and include mature natural killer cells as well as NK progenitor cells.

As used herein, the term “NK progenitor cell population” refers to a population of cells comprising cells of the natural killer cell lineage that have yet to develop into mature NK cells, as ted by, e.g., the level(s) of expression one or more phenotypic markers, e.g., CD56, CD16, and KIRs. In one embodiment, the NK progenitor cell population comprises cells with low CD16 and high CD56.

As used herein, “PiNK” and “PiNK cells” refer to placental intermediate natural killer cells that are obtained from human placenta, e.g., human placental perfusate or placental tissue that has been mechanically and/or enzymatically disrupted. The cells are CD56+ and CD16‘, e. g., as determined by flow cytometry, e.g., cence-activated cell sorting using antibodies to CD56 and CD16.

As used herein, “placental perfusate” means perfusion on that has been passed through at least part of a placenta, e.g., a human placenta, e.g., through the placental vasculature, and includes a ity of cells collected by the perfusion solution during passage through the placenta.

As used herein, “placental perfusate cells” means nucleated cells, e.g., total nucleated cells, ed from, or isolatable from, placental perfusate.

As used herein, “feeder cells” refers to cells of one type that are co—cultured with cells of a second type, to provide an environment in which the cells of the second type can be 2015/068069 maintained, and perhaps proliferate. Without being bound by any theory, feeder cells can provide, for e, peptides, ptides, electrical signals, organic molecules (e.g., steroids), nucleic acid molecules, growth factors (e.g., bFGF), other factors (e.g., cytokines), and metabolic nutrients to target cells. In certain embodiments, feeder cells grow in a mono-layer.

As used herein, the term “hematopoietic cells” includes hematopoietic stem cells and hematopoietic itor cells.

As used herein, the “undefined component” is a term of art in the culture medium field that refers to components whose constituents are not generally provided or quantified.

Examples of an “undefined component” include, without limitation, human serum (e.g., human serum AB) and fetal serum (e.g, fetal bovine serum or fetal calf serum).

As used herein, “+”, when used to indicate the presence of a particular cellular marker, means that the cellular marker is detectably present in fluorescence activated cell g over an isotype control, or is detectable above background in quantitative or semi-quantitative As used herein, “—”, when used to indicate the presence of a particular cellular marker, means that the cellular marker is not detectably present in fluorescence ted cell sorting over an isotype control, or is not detectable above background in quantitative or semi- quantitative RT-PCR.

As used herein, “cancer” refers to a hematological cancer or a solid tumor. 4. BRIEF DESCRIPTION OF S Fig. 1 depicts the antibody-dependent cellular cytotoxicity (ADCC) activities of PiNK cells against Daudi cells at ent trations of rituximab.

Fig. 2 depicts the expression of PD-Ll and CS-l on the MM cells lines MM285, MMZ93, RPM18226, and OPM2. Cells were stained with D-Ll APC (Biolegend, Cat# ), anti-CS1 PE—Cy7 (Biolegend, Cat# 331816), and 7-AAD (BD Bioscience, Cat# 559925) according to the manufacturer’s protocol. Data were acquired on BD LSRFortessa (BD Biosciences) and analyzed using FLOWJO® re (Tree Star). Data were expressed as % positive cells gated under 7—AAD- single cells. Setting of the % positive gate was done using unstained sample as control. The left—most peak in the panels indicates the control, whereas the right-most peak indicates the sample. The percentage of cells positive for PD-Ll was as follows: 71.6% MIVI285, 70.7% MM293, 66.2% OPM-2, and 94.4% RPMI8226. The percentage of cells positive for CS—l was as follows: 31.8% MMZSS, 58.8% MM293, 93.4% OPM-2, and 29.5% RPM18226.

Fig. 3 depicts the 24-hour cytotoxicity assay of three-stage NK cells against the indicated MM cell lines and primary MM samples at a 3:1 effector-to-target ratio. The number of viable target cells (PKH26+TO-PRO-3') in each sample was quantified by flow cytometry using ng beads following the protocol provided by the manufacturer (Invitrogen, Cat# C3 6950). Counting beads were introduced in this assay in order to account for any ial proliferation of tumor cells during the prolonged 24 hour culture After incubation for 24 hours at 37° C and 5% C02, cells were harvested, followed by staining with 1 uM TO-PRO-3 to identify the dead cells. Results are depicted as mean i rd deviation of the mean.

Fig. 4 depicts the 24-hour cytotoxicity assay of three-stage NK cells against OPM2 cells at a 3:1 effector-to—target ratio, along with the following additional conditions: IL-15 (5 ng/mL) (Invitrogen, Cat# PHC9153); IL-2 (200 IU/mL) (Invitrogen, Cat# PHC0023), anti-PD- L1 (lOng/mL) (Affymetrix, Cat# 1682), anti-IgG(10ng/mL) (AffymetriX, Cat# 16 82); REVLIMID® (lenalidomide, luM), or DMSO (0.1%) in 48-well . Target cells alone were plated as controls. After incubation for 24 hours at 37° C and 5% C02, cells were ted, followed by staining with 1 uM TO-PRO-3 to identify the dead cells. Results are depicted as mean :: standard deviation of the mean.

. DETAILED DESCRIPTION Provided herein are methods of ng a disease (e.g., a hematological disorder, a solid tumor, or an ious disease) in a subject in need thereof, using natural killer (NK) cells in combination with a second agent that can be used to treat the disease. Also provided herein are methods of treating a disease (e.g., a hematological disorder, a solid tumor, or an infectious disease) in a t in need thereof, using NK cells with genetic modifications (e.g., NK cells that comprise a chimeric antigen receptor (CAR) and/or a homing receptor) for target specificity and/or homing city. Kits for treating a disease (e.g., a logical disorder, a solid tumor, or an infectious e) in a subject in need thereof, which comprise an isolated population ofNK cells and a second agent that can be used to treat the disease, or which comprise an isolated population ofNK cells with c modifications (e.g, NK cells that se a chimeric antigen receptor (CAR) and/or a homing receptor) are also provided herein. 2015/068069 .1. NK Cells bed herein are NK cells, including PiNK cells, activated NK cells, TSPNK cells, and NK cells produced by the three-stage method. .1.1. Placental Intermediate Natural Killer (PiNK) Cells In some embodiments, natural killer cells are placental intermediate natural killer (PiNK) cells (see also US. Patent No. 8,263,065, the disclosure of which is hereby orated by reference in its entirety). In various embodiments, PiNK cells are derived from placental cells. In specific embodiments, the placental cells are obtained from placental perfusate, e.g., human placental perfusate. In specific embodiments, the placental cells are obtained from tal tissue that has been mechanically and/or enzymatically disrupted.

PiNK cells are characterized as being CD56+CD16_, 1'.e., ying the CD56 cellular marker and lacking the CD16 cellular marker, e.g., as determined by flow cytometry, e.g, fluorescence-activated cell sorting using antibodies against CD16 and CD56, as described above.

In n embodiments, the PiNK cells are CD3‘.

In other embodiments, the PiNK cells do not exhibit one or more ar markers exhibited by fully mature natural killer cells (e.g., CD16), or t such one or more markers at a detectably reduced level ed to fully mature natural killer cells, or exhibit one or more cellular markers associated with natural killer cell precursors but not fully mature natural killer cells. In a specific embodiment, a PiNK cell described herein expresses NKGZD, CD94 and/or NKp46 at a ably lower level than a fully mature NK cell. In another specific embodiment, a plurality of PiNK cells bed herein expresses, in total, NKGZD, CD94 and/or NKp46 at a detectably lower level than an equivalent number of fully mature NK cells.

In certain embodiments, PiNK cells express one or more of the microRNAs hsa—miR— 100, hsa-miR—127, hsa-miR—Zl l, hsa—miR-302c, hsa—miR-326, hsa-miR-337, hsa-miR-497, hsa- miR3p, hsa-miR5p, hsa-miR-517b, hsa-miR-517c, hsa-miR-518a, hsa-miR-518e, hsa- miR-5l9d, R-520g, hsa—miR-520h, R-564, hsa—miR-566, hsa-miR-6l8, and/or hsa- miR—99a at a detectably higher level than peripheral blood natural killer cells.

Because the post-partum placenta comprises tissue and cells from the fetus and from the mother placental perfusate, depending upon the method of collection, PiNK cells can comprise fetal cells only, or a substantial majority of fetal cells (e.g, greater than about 90%, 95%, 98% or 99%), or can comprise a mixture of fetal and maternal cells (e.g., the fetal cells WO 09668 comprise less than about 90%, 80%, 70%, 60%, or 50% of the total nucleated cells of the perfusate). In one embodiment, the PiNK cells are derived only from fetal placental cells, e.g, cells obtained from -circuit perfusion of the placenta (see above) wherein the perfusion produces perfusate comprising a substantial majority, or only, fetal placental cells. In another embodiment, the PiNK cells are derived from fetal and maternal cells, e.g., cells ed by perfusion by the pan method (see above), n the perfusion produced perfusate comprising a mix of fetal and maternal placental cells. Thus, in one ment, the NK cells are a population of placenta-derived intermediate natural killer cells, the substantial majority of which have the fetal genotype. In another embodiment, the NK cells are a population of placenta— derived ediate natural killer cells that comprise natural killer cells having the fetal genotype and l killer cells having the maternal phenotype. .1.2. Activated NK Cells In some embodiments, l killer cells are activated NK cells (1'. e., Two-Step NK cells, or TSNK cells) (see also U. S. Patent ation Publication No. 2012/0148553, the disclosure of which is hereby incorporated by reference in its entirety), which are NK cells produced by any method/process described below in Section 5.2.4.

In a specific embodiment, the activated NK cells are CD3‘CD56+. In a specific ment, the activated NK cells are CD3‘CD56+CD16_. In another specific embodiment, the ted I\K cells are additionally CD94+CD117+. In another specific embodiment, the activated I\K cells are additionally CD161‘. In another specific embodiment, the activated NK cells are additionally NKG2D+. In another specific embodiment, the activated NK cells are additionally NKp46+. In another specific embodiment, the activated NK cells are additionally CD226] In certain embodiments, greater than 50%, 60%, 70%, 80%, 90%, 92%, 94%, 96%, 98% of said activated NK cells are CD56+ and CD16‘. In other embodiments, at least 50%, 60%, 70%, 80%, 82%, 84%, 86%, 88% or 90% of said activated NK cells are CD3‘ and CD56+.

In other embodiments, at least 50%, 52%, 54%, 56%, 58% or 60% of said activated NK cells are NKG2D+. In other embodiments, fewer than 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4% or 3% of said cells are NKB1+. In certain other ments, fewer than 30%, 20%, 10%, 8%, 6%, 4% or 2% of said activated NK cells are NKAT2+. In certain other embodiments, fewer than %, 20%, 10%, 8%, 6%, 4% or 2% of said activated NK cells are CD56+ and CD16+. In more specific embodiments, at least 10%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65% or 70% of said CD3‘, CD56+ activated NK cells are NKp46+. In other more specific embodiments, at least 10%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or 85% of said CD3‘, CD56" activated NK cells are CD117+. In other more specific embodiments, at least 10%, %, 25%, 30%, 35%, 40%, 45% or 50% of said CD3‘, CD56+ activated NK cells are CD94+.

In other more specific embodiments, at least 10%, 20%, 25%, 30%, 35%, 40%, 45% or 50% of said CD3‘, CD56+ activated NK cells are CD161‘. In other more specific embodiments, at least %, 12%, 14%, 16%, 18% or 20% of said CD3‘, CD56+ activated NK cells are CD226+. In more specific embodiments, at least 20%, 25%, 30%, 35% or 40% of said CD3‘, CD56+ activated NK cells are CD7+. In more c embodiments, at least 30%, 35%, 40%, 45%, 50%, 55% or 60% of said CD3‘, CD56+ activated NK cells are CD5+.

Activated NK cells can have a fetal genotype or a maternal genotype. For example, because the artum placenta, as a source of hematopoietic cells suitable for producing activated NK cells, comprises tissue and cells from the fetus and from the mother, placental perfusate can comprise fetal cells only, or a substantial majority of fetal cells (e.g., r than about 90%, 95%, 98% or 99%), or can comprise a mixture of fetal and maternal cells (e.g., the fetal cells comprise less than about 90%, 80%, 70%, 60%, or 50% of the total nucleated cells of the perfusate). In one embodiment, the ted NK cells are derived only from fetal placental hematopoietic cells, e.g., cells obtained from closed-circuit perfusion of the placenta wherein the perfusion produces perfusate comprising a ntial majority, or only, fetal placental hematopoietic cells. In another embodiment, the activated NK cells are d from fetal and al cells, e.g., cells obtained by perfusion by the pan method (see above), wherein the perfusion produced perfusate comprising a mix of fetal and maternal placental cells. Thus, in one embodiment, the activated NK cells are derived from a population of placenta-derived intermediate natural killer cells, the substantial majority of which have the fetal genotype. In another embodiment, the activated NK cells are derived from a population of placenta-derived intermediate l killer cells that comprise natural killer cells having the fetal genotype and natural killer cells having the maternal ype.

In certain ments, the activated NK cells or populations enriched for activated NK cells can be assessed by detecting one or more functionally relevant markers, for example, CD94, CD161, NKp44, DNAM-l, 2B4, NKp46, CD94, KIR, and the NKG2 family of activating receptors (e.g, NKGZD). ally, the xic activity of isolated or enriched natural killer cells can be assessed, e.g., in a cytotoxicity assay using tumor cells, e.g., cultured K562, LN-18, U937, WERI-RB-l, U-1 18MG, HT-29, HCC2218, KG—l, or U266 tumor cells, or the like as target cells. .1.3. Three-Step Process NK (TSPNK) Cells In some ments, natural killer cells are Three-Step Process NK (TSPNK) cells, which are NK cells produced by any method/process described below in Section 5.2.5. In specific embodiments, the TSPNK cells are NK progenitor cells (see also U. S. Patent Application Publication No. 2012/0148553, the disclosure of which is hereby incorporated by reference in its ty). .1.3.1. TSPNK Cells In one embodiment, said isolated TSPNK cell population produced by a three-step process described herein comprises a greater percentage of CD3—CD56+ cells than an NK progenitor cell population produced by a three-step process described herein, e.g., an NK progenitor cell population produced by the same three-step process with the exception that the third culture step used to produce the NK progenitor cell population was of r duration than the third culture step used to produce the TSPNK cell tion. In a specific embodiment, said TSPNK cell population comprises about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% CD3—CD56+ cells. In another specific embodiment, said TSPNK cell population comprises no less than 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% CD3—CD56+ cells. In another specific embodiment, said TSPNK cell tion comprises between 65%-70%, 70%-75%, 75%-80%, 80%-85%, 85%-90%, %, or 95%-99% CD3—CD56+ cells. In r specific embodiment, said TSPNK cell population produced by a three-step process bed herein is produced using a three-step process that ses a long third culture step, e.g., a third culture step of 18-20, 19-21, 20-22, or 21-23 days.

In certain embodiments, said CD3‘CD56Jr cells in said TSPNK cell population comprises CD3‘CD56+ cells that are additionally CD117+, n said TSPNK cell population comprises a lesser percentage of CD3‘CD56+CD117+ cells than an NK progenitor cell population produced by a three-step process described herein, e.g., an NK progenitor cell population produced by the same three-step process with the ion that the third culture step used to produce the NK progenitor cell population was of shorter on than the third culture step used to produce the TSPNK cell population.

In certain embodiments, said 56+ cells in said TSPNK cell population comprises CD3‘CD56+ cells that are onally CD161+, wherein said TSPNK cell population comprises a lesser tage of CD3‘CD56+CD161+ cells than an NK itor cell population produced by a three-step process described herein, e.g., an NK progenitor cell population produced by the same three-step process with the exception that the third culture step used to produce the NK progenitor cell population was of shorter duration than the third culture step used to produce the TSPNK cell population.

In certain embodiments, said CD3‘CD56+ cells in said TSPNK cell population comprises CD3‘CD56+ cells that are onally NKp46+, wherein said TSPNK cell population comprises a greater percentage of CD3'CD56WKp46+ cells than an NK progenitor cell population produced by a step process described herein, e.g., an NK itor cell tion produced by the same three-step process with the exception that the third culture step used to produce the NK progenitor cell population was of shorter duration than the third culture step used to produce the TSPNK cell population.

In certain embodiments, said CD3‘CD56+ cells in said TSPNK cell tion comprises CD3‘CD56+ cells that are additionally CD16-, n said TSPNK cell population comprises a greater percentage of 56+CD16- cells than an NK progenitor cell population produced by a three-step process described herein, e.g., an NK progenitor cell population produced by the same three-step process with the exception that the third culture step used to produce the NK itor cell population was of shorter duration than the third culture step used to produce the TSPNK cell tion. In another embodiment, the TSPNK cells produced using the three-step process described herein possess longer telomeres than peripheral blood (PB) derived NK cells.

In one embodiment, a TSPNK cell population produced by a three-step process described herein comprises cells which are CD117+. In a specific embodiment, said TSPNK cell populations comprise no more than about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% CD117+ cells. In one embodiment, a TSPNK cell population produced by a three—step process described herein comprises cells which are . In a c ment, said TSPNK cell populations comprise no more than about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% NKGZD+ cells. In one embodiment, a TSPNK cell population produced by a three—step process described herein comprises cells which are NKp44+. In a specific embodiment, said TSPNK cell populations comprise no more than about 5%, 10%, 15%, 20%, %, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% NKp44+ cells. In one embodiment, a TSPNK cell population produced by a three-step process described herein comprises cells which are CD52+. In a specific embodiment, said TSPNK cell populations comprise no more than about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% CD52+ cells. In a particular embodiment, said TSPNK cell population produced by a three-step process described herein comprises cells which are CD52+ CD117+. In one embodiment, a TSPNK cell population produced by a three- step process described herein comprises cells which are CD244+. In a c embodiment, said TSPNK cell populations se no more than about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% CD244+ cells. In a particular embodiment, said TSPNK cell tion produced by a step process described herein comprises cells which are CD244+ CD117+. In one embodiment, a TSPNK cell population produced by a three-step process described herein comprises cells which are LFA-1+. In a specific embodiment, said TSPNK cell populations comprise no more than about 5%, 10%, 15%, %, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% LFA- 1+ cells. In one embodiment, a TSPNK cell population produced by a three-step process described herein comprises cells which are CD94+. In a specific embodiment, said TSPNK cell populations comprise no more than about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% CD94+ cells. 2. NK Progenitor Cells In one ment, said isolated NK progenitor cell population comprises a low percentage of CD3—CD56+ cells as compared to the percentage of CD3—CD56+ cells associated with non-progenitor NK cell populations, such as non-progenitor NK cell populations produced by the three-step methods described , e.g., the NK progenitor cell population comprises about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% CD3—CD56+ cells. In another specific embodiment, said NK progenitor cell population comprises no more than 5%, 10%, %, 20%, 25%, 30%, 35%, 40%, 45%, or 50% CD3—CD56+ cells. In another specific embodiment, said NK progenitor cell population comprises between 0%-5%, 5%-10%, 10%— %, 15%-20%, 20%—25%, 25%-30%, %, 35%-40%, 40%-45%, or 45%-50% CD3— CD56+ cells. In some embodiments, said NK progenitor cell tions, e.g., a NK progenitor cell tions that comprise a low percentage of CD3—CD56+ cells as compared to the tage of CD3—CD56+ cells associated with non-progenitor NK cell populations, comprise no more than 1%, no more than 2%, no more than 3%, no more than 4%, no more than 5%, no more than 10%, or no more than 15% CD3—CD56+ cells. In another specific embodiment, said NK progenitor cell populations produced by a three-step s described herein are produced using a three-step s that comprises a short third culture step, e.g., a third culture step of 4- 6, 5-7, 6-8, or 7-9 days.

In certain embodiments, said CD3‘CD56+ cells in said NK progenitor cell populations are additionally CD117+. In a specific embodiment, about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of said CD3‘CD56+ cells in said NK progenitor cell populations are CD117+.

In another specific embodiment, no less than 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of said CD3‘CD56+ cells in said NK progenitor cell populations are CD117+. In another specific ment, between 65%-70%, 70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%- 95%, or 95%-99% of said 56+ cells in said NK progenitor cell populations are CD1 17+.

In certain embodiments, said CD3—CD56+ cells in said NK itor cell populations are additionally . In a specific embodiment, about 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% of said CD3—CD56+ cells in said NK progenitor cell populations are CDl6l+. In another specific embodiment, no less than 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% of said CD3—CD56+ cells in said NK progenitor cell populations are CDl6l+. In another specific embodiment, between 40%-45%, 45%-50%, 50%-55%, 55%-60%, 60%-65%, %, or 70%-75% of said CD3—CD56+ cells in said NK progenitor cell populations are CDl6l+.

In certain embodiments, said CD3—CD56+ cells in said NK progenitor cell populations are additionally NKp46+. In a specific embodiment, about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more of said CD3—CD56+ cells in said NK progenitor cell populations are NKp46+. In a more specific ment, about 25%, %, 35%, 40%, 45%, 50%, or 55% of said CD3—CD56+ cells in said NK progenitor cell populations are NKp46+. In another specific embodiment, no more than 25%, 30%, 35%, 40%, 45%, 50%, or 55% of said CD3—CD56+ cells in said NK progenitor cell populations are NKp46+. In another specific embodiment, between 25%-3 0%, %, 35%-40%, 40%-45%, 45%—50%, 50%—55%, 55%-60%, 60%-65%, 65%-70%, 70%—75%, 75%-80%, 80%-85%, 85%- 90% or more of said 56+ cells in said NK progenitor cell populations are NKp46+. In a more specific embodiment, between 25%—30%, 30%-35%, 35%-40%, 40%-45%, %, or 50%-55% of said CD3—CD56+ cells in said NK progenitor cell populations are NKp46+.

In certain embodiments, said NK progenitor cell population contains cells that are CD56+CD16_. In certain ments, CD3‘CD56+ cells in said NK progenitor cell populations are CD167 In certain embodiments, CD3‘CD56+ cells in said NK progenitor cell tions are CD16+. In a specific embodiment, said NK progenitor cell populations comprise no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% CD16+ cells. In another specific embodiment, said NK progenitor cell populations comprise between O%-5%, 5%-10%, 10%- %, 15%-20%, or 20%-25% CD16Jr cells. In some embodiments, said NK progenitor cell populations se no more than 1%, no more than 2%, no more than 3%, no more than 4%, no more than 5%, no more than 10%, or no more than 15% CD16+ cells.

] In certain ments, said CD3—CD56+ cells in said NK progenitor cell populations are additionally CD16-. In certain embodiments, said 56+ cells in said NK progenitor cell populations are additionally CD117+ and CD161+. In certain embodiments, said CD3—CD56+ cells in said NK progenitor cell populations are additionally CD16-, CD117+ and CD161+. In certain embodiments, said CD3—CD56+ cells in said NK itor cell populations are additionally CD16-, CD117+, CD161+, and NKp46+.

In one embodiment, an NK progenitor cell population produced by a three-step process described herein comprises no more than about 40% CD3—CD56+ cells. In one ment, an NK progenitor cell population produced by a three-step process described herein comprises cells which are CD117+. In a specific embodiment, said NK progenitor cell populations comprise no more than about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% CD117+ cells. In one embodiment, an NK progenitor cell population produced by a three-step process described herein ses cells which are CD52+. In a specific ment, said NK itor cell populations comprise no more than about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% CD52+ cells. In a particular embodiment, said NK progenitor cell population produced by a three—step process described herein ses cells which are CD52+ CD117+. In one ment, an NK progenitor cell population ed by a three-step process described herein ses cells which are CD244+. In a c embodiment, said NK progenitor cell populations comprise no more than about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% CD244+ cells. In a particular embodiment, said NK itor cell population produced by a three—step process described herein comprises cells which are CD244+ CD117+. In one embodiment, an NK progenitor cell population produced by a three—step process described herein comprises cells which are .

In a specific embodiment, said NK progenitor cell populations comprise no more than about 5%, %, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% LFA-1+ cells. In one embodiment, an NK progenitor cell population produced by a three- step process described herein ses cells which are CD94+. In a specific embodiment, said NK progenitor cell populations comprise no more than about 5%, 10%, 15%, 20%, 25%, 30%, %, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% CD94+ cells.

In particular embodiments, an NK progenitor cell population produced by a three—step process described herein comprises a greater proportion of CD56- cells than CD56+ cells. In particular embodiments, an NK progenitor cell population produced by a three-step process described herein entiates in Vivo or eX Vivo into a population with an increased proportion of CD56+ cells.

In a specific embodiment, an NK progenitor cell population ed by a step process described herein comprises a low percentage of CD34‘CD117+ cells as ed to the percentage of CD34‘CD1 17+ cells associated with a non-progenitor NK cell population, e.g., the NK progenitor cell population comprises about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% CD34‘CD117+ cells. In another specific embodiment, said NK progenitor cell population comprises no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% CD34'CD117+ cells. In another specific embodiment, said NK progenitor cell population comprises between 0%-5%, 5%-10%, 10%-15%, %, 20%-25%, 25%-30%, 30%-3 5%, %-40%, 40%-45%, or 45%-50% CD34‘CD117+ cells. In some embodiments, said NK progenitor cell population comprises no more than 1%, no more than 2%, no more than 3%, no more than 4%, no more than 5%, no more than 10%, or no more than 15% CD34‘CD117+ cells.

WO 09668 In another specific embodiment, said NK progenitor cell population produced by a three-step process described herein is produced using a three-step process that comprises a short third culture step, e.g., a third culture step of 4-6, 5-7, 6-8, or 7-9 days.

In a specific embodiment, an NK progenitor cell population produced by a three-step process described herein comprises a low percentage of CD161+ cells as compared to the percentage of CD161+ cells associated with a non-progenitor NK cell population, e.g, the NK progenitor cell population comprises about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% CD161+ cells. In r specific embodiment, said NK progenitor cell population comprises no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% CD161+ cells. In another c embodiment, said NK progenitor cell population comprises between 0%—5%, 5%—10%, 10%-15%, 15%—20%, 20%—25%, 25%—30%, 5%, 35%,—40%, 40%,—45%, or 45%-50% CD1614r cells. In some ments, said NK progenitor cell tion comprises no more than 1%, no more than 2%, no more than 3%, no more than 4%, no more than 5%, no more than 10%, or no more than 15% CD161+ cells. In another specific embodiment, said NK itor cell population produced by a three-step process bed herein is produced using a three-step process that ses a short third culture step, e.g., a third culture step of 4-6, 5-7, 6-8, or 7-9 days.

In a specific embodiment, an NK progenitor cell population produced by a three-step process described herein comprises a low percentage of NKp46+ cells as compared to the tage ofNKp46+ cells associated with a non-progenitor NK cell population, e.g., the NK progenitor cell population comprises about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% NKp46+ cells. In another specific embodiment, said NK progenitor cell population comprises no more than 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% NKp46+ cells. In another specific embodiment, said NK progenitor cell population comprises between O%-5%, 5%-10%, 10%-15%, 15%-20%, 20%-25%, 25%-30%, 30%-35%, 35%-40%, 40%-45%, or 45%-50% NKp46Jr cells. In some embodiments, said NK progenitor cell population comprises no more than 1%, no more than 2%, no more than 3%, no more than 4%, no more than 5%, no more than 10%, or no more than 15% NKp46+ cells. In another specific embodiment, said NK progenitor cell population produced by a three-step process bed herein is produced using a step s that comprises a short third culture step, e.g., a third culture step of 4-6, 5-7, 6-8, or 7-9 days.

In a specific embodiment, an NK progenitor cell population produced by a three-step process described herein comprises a low percentage of CD56+CD16— cells as compared to the percentage of D16- cells associated with a non-progenitor NK cell population, e.g., the NK progenitor cell population ses about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% CD56+CD16- cells. In another c embodiment, said NK progenitor cell population comprises no more than 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% D16- cells. In another c embodiment, said NK progenitor cell population ses between 0%-5%, 5%-10%, 10%-15%, 15%-20%, 20%-25%, 25%-30%, 30%-3 5%, %-40%, 40%—45%, or 45%—50% CD56+CD16- cells. In some embodiments, said NK progenitor cell tion comprises no more than 1%, no more than 2%, no more than 3%, no more than 4%, no more than 5%, no more than 10%, or no more than 15% CD56+CD16- cells.

In another specific embodiment, said NK itor cell population produced by a three-step process described herein is produced using a step process that comprises a short third culture step, e.g., a third e step of 4-6, 5—7, 6-8, or 7-9 days.

In one embodiment, an NK progenitor cell population produced by a three-step process bed herein comprises cells that are CD52+CD117+. In a specific embodiment, an NK progenitor cell population produced by a three-step s described herein comprises a higher percentage of CD52+CD117+ cells as compared to the percentage of CD52+CD1 17+ cells associated with a hematopoietic progenitor cell population. In a specific ment, an NK progenitor cell population produced by a three-step process described herein comprises a higher percentage of CD52+CD1 17+ cells as compared to the percentage of CD52+CD117+ cells associated with a non-progenitor NK cell population, e.g., the NK progenitor cell population comprises about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more CD52+CD117+ cells. In another specific embodiment, said NK progenitor cell population comprises no less than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% CD52+CD117+ cells. In another Specific embodiment, said NK progenitor cell tion comprises n 50%-55%, 55%-60%, 60%- 65%, %, 70%—75%, 75%-80%, 80%-85%, 85%-90%, 90%—95% or more CD52+CD117+ cells. In another specific embodiment, said NK progenitor cell population which comprises CD52+CD117+ cells produced by a three-step process described herein is produced using a three-step process that comprises a short third culture step, e.g., a third culture step of 4-6, 5—7, 6- 8, or 7-9 days. In a specific embodiment, said NK progenitor cell population which comprises CD52+CD117+ cells is produced using a three-step process that comprises a total of 12 days or more, 13 days or more, 14 days or more, 15 days or more, 16 days or more, 17 days or more, 18 days or more, 19 days or more, 20 days or more, or 21 days or more of culture. In a specific embodiment, said NK progenitor cell population which comprises CD52+CD117+ cells is produced using a three-step process that comprises a total of at least 12 days, 13 days, or 14 days of culture but not more than 21-25 days, 25—30 days, or 30—35 days of culture. In a c embodiment, said NK progenitor cell population which comprises CD52+CD117+ cells is produced using a three-step process that comprises a total of 21 days of culture.

In a specific embodiment, the NK progenitor cells described herein possess a greater ability to engraft bone marrow (e.g., in vivo) than non-progenitor NK cells, e.g., non-progenitor NK cells produced using a comparable method. For example, in certain embodiments, NK progenitor cells produced using a three-step process that comprises a short third culture step, e.g., a third culture step of 4-6, 5-7, 6-8, or 7-9 days t bone marrow (e.g., in vivo) at a higher efficiency than non-progenitor NK cells produced using a step process that comprises a longer third culture step, e.g., a third culture step of 18-20, 19-21, 20-22, or 21-23 days. In another embodiment, the NK progenitor cells bed herein s longer res than peripheral blood (PB) derived NK cells. .1.4. NK Cells Produced by Three-Stage Method In one embodiment, provided herein is an isolated NK cell population, wherein said NK cells are produced according to the three-stage method described below.

In one embodiment, provided herein is an isolated NK cell population produced by a three-stage method described herein, n said NK cell population comprises a greater percentage of CD3—CD56+ cells than an NK progenitor cell population ed by a three- stage method described herein, e.g., an NK progenitor cell population produced by the same three-stage method with the exception that the third culture step used to produce the NK progenitor cell population was of shorter on than the third e step used to e the NK cell population. In a specific embodiment, said NK cell tion comprises about 70% or more, in some embodiments, 75%, 80%, 85%, 90%, 95%, 98%, or 99% CD3—CD56+ cells. In another specific embodiment, said NK cell population comprises no less than 80%, 85%, 90%, 95%, 98%, or 99% CD3—CD56+ cells. In another specific embodiment, said NK cell population comprises between 70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95%, or 95%-99% CD3— CD56+ cells.

In certain embodiments, said CD3—CD56+ cells in said NK cell population comprises CD3—CD56+ cells that are additionally NKp46+. In certain embodiments, said 56+ cells in said NK cell population comprises CD3—CD56+ cells that are additionally CD16-. In certain embodiments, said CD3—CD56+ cells in said NK cell population comprises CD3—CD56+ cells that are additionally CD16+. In certain embodiments, said CD3—CD56+ cells in said NK cell population comprises CD3—CD56+ cells that are additionally CD94-. In certain embodiments, said CD3—CD56+ cells in said NK cell population comprises CD3—CD56+ cells that are additionally CD94+.

In one embodiment, an NK cell population produced by a three-stage method described herein comprises cells which are CD117+. In one embodiment, an NK cell population produced by a three-stage method described herein comprises cells which are NKG2D+. In one ment, an NK cell population produced by a three-stage method described herein comprises cells which are NKp44+. In one embodiment, an NK cell population produced by a three-stage method described herein comprises cells which are CD244+. .1.5. Cell Combinations and Cell/Perfusate Combinations The NK cells, e.g., activated NK cells and/or TSPNK cells can further be ed with placental perfusate, placental perfusate cells and/or adherent tal cells in the t invention. .1.5.1. ations ofNK Cells and Perfusate or Perfusate Cells In specific embodiments, the natural killer cells comprise CD56+CD16_ PiNK cells in combination with CD56+CD16+ natural killer cells. In more specific embodiments, the CD56+CD16Jr natural killer cells can be isolated from placenta, or from another source, e.g., peripheral blood, umbilical cord blood, bone marrow, or the like. Thus, in various other ments, PiNK cells can be combined with CD56+CD16+ natural killer cells, e.g, in ratios of, for example, about 1:10, 2:9, 3:8, 4:7:, 5:6, 6:5, 7:4, 8:3, 9:2,1:10,1:9,1:8,1:7,l:6,1:5,1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1 or about 9:1. As used in this t, “isolated” means that the cells have been removed from their normal nment, e.g, the ta.

In various specific embodiments, the isolated population ofNK cells comprises at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or at least about 99% PiNK cells. In r embodiment, the plurality ofPiNK cells comprises, or consists of, PiNK cells that have not been expanded; e.g, are as collected from placental ate. In another embodiment, the plurality of PiNK cells comprises, or consists of, PiNK cells that have been ed. Methods of expanding natural killer cells are described elsewhere herein, and have been described, e.g., in Ohno el al., US. Patent Application Publication No. 2003/0157713; see also Yssel et 01]., J. Immunol. Methods 72(1):2l9-227 (1984) and Litwin et 0]., J. Exp. Med. 178(4):1321-1326 (1993).

] In specific embodiments, the isolated population ofNK cells is a tion of placental cells comprising PiNK cells. In a specific embodiment, the isolated population ofNK cells is total nucleated cells from placental perfusate, e.g., placental ate cells, comprising autologous, isolated PiNK cells. In various other embodiments, activated NK cells can be combined with, e.g., NK cells, wherein said NK cells have been isolated from a tissue source and have not been expanded, NK cells isolated from a tissue source and expanded, or NK cells produced by a different method, e.g, CD56+CD16+ l killer cells, e.g, in ratios of, for example, about 1:10, 2:9, 3:8, 4:7:, 5:6, 6:5, 7:4, 8:3, 9:2,1:10,1:9,1:8,1:7,1:6,1:5,1:4,1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1 or about 9:1. As used in this t, “isolated” means that the cells have been removed from their normal tissue environment.

In specific ments, activated NK cells can also be combined with, e.g., NK cells, wherein said NK cells have been isolated from a tissue source and have not been expanded, NK cells isolated from a tissue source and expanded, or NK cells produced by a different method, e.g., CD56+CD16+ natural killer cells, e.g., in ratios of, for example, about 1:10, 2:9, 3:8, 4:7:, 5:6, 6:5, 7:4, 8:3, 9:2,1:10,1:9,1:8,1:7,1:6,1:5,1:4,1:3,1:2,1:1, 2:1, 3:1, 4:1, 5:1, 6: 1, 7: 1, 8:1 or about 9: 1. As used in this context, “isolated” means that the cells have been removed from their normal tissue nment.

] In one embodiment, for example, a volume of placental ate supplemented with NK cells produced using the processes described herein, e.g., activated NK cells or TSPNK cells (e.g., NK progenitor cells), is used. In specific embodiments, for example, each milliliter of placental perfusate is supplemented with about 1 x 104, 5 x 104, 1 x 105, 5 x 105, 1 x 106, 5 x 106, 1 x 107, 5 x 107, l x 108, 5 x 108 or more NK cells produced using the processes described herein, e. g., activated NK cells or TSPNK cells (e.g., NK progenitor cells). In another embodiment, placental perfusate cells are supplemented with NK cells produced using the 2015/068069 processes described herein, e.g., activated NK cells or TSPNK cells (e.g., NK progenitor cells).

In certain other embodiments, when placental ate cells are combined with NK cells produced using the processes described herein, e.g., activated NK cells or TSPNK cells (e.g., NK progenitor cells), the placental perfusate cells lly se about, r than about, or fewer than about, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 8%, 6%, 4%, 2% or 1% of the total number of cells. In certain other embodiments, when NK cells produced using the processes described herein, e.g., activated NK cells or TSPNK cells (e.g., NK progenitor cells), are combined with a plurality of placental ate cells and/or combined natural killer cells, the NK cells generally comprise about, greater than about, or fewer than about, 50%, 45%, 40%, %, 30%, 25%, 20%, 15%, 10%, 8%, 6%, 4%, 2% or 1% of the total number of cells. In certain other embodiments, when NK cells produced using the processes described herein, e.g., activated NK cells or TSPNK cells (e.g., NK progenitor cells), are used to ment tal perfusate, the volume of solution (e.g., saline solution, culture medium or the like) in which the cells are suspended comprises about, greater than about, or less than about, 50%, 45%, 40%, %, 30%, 25%, 20%, 15%, 10%, 8%, 6%, 4%, 2% or 1% of the total volume of perfusate plus cells, where the NK cells are suspended to about 1 X 104, 5 X 104, 1 X 105, 5 X 105, 1 X 106, 5 X 106, 1 X 107, 5 X 107, 1 X 108, 5 X 108 or more cells per milliliter prior to supplementation.

In other embodiments, any of the above combinations of cells is, in turn, combined with umbilical cord blood or nucleated cells from umbilical cord blood.

Pooled tal perfusate that is obtained from two or more sources, e.g, two or more placentas, and combined, e.g., pooled, can further be used in the present ion. Such pooled perfusate can comprise imately equal volumes of perfusate from each source, or can comprise different volumes from each source. The relative volumes from each source can be randomly selected, or can be based upon, e.g., a concentration or amount of one or more ar factors, e.g, cytokines, growth factors, hormones, or the like; the number of placental cells in perfusate from each source, or other characteristics of the perfusate from each . Perfusate from multiple perfusions of the same placenta can similarly be pooled.

Similarly, placental perfusate cells, and placenta-derived intermediate natural killer cells, that are obtained from two or more sources, e.g., two or more placentas, and pooled, can also be used in the present invention. Such pooled cells can comprise approximately equal numbers of cells from the two or more sources, or different numbers of cells from one or more of the pooled sources. The relative numbers of cells from each source can be selected based on, e.g., the number of one or more specific cell types in the cells to be pooled, e.g‘, the number of CD34+ cells, etc.

NK cells produced using the processes described herein, e.g., activated NK cells or TSPNK cells (e.g., NK progenitor cells), and combinations of such cells with placental perfusate and/or placental perfusate cells can be assayed to determine the degree or amount of tumor/infection suppression (that is, the potency) to be expected from, e.g, a given number of the NK cells, or a given volume of perfusate. For example, an aliquot or sample number of cells is contacted or brought into proximity with a known number of tumor/infected cells under conditions in which the infected cells would otherwise proliferate, and the rate of proliferation of the tumor/infected cells in the presence of placental ate, perfusate cells, placental natural killer cells, or combinations thereof, over time (e.g, 1, 2, 3, 4, 5, 6, 7, 8, 9, or weeks, or longer) is compared to the proliferation of an equivalent number of the tumor/infected cells in the absence of perfusate, perfusate cells, placental natural killer cells, or combinations thereof. The y of the cells can be expressed, e.g, as the number of cells or volume of solution required to suppress tumor cell growth/infection spread, e.g., by about 10%, %, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or the like.

] In n embodiments, NK cells produced using the processes described herein, e.g., activated NK cells or TSPNK cells (e.g., NK progenitor cells), are provided as pharmaceutical grade administrable units. Such units can be provided in discrete volumes, e.g., 15 mL, 20 mL, mL, 30 nL. 35 mL, 40 mL, 45 mL, 50 mL, 55 mL, 60 mL, 65 mL, 70 mL, 75 mL, 80 mL, 85 mL, 90 mL, 95 mL, 100 mL, 150 mL, 200 mL, 250 mL, 300 mL, 350 mL, 400 mL, 450 mL, 500 mL, or the like. Such units can be provided so as to n a specified number of cells, e.g., NK cells or NK cell populations, or NK progenitor cell populations in combination with other NK cells or perfusate cells, e.g., 1 x 104, 5 x104, 1x105,5 x105,1x106,5 x106,1x107,5 x 107,1 x 108, 5 x 108 or more cells per milliliter, or 1 x 104, 5 x104,lx105,5 x105, 1x106,5 x106, lx 107, 5 x107,1x108,5 x108, 5 x109, 1x1010,5 lx10110r more cells per unit.

In specific embodiments, the units can comprise about, at least about, or at most about 1 x 104, 5 x104, 1x105,5 x105, 1x106,5 x106 or more NK cells per milliliter, or 1 x 104, 5 x104, 1x 105, 5 x105, 5 x106, 1x107,5 x107, 1x108,5 x108, 5 x109, 1x1010,5 x1010, 1 x 1011 or more cells per unit. Such units can be provided to contain specified numbers ofNK cells, and/or any of the other cells.

In the above embodiments, the NK cells or combinations ofNK cells with perfusate cells or perfusate can be autologous to a recipient (that is, obtained from the recipient), or allogeneic to a recipient (that is, obtained from at last one other individual from said recipient).

In certain embodiments, each unit of cells is labeled to y one or more of volume, number of cells, type of cells, whether the unit has been enriched for a particular type of cell, and/or potency of a given number of cells in the unit, or a given number of milliliters of the unit, that is, whether the cells in the unit cause a measurable suppression of proliferation of a particular type or types of tumor cell. .1.5.2. Combination of NK Cells from Matched Perfusate and Cord Blood Natural Killer Cells can be further obtained from combinations of matched units of placental perfusate and umbilical cord blood in the present ion, and are referred to herein as combined natural killer cells. “Matched units,” as used , indicates that the NK cells are obtained from tal perfusate cells, and umbilical cord blood cells, wherein the cal cord blood cells are obtained from umbilical cord blood from the placenta from which the placental perfusate is obtained, i.e., the placental perfusate cells and umbilical cord blood cells, and thus the natural killer cells from each, are from the same dual.

In certain embodiments, the combined placental killer cells comprise only, or substantially only, natural killer cells that are CD56“ and CD16‘. In certain other embodiments, the combined placental killer cells comprise NK cells that are CD56+ and CD16‘, and NK cells that are CD56+ and CD16+. In certain specific embodiments, the combined placental killer cells comprise at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% D16' natural killer cells (PiNK cells).

In one embodiment, the combined natural killer cells have not been cultured. In a specific embodiment, the combined natural killer cells se a detectably higher number of 56+CD16_ natural killer cells than an equivalent number of natural killer cells from peripheral blood. In another ic embodiment, the combined natural killer cells comprise a detectably lower number of 56+CD16_ natural killer cells than an equivalent number of l killer cells from peripheral blood. In another specific embodiment, the combined l killer cells comprise a detectably higher number of CD3’CD56+KIR2DL2/L3+ natural killer cells than an equivalent number of natural killer cells from peripheral blood. In another specific WO 09668 embodiment, the combined natural killer cells comprise a detectably lower number of CD3— CD56+ NKp46+ natural killer cells than an equivalent number of natural killer cells from peripheral blood. In another specific ment, the combined natural killer cells se a detectably lower number of CD3‘CD56+ NKp3 0+ natural killer cells than an equivalent number of natural killer cells from peripheral blood. In another specific embodiment, the combined natural killer cells comprise a detectably lower number of CD3‘CD56+2B4+ natural killer cells than an equivalent number of natural killer cells from peripheral blood. In another specific ment, the combined natural killer cells comprise a detectably lower number of CD3— CD56+CD94+ natural killer cells than an equivalent number of natural killer cells from peripheral blood.

In another embodiment, the ed natural killer cells have been cultured, e.g, for 21 days. In a specific embodiment, the combined natural killer cells comprise a detectably lower number of CD3'CD56+KIR2DL2/L3+ natural killer cells than an lent number of natural killer cells from peripheral blood. In another specific embodiment, the combined natural killer cells have not been cultured. In another specific embodiment, the combined natural killer cells se a detectably higher number of CD3‘CD56WKp44+ natural killer cells than an equivalent number of natural killer cells from peripheral blood. In a specific ment, the combined natural killer cells se a detectably higher number of CD3‘CD56lNKp30+ natural killer cells than an equivalent number of natural killer cells from peripheral blood.

In another embodiment, the combined natural killer cells express a detectably higher amount of granzyme B than an equivalent number of peripheral blood natural killer cells.

Combined natural killer cells can further be combined with umbilical cord blood. In various embodiments, cord blood is combined with combined natural killer cells at about 1 x 104, x104,1x105,5 x105,1x106,5 x106,1x107,5 X108,5 x108 combined l killer cells per milliliter of cord blood. .1.5.3. Combinations ofNK Cells with Adherent Placental Stem Cells In other embodiments, the NK cells produced using the processes described herein, e. g., activated NK cells or TSPNK cells (e.g, NK progenitor cells) produced using the three-step process described herein, either alone or in combination with placental perfusate or placental perfusate cells, are supplemented with isolated adherent placental cells, e.g, placental stem cells and tal otent cells as described, e.g., in Hariri US. Patent Nos. 7,045,148 and 7,255,879, and in US. Patent ation Publication No. 2007/0275362, the disclosures of which are incorporated herein by reference in their ties. “Adherent placental cells” means that the cells are nt to a tissue culture surface, e.g., tissue culture plastic. The adherent placental cells useful in the compositions and methods disclosed herein are not trophoblasts, embryonic germ cells or embryonic stem cells. In certain embodiments, adherent placental stem cells are used as feeder cells during the processes (e.g, two—step method) as described above.

The NK cells produced using the processes described herein, e.g, activated NK cells or TSPNK cells (e.g., NK progenitor cells), either alone or in combination with placental perfusate or placental perfusate cells can be supplemented with, e.g., 1 X 104, 5 X 104, 1 X 105, 5 X 105, 1 x 106, 5 x 10", 1 x 107, 5 x 107, 1 x 108, 5 x 108 or more adherent placental cells per iter, or 1 x 104, 5 x104, 1x105,5 x105,1x106,5 x106,1x107,5 x107,1x108,5 x108,1 X 109, 5 X 109, 1 X 1010, 5 X 1010, 1 X 1011 or more adherent placental cells. The adherent placental cells in the combinations can be, e.g., adherent placental cells that have been cultured for, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 population doublings, or more. ed nt tal cells, when cultured in primary cultures or expanded in cell culture, adhere to the tissue culture substrate, e.g., tissue culture container surface (e.g., tissue culture plastic). Adherent placental cells in culture assume a generally fibroblastoid, stellate appearance, with a number of cytoplasmic processes ing from the central cell body. Adherent placental cells are, however, morphologically distinguishable from fibroblasts cultured under the same conditions, as the nt placental cells eXhibit a greater number of such processes than do fibroblasts. logically, adherent placental cells are also distinguishable from hematopoietic stem cells, which generally assume a more rounded, or stone, morphology in e.

The isolated adherent placental cells, and populations of adherent placental cells, useful in the compositions and methods provided herein, eXpress a plurality of markers that can be used to identify and/or isolate the cells, or populations of cells that comprise the adherent placental cells. The nt placental cells, and adherent placental cell tions useful in the compositions and methods ed herein include adherent placental cells and adherent placental cell—containing cell populations obtained directly from the placenta, or any part thereof (e. g., amnion, chorion, amnion-chorion plate, placental cotyledons, umbilical cord, and the like).

The adherent placental stem cell population, in one embodiment, is a population (that is, two or more) of adherent placental stem cells in e, e.g., a population in a container, e.g., a bag.

The adherent placental cells generally express the markers CD73, CD105, and CD200, and/or OCT-4, and do not s CD34, CD38, or CD45. Adherent placental stem cells can also express HLA-ABC (MHC-l) and HLA-DR. These markers can be used to identify adherent placental cells, and to distinguish the adherent placental cells from other cell types.

Because the adherent placental cells can express CD73 and CD105, they can have mesenchymal stem cell-like characteristics. Lack of expression of CD34, CD38 and/or CD45 identifies the adherent placental stem cells as matopoietic stem cells.

] In certain ments, the isolated nt placental cells described herein detectably suppress cancer cell proliferation or tumor growth.

In n embodiments, the isolated adherent placental cells are isolated placental stem cells. In certain other embodiments, the isolated adherent placental cells are isolated placental multipotent cells. In a specific embodiment, the isolated adherent placental cells are CD34‘, CD10+ and CD105+ as detected by flow cytometry. In a more specific embodiment, the isolated CD34‘, CD10+, CD105+ adherent placental cells are placental stem cells. In another more specific embodiment, the isolated CD34‘, CD10+, CD105+ placental cells are multipotent adherent placental cells. In another specific embodiment, the isolated CD34‘, CD10+, CD105+ tal cells have the potential to differentiate into cells of a neural phenotype, cells of an osteogenic phenotype, or cells of a chondrogenic phenotype. In a more specific embodiment, the isolated CD34‘, CD10+, CD105+ nt placental cells are onally CD200] In another more specific embodiment, the isolated CD34‘, CD10+, CD105+ adherent placental cells are additionally CD90+ or CD45‘, as ed by flow cytometry. In another more specific embodiment, the isolated CD34‘, CD10+, CD105+ adherent placental cells are additionally CD90" or CD45‘, as detected by flow cytometry. In a more specific embodiment, the CD34‘, CD10", CD105+, CD200+ adherent placental cells are additionally CD90+ or CD45‘, as detected by flow cytometry. In r more specific embodiment, the CD34", CD10+, CDIOSI, CD200+ adherent placental cells are additionally CD90+ and CD45‘, as detected by flow cytometry. In another more specific embodiment, the CD34‘, CD101 CD105: CD200: CD901 CD45— adherent tal cells are additionally CD80‘ and CD86‘, as detected by flow cytometry.

In one embodiment, the isolated adherent placental cells are CD200+, HLA-G] In a specific embodiment, said isolated adherent placental cells are also CD73+ and CD105+. In another specific embodiment, said isolated adherent placental cells are also CD34‘, CD3 8— or CD45‘. In a more specific embodiment, said ed adherent placental cells are also CD34‘, CD3 8‘, CD45‘, CD73+ and CD105+. In another embodiment, said isolated adherent tal cells produce one or more embryoid-like bodies when ed under conditions that allow the formation of embryoid-like bodies.

In another embodiment, the isolated adherent placental cells are CD73+, , CD200+. In a specific embodiment, said isolated adherent placental cells are also . In another specific embodiment, said isolated adherent placental cells are also CD34‘, CD3 8— or CD45‘. In another specific embodiment, said isolated adherent placental cells are also CD34‘, CD3 8— and CD45‘. In a more specific embodiment, said isolated adherent placental cells are also CD34‘, CD3 8—, CD45‘, and HLA-G+. In another specific embodiment, said isolated adherent placental cells produce one or more embryoid-like bodies when cultured under conditions that allow the formation of embryoid-like bodies.

In another embodiment, the isolated adherent placental cells are CD200+, OCT-4+. In a specific ment, said ed adherent placental cells are also CD73+ and CD105+. In another specific embodiment, said isolated nt placental cells are also HLA-G+. In another specific embodiment, said isolated adherent placental cells are also CD34‘, CD3 8— and CD45‘.

In a more specific embodiment, said ed adherent placental cells are also CD34‘, CD3 8—, CD45", CD73+, CD105+ and PEA-G“. In r specific ment, the isolated adherent placental cells also produce one or more embryoid-like bodies when cultured under conditions that allow the formation of embryoid-like bodies.

In another embodiment, the isolated adherent placental cells are CD73+, CD105Jr and HLA-G+. In a specific embodiment, said isolated adherent tal cells are also CD34‘, CD3 8— or CD45‘. In r specific embodiment, said isolated adherent placental cells also CD34‘, CD38‘ and CD45‘. In another specific embodiment, said adherent stem cells are also . In another specific embodiment, said adherent stem cells are also CD200+. In a more specific embodiment, said adherent stem cells are also CD34‘, CD38‘, CD45‘, OCT-4+ and CD200] In another embodiment, the isolated adherent placental cells are CD73+, CD105Jr stem cells, wherein said cells produce one or more embryoid-like bodies under conditions that allow ion of embryoid-like bodies. In a specific embodiment, said isolated adherent placental cells are also CD34‘, CD3 8‘ or CD45‘. In another specific embodiment, isolated adherent placental cells are also CD34: CD3 8‘ and CD45‘. In r c embodiment, isolated adherent placental cells are also OCT-4+. In a more specific embodiment, said isolated nt placental cells are also OCT-4+, CD34: CD38‘ and CD45‘.

In another embodiment, the adherent tal stem cells are OCT-4+ stem cells, wherein said adherent tal stem cells produce one or more embryoid-like bodies when cultured under conditions that allow the formation of embryoid-like bodies, and wherein said stem cells have been fied as detectably suppressing cancer cell proliferation or tumor growth.

In various embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50% at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of said isolated adherent placental cells are . In a specific embodiment, said ed adherent placental cells are also CD73+ and CD105+. In r specific embodiment, said isolated adherent placental cells are also CD34‘, CD3 8‘, or CD45‘. In another specific embodiment, said stem cells are CD200+. In a more specific embodiment, said ed adherent placental cells are also CD73+, , CD200+, CD34‘, CD3 8‘, and CD45‘. In another specific embodiment, said isolated adherent placental cells have been expanded, for example, passaged at least once, at least three times, at least five times, at least 10 times, at least 15 times, or at least 20 times.

In a more specific embodiment of any of the above embodiments, the isolated adherent placental cells express ABC—p (a placenta-specific ABC transporter protein, see, e.g., Allikmets et al., Cancer Res. 58(23):5337-9 (1998)).

] In another embodiment, the isolated adherent placental cells CD29+, CD44: CD73+, CD90+, CD105+, CD200+, CD34‘ and CD133‘. In another embodiment, the isolated adherent placental cells constitutively secrete IL-6, IL-8 and monocyte chemoattractant protein (MCP-l).

Each of the referenced isolated adherent placental cells can comprise cells obtained and isolated directly from a mammalian placenta, or cells that have been cultured and passaged at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, l4, l6, 18, 20, 25, 30 or more times, or a combination thereof. Tumor cell suppressive pluralities of the isolated adherent placental cells described above can comprise about, at least, or no more than, 1 x 105, 5 x 105, l x 106, 5 x 106, l x 107, 5 x107, 1x108,5 x108, 5 x109, 1x1010,5 x1010, 1x1011or more isolated adherent placental cells. .1.5.4. Compositions Comprising Adherent Placental Cell Conditioned Media Also can be used in the present invention is a composition comprising NK cells ed using the processes described herein, e.g., activated NK cells or TSPNK cells (e.g, NK progenitor cells) produced using the three-step process described , and additionally ioned medium, wherein said composition is tumor suppressive, or is effective in the treatment of cancer or viral infection. Adherent placental cells as described herein can be used to e conditioned medium that is tumor cell suppressive, anti-cancer or iral that is, medium comprising one or more biomolecules secreted or excreted by the cells that have a detectable tumor cell suppressive effect, anti—cancer effect or antiviral effect. In various embodiments, the conditioned medium comprises medium in which the cells have proliferated (that is, have been cultured) for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days. In other embodiments, the conditioned medium comprises medium in which such cells have grown to at least 30%, 40%, 50%, 60%, 70%, 80%, 90% confluence, or up to 100% confluence. Such conditioned medium can be used to support the culture of a separate population of cells, e.g., placental cells, or cells of another kind. In another embodiment, the conditioned medium provided herein comprises medium in which isolated adherent placental cells, e.g., isolated adherent tal stem cells or isolated adherent placental multipotent cells, and cells other than isolated adherent placental cells, e.g., non-placental stem cells or otent cells, have been cultured.

] Such conditioned medium can be combined with any of, or any combination ofNK cells produced using the processes described herein, e.g., activated NK cells or TSPNK cells (e.g., NK progenitor cells), placental perfusate, placental perfusate cells to form a ition that is tumor cell suppressive, anticancer or antiviral. In certain embodiments, the composition ses less than half conditioned medium by volume, e.g, about, or less than about, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% by volume.

Thus, in one ment, used in the present invention is a ition comprising NK cells produced using the processes described herein, e.g., activated NK cells or TSPNK cells (e.g., NK progenitor cells), and culture medium from a culture of isolated adherent placental cells, wherein said isolated adherent tal cells (a) adhere to a substrate; and (b) are CD34", CD10+ and CD105+, n said composition detectably suppresses the growth or proliferation WO 09668 of tumor cells, or is anti-cancer or antiviral. In a specific embodiment, the isolated adherent placental cells are CD342 CD10+ and CD105+ as detected by flow cytometry. In a more specific ment, the isolated CD34‘, CDlO+, CD105+ adherent placental cells are placental stem cells. In another more specific embodiment, the isolated CD34‘, CDlO+, CD105+ placental cells are multipotent nt placental cells. In another specific embodiment, the isolated CD34: CD10+, CD105+ placental cells have the potential to differentiate into cells of a neural ype, cells of an osteogenic phenotype, or cells of a chondrogenic phenotype. In a more specific embodiment, the isolated CD34‘, CDlO+, CD105+ adherent placental cells are additionally CD200+. In another more specific embodiment, the isolated CD34: CDlO+, CD105+ adherent placental cells are additionally CD90+ or CD45: as detected by flow cytometry. In another more c embodiment, the isolated CD34‘, CD10+, CD105+ adherent placental cells are onally CD90+ or CD45‘, as detected by flow cytometry. In a more specific ment, the CD34‘, CDlO+, CD105+, CD200+ adherent placental cells are additionally CD90+ or CD45: as detected by flow cytometry. In another more specific embodiment, the CD34‘, CD10+, CD105+, CD200+ adherent placental cells are additionally CD90+ and CD45: as ed by flow cytometry. In another more specific embodiment, the CD34‘, CD10+, CD105+, CD200+, CD901 CD45‘ adherent placental cells are additionally CD80‘ and CD86: as detected by flow cytometry.

In another embodiment, used in the present invention is a composition comprising NK cells produced using the processes bed , e.g., ted NK cells or TSPNK cells (e.g., NK progenitor cells), and culture medium from a culture of isolated adherent placental cells, wherein said isolated nt placental cells (a) adhere to a substrate; and (b) express CD200 and HLA-G, or express CD73, CD105, and CD200, or express CD200 and OCT-4, or express CD73, CD105, and HLA-G, or express CD73 and CD105 and facilitate the formation of one or more embryoid-like bodies in a population of placental cells that comprise the placental stem cells when said population is cultured under conditions that allow formation of embryoid- like bodies, or express OCT—4 and facilitate the formation of one or more embryoid-like bodies in a population of placental cells that comprise the tal stem cells when said population is cultured under conditions that allow formation of embryoid—like bodies; wherein said composition detectably suppresses the growth or proliferation of tumor cells, or is anti-cancer or antiviral. In a specific embodiment, the composition further comprises a plurality of said WO 09668 isolated placental adherent cells. In another specific embodiment, the composition ses a plurality of non—placental cells. In a more specific embodiment, said non-placental cells comprise CD34+ cells, e.g., poietic progenitor cells, such as peripheral blood poietic progenitor cells, cord blood hematopoietic progenitor cells, or placental blood hematopoietic progenitor cells. The non-placental cells can also comprise stem cells, such as mesenchymal stem cells, e.g., bone marrow-derived mesenchymal stem cells. The non-placental cells can also be one or more types of adult cells or cell lines. In another specific embodiment, the composition comprises an anti-proliferative agent, e.g., an anti-MIP-lot or anti-MIP-lB antibody.

In a specific embodiment, culture medium ioned by one of the cells or cell combinations described above is obtained from a plurality of isolated adherent placental cells co- cultured with a plurality of tumor cells at a ratio of about 1:1, about 2: 1, about 3:1, about 4: 1, or about 5:1 isolated adherent placental cells to tumor cells. For example, the conditioned e medium or supernatant can be obtained from a culture comprising about 1 X 105 isolated adherent placental cells, about 1 X 106 ed adherent placental cells, about 1 X 107 isolated adherent placental cells, or about 1 X 108 isolated adherent placental cells, or more. In r c embodiment, the conditioned culture medium or atant is obtained from a co- culture comprising about 1 X 105 to about 5 X 105 isolated adherent placental cells and about 1 X 105 tumor cells, about 1 X 106 to about 5 X 106 isolated adherent tal cells and about 1 X 106 tumor cells; about 1 X 107 to about 5 X 107 isolated adherent placental cells and about 1 X 107 tumor cells, or about 1 X 108 to about 5 X 108isolated adherent placental cells and about 1 X 108 tumor cells. .2. Methods of Producing NK Cells NK cells may be produced from hematopoietic cells, e. g., hematopoietic stem or progenitors from any source, e. g., placental , placental ate, umbilical cord blood, placental blood, peripheral blood, spleen, liver, or the like.

One important source of natural killer cells and cells that can be used to derive natural killer cells as described above is the placenta, for example, full-term placenta, e. g., full-term human placenta. Placental perfusate comprising placental perfusate cells that can be obtained, for example, by the methods disclosed in US. Patent Nos. 7,045,148 and 7,468,276 and US.

Patent Application Publication No. 2009/0104164, the disclosures of each of which are hereby incorporated in their entireties. .2.1. Cell Collection Composition The placental perfusate and perfusate cells, from which hematopoietic stem or progenitors may be isolated, or useful in tumor ssion or the treatment of an individual having tumor cells, cancer or a viral infection, e.g., in combination with the NK cells, e.g., NK cell populations produced ing to the three-stage method provided herein, can be ted by perfusion of a mammalian, e.g., human post-partum placenta using a placental cell collection composition. Perfusate can be collected from the placenta by perfusion of the placenta with any physiologically-acceptable solution, e.g., a saline solution, culture medium, or a more complex cell collection composition, A cell collection ition suitable for perfusing a placenta, and for the collection and preservation of perfusate cells is described in detail in related US.

Application Publication No. 2007/0190042, which is incorporated herein by reference in its entirety.

The cell collection composition can se any physiologically-acceptable solution suitable for the collection and/or culture of stem cells, for example, a saline solution (e.g., ate-buffered saline, Kreb’s solution, d Kreb’s on, Eagle’s solution, 0.9% NaCl. etc), a culture medium (e.g., DMEM, , etc), and the like, The cell collection composition can comprise one or more components that tend to preserve placental cells, that is, prevent the placental cells from dying, or delay the death of the placental cells, reduce the number of placental cells in a tion of cells that die, or the like, from the time of collection to the time of culturing. Such ents can be, e.g, an apoptosis tor (e.g, a caspase inhibitor or INK inhibitor); a vasodilator (e.g., magnesium sulfate, an antihypertensive drug, atrial natriuretic peptide (ANP), adrenocorticotropin, corticotropin- releasing e, sodium nitroprusside, hydralazine, adenosine triphosphate, ine, indomethacin or magnesium sulfate, a phosphodiesterase inhibitor, etc), a necrosis inhibitor (e.g, 2-(1H—Indolyl)pentylamino-maleimide, pyrrolidine dithiocarbamate, or clonazepam), a TNF-d inhibitor, and/or an oxygen-carrying perfluorocarbon (e.g., perfluorooctyl bromide, perfluorodecyl bromide, etc).

The cell collection composition can comprise one or more tissue-degrading enzymes, e.g., a metalloprotease, a serine protease, a neutral protease, a hyaluronidase, an RNase, or a DNase, or the like. Such enzymes include, but are not limited to, enases (e.g., collagenase I, II, III or IV, a collagenase from idium histolyticum, etc), dispase, thermolysin, elastase, trypsin, LIBERASE, hyaluronidase, and the like.

The cell collection composition can comprise a bacteriocidally or iostatically effective amount of an antibiotic. In certain non-limiting embodiments, the antibiotic is a macrolide (e.g., tobramycin), a cephalosporin (e.g., cephalexin, cephradine, cefuroxime, cefprozil, cefaclor, cefiXime or cefadroxil), a clarithromycin, an erythromycin, a penicillin (e.g., penicillin V) or a quinolone (e.g., ofloxacin, ciprofloxacin or xacin), a tetracycline, a streptomycin, etc. In a particular embodiment, the antibiotic is active against Gram(+) and/or Gram(—) bacteria, e.g, Pseudomonas aeruginosa, Staphylococcus aureus, and the like.

The cell collection composition can also comprise one or more of the following compounds: adenosine (about 1 mM to about 50 mM); D-glucose (about 20 mM to about 100 mM); ium ions (about 1 mM to about 50 mM), a macromolecule of lar weight greater than 20,000 daltons, in one embodiment, present in an amount sufficient to maintain endothelial integrity and cellular viability (e.g., a synthetic or naturally occurring colloid, a polysaccharide such as dextran or a hylene glycol present at about 25 g/l to about 100 g/l, or about 40 g/l to about 60 g/l); an antioxidant (e.g., butylated hydroxyanisole, butylated hydroxytoluene, glutathione, vitamin C or vitamin E present at about 25 uM to about 100 HM), a reducing agent (e.g, N—acetylcysteine present at about 0.1 mM to about 5 mM); an agent that prevents calcium entry into cells (e.g., verapamil present at about 2 uM to about 25 uM), lycerin (e.g., about 0.05 g/L to about 0.2 g/L); an anticoagulant, in one embodiment, present in an amount sufficient to help prevent clotting of residual blood (e.g, n or hirudin present at a concentration of about 1000 units/l to about 100,000 1), or an amiloride containing compound (e.g., amiloride, ethyl isopropyl amiloride, thylene amiloride, dimethyl amiloride or isobutyl amiloride present at about 1.0 uM to about 5 uM). .2.2. Collection and Handling of Placenta Generally, a human ta is recovered shortly after its expulsion after birth. In one embodiment, the placenta is recovered from a patient after informed consent and after a te medical history of the patient is taken and is associated with the ta. In one ment, the medical history continues after delivery.

Prior to ry of perfusate, the umbilical cord blood and placental blood are removed. In certain embodiments, after ry, the cord blood in the placenta is recovered.

The placenta can be subjected to a conventional cord blood recovery process. Typically a needle or cannula is used, with the aid of gravity, to exsanguinate the placenta (see, e. g., Anderson, US. Patent No. 5,372,581; Hessel et 611., US. Patent No. 5,415,665). The needle or cannula is usually placed in the umbilical vein and the placenta can be gently massaged to aid in draining cord blood from the placenta. Such cord blood recovery may be performed commercially, e.g., LifeBank Inc., Cedar Knolls, N.J., ViaCord, Cord Blood Registry and CryoCell. In one embodiment, the placenta is gravity drained without further manipulation so as to minimize tissue disruption during cord blood recovery.

Typically, a ta is transported from the delivery or birthing room to r location, e. g., a laboratory, for recovery of cord blood and tion of perfusate. The placenta can be transported in a sterile, thermally insulated transport device (maintaining the ature of the placenta between 20-28 CC), for example, by placing the placenta, with clamped proximal umbilical cord, in a sterile zip—lock plastic bag, which is then placed in an insulated container. In another embodiment, the placenta is orted in a cord blood collection kit substantially as described in US. Patent No. 7,147,626. In one ment, the ta is delivered to the laboratory four to twenty-four hours following delivery. In certain embodiments, the proximal umbilical cord is clamped, for example within 4-5 cm (centimeter) of the insertion into the placental disc prior to cord blood recovery. In other embodiments, the proximal umbilical cord is clamped after cord blood recovery but prior to r processing of the placenta.

The placenta, prior to collection of the perfusate, can be stored under sterile ions and at either room temperature or at a temperature of 5 to 25 °C (centigrade). The placenta may be stored for a period of longer than forty eight hours, or for a period of four to twenty-four hours prior to perfusing the placenta to remove any residual cord blood. The placenta can be stored in an agulant solution at a temperature of 5 °C to 25 °C (centigrade).

Suitable anticoagulant solutions are well known in the art. For example, a solution of heparin or warfaiin sodium can be used. In one embodiment, the agulant solution ses a solution of heparin (e.g., 1% w/w in 121000 solution). In some embodiments, the exsanguinated placenta is stored for no more than 36 hours before placental perfusate is collected. .2.3. Placental Perfusion ] Methods of perfusing mammalian placentae and obtaining placental perfusate are disclosed, e.g., in Hariri, US. Patent Nos. 7,045,148 and 7,255,879, and in US. Application Publication Nos. 104164, 2007/0190042 and 20070275362, issued as US. Pat No. 8,057,788, the disclosures of each of which are hereby incorporated by reference herein in their entireties.

Perfusate can be obtained by passage of perfusion solution, e.g., saline solution, culture medium or cell collection compositions described above, through the placental vasculature. In one ment, a mammalian placenta is ed by passage of perfusion solution through either or both of the umbilical artery and umbilical vein. The flow of perfusion solution through the placenta may be accomplished using, e.g., gravity flow into the placenta.

For example, the perfusion solution is forced through the placenta using a pump, e.g, a altic pump. The umbilical vein can be, e.g., cannulated with a cannula, e.g., a TEFLON® or plastic cannula, that is ted to a sterile tion apparatus, such as e tubing. The sterile connection apparatus is connected to a perfusion manifold.

In preparation for perfusion, the placenta can be oriented in such a manner that the umbilical artery and cal vein are d at the highest point of the placenta. The placenta can be perfused by passage of a perfusion solution through the placental vasculature, or through the tal vasculature and surrounding tissue. In one embodiment, the umbilical artery and the umbilical vein are connected simultaneously to a pipette that is connected via a flexible connector to a reservoir of the perfusion on. The perfusion solution is passed into the umbilical vein and . The perfusion solution exudes from and/or passes through the walls of the blood vessels into the surrounding tissues of the placenta, and is collected in a suitable open vessel from the surface of the placenta that was attached to the uterus of the mother during ion. The ion solution may also be introduced through the umbilical cord opening and d to flow or percolate out of openings in the wall of the placenta which aced with the maternal uterine wall. In another embodiment, the perfusion solution is passed through the umbilical veins and collected from the umbilical artery, or is passed through the umbilical artery and collected from the cal veins, that is, is passed through only the placental vasculature (fetal tissue).

In one embodiment, for example, the umbilical artery and the umbilical vein are connected simultaneously, e.g., to a pipette that is connected via a flexible connector to a reservoir of the perfusion solution. The perfusion solution is passed into the umbilical vein and 2015/068069 artery. The perfusion solution exudes from and/or passes h the walls of the blood vessels into the nding tissues of the placenta, and is collected in a suitable open vessel from the surface of the ta that was attached to the uterus of the mother during gestation. The perfusion solution may also be introduced through the umbilical cord g and allowed to flow or ate out of openings in the wall of the placenta which interfaced with the maternal uterine wall. Placental cells that are collected by this , which can be referred to as a “pan” method, are typically a mixture of fetal and maternal cells.

In another embodiment, the perfusion solution is passed through the umbilical veins and collected from the umbilical artery, or is passed through the umbilical artery and collected from the umbilical veins. Placental cells collected by this method, which can be ed to as a “closed circuit” method, are typically almost exclusively fetal.

The closed t perfusion method can, in one embodiment, be performed as follows. A post-partum placenta is obtained within about 48 hours after birth. The umbilical cord is d and cut above the clamp. The umbilical cord can be discarded, or can processed to recover, e.g., cal cord stem cells, and/or to process the umbilical cord membrane for the production of a biomaterial. The amniotic membrane can be retained during perfusion, or can be separated from the chorion, e.g., using blunt dissection with the . If the amniotic membrane is separated from the chorion prior to perfusion, it can be, e.g., discarded, or processed, e.g., to obtain stem cells by enzymatic digestion, or to produce, e.g., an amniotic membrane biomaterial, e.g., the biomaterial described in US. ation Publication No. 2004/0048796. After cleaning the placenta of all visible blood clots and residual blood, e. g., using sterile gauze, the umbilical cord vessels are exposed, e.g., by partially cutting the umbilical cord membrane to expose a cross-section of the cord. The vessels are identified, and opened, e. g., by advancing a closed alligator clamp through the cut end of each vessel. The apparatus, e. g., plastic tubing connected to a perfusion device or peristaltic pump, is then inserted into each of the tal arteries. The pump can be any pump suitable for the purpose, e.g, a peristaltic pump. Plastic tubing, connected to a sterile collection reservoir, e.g, a blood bag such as a 250 mL collection bag, is then inserted into the placental vein. Alternatively, the tubing connected to the pump is inserted into the placental vein, and tubes to a collection reservoir(s) are inserted into one or both of the placental arteries. The placenta is then perfused with a volume of perfusion solution, e. g., about 750 ml of ion solution. Cells in the perfusate are then collected, e.g., by centrifugation.

In one embodiment, the proximal umbilical cord is clamped during perfusion, and, more specifically, can be clamped within 4-5 cm (centimeter) of the cord’s insertion into the placental disc.

The first collection of perfusion fluid from a mammalian placenta during the uination process is generally colored with residual red blood cells of the cord blood and/or placental blood. The perfusion fluid becomes more colorless as ion proceeds and the al cord blood cells are washed out of the placenta. Generally from 30 to 100 mL of perfusion fluid is adequate to initially flush blood from the placenta, but more or less perfusion fluid may be used depending on the ed results.

In certain embodiments, cord blood is removed from the placenta prior to perfusion (e.g., by y drainage), but the ta is not flushed (e.g., perfused) with solution to remove residual blood. In certain embodiments, cord blood is d from the placenta prior to perfusion (e.g, by gravity drainage), and the placenta is flushed (e.g., perfused) with solution to remove residual blood.

The volume of perfusion liquid used to perfuse the placenta may vary depending upon the number of placental cells to be collected, the size of the placenta, the number of collections to be made from a single placenta, etc. In various embodiments, the volume of perfusion liquid may be from 50 mL to 5000 mL, 50 mL to 4000 mL, 50 mL to 3000 mL, 100 mL to 2000 mL, 250 mL to 2000 mL, 500 mL to 2000 mL, or 750 mL to 2000 mL. Typically, the placenta is perfused with 0 mL of perfusion liquid following exsanguination.

The placenta can be ed a plurality of times over the course of several hours or several days. Where the placenta is to be perfused a plurality of times, it may be maintained or cultured under aseptic conditions in a container or other suitable vessel, and ed with a cell collection composition, or a standard perfusion solution (e.g, a normal saline solution such as phosphate buffered saline (“PB S”) with or without an anticoagulant (e.g., heparin, warfarin sodium, in, bishydroxycoumarin), and/or with or without an antimicrobial agent (e.g., [3- mercaptoethanol (0.1 mM); antibiotics such as streptomycin (e.g., at 40-100 , penicillin (e.g., at 40 U/ml), amphotericin B (e.g., at 05 ug/ml). In one embodiment, an isolated placenta is maintained or cultured for a period of time without collecting the perfusate, such that the placenta is maintained or cultured for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, , 21, 22, 23, or 24 hours, or 2 or 3 or more days before perfusion and collection of perfusate.

The perfused placenta can be ined for one or more additional time(s), e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13,14, 15,16, 17,18, 19, 20, 21, 22, 23, 24 or more hours, and perfused a second time with, e.g, 700—800 mL perfusion fluid. The placenta can be perfused 1, 2, 3, 4, 5 or more times, for example, once every 1, 2, 3, 4, 5 or 6 hours. In one embodiment, perfusion of the placenta and collection of perfusion solution, e.g, placental cell collection composition, is repeated until the number of recovered nucleated cells falls below 100 cells/ml. The perfusates at different time points can be further processed individually to recover time-dependent populations of cells, e.g., total nucleated cells. Perfusates from ent time points can also be pooled. .2.4. Placental Perfusate and Placental Perfusate Cells ] Typically, placental ate from a single placental perfusion comprises about 100 million to about 500 million nucleated cells, including hematopoietic cells from which NK cells, e. g., NK cells produced according to the three-stage method described herein, may be produced by the method disclosed herein. In certain embodiments, the placental perfusate or perfusate cells se CD34+ cells, e.g., hematopoietic stem or progenitor cells. Such cells can, in a more specific embodiment, comprise CD34+CD45_ stem or progenitor cells, D45+ stem or progenitor cells, or the like. In certain embodiments, the perfusate or perfusate cells are eserved prior to isolation of hematopoietic cells therefrom. In certain other embodiments, the placental ate ses, or the perfusate cells comprise, only fetal cells, or a combination of fetal cells and maternal cells. .2.5. Hematopoietic Cells In various embodiments, NK cells are produced from hematopoietic cells, e.g., hematopoietic stem cells or progenitor cells.

Hematopoietic cells as used herein can be any poietic cells able to differentiate into NK cells, e.g., precursor cells, hematopoietic itor cells, hematopoietic stem cells, or the like. Hematopoietic cells can be obtained from tissue sources such as, e.g., bone marrow, cord blood, placental blood, peripheral blood, liver or the like, or combinations f.

Hematopoietic cells can be obtained from placenta. In a specific embodiment, the hematopoietic cells are obtained from placental perfusate. Hematopoietic cells from placental perfusate can comprise a mixture of fetal and maternal hematopoietic cells, e.g., a mixture in which al cells comprise greater than 5% of the total number of hematopoietic cells. In one embodiment, hematopoietic cells from placental perfusate comprise at least about 90%, 95%, 98%, 99% or 99.5% fetal cells.

In another specific embodiment, the hematopoietic cells, e.g., hematopoietic stem cells or progenitor cells, are obtained from placental perfusate, umbilical cord blood or peripheral blood. In another specific embodiment, the hematopoietic cells, e.g., hematopoietic stem cells or itor cells, are combined cells from placental perfusate and cord blood, e. g., cord blood from the same placenta as the perfusate. In r specific embodiment, said umbilical cord blood is isolated from a placenta other than the placenta from which said placental perfusate is ed. In certain embodiments, the ed cells can be obtained by pooling or combining the cord blood and placental ate. In certain embodiments, the cord blood and placental perfusate are combined at a ratio of 100: 1, 95:5, 90:10, 85: 15, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45: 50:50, 45:55, 40:60, 35:65, 30:70, 25:75, 20:80, 15:85, 10:90, 5:95, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1 7 :1,10:1,5:1,1:1,1:5,1:10,1:15,1:20,1:25,1:30,1:35,1:40,1:45,1:50,1:55,]:60,1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, or the like by volume to obtain the combined cells. In a c embodiment, the cord blood and placental perfusate are combined at a ratio of from :1 to 1:10, from 5:1 to 1:5, or from 3:1 to 1:3. In another specific embodiment, the cord blood and placental perfusate are ed at a ratio of 10:1, 5:1, 3:1, 1:1, 1:3, 1:5 or 1:10. In a more specific embodiment, the cord blood and placental perfusate are combined at a ratio of 8.5: 1.5 (85%: 15%).

In certain embodiments, the cord blood and placental perfusate are combined at a ratio of 100:1, 95:5, 90:10, 85:15, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45: 50:50, 45:55, 40:60, :65, 30:70, 25:75, 20:80, 15:85, 10:90, 5:95, 1001, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, 10:1, 5:1, 1:1, 1:5, 1:10, 1:15, 1:20, 1:25,1:30,1:35,1:40,1:45,1:50,1:55,1:60,1:65,1:70,1:75,1:80,l:85,1:90,1:95,1:100,or the like by total nucleated cells (INC) content to obtain the combined cells. In a specific embodiment, the cord blood and placental perfusate are combined at a ratio of from 10:1 to 10: 1, from 5:1 to 1:5, or from 3:1 to 1: 3. In r specific embodiment, the cord blood and placental perfusate are combined at a ratio of 10:1, 5:1, 3:1, 1:1, 1:3,1:5 or 1:10.

WO 09668 In another specific embodiment, the hematopoietic cells, e.g., hematopoietic stem cells or itor cells, are from both umbilical cord blood and placental perfusate, but wherein said umbilical cord blood is ed from a placenta other than the placenta from which said placental perfusate is obtained.

In certain ments, the hematopoietic cells are CD34+ cells. In specific embodiments, the hematopoietic cells useful in the methods sed herein are CD34+CD3 8+ or CD34+CD3 8—. In a more specific embodiment, the hematopoietic cells are CD34+CD3 8‘Lin‘. In another specific embodiment, the hematopoietic cells are one or more of CD2‘, CD3‘, , CD11c‘, CD14_, CD16‘, CD19‘, CD24‘, CD56: CD66b‘ and/or glycophofin A‘. In another specific embodiment, the hematopoietic cells are CD2‘, CD3‘, CD11b‘, CD11c‘, CD14‘, CD16‘, CD19‘, CD24‘, CD56‘, CD66b‘ and glycophon'n A‘. In another more specific ment, the hematopoietic cells are CD34+CD38_CD33_CD117_. In another more specific embodiment, the hematopoietic cells are CD34+CD38_CD33_CD117‘CD235‘CD36‘.

In another embodiment, the hematopoietic cells are CD45+. In another specific embodiment, the hematopoietic cells are CD34+CD45+. In another embodiment, the hematopoietic cell is . In a specific embodiment, the hematopoietic cell is CD34+Thy—1+, In another embodiment, the poietic cells are CD133+. In specific embodiments, the hematopoietic cells are CD34+CD133+ or CD133+Thy-1+. In another specific embodiment, the CD34+ poietic cells are CXCR4+. In another specific embodiment, the CD34+ poietic cells are CXCR4'. In another embodiment, the hematopoietic cells are positive for KDR (vascular growth factor receptor 2). In specific embodiments, the hematopoietic cells are CD34+KDR+, CD133+KDR+ or Thy-1+KDR+. In certain other embodiments, the hematopoietic cells are positive for de dehydrogenase (ALDH+), e.g, the cells are CD34+ALDH+.

In certain other ments, the CD34+ cells are CD45‘. In specific embodiments, the CD34+ cells, e.g., CD34+, CD45‘ cells express one or more, or all, of the miRNAs hsa-miR- 380, hsa-miR—S 12, hsa-miR-517, hsa—miR—S 18c, hsa—miR—S 19b, and/or hsa—miR-520a.

In certain embodiments, the hematopoietic cells are CD347 The poietic cells can also lack certain markers that indicate lineage commitment, or a lack of developmental naiveté. For example, in another embodiment, the hematopoietic cells are HLA—DR‘. In specific embodiments, the hematopoietic cells are CD34+HLA-DR‘, CD133+HLA—DR‘, HLA-DR‘ or LA-DR‘ In another embodiment, the hematopoietic cells are negative for one or more, preferably all, of lineage markers CD2, CD3, CD1 1b, CD1 lc, CD14, CD16, CD19, CD24, CD56, CD66b and glycophorin A.

Thus, hematopoietic cells can be selected for use in the methods disclosed herein on the basis of the presence of markers that indicate an undifferentiated state, or on the basis of the absence of e markers indicating that at least some lineage differentiation has taken place.

Methods of isolating cells, including hematopoietic cells, on the basis of the presence or absence of specific markers are discussed in detail below.

Hematopoietic cells as used herein can be a substantially homogeneous population, e.g., a population comprising at least about 95%, at least about 98% or at least about 99% hematopoietic cells from a single tissue source, or a population comprising hematopoietic cells exhibiting the same hematopoietic ssociated cellular markers. For example, in various ments, the hematopoietic cells can comprise at least about 95%, 98% or 99% hematopoietic cells from bone marrow, cord blood, placental blood, peripheral blood, or placenta, e.g., placenta perfusate.

Hematopoietic cells as used herein can be obtained from a single individual, e.g., from a single placenta, or from a plurality of individuals, e.g., can be pooled. Where the hematopoietic cells are obtained from a plurality of individuals and pooled, the hematopoietic cells may be obtained from the same tissue source. Thus, in various ments, the pooled poietic cells are all from ta, e.g, placental perfusate, all from placental blood, all from umbilical cord blood, all from eral blood, and the like.

Hematopoietic cells as used herein can, in certain embodiments, comprise hematopoietic cells from two or more tissue sources. For example, in certain embodiments, when hematopoietic cells from two or more sources are combined for use in the methods , a plurality of the hematopoietic cells used to e NK cells comprise hematopoietic cells from placenta, e.g., placenta perfusate. In various embodiments, the hematopoietic cells used to produce NK cells comprise hematopoietic cells from placenta and from cord blood; from placenta and peripheral blood; from placenta and tal blood, or placenta and bone .

In a preferred embodiment, the hematopoietic cells comprise hematopoietic cells from placental perfusate in combination with hematopoietic cells from cord blood, wherein the cord blood and placenta are from the same individual, 1'.e., wherein the perfusate and cord blood are matched. In embodiments in which the hematopoietic cells comprise hematopoietic cells from two tissue sources, the hematopoietic cells from the s can be combined in a ratio of, for example, 1:10, 2:9, 3:8, 4:7:, 5:6, 6:5, 7:4, 8:3, 9:2,1:10,1:9,1:8,1:7,1:6,1:5,1:4,1:3,1:2,1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1 or 9:1. .2.5.1. Placental Hematopoietic Stem Cells In certain embodiments, the hematopoietic cells are placental hematopoietic cells. As used herein, “placental hematopoietic cells” means hematopoietic cells obtained from the placenta itself, and not from placental blood or from cal cord blood. In one embodiment, placental hematopoietic cells are CD34+. In a specific embodiment, the placental hematopoietic cells are predominantly (e.g., at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98%) CD34+CD3 8‘ cells. In another specific ment, the placental hematopoietic cells are predominantly (e.g., at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98%) CD34+CD3 8+ cells. Placental hematopoietic cells can be obtained from a post- partum mammalian (e.g., human) ta by any means known to those of skill in the art, e.g., by perfusion.

In another embodiment, the placental hematopoietic cell is CD457 In a specific embodiment, the hematopoietic cell is CD34+CD45_. In another specific embodiment, the placental poietic cells are CD34+CD45+. .2.6. Methods of Producing PiNK Cells In various embodiments, PiNK cells are derived from placental cells. In c embodiments, the placental cells are obtained from placental ate, e.g., human placental ate. In c embodiments, the placental cells are obtained from placental tissue that has been mechanically and/or enzymatically disrupted. .2.6.1. Obtaining PiNK Cells from Placental Perfusate In one embodiment, PiNK cells are collected by obtaining placental ate, then contacting the placental perfusate with a composition that specifically binds to CD56+ cells, e.g., an dy against CD56, followed by isolating of CD56+ cells on the basis of said binding to form a population of CD56+ cells. The population of CD56+ cells comprises an isolated population of natural killer cells. In a specific embodiment, CD56+ cells are contacted with a composition that specifically binds to CD16" cells, e.g., an antibody against CD16, and the CD16+ cells are excluded from the population of CD56+ cells. In another specific embodiment, CD3+ cells are also excluded from the population of CD56+ cells.

In one embodiment, PiNK cells are obtained from placental perfusate as follows.

Post-partum human placenta is exsanguinated and perfused, e.g., with about 200-800 mL of perfusion solution, through the placental vasculature only. In a specific embodiment, the placenta is drained of cord blood and flushed, e.g., with perfusion solution, through the placental vasculature to remove residual blood prior to said perfusing. The perfusate is collected and processed to remove any residual erythrocytes. Natural killer cells in the total nucleated cells in the perfusate can be isolated on the basis of expression of CD56 and CD16. In certain embodiments, the isolation of PiNK cells comprises isolation using an antibody to CD56, wherein the isolated cells are CD56+. In another embodiment, the isolation of PiNK cells comprises isolation using an antibody to CD16, wherein the isolated cells are CD167 In another embodiment, the isolation of PiNK cells comprises isolation using an antibody to CD56, and exclusion of a ity of non—PiNK cells using an antibody to CD16, wherein the ed cells comprise CD56+, CD16_ cells.

Cell tion can be accomplished by any method known in the art, e.g, fluorescence-activated cell sorting (FACS), or, preferably, magnetic cell sorting using microbeads conjugated with specific antibodies. Magnetic cell separation can be performed and automated using, e.g, an C STM Separator (Miltenyi).

In another aspect, the process of isolating placental natural killer cells (e.g., PiNK cells) comprises obtaining a plurality of tal cells, and isolating natural killer cells from said plurality of placental cells. In a specific embodiment, the placental cells are, or comprise, placental perfusate cells, e.g., total nucleated cells from placental ate. In another specific embodiment, said plurality of tal cells are, or comprise, placental cells obtained by mechanical and/or enzymatic digestion of placental tissue. In r ment, said isolating is med using one or more antibodies. In a more specific embodiment, said one or more antibodies comprises one or more of antibodies to CD3, CD16 or CD56. In a more specific ment, said isolating ses isolating CD56+ cells from CD56‘ cells in said plurality of placental cells. In a more c embodiment, said isolating ses isolating CD56+, CD16— 2015/068069 placental cells, e. g., placental l killer cells, e.g, PiNK cells, from placental cells that are CD56‘ or CD16+. In a more specific embodiment, said isolating comprises isolating CD56“, CD16‘, CD3‘ placental cells from placental cells that are CD56‘, CD16+, or CD3+. In another ment, said process of isolating placental natural killer cells results in a population of placental cells that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or at least 99% CD56+, CDl6_ l killer cells.

In certain embodiments, the placental natural killer cells, e.g., PiNK cells, have been expanded in culture. In certain other embodiments, the placental perfusate cells have been expanded in culture. In a specific embodiment, said placental perfusate cells have been expanded in the ce of a feeder layer and/or in the presence of at least one ne. In a more specific ment, said feeder layer comprises K562 cells or peripheral blood mononuclear cells. In another more specific embodiment, said at least one cytokine is interleukin-2. In specific ments, the PiNK cells have been cultured, e.g., expanded in culture, for atleast, about, or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ll, 12, 13, 14, 15, 16, 17, 18, 19, , 21, 22, 23, 24, 25, 26, 27 or 28 days. In a specific embodiment, the PiNK cells are cultured for about 21 days. .2.6.2. tion and Digestion of Placental Tissue to Obtain PiNK Cells Placental natural killer cells, e.g., PiNK cells, can also be ed from placental tissue that has been mechanically and/or enzymatically disrupted.

Placental tissue can be disrupted using one or more tissue-degrading enzymes, e.g., a metalloprotease, a serine protease, a l protease, an RNase, or a DNase, or the like. Such enzymes include, but are not limited to, enases (e.g., collagenase I, II, III or IV, a collagenase from Clostridium histolyticum, etc), dispase, thermolysin, elastase, trypsin, LIBERASE, hyaluronidase, and the like. Typically after digestion, the digested tissue is passed h a strainer or filter to remove partially-digested cell clumps, leaving a substantially single—celled suspension.

After a suspension of placental cells is obtained, natural killer cells can be isolated using, e.g., antibodies to CD3 and CD56. In a specific embodiment, placental natural killer cells are isolated by selecting for cells that are CD56+ to produce a first cell population, contacting said first cell population with antibodies specific for CD3 and/or CD16; and removing cells from said first cell population that are CD3+ or CD56+, thereby producing a second population of cells that is substantially CD56+ and CD3‘, CD56+ and CD16‘, or CD56, CD3‘ and CD16‘.

In one embodiment, magnetic beads are used to isolate placental natural killer cells from a suspension of placental cells. The cells may be isolated, e.g., using a magnetic activated cell sorting (MACS) que, a method for separating les based on their ability to bind magnetic beads (e.g., about 05-100 um diameter) that comprise one or more specific antibodies, e.g, anti-CD56 antibodies. A variety of useful modifications can be performed on the magnetic microspheres, including covalent addition of antibody that specifically recognizes a particular cell surface molecule or . The beads are then mixed with the cells to allow binding. Cells are then passed through a magnetic field to separate out cells having the specific cell surface marker. In one embodiment, these cells can then isolated and re-mixed with magnetic beads coupled to an antibody against additional cell surface markers. The cells are again passed h a magnetic field, isolating cells that bound both the antibodies. Such cells can then be diluted into separate dishes, such as microtiter dishes for clonal isolation. .2.7. Methods of Producing ted NK Cells ted NK cells may be produced from hematopoietic cells, which are bed above. In certain embodiment, the activated NK cells are produced from expanded hematopoietic cells, e.g., poietic stem cells and/or hematopoietic progenitor cells. In a specific embodiment, the hematopoietic cells are expanded and differentiated, continuously, in a first medium without the use of feeder cells. The cells are then cultured in a second medium in the presence of feeder cells. Such isolation, expansion and differentiation can be performed in a central facility, which provides expanded hematopoietic cells for shipment to decentralized expansion and differentiation at points of use, e.g., hospital, ry base, military front line, or the like.

In some embodiments, production of activated NK cells comprises expanding a population of hematopoietic cells. During cell expansion, a ity of hematopoietic cells within the hematopoietic cell population differentiate into NK cells.

In one embodiment, the process of producing a population of activated natural killer (NK) cells comprises: (a) seeding a population of hematopoietic stem or itor cells in a first medium comprising interleukin-15 (IL-15) and, optionally, one or more of stem cell factor (SCF) and interleukin-7 (IL-7), n said IL-15 and optional SCF and lL-7 are not comprised within an undefined component of said medium, such that the population expands, and a plurality of hematopoietic stem or progenitor cells within said tion of hematopoietic stem or progenitor cells entiate into NK cells during said expanding; and (b) ing the cells from step (a) in a second medium comprising interleukin-2 (IL-2), to produce a population of activated NK cells.

] In another embodiment, activated NK cells as described herein are produced by a two-step process of expansion/ differentiation and maturation ofNK cells. The first and second steps comprise culturing the cells in media with a unique combination of cellular factors. In certain ments, the process involves (a) culturing and expanding a population of hematopoietic cells in a first medium, wherein a plurality of hematopoietic stem or progenitor cells within the hematopoietic cell population differentiate into NK cells; and (b) ing the NK cells from step (a) in a second medium, wherein the NK cells are further ed and differentiated, and wherein the NK cells are maturated (e.g., activated or otherwise possessing cytotoxic activity). In certain embodiments, the process includes no intermediary steps between step (a) and (b), no additional culturing steps prior to step (a), and/or no additional steps (e.g., maturation step) after step (b). .2.7.1. First Step In certain embodiments, the process of producing ted NK cells comprises a first step of ing and expanding a population of poietic cells in a first medium, wherein a plurality of hematopoietic stem or progenitor cells within the hematopoietic cell population differentiate into NK cells.

Without wishing to be bound by any parameter, mechanism or theory, e of the hematopoietic cells as described herein results in continuous expansion of the hematopoietic cells and differentiation ofNK cells from said cells. In certain embodiments, hematopoietic cells, e. g., stem cells or itor cells, used in the processes described herein are expanded and differentiated in the first step using a feeder layer. In other embodiments, hematopoietic cells, e. g., stem cells or progenitor cells, are expanded and differentiated in the first step without the use of a feeder layer.

Feeder cell-independent expansion and differentiation of hematopoietic cells can take place in any container compatible with cell culture and expansion, e.g., flask, tube, beaker, dish, multiwell plate, bag or the like. In a c embodiment, feeder cell-independent ion of hematopoietic cells takes place in a bag, e.g, a flexible, gas-permeable fluorocarbon culture bag (for example, from American Fluoroseal). In a specific embodiment, the container in which the hematopoietic cells are expanded is suitable for ng, e.g, to a site such as a hospital or military zone wherein the expanded NK cells are further expanded and differentiated.

In certain embodiments, hematopoietic cells are ed and differentiated, e.g, in a continuous fashion, in a first culture medium. In one embodiment, the first e medium is an animal-component free medium. Exemplary animal component-free media useful in the processes described herein include, but are not limited to, Basal Medium Eagle (BME), Dulbecco’s d Eagle’s Medium , Glasgow Minimum Essential Medium (GMEM), Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-l2 Ham (DMEM/F-l2), Minimum Essential Medium (MEM), Iscove’s d co’s Medium (IMDM), Nutrient Mixture F-10 Ham (Ham’s F-lO), Nutrient Mixture F-l2 Ham (Ham’s F-12), RPMI-1640 Medium, Williams’ Medium E, STEMSPAN® (Cat. No. Stem Cell Technologies, Vancouver, Canada), Glycostem Basal Growth Medium (GBGM®), AIM-V® medium (Invitrogen), XVIVOTM (Lonza), x—v1voTM 15 (Lonza), OPTMIZER (Invitrogen), STEMSPAN® H3000 (STEMCELL Technologies), CELLGRO COMPLETETM (Mediatech), or any modified variants or combinations thereof. In a specific embodiment of any of the embodiments herein, the medium is not GBGM®.

In preferred embodiments, the first culture medium comprises one or more of medium supplements (e.g., nutrients, cytokines and/or factors). Medium supplements suitable for use in the processes bed herein include, for example without limitation, serum such as human serum AB, fetal bovine serum (PBS) or fetal calf serum (FCS), ns, bovine serum albumin (BSA), amino acids (e.g., L-glutamine), fatty acids (e.g., oleic acid, linoleic acid or palmitic acid), insulin (e.g., recombinant human insulin), transferrin (iron ted human transferrin), B-mercaptoethanol, stem cell factor (SCF), Fms-like-tyrosine kinase 3 ligand (Flt3- L), nes such as interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-15 (IL-15), thrombopoietin (Tpo), heparin, or yl-carnitine (also referred to as acetylcamitine, O- acetyl-L-camitine or OAC). In a specific ment, the medium used herein comprises human serum AB. In another specific embodiment, the medium used herein comprises FBS. In another specific embodiment, the medium used herein comprises OAC.

In certain embodiments, the first medium does not comprise one or more of, granulocyte colony-stimulating factor (G—CSF), granulocyte/macrophage colony stimulating factor (GM-CSF), interleukin-6 (IL-6), macrophage inflammatory Protein 1 0t (MIPlOt), or leukemia inhibitory factor (LIF).

Thus, in one aspect, described herein is a ep process of producing NK cells, wherein said first step comprises expanding and differentiating a population of hematopoietic cells in a first culture medium in the absence of feeder cells, wherein a plurality of hematopoietic cells within said population of hematopoietic cells differentiate into NK cells during said expanding, and wherein the medium comprises SCF at a concentration of about 1 to about 150 ng/mL, IL-2 at a concentration of about 50 to about 1500 IU/mL, E-7 at a concentration of about 1 to about 150 ng/mL, IL—15 at a concentration 1 to about 150 ng/mL and heparin at a concentration of about 0.1 to about 30 IU/mL, and wherein said SCF, IL-2, IL-7, IL-15 and heparin are not comprised within an undefined component of said medium (e.g., serum). In certain embodiments, said medium comprises one or more of O-acetyl-carnitine (also referred to as carnitine, O—acetyl-L-carnitine or OAC), or a nd that affects acetyl-CoA g in onia, thiazovivin, Y-27632, pyintegrin, Rho kinase (ROCK) inhibitors, caspase inhibitors or other anti-apoptotic compounds/peptides, NOVA-RS (Sheffield Bio-Science) or other small-molecule growth enhancers. In certain embodiments, said medium comprises nicotinamide. In certain embodiments, said medium comprises about 0.5 mM-10 mM OAC. In one embodiment, said medium comprises an® H3000, and/or 12 and about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mM OAC. In a specific embodiment, said medium is GBGM®. In another specific embodiment, the medium is not GBGM®. In another specific embodiment, said medium comprises an® H3000 and about 5 mM of OAC. In another specific embodiment, said medium comprises DMEM:F12 and about 5 mM of OAC. The OAC can be added anytime during the culturing processes described herein. In certain embodiments, said OAC is added to the first medium and/or during the first culturing step. In some embodiments, said OAC is added to the first medium on Day 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 of the culture. In a c embodiment, said OAC is added to the first medium on Day 7 of the first culturing step. In a more c embodiment, said OAC is added to the first medium on Day 7 of the e and is t throughout the first and second culturing steps. In certain embodiments, said OAC is added to the second medium and/or during the second culturing step. In some embodiments, said OAC is added to the second medium on Day 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 ofthe culture.

] In r c embodiment, said medium is HVIDM supplemented with about 5- % BSA, about 1-10 ug/mL inant human insulin, about 10—50 ug/mL iron saturated human transferrin and about 10-50 uM B-mercaptoethanol. In another specific embodiment, said medium does not se one or more, or any, of IL-1 1, IL-3, homeoboX-B4 (HoxB4), and/or methylcellulose.

In other specific embodiments, said medium comprises SCF at a concentration of about 0.1 to about 500 ng/mL; about 5 to about 100 ng/mL; or about 20 ng/mL. In other specific embodiments, said medium comprises IL-2 at a concentration of about 10 to about 2000 IU/mL, or about 100 to about 500 IU/mL, or about 200 IU/mL. In other specific embodiments, said medium comprises IL—7 at a concentration of about 0.1 to about 500 ng/mL; about 5 to about 100 ng/mL; or about 20 ng/mL. In other specific embodiments, said medium comprises IL-15 at a concentration of about 0.1 to about 500 ng/mL, about 5 to about 100 ng/mL; or about 10 ng/mL.

In other specific embodiments, said medium comprises heparin at concentration of about 0.05 to about 100 U/mL, or about 0.5 to about 20 U/ml, or about 1.5 U/mL.

In yet other specific embodiment, said medium further comprises Fms-like-tyrosine kinase 3 ligand (Flt-3L) at a concentration of about 1 to about 150 ng/mL, thrombopoietin (Tpo) at a tration of about 1 to about 150 ng/mL, or a combination of both. In other specific embodiments, said medium comprises Flt-3L at a concentration of about 0.1 to about 500 ng/mL; about 5 to about 100 ng/mL, or about 20 ng/mL. In other specific embodiments, said medium comprises Tpo at a concentration of about 0.1 to about 500 ng/mL; about 5 to about 100 ng/mL, or about 20 ng/mL. [0023 7] In a more specific embodiment, the first culture medium is GBGM®, which comprises about 20 ng/mL SCF, about 20 ng/mL IL-7, about 10 ng/mL IL-15. In another more c embodiment, the first culture medium is GBGM®, which ses about 20 ng/mL SCF, about 20 ng/mL Flt3-L, about 200 IU/mL IL-2, about 20 ng/mL IL-7, about 10 ng/mL IL- , about 20 ng/mL Tpo, and about 1.5 U/mL heparin. In another specific embodiment, said first e medium further comprises 10% human serum (e.g., human serum AB) or fetal serum (e. g., FB S). In a specific embodiment of any of the embodiments herein, the medium is not GBGM®.

In another embodiment, hematopoietic cells are expanded by culturing said cells, e.g., in said first medium, in contact with an immunomodulatory compound, e.g., a TNF-or inhibitory compound, for a time and in an amount sufficient to cause a detectable increase in the proliferation of the hematopoietic cells over a given time, compared to an equivalent number of hematopoietic cells not contacted with the immunomodulatory compound. See, e. g., US. Patent Application Publication No. 2003/023 5909, the disclosure of which is hereby incorporated by reference in its entirety. In certain embodiments, the immunomodulatory compound is an amino-substituted isoindoline. In a preferred embodiment, the immunomodulatory compound is 3-(4-amino-1—oxo-1,3—dihydroisoindolyl)—piperidine-2,6-dione, 3—(4'aminoisolindoline-1'— one)piperidine-2,6-dione, 4-(amino)(2,6-dioxo(3-piperidyl))—isoindoline-1,3-dione, or 4- Amino(2,6—dioxopiperidin—3—yl)isoindole-1,3-dione. In another preferred embodiment, the modulatory compound is pomalidomide, or lenalidomide.

Specific examples of immunomodulatory nds include, but are not limited to, cyano and carboxy derivatives of substituted es such as those disclosed in US. patent no. ,929,117, 2-(2,6-dioxofluoropiperidin-3yl) isoindolines and 1,3—dioxo(2,6-dioxo fiuoropiperidineyl) olines such as those described in US. patent no. 5,874,448, the tetra substituted -dioxopiperdinyl)—1-oxoisoindolines described in US. patent no. 5,798,368, 1-oxo and 1,3—dioxo-2—(2,6-dioxopiperidin-3—yl) isoindolines (e.g., 4—methyl derivatives of thalidomide and EM-12), including, but not d to, those disclosed in US. patent no. ,63 5,517; and a class of non-polypeptide cyclic amides disclosed in US. patent nos. 5,698,579 and 5,877,200, analogs and derivatives of thalidomide, including hydrolysis products, metabolites, derivatives and precursors of thalidomide, such as those described in US. patent nos. 5,593,990, 5,629,327, and 6,071,948 to D’Amato, aminothalidomide, as well as analogs, ysis products, lites, derivatives and precursors of aminothalidomide, and substituted 2-(2,6-dioxopiperidinyl) imides and substituted 2-(2,6-dioxopiperidin-3 —yl)- 1-oxoisoindoles such as those described in US. patent nos. 6,281,230 and 6,316,471, isoindole- imide compounds such as those described in US. patent application no. 09/972,487 filed on October 5, 2001, US. patent application no. 10/032,286 filed on December 21, 2001, and ational Application No. 01/50401 (International Publication No. W0 02/059106).

The entireties of each of the patents and patent applications identified herein are incorporated herein by reference. Immunomodulatory compounds do not include thalidomide.

In another ment, immunomodulatory compounds include, but are not limited to, 1-oxo-and 1,3 dioxo(2,6-dioxopiperidinyl) isoindolines substituted with amino in the 2015/068069 benzo ring as described in US. Patent no. 5,635,517 which is incorporated herein by reference.

These compounds have the structure or.»X\ R2 NH wherein one of X and Y is C=O, the other ofX and Y is C=O or CH2, and R2 is hydrogen or lower alkyl, or a pharmaceutically acceptable salt, e, solvate, clathrate, enantiomer, diastereomer, racemate, or mixture of stereoisomers thereof.

In another embodiment, specific immunomodulatory compounds include, but are not limited to: 1-oxo(2,6-dioxopiperidinyl)—4—aminoisoindoline, 1-oxo—2—(2,6-dioxopiperidin-3—yl)—5-aminoisoindoline; 1-oxo(2,6-dioxopiperidinyl)—6-aminoisoindoline, 1-oxo(2,6-dioxopiperidiny1)—7-aminoisoindoline; 1,3-dioxo(2,6-dioxopiperidin-3 -yl)—4-aminoisoindoline, and 1,3-dioxo-2—(2,6-dioxopiperidin-3 -y1)aminoisoindoline.

Other specific modulatory compounds belong to a class of substituted 2—(2,6- dioxopiperidin-3 -yl) phthalimides and tuted -dioxopiperidinyl)—1—oxoisoindoles, such as those described in US. patent nos. 6,281,230; 6,316,471, 6,335,349; and 6,476,052, and International Patent Application No. 97/13375 (International Publication No. WO 98/03502), each of which is incorporated herein by reference. Compounds representative of this class are of the formulas: S? o I :N N'H 7‘ C H2N ll 9d 0 H O (PI 1 O @ifil @961: wherein R is hydrogen or methyl. In a te embodiment, the invention asses the use of enantiomerically pure forms (e.g. optically pure (R) or (S) enantiomers) of these compounds.

Still other specific immunomodulatory nds belong to a class of isoindole-imides disclosed in US. patent application nos. 10/032,286 and 09/972,487, and ational Application No. PCT/U801/50401 (International Publication No. WO 02/059106), each of which are incorporated herein by reference. In one representative embodiment, said immunomodulatory compound is a compound having the structure Y\ NH N 0 X R2 R1\N )1] wherein one of X and Y is C=O and the other is CH2 or C=O; R1 is H, (C1—C3 )alkyl, (C3—C7)cycloalkyl, )alkenyl, (C2-C8)alkynyl, benzyl, aryl, (C0-C4)alkyl—(C1-C6)heterocycloalkyl, (C0-C4)alkyl—(Cz-C5)heteroaryl, C(O)R3, C(S)R3, C(O)OR4, (C1—C8)alkyl—N(R6)2, (C1—C8)alky1—OR5, )alkyl—C(O)OR5, C(O)NHR3, C(S)NHR3, C(O)NR3R3’, C(S)NR3R3’ or (C1—C8)alkyl—O(CO)R5, R2 is H, F, benzyl, (Cl-Cg)alkyl, (C2-C8)alkenyl, or )alkynyl; R3 and R3, are independently (C1-C8)alkyl, (C3-C7)cycloalkyl, (C2-C3)alkenyl, (C2- Cg)a1kyny1, benzyl, aryl, (C0-C4)alkyl—(C1-C6)heterocycloalkyl, (C0-C4)alkyl—(C2-C5)heteroaryl, (Co-Cg)alkyl—N(R6)2, (Cl-Cg)alkyl—OR5, (Cl—Cg)alkyl—C(O)OR5, (C1—C8)alkyl—O(CO)R5, or C(O)OR5, R4 is (C1-C8)alkyl, (C2—Cg)alkenyl, (CZ—Cg)alkynyl, (C1—C4)alkyl—OR5, benzyl, aryl, (C0- C4)alkyl—(C1-C6)heterocycloalkyl, or (C0-C4)alkyl—(C2-C5)heteroaryl, R5 is (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C3)alkynyl, benzyl, aryl, or (C2-C5)heteroaryl; each ence of R6 is independently H, (C1—C8)alkyl, (C2-C8)alkenyl, (C2—C3)alkynyl, , aryl, )heteroaryl, or (C0-Cg)alkyl—C(O)O—R5 or the R6 groups can join to form a heterocycloalkyl group; n is O or 1; and * represents a chiral-carbon center; or a pharmaceutically acceptable salt, hydrate, solvate, clathrate, enantiomer, diastereomer, racemate, or mixture of stereoisomers thereof.

In specific compounds of the above formula, when n is 0 then R1 is (C3-C7)cycloalkyl, )alkenyl, (C2-C8)alkynyl, benzyl, aryl, (C0-C4)alkyl—(C1-C6)heterocycloalkyl, (C0- C4)alkyl-(Cz-C5)heteroaryl, , C(O)OR4, (C1-C8)alkyl—N(R6)2, (C1-C3)a1kyl—OR5, (C1- Cg)alky1—C(O)OR5, C(S)NHR3, or (C1-C8)alkyl—O(CO)R5; R2 is H or (C1-C8)alkyl; and R3 is (C1-C8)alkyl, (C3—C7)cycloalkyl, (C2-C3)alkenyl, (C2-Cg)alkynyl, benzyl, aryl, (C0- C4)alkyl—(C1—C6)heterocycloalkyl, (C0-C4)alkyl—(C2-C5)heteroaryl, (C5-C8)alkyl—N(R6)2 ; (C0- Cg)alky1—NH—C(O)O—R5; (Cl—Cg)alkyl—OR5,(C1-C3)alkyl—C(O)OR5, (C1-C8)alkyl—O(CO)R5, or C(O)OR5; and the other les have the same definitions.

In other specific compounds of the above formula, R2 is H or (C1-C4)alkyl.

In other c compounds of the above formula, R1 is (C1-C8)alkyl or benzyl.

In other specific compounds of the above formula, R1 is H, (C1-C8)alkyl, benzyl, CH20CH3, CH2CH20CH3, or “""CHzfl In another embodiment of the compounds of the above formula, R1 is R7 R7 n n m w. W {I wherein Q is O or S, and each occurrence of R7 is independently H, (Cl-Cg)alkyl, benzyl, 3, or 0CH3.

In other specific compounds of the above formula, R1 is C(O)R3.

In other specific compounds of the above formula, R3 is (C0—C4)alkyl—(Cz—Cs)heteroaryl, )a1kyl, aryl, or (C0-C4)alkyl—OR5.

In other specific compounds of the above formula, heteroaryl is pyridyl, furyl, or thienyl.

In other c compounds of the above formula, R1 is C(O)OR4.

In other specific compounds of the above formula, the H of C(O) can be ed with (C1-C4)alkyl, aryl, or benzyl.

In another embodiment, said immunomodulatory compound is a compound having the structure 0 IR Y\ N N o R3 X R6 wherein: one ofX and Y is C=O and the other is CH2 or C=O; R is H or CHzOCOR’; (i) each of R1, R2, R3, or R4, independently of the others, is halo, alkyl of l to 4 carbon atoms, or alkoxy of l to 4 carbon atoms or (ii) one of R1, R2, R3, or R4 is nitro or -NHR5 and the remaining of R1, R2, R3, or R4 are hydrogen; R5 is hydrogen or alkyl of l to 8 carbons R6 hydrogen, alkyl of l to 8 carbon atoms, benzo, , or fluoro; R’ is R7-CHR10-N(R8R9), R7 is m-phenylene or p-phenylene or -(CnH2n)- in which n has a value of 0 to 4, each of R8 and R9 taken independently of the other is hydrogen or alkyl of l to 8 carbon atoms, or R8 and R9 taken together are ethylene, pentamethylene, hexamethylene, or -CH2CH2X1CH2CH2— in which X1 is -O-, -S—, or -NH—; R10 is hydrogen, alkyl of to 8 carbon atoms, or phenyl, and * represents a chiral-carbon center; or a pharmaceutically acceptable salt, hydrate, solvate, clathrate, enantiomer, reomer, racemate, or mixture of stereoisomers thereof.

In a specific embodiment, expansion of the hematopoietic cells is performed in IMDM supplemented with 20% BITS (bovine serum albumin, recombinant human insulin and transferrin), SCF, Flt—3 ligand, IL-3, and 4-(Amino)(2,6-dioxo(3-piperidyl))—isoindoline—l,3- dione (10 pM in 0.05% DMSO). In a more specific embodiment, about 5 X 107 hematopoietic cells, e.g, CD34+ cells, are expanded in the medium to from about 5 X 1010 cells to about 5 X 1012 cells, which are resuspended in 100 mL of IMDM to produce a population of expanded hematopoietic cells. The population of ed hematopoietic cells is preferably cryopreserved to facilitate shipping.

In various specific embodiments, at least 50%, 55%, 60%, 65%, 70%. 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% of the hematopoietic cells are entiated to NK cells.

In certain embodiments, the process of expansion and differentiation of the hematopoietic cells, as bed herein, comprises ining the cell population comprising said hematopoietic cells at between about 2 x 104 and about 2 x 105 cells per iter during expansion and differentiation. In certain other embodiments, the process of ion and differentiation of the hematopoietic cells, as described herein, comprises maintaining the cell population sing said hematopoietic cells at no more than about 1 x 105 cells per milliliter.

The time for expansion and differentiation of hematopoietic cells into NK cells can be, for e, from about 3 days to about 120 days. In one embodiment, the differentiation time is about 7 days to about 75 days. In r embodiment, the differentiation time is about 14 days to about 50 days. In a specific embodiment, the differentiation time is about 21 days to about 28 days. .2.7.2. Second Step The hematopoietic cells, e.g., stem cells or progenitor cells, and l killer cells, resulting from the first step, are further expanded and differentiated in a second step, e.g, without the use of feeder layer or in the presence of feeder cells. Culture of the cells as described herein results in continuous expansion, differentiation as well as maturation of the NK cells from the first step. In the second step, the NK cells are expanded, differentiated and maturated, in a continuous fashion, in a second culture medium, e.g, comprising different nes and/or bioactive molecules than said first medium. In certain embodiments, the second culture medium is an animal component-free medium. Exemplary animal component-free cell culture media are described in the disclosure.

Thus, in one aspect, bed herein is a process of producing activated NK cells, comprising expanding the NK cells from the first step, described above, in a second medium in the presence of feeder cells and in contact with interleukin-2 (IL-2). In specific embodiments, said second medium comprises cell growth medium sing IL-2, e.g., 10 IU/mL to 1000 IU/mL, and one or more of: human serum (e.g., human serum AB), fetal bovine serum (PBS) or fetal calf serum (FCS), e.g., 5%-15% FCS V/v; transferrin, e.g., 10 ug/mL to 50 ug/mL; insulin, e.g., 5 ug/mL to 20 ug/mL, lamine, e.g., 5 X 10‘4 to 5 X 10‘5 M, oleic acid, e.g., 0.l ug/mL to 5 ug/mL, linoleic acid, e.g., 0.l ug/mL to 5 ug/mL, palmitic acid, e.g., 0.05 ug/mL to 2 ug/mL; bovine serum albumin (BSA), e.g., l ug/mL to 5 ug/mL, and/or phytohemagglutinin, e. g., 0.01 ug/mL to 1 ug/mL. In a more specific embodiment, said second medium comprises cell growth medium comprising PBS or FCS, e.g‘, 10% FCS V/V, IL—2, transferrin, insulin, ethanolamine, oleic acid, linoleic acid, palmitic acid, bovine serum albumin (BSA) and phytohemagglutinin. In a more c embodiment, said second medium comprises Iscove’s Modified Dulbecco’s Medium (IMDM), 10% PBS or FCS, 400 IU IL-2, 35 ug/mL transferrin, 5 ug/mL insulin, 2 X 10‘5 M lamine, 1 ug/mL oleic acid, 1 ug/mL linoleic acid (Sigma- Aldrich), 0.2 ug/mL palmitic acid (Sigma-Aldrich), 2.5 ug/mL BSA (Sigma—Aldrich) and 0.1 ug/mL phytohemagglutinin.

In certain embodiments, the second medium does not comprise one or more of, granulocyte colony-stimulating factor (G—CSF), granulocyte/macrophage colony stimulating factor (GM-CSF), interleukin-6 (IL-6), macrophage inflammatory Protein 1 (X (MIPlOL), or leukemia inhibitory factor (LIF).

] Feeder cells, when used, can be established from various cell types. Examples of these cell types include, without limitation, fibroblasts, stem cells (e.g., tissue culture-adherent placental stem cells), blood cells (e.g., peripheral blood clear cells (PBMC)), and cancerous cells (e.g, chronic myelogenous leukemia (CML) cells such as K562). In a specific embodiment, said culturing in said second medium comprises culturing using feeder cells, e.g., K562 cells and/or peripheral blood mononuclear cells (PBMCs), e.g., at the time the cells are started in said second medium, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days fter. In certain embodiments, feeder cells are optionally from a different species as the cells they are ting.

For example, human NK cells can be supported by mouse embryonic fibroblasts (from primary culture or a telomerized line).

In certain embodiments, feeder cells are optionally inactivated by irradiation (e.g., y- ation) or ent with an anti-mitotic agent such as mitomycin C, to prevent them from outgrowing the cells they are supporting, but permit synthesis of important factors that support the NK cells. For example, cells can be irradiated at a dose to inhibit proliferation but permit synthesis of important factors that support human embryonic stem (hES) cells (about 4000 rads gamma irradiation).

Culture ofNK cells for the second step can take place in any container compatible with cell culture and expansion, e.g., flask, tube, beaker, dish, multiwell plate, bag or the like. In a specific embodiment, feeder cell-dependent culture ofNK cells takes place in a bag, e.g, a flexible, gas-permeable fluorocarbon e bag (for example, from American Fluoroseal). In a specific embodiment, the container in which the NK cells are cultured is suitable for shipping, e. g., to a site such as a hospital or military zone n the expanded NK cells are further expanded, differentiated and maturated.

Differentiation of the cells from step 1 into activated NK cells can be assessed by detecting NK cell-specific markers, e.g., by flow cytometry. NK pecific s include, but are not limited to, CD56, CD94, CD1 17 and NKp46. Differentiation can also be assessed by the morphological characteristics ofNK cells, e.g., large size, high protein sis activity in the abundant asmic reticulum (ER), and/or preformed granules.

The time for expansion and differentiation of cells from step 1 into activated NK cells can be, for example, from about 3 days to about 120 days. In one embodiment, the entiation time is about 7 days to about 75 days. In another embodiment, the differentiation time is about 14 days to about 50 days. In a specific embodiment, the differentiation time is about 10 days to about 21 days.

] Differentiation of hematopoietic cells into NK cells can be assessed by detecting markers, e.g, CD56, CD94, CD117, NKG2D, DNAM-l and NKp46, by, for example, flow cytometry. Differentiation can also be assessed by the logical characteristics ofNK cells, e. g., large size, high protein synthesis activity in the abundant endoplasmic reticulum (ER), and/or preformed granules. Maturation ofNK cells (e.g., activated NK cells) can be ed by detecting one or more functionally relevant makers, for example, CD94, CD161, NKp44, DNAM-l, 2B4, NKp46, CD94, KIR, and the NKG2 family of ting receptors (e.g., NKG2D). Maturation ofNK cells (e.g., ted NK cells) can also be assessed by detecting specific markers during different pmental stages. For example, in one embodiment, pro- NK cells are CD34+, CD45RA+, CD10+, CD1 17— and/or CDl6l‘. In another embodiment, pre- NK cells are CD34+, +, CD10_, CD117+, and/or CD161‘. In another embodiment, immature NK cells are CD34‘, CD117+, CD161+, NKp46‘ and/or CD94/NKG2A‘. In another embodiment, CD56bright NK cells are CD117+, NKp46+, CD94/NKG2A+, CD16‘, and/or KIR+/_.

In another embodiment, CD56dim NK cells are CD117‘, NKp46+, CD94/NKG2A+/‘, CD16+, and/or KIR+. In a specific embodiment, maturation ofNK cells (e.g., activated NK cells) is determined by the percentage ofNK cells (e.g., activated NK cells) that are , CD94+ and/or NKp46+. In a more specific embodiment, at least 10%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65% or 70% of mature NK cells (e.g., activated NK cells) are NKp46+. In another more specific embodiment, at least 10%, 20%, 25%, 30%, 35%, 40%, 45% or 50% of mature NK cells (e.g., activated NK cells) are CD94+. In another more specific embodiment, at least %, 20%, 25%, 30%, 35%, 40%, 45% or 50% of mature NK cells (e.g., activated NK cells) are CD161_.

] In certain embodiments, the differentiation of hematopoietic cells into NK cells are assessed by detecting the expression level of, e.g., CD3, CD7 or CD127, CD10, CD14, CD15, CD16, CD33, CD34, CD56, CD94, CD117, CD161, NKp44, NKp46, NKG2D, DNAM-l, 234 or TO—PRO-3, using, e.g., antibodies to one or more of these cell markers. Such antibodies can be conjugated to a detectable label, for example, as fluorescent label, e.g, FITC, R—PE, PerCP, PerCP-Cy5.5, APC, APC-Cy7 or APC-H7. .2.8. Methods of Producing TSPNK Cells TSPNK cells may be ed from hematopoietic cells, which are described above.

In certain embodiment, the TSPNK cells are ed from expanded hematopoietic cells, e.g., poietic stem cells and/or hematopoietic progenitor cells.

In one embodiment, the TSPNK cells are produced by a three-step process. In certain ments, the process of expansion and entiation of the hematopoietic cells, as described herein, to produce NK progenitor cell populations or NK cell populations according to a three—step s described herein comprises maintaining the cell population comprising said hematopoietic cells at n about 2 x 104 and about 6 x 106 cells per milliliter, e.g., n about 2 x 104 and about 2 x 105 cells per milliliter, during expansion and differentiation. In certain other embodiments, the process of ion and differentiation of the hematopoietic cells, as described herein, comprises maintaining the cell population comprising said hematopoietic cells at no more than about 1 x 105 cells per milliliter. In certain other ments, the process of expansion and differentiation of the hematopoietic cells, as described herein, comprises maintaining the cell tion comprising said hematopoietic cells at no more than about 1 X 105 cells per milliliter, 2 X 105 cells per milliliter, 3 X 105 cells per iter, 4 X 105 cells per milliliter, 5 X 105 cells per milliliter, 6 X 105 cells per milliliter, 7 X 105 cells per milliliter, 8 X 105 cells per milliliter, 9 X 105 cells per milliliter, l X 106 cells per milliliter, 2 X 106 cells per milliliter, 3 X 106 cells per milliliter, 4 X 106 cells per milliliter, 5 X 106 cells per milliliter, 6 X 106 cells per milliliter, 7 X 106 cells per milliliter, 8 X 106 cells per iter, or 9 X 106 cells per milliliter.

In a certain embodiment, the three—step process comprises a first step (“step 1”) comprising culturing poietic stem cells or progenitor cells, e.g., CD34+ stem cells or progenitor cells, in a first medium for a specified time period, e.g., as described herein. In certain embodiments, the first medium contains one or more factors that promote expansion of hematopoietic progenitor cells, one or more factors for initiation of lymphoid differentiation within the expanding hematopoietic progenitor population, and/or one or more s that mimic stromal feeder support. In certain ments, the first medium comprises one or more cytokines (for example, Flt3L, TPO, SCF). In certain embodiments, the first medium comprises IL-7. In certain embodiments, the first medium comprises sub-ng/mL concentrations of G—CSF, IL-6 and/or GM-CSF. In a specific embodiment, the first medium comprises the cytokines Flt3L, TPO, and SCF, IL-7, and sub-ng/mL concentrations of G—CSF, IL-6 and GM-CSF. In specific ments, in the first , CD34+ cells undergo expansion into lineage specific progenitors, which then become CD34-. In n embodiments, this expansion occurs y.

In certain embodiments, the CD34- cells se more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, or more of the total population at the end of step 1. In a more specific embodiment, CD34- cells comprise more than 80% of the total population at the end of step 1.

In certain embodiments, subsequently, in “step 2” said cells are cultured in a second medium for a specified time period, e.g., as described herein. In certain embodiments, the second medium contains factors that may promote further expansion of lymphoid progenitors, factors that may contribute to development along the NK lineage, and/or factors that mimic stromal feeder support. In n embodiments, the second medium comprises one or more cytokines (e.g., Flt3L, SCF, IL-15, and/or IL-7). In certain embodiments, the second medium comprises IL-l7 and/or IL-15. In certain embodiments, the second medium comprises sub-ng/mL concentrations of G—CSF, IL—6 and/or GM-C SF. In a specific embodiment, the second medium comprises the cytokines Flt3L, SCF, IL-15, and IL-7, IL-17 and IL-15, and sub-ng/mL concentrations of G—CSF, 1L-6 and .

In certain embodiments, subsequently, in “step 3” said cells are cultured in a third medium for a specified time , e.g., as described herein. In certain embodiments, the third medium comprises factors that promote differentiation and onal activation of CD56+CD3- CDl6- cells, which may be NK progenitor cells. In one embodiment, such s comprise IL2 and ILlZ and IL18, IL12 and ILlS, IL12 and E18, ILZ and IL12 and ILIS and ILl8, or IL2 and ILlS and IL18. In certain embodiments, the third medium comprises factors that mimic stromal feeder t. In certain embodiments, the third medium comprises one or more cytokines (e.g., SCF, IL-15, IL-7, IL-2). In certain embodiments, the third medium comprises sub-ng/mL concentrations of G—CSF, 1L-6 and/or GM-C SF. In a specific embodiment, the third medium comprises the cytokines SCF, IL-15, IL-7, IL—2, and sub-ng/mL concentrations of G—CSF, E-6 and GM-CSF.

In specific embodiments, the three—step s is used to produce NK cell (e.g., mature NK cell) populations. In specific embodiments, the three-step process is used to produce NK progenitor cell populations. In certain embodiments, the step process is conducted in the absence of stromal feeder cell support. In certain embodiments, the three-step process is conducted in the absence of exogenously added steroids (e.g, cortisone, hydrocortisone, or derivatives thereof).

In certain embodiments, the first medium used in the three-step processes described herein may contain any of the components of the first or second medium described in Section .2.4 in connection with the two-step method. In certain ments, said first medium used in the three-step process comprises medium sing one or more of: animal serum, e.g., human serum (e.g., human serum AB), fetal bovine serum (FBS) or fetal calf serum (FCS), e.g., 1% to % v/v serum, e.g., 5% to 20% v/v serum; stem cell factor (SCF), e.g, 1 ng/mL to 50 ng/mL SCF, FMS-like tyrosine kinase-3 ligand (Flt-3 ligand), e.g, 1 ng/ml to 30 ng/mL Flt-3 ligand, interleukin-7 (IL-7), e.g, 1 ng/mL to 50 ng/mL IL-7; opoietin (TPO), e.g., 1 ng/mL to 100 ng/mL, for example, 1 ng/mL to 50 PO; interleukin-2 (IL-2), e.g., up to 2000 IU/mL, for example, 50 IU/mL to 500 IU/mL; and/or heparin, e.g., low-weight heparin (LWH), WO 09668 e. g., 0.1 IU/mL to 10 IU/mL heparin. In certain embodiments, said first medium additionally comprises one or more of the following: antibiotics such as ycin; antioxidants such as transferrin, insulin, and/or beta-mercaptoethanol, sodium selenite, ascorbic acid; ethanolamine, and glutathione. In certain embodiments, said first medium additionally comprises OAC. In certain ments, said first medium additionally ses interleukin-6 (IL-6), leukemia inhibitory factor (LIF), G—CSF, GM-CSF, and/or MIP-ld. In certain embodiments, said first medium additionally comprises one or more anti-oxidants, e.g., holo—transferrin, insulin solution, reduced glutathione, sodium selenite, ethanolamine, ascorbic acid, aptoethanol, O-acetyl- L-camitine, N—acetylcysteine, (+/-) lipoic acid, nicotinamide, or resveratrol. In certain embodiments, the medium that provides the base for the first medium is a cell/tissue culture medium known to those of skill in the art, e.g., a commercially ble cell/tissue culture medium such as GBGM®, AIM-V®, TM 10, X—vrvoTM 15, OPTMIZER, STEMSPAN® H3000, O COMPLETETM, DMEM:Ham’s F12 (“F12”) (e.g., 2:1 ratio, or high glucose or low glucose DMEM), ed DMEM (Gibco), EL08-1D2, MyelocultTM H5100, IMDM, and/or RPMI-1640, or is a medium that comprises components lly included in known cell/tissue culture media, such as the components included in GBGM®, AHVI- V®, X-VIVOTM lO, X-VIVOTM 15, OPTMIZER, STEMSPAN® H3 000, CELLGRO COMPLETETM, zHam’s F12 (“F12”) (e.g., 2:1 ratio, or high glucose or low glucose DMEM), Advanced DMEM (Gibco), EL08-1D2, MyelocultTM H5100, IMDM, and/or RPMI- 1640. In a specific embodiment of any of the embodiments herein, the medium is not GBGM®.

In certain embodiments, the second medium used in the three-step processes described herein may contain any of the components of the first or second medium described in Section 5.24 in tion with the two-step method. In certain embodiments, said second medium used in the three-step process comprises medium comprising one or more of: animal serum, e.g., human serum (e.g., human serum AB), FBS or FCS, e.g., 5% to 20% v/v serum; SCF, e.g., 1 ng/mL to 50 ng/mL SCF, Flt-3 ligand, e.g., 1 ng/ml to 30 ng/mL Flt-3 ligand, IL-7, e.g., 1 ng/mL to 50 ng/mL IL-7; interleukin-15 (IL-15), e.g., 1 ng/mL to 50 ng/mL IL-15; and/or heparin, e.g., LWH, e.g., 0.1 IU/mL to 10 IU/mL heparin. In certain embodiments, said second medium additionally comprises one or more of the following: otics such as gentamycin; antioxidants such as transferrin, insulin, and/or beta-mercaptoethanol; sodium selenite, ascorbic acid, ethanolamine, and glutathione. In certain embodiments, said second medium additionally comprises OAC. In certain embodiments, said second medium additionally ses interleukin-6 (IL-6), leukemia inhibitory factor (LIF), G—CSF, GM-CSF, and/or MIP—la. In certain ments, said second medium additionally comprises one or more xidants, e. g., ransferrin, insulin solution, d glutathione, sodium selenite, ethanolamine, ascorbic acid, b-mercaptoethanol, O-acetyl-L-camitine, N—acetylcysteine, (+/-) lipoic acid, nicotinamide, or resveratrol. In certain embodiments, the medium that provides the base for the second medium is a cell/tissue culture medium known to those of skill in the art, e.g., a commercially available cell/tissue culture medium such as GBGM®, AH\/I-V®, X-VIVOTM 10, X-VIVOTM 15, OPTMIZER, STEMSPAN® H3000, CELLGRO COMPLETETM, DMEM:Ham’s F12 (“F 12”) (e.g., 2:] ratio, or high glucose or low glucose DMEM), Advanced DMEM (Gibco), EL08-1D2, ultTM H5100, IMDM, and/or RPMI-1640; or is a medium that comprises components generally included in known cell/tissue culture media, such as the components included in GBGM®, AIM-V®, TM 10, X-VIVOTM 15, ER, STEMSPAN® H3000, CELLGRO COMPLETETM, DMEM:Ham’s F12 (“F12”) (e.g., 2:1 ratio, or high glucose or low glucose DMEM), Advanced DMEM (Gibco), EL08-1D2, MyelocultTM H5100, IMDM, and/or RPMI—l640. In a specific embodiment of any of the embodiments herein, the medium is not GBGM®.

In certain embodiments, the third medium used in the three-step processes described herein may contain any of the components of the first or second medium described in n .2.4 in connection with the two-step method. In certain embodiments, said third medium used in the three-step process comprises medium comprising one or more of: animal serum, e.g., human serum (e.g., human serum AB), FBS or FCS, e.g., 5% to 20% v/v serum, SCF, e.g., 1 ng/mL to 50 ng/mL SCF, Flt—3 ligand, e.g., 1 ng/ml to 30 ng/mL Flt—3 ligand, IL-7, e.g., 1 ng/mL to 50 ng/mL IL-7, IL—15, e.g., 1 ng/mL to 50 ng/mL IL-15, and interleukin-2 (IL-2), e.g., in the range from 0 to 2000 IU/mL, for example, 50 IU/mL to 1000 IU/mL 1L-2. In certain embodiments, said third medium additionally comprises one or more of the following: antibiotics such as gentamycin; antioxidants such as transferrin, insulin, and/or beta-mercaptoethanol; sodium selenite, ascorbic acid, ethanolamine; and hione. In certain embodiments, said third medium additionally comprises OAC. In n embodiments, said third medium additionally comprises interleukin-6 (IL-6), leukemia inhibitory factor (LIF), G—CSF, GM-CSF, and/or MIP-loc. In certain ments, said third medium additionally comprises one or more anti-oxidants, e.g., ransferrin, insulin solution, reduced glutathione, sodium selenite, ethanolamine, ascorbic acid, aptoethanol, O-acetyl-L—carnitine, N—acetylcysteine, (+/—) lipoic acid, nicotinamide, or resveratrol. In certain embodiments, the medium that provides the base for the third medium is a cell/tissue e medium known to those of skill in the art, e.g., a commercially available cell/tissue culture medium such as GBGM®, AH\/I-V®, X-VIVOTM 10, X-VIVOTM 15, OPTMIZER, STEMSPAN® H3000, CELLGRO COMPLETETM, DMEM:Ham’s F12 (“F12”) (e.g., 2:1 ratio, or high glucose or low e DMEM), Advanced DMEM (Gibco), ELO8-1D2, MyelocultTM H5100, lMDM, and/or RPMI-l640; or is a medium that comprises components generally included in known cell/tissue culture media, such as the components included in GBGM®, AlM-V®, TM 10, x—vrvoTM 15, OPTMIZER, STEMSPAN® H3000, CELLGRO COMPLETETM, DMEM:Ham’s F12 ) (e.g., 2:1 ratio, or high glucose or low glucose DMEM), Advanced DMEM (Gibco), ELO8-1D2, MyelocultTM H5100, HVIDM, and/or RPMI-l640. In a specific embodiment of any of the embodiments herein, the medium is not GBGM®, In certain embodiments, in the three-step processes described herein, said poietic stem or progenitor cells are cultured in said first medium for l, 2, 3, 4, 5, 6, 7, 8, 9, 10, ll, l2, l3, 14, 15, 16, 17, 18, 19, or 20 days before said culturing in said second medium.

In certain embodiments, cells cultured in said first medium are cultured in said second medium for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12,13, 14,15, 16, 17, 18, 19, or 20 days before said culturing in said third medium. In certain embodiments, cells cultured in said first medium and said second medium are ed in said third medium for l, 2, 3, 4, 5, 6, 7, 8, 9, 10, ll, l2, l3, 14, , 16, 17, 18, 19, 20,21, 22,23, 24,25, 26, 27, 28, 29, or 30 days, or for more than 30 days.

In n embodiments, in the three-step processes described herein, said poietic stem or progenitor cells are cultured in said first medium for 2-12 days, 3-11 days, for example, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, or 9-11 days, before said culturing in said second medium. In certain embodiments, cells cultured in said first medium are cultured in said second medium for 1-10 days, for example, 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, or 7-9 days, before said culturing in said third medium. In n embodiments, cells cultured in said first medium and said second medium are cultured in said third medium for 2-27 days, for example, 3-25 days, e.g., for 3-5,4-6,5-7,6-8,7-9,8-10,9-11,10-12,11-13,12-14,13-15,14-16,15-17,16-18,17-19,18- , 19—21, 20—22, 21—23, 22—24, or 23—25 days.

In a specific embodiment, in the three-step processes described herein, said hematopoietic stem or progenitor cells are cultured in said first medium for 9 days before said culturing in said second medium; cultured in said second medium for 5 days before said culturing in said third medium; and cultured in said third medium for 7 days, i.e., the cells are cultured a total of 21 days.

In a specific embodiment, in the three-step processes described herein, said hematopoietic stem or progenitor cells are cultured in said first medium for 7-9 days before said culturing in said second medium; cultured in said second medium for 5-7 days before said culturing in said third medium; and cultured in said third medium for 21-35 days, i.e., the cells are cultured a total of 35 days. In a more specific embodiment, in the three-step processes bed herein, said hematopoietic stem or progenitor cells are ed in said first medium for 9 days before said culturing in said second medium; cultured in said second medium for 5 days before said culturing in said third medium; and cultured in said third medium for 21 days, 1'. e., the cells are cultured a total of 35 days. .2.9. Methods of Producing Three-Stage NK Cells Production ofNK cells and NK cell populations by the three-stage method comprises expanding a population of hematopoietic cells. During cell ion, a ity of poietic cells within the hematopoietic cell population differentiate into NK cells. In one aspect, provided herein is a method of producing NK cells comprising culturing hematopoietic stem cells or itor cells, e.g., CD34+ stem cells or progenitor cells, in a first medium comprising a stem cell mobilizing agent and thrombopoietin (Tpo) to produce a first population of cells, subsequently culturing said first population of cells in a second medium comprising a stem cell mobilizing agent and interleukin-15 (IL-15), and lacking Tpo, to produce a second tion of cells, and subsequently culturing said second tion of cells in a third medium comprising IL-2 and IL-15, and lacking a stem cell zing agent and LMWH, to produce a third population of cells, wherein the third population of cells comprises natural killer cells that are CD56+, CD3-, and wherein at least 70%, for example 80%, of the natural killer cells are Viable with certain ments, such natural killer cells se natural killer cells that are CD16—. In certain embodiments, such l killer cells comprise natural killer cells that are CD94—.

In one embodiment, provided herein is a three-stage method of producing NK cell populations. In certain embodiments, the method of ion and differentiation of the hematopoietic cells, as described , to produce NK cell populations according to a three— stage method described herein comprises maintaining the cell population comprising said hematopoietic cells at between about 2 X 104 and about 6 X 106 cells per milliliter. In certain aspects, said hematopoietic stem or progenitor cells are initially inoculated into said first medium from 1 X 104 to 1 X 105 mL. In a specific aspect, said hematopoietic stem or progenitor cells are initially inoculated into said first medium at about 3 X 104 mL.

In certain embodiments, said hematopoietic stem or progenitor cells are mammalian cells. In specific embodiments, said hematopoietic stem or progenitor cells are human cells. In specific embodiments, said poietic stem or progenitor cells are primate cells. In c embodiments, said hematopoietic stem or progenitor cells are canine cells. In specific embodiments, said hematopoietic stem or progenitor cells are rodent cells.

In certain aspects, said first population of cells are initially inoculated into said second medium from 5 X 104 to 5 X 105 mL. In a specific aspect, said first tion of cells is initially inoculated into said second medium at about 1 X 105 cells/mL.

In certain aspects said second population of cells is initially inoculated into said third medium from 1 X 105 to 5 X 106 mL. In n s, said second population of cells is initially inoculated into said third medium from 1 X 105 to 1 X 106 cells/mL. In a specific aspect, said second population of cells is initially inoculated into said third medium at about 5 X 105 cells/mL. In a more specific aspect, said second tion of cells is initially inoculated into said third medium at about 5 X 105 cells/mL in a spinner flask. In a specific aspect, said second population of cells is initially inoculated into said third medium at about 3 X 105 cells/mL. In a more specific aspect, said second population of cells is initially inoculated into said third medium at about 3 X 105 cells/mL in a static culture.

In a certain embodiment, the three-stage method comprises a first stage (“stage 1”) comprising culturing hematopoietic stem cells or progenitor cells, e.g, CD34+ stem cells or progenitor cells, in a first medium for a specified time period, e.g., as described herein, to produce a first population of cells. In certain embodiments, the first medium comprises a stem cell mobilizing agent and thrombopoietin (Tpo). In n embodiments, the first medium ses in addition to a stem cell mobilizing agent and Tpo, one or more of LMWH, Flt-3L, SCF, IL-6, IL-7, G—CSF, and GM-CSF.

In a specific embodiment, the first medium comprises each of the first medium ses in on to a stem cell mobilizing agent and Tpo, each of LMWH, Flt-3L, SCF, IL-6, IL—7, G-CSF, and GM-CSF.

In certain embodiments, subsequently, in “stage 2” said cells are cultured in a second medium for a specified time period, e.g., as bed herein, to produce a second population of cells. In certain embodiments, the second medium ses a stem cell mobilizing agent and interleukin-15 (IL-15), and lacks Tpo. In certain embodiments, the second medium comprises, in on to a stem cell mobilizing agent and IL-15, one or more of LMWH, Flt-3, SCF, IL-6, IL-7, G—CSF, and GM-CSF. In certain embodiments, the second medium ses, in addition to a stem cell mobilizing agent and IL—15, each of LMWH, Flt-3, SCF, IL-6, IL-7, G—CSF, and GM-CSF.

In n embodiments, subsequently, in “stage 3” said cells are cultured in a third medium for a specified time period, e. g., as described herein, to produce a third tion of cell, e.g., natural killer cells. In certain embodiments, the third medium comprises IL-2 and IL- , and lacks a stem cell mobilizing agent and LMWH. In certain embodiments, the third medium comprises in addition to IL-2 and IL-15, one or more of SCF, IL-6, IL-7, G—CSF, and GM-CSF. In certain embodiments, the third medium comprises in addition to IL-2 and IL-15, each of SCF, IL-6, IL-7, G—CSF, and GM-CSF.

In a specific embodiment, the three-stage method is used to produce NK cell tions. In certain embodiments, the three-stage method is conducted in the absence of l feeder cell support. In certain embodiments, the three-stage method is conducted in the absence of exogenously added steroids (e.g., cortisone, hydrocortisone, or derivatives thereof).

In certain aspects, said first medium used in the three-stage method comprises a stem cell mobilizing agent and thrombopoietin (Tpo). In certain aspects, the first medium used in the three-stage method comprises, in addition to a stem cell mobilizing agent and Tpo, one or more of Low Molecular Weight Heparin (LMWH), Flt-3 Ligand L), stem cell factor (SCF), IL-6, IL-7, granulocyte colony-stimulating factor (G—CSF), or granulocyte—macrophage-stimulating factor (GM-CSF). In certain aspects, the first medium used in the three-stage method comprises, in addition to a stem cell mobilizing agent and Tpo, each of LMWH, Flt-3L, SCF, IL-6, IL-7, G- CSF, and GM-CSF. In certain aspects, said Tpo is present in the first medium at a concentration of from 1 ng/mL to 100 ng/mL, from 1 ng/mL to 50 ng/mL, from 20 ng/mL to 30 ng/mL, or about 25 ng/mL. In n aspects, in the first medium, the LMWH is present at a concentration of from 1U/mL to lOU/mL, the Flt-3L is present at a concentration of from 1 ng/mL to 50 ng/mL; the SCF is t at a concentration of from 1 ng/mL to 50 ng/mL; the IL-6 is present at a concentration of from 0.01 ng/mL to 0.1 ng/mL, the IL-7 is t at a concentration of from 1 ng/mL to 50 ng/mL; the G—CSF is present at a concentration of from 0.01 ng/mL to 0.50 ng/mL; and the GM-CSF is t at a concentration of from 0.005 ng/mL to 0.1 ng/mL. In certain aspects, in the first medium, the LMWH is present at a concentration of from 4U/mL to 5U/mL, the Flt—3L is present at a concentration of from 20 ng/mL to 30 ng/mL; the SCF is present at a concentration of from 20 ng/mL to 30 ng/mL; the IL-6 is present at a concentration of from 0.04 ng/mL to 0.06 ng/mL; the IL—7 is present at a concentration of from 20 ng/mL to 30 ng/mL; the G—CSF is present at a concentration of from 0.20 ng/mL to 0.30 ng/mL, and the GM-CSF is present at a concentration of from 0.005 ng/mL to 0.5 ng/mL. In certain aspects, in the first medium, the LMWH is t at a concentration of about 4.5U/mL; the Flt-3L is present at a concentration of about 25 ng/mL, the SCF is present at a concentration of about 27 ng/mL; the IL-6 is present at a concentration of about 0.05 ng/mL, the IL-7 is present at a concentration of about 25 ng/mL, the G—C SF is present at a tration of about .25 ng/mL; and the GM-CSF is present at a concentration of about 0.01 ng/mL. In certain embodiments, said first medium additionally comprises one or more of the following: antibiotics such as gentamycin; antioxidants such as transferrin, insulin, and/or ercaptoethanol; sodium selenite, ascorbic acid, ethanolamine, and glutathione. In certain embodiments, the medium that es the base for the first medium is a cell/tissue culture medium known to those of skill in the art, e.g., a cially available cell/tissue culture medium such as , STEMMACSTM, GBGM®, AIM-V®, TM 10, X—vrvoTM 15, OPTMIZER, STEMSPAN® H3 000, CELLGRO COMPLETETM, DMEMzHam’s F12 (“F12”) (e.g., 2:1 ratio, or high glucose or low glucose DMEM), Advanced DMEM (Gibco), EL08-1D2, MyelocultTM H5100, IMDM, and/or RPMI- 1640; or is a medium that comprises components generally included in known cell/tissue e media, such as the components included in GBGM®, AIM-V®, X-VIVOTM 10, X-VIVOTM 15, OPTMIZER, STEMSPAN® H3000, O COMPLETETM, DMEM:Ham’s F12 (“Fl2”) (e.g., 2:1 ratio, or high glucose or low glucose DMEM), Advanced DMEM (Gibco), EL08-1D2, MyelocultTM H5100, IMDM, and/or RPMI—l640. In a specific embodiment of any of the embodiments herein, the medium is not GBGM® In certain aspects, said second medium used in the three-stage method comprises a stem cell mobilizing agent and interleukin-15 (IL-15), and lacks Tpo. In certain aspects, the second medium used in the three-stage method comprises, in on to a stem cell mobilizing agent and IL-15, one or more of LMWH, Flt—3, SCF, IL-6, IL-7, G—CSF, and GM-CSF. In certain aspects, the second medium used in the three-stage method comprises, in addition to a stem cell mobilizing agent and IL-15, each of LMWH, Flt-3, SCF, IL-6, IL-7, G—CSF, and GM- CSF. In certain aspects, said lL-15 is present in said second medium at a concentration of from 1 ng/mL to 50 ng/mL, from 10 ng/mL to 30 ng/mL, or about 20 ng/mL. In certain aspects, in said second medium, the LMWH is present at a concentration of from lU/mL to lOU/mL, the Flt-3L is present at a concentration of from 1 ng/mL to 50 ng/mL; the SCF is present at a concentration of from 1 ng/mL to 50 ng/mL, the IL-6 is present at a concentration of from 0.01 ng/mL to 0.1 ng/mL; the IL—7 is present at a concentration of from 1 ng/mL to 50 ng/mL; the G—CSF is t at a concentration of from 0.01 ng/mL to 0.50 ng/mL, and the GM-C SF is t at a concentration of from 0.005 ng/mL to 0.1 ng/mL. In certain aspects, in the second medium, the LMWH is present in the second medium at a concentration of from 4U/mL to 5U/mL, the Flt-3L is present at a concentration of from 20 ng/mL to 30 ng/mL; the SCF is present at a concentration of from 20 ng/mL to 30 ng/mL; the IL—6 is present at a concentration of from 0.04 ng/mL to 0.06 ng/mL, the IL-7 is present at a concentration of from 20 ng/mL to 30 ng/mL, the G—CSF is present at a concentration of from 0.20 ng/mL to 0.30 ng/mL; and the GM-C SF is present at a tration of from 0.005 ng/mL to 0.5 ng/mL. In certain aspects, in the second medium, the LMWH is present in the second medium at a concentration of from 4U/mL to 5U/mL; the Flt-3L is present at a concentration of from 20 ng/mL to 30 ng/mL, the SCF is present at a concentration of from 20 ng/mL to 30 ng/mL, the IL-6 is t at a concentration of from 0.04 ng/mL to 0.06 ng/mL; the IL—7 is t at a concentration of from 20 ng/mL to 30 ng/mL; the G—CSF is present at a concentration of from 0.20 ng/mL to 0.30 ng/mL; and the GM-C SF is present at a concentration of from 0.005 ng/mL to 0.5 ng/mL. In certain aspects, in the second medium, the LMWH is present in the second medium at a concentration of about L, the Flt-3L is present at a concentration of about 25 ng/mL; the SCF is present at a concentration of about 27 ng/mL; the IL—6 is present at a concentration of about 0.05 ng/mL, the IL-7 is t at a concentration of about 25 ng/mL, the G—CSF is present at a concentration of about 0.25 ng/mL, and the GM-CSF is t at a concentration of about 0.01 ng/mL. In certain embodiments, said second medium additionally comprises one or more of the following: antibiotics such as gentamycin, antioxidants such as transferrin, insulin, and/or beta-mercaptoethanol; sodium selenite; ascorbic acid; ethanolamine; and glutathione. In certain embodiments, the medium that provides the base for the second medium is a cell/tissue culture medium known to those of skill in the art, e.g., a commercially ble cell/tissue e medium such as SCGMTM, STEMMACSTM, GBGM®, , X-VIVOTM 10, X—vrvoTM 15, OPTMIZER, STEMSPAN® H3000, CELLGRO COMPLETETM, DMEM:Ham’s F12 ) (e.g., 2:1 ratio, or high glucose or low glucose DMEM), Advanced DMEM (Gibco), EL08-1D2, MyelocultTM H5100, IMDM, and/or RPMI-1640; or is a medium that comprises components generally included in known issue culture media, such as the components included in GBGM®, AIM- V®, X-VIVOTM 10, X-VIVOTM 15, ER, STEMSPAN® H3000, CELLGRO TETM, am’s F12 (“F12”) (e.g., 2:1 ratio, or high glucose or low glucose DMEM), Advanced DMEM (Gibco), EL08-1D2, MyelocultTM H5100, IMDM, and/or RPMI- 1640. In a specific embodiment of any of the embodiments herein, the medium is not GBGM®.

In certain embodiments, the third medium used in the three-stage method comprises medium comprising In certain aspects, said third medium used in the three-stage method comprises IL-2 and IL—15, and lacks a stem cell mobilizing agent and LMWH. In certain aspects, the third medium used in the three-stage method comprises, in addition to IL-2 and IL- , one or more of SCF, IL-6, IL-7, G—CSF, or GM-CSF. In certain aspects, the third medium used in the three-stage method comprises, in addition to IL-2 and 1L-15, each of SCF, IL-6, IL-7, G—CSF, and GM-CSF. In certain aspects, said IL-2 is present in said third medium at a concentration of from 10 U/mL to 10,000 U/mL and said 1L—15 is present in said third medium at a concentration of from 1 ng/mL to 50 ng/mL. In certain aspects, said 1L-2 is t in said third medium at a concentration of from 100 U/mL to 10,000 U/mL and said IL-lS is present in said third medium at a concentration of from 1 ng/mL to 50 ng/mL. In n aspects, said IL-2 is present in said third medium at a tration of from 300 U/mL to 3,000 U/mL and said IL- is present in said third medium at a concentration of from 10 ng/mL to 30 ng/mL. In certain aspects, said IL-2 is present in said third medium at a concentration of about 1,000 U/mL and said IL-15 is present in said third medium at a concentration of about 20 ng/mL. In certain s, in said third medium, the SCF is present at a concentration of from 1 ng/mL to 50 ng/mL; the IL—6 is present at a concentration of from 0.01 ng/mL to 0.1 ng/mL; the 1L-7 is present at a concentration of from 1 ng/mL to 50 ng/mL, the G—C SF is present at a concentration of from 0.01 ng/mL to 0.50 ng/mL; and the GM-C SF is present at a concentration of from 0.005 ng/mL to 0.1 ng/mL. In certain aspects, in said third medium, the SCF is present at a concentration of from 20 ng/mL to 30 ng/mL; the IL-6 is present at a concentration of from 0.04 ng/mL to 0.06 ng/mL; the IL-7 is present at a concentration of from 20 ng/mL to 30 ng/mL; the G—CSF is present at a concentration of from 0.20 ng/mL to 0.30 ng/mL; and the GM-CSF is present at a concentration of from 0.005 ng/mL to 0.5 ng/mL. In certain aspects, in said third medium, the SCF is present at a concentration of about 22 ng/mL, the IL-6 is present at a concentration of about 0.05 ng/mL; the IL-7 is present at a concentration of about 20 ng/mL; the G—CSF is present at a concentration of about 0.25 ng/mL, and the GM—CSF is present at a concentration of about 0.01 ng/mL. In certain ments, said third medium additionally comprises one or more of the following: antibiotics such as gentamycin; antioxidants such as errin, insulin, and/or beta-mercaptoethanol; sodium selenite; ascorbic acid; ethanolamine; and glutathione. In certain embodiments, the medium that provides the base for the third medium is a cell/tissue culture medium known to those of skill in the art, e.g., a commercially ble cell/tissue e medium such as SCGMTM, STEMMACSTM, GBGM®, AIM-V®, X- v1voTM 10, X—VIvoTM 15, OPTMIZER, STEMSPAN® H3000, CELLGRO COMPLETETM, DMEM:Ham’s F12 (“F12”) (e.g., 2:1 ratio, or high glucose or low glucose DMEM), Advanced DMEM ), EL08-1D2, MyelocultTM H5100, IlVIDM, and/or RPMI-1640; or is a medium that comprises components generally included in known issue culture media, such as the components included in GBGM®, AIM-V®, X—VIVOTM 10, X—VIVOTM 15, ER, STEMSPAN® H3000, CELLGRO COMPLETETM, DMEM1Ham’s F12 (“F12”) (e.g., 2:1 ratio, or high glucose or low glucose DMEM), Advanced DIVIEM (Gibco), EL08-1D2, MyelocultTM H5100, IMDM, and/or RPMI—1640. In a specific ment of any of the embodiments herein the medium is not GBGM® Generally, the particularly recited medium components do not refer to possible tuents in an undefined component of said medium. For example, said Tpo, IL-2, and IL-15 are not comprised within an undefined component of the first medium, second medium or third , e.g., said Tpo, IL-2, and IL-15 are not comprised within serum. Further, said LMWH, Flt-3, SCF, IL-6, IL-7, G—CSF, and/or GM-C SF are not comprised within an undefined component of the first , second medium or third medium, e.g, said LMWH, Flt-3, SCF, IL-6, IL-7, G—C SF, and/or GM-CSF are not comprised within serum.

In certain aspects, said first medium, second medium or third medium ses human serum—AB. In certain aspects, any of said first medium, second medium or third medium comprises 1% to 20% human serum-AB, 5% to 15% human serum-AB, or about 2, 5, or 10% human serum-AB.

In certain embodiments, in the three-stage methods described herein, said poietic stem or progenitor cells are cultured in said first medium for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ll, 12, l3, 14, 15, 16, 17, 18, 19, or 20 days, In certain ments, in the three-stage methods bed herein, cells are cultured in said second medium for l, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, l3, 14, 15, l6, 17, 18, 19, or 20 days. In certain embodiments, in the three-stage methods described herein, cells are cultured in said third medium for l, 2, 3, 4, 5, 6, 7, 8, 9, 10, ll, l2, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days, or for more than 30 days.

In a specific embodiment, in the three-stage methods described , said hematopoietic stem or progenitor cells are cultured in said first medium for 7-13 days to produce a first population of cells, before said culturing in said second medium, said first population of cells are ed in said second medium for 2-6 days to produce a second population of cells before said culturing in said third medium; and said second population of cells are cultured in said third medium for 10-30 days, 1'.e., the cells are cultured a total of 19-49 days.

In a specific embodiment, in the three-stage methods described herein, in the three- stage methods bed herein, said hematopoietic stem or progenitor cells are cultured in said first medium for 8-12 days to produce a first population of cells, before said culturing in said second medium, said first population of cells are cultured in said second medium for 3-5 days to produce a second tion of cells before said culturing in said third ; and said second population of cells are cultured in said third medium for 15-25 days, i.e., the cells are cultured a total of 26-42 days.

In a specific embodiment, in the three-stage methods described , said hematopoietic stem or progenitor cells are cultured in said first medium for about 10 days to e a first population of cells, before said culturing in said second medium; said first population of cells are cultured in said second medium for about 4 days to produce a second population of cells before said culturing in said third medium; and said second population of cells are cultured in said third medium for about 21 days, 1'. e., the cells are cultured a total of about 35 days. 2015/068069 In certain aspects, said culturing in said first medium, second medium and third medium are all performed under static culture conditions, e.g., in a culture dish or culture flask.

In certain aspects, said culturing in at least one of said first medium, second medium or third medium are med in a spinner flask. In certain aspects, said culturing in said first medium and said second medium is performed under static culture conditions, and said culturing in said third medium is performed in a spinner flask.

In n aspects, said culturing is performed in a r flask. In other aspects, said culturing is med in a G—ReX device. In yet other aspects, said culturing is performed in a WAVE bioreactor.

In certain aspects, said hematopoietic stem or progenitor cells are initially inoculated into said first medium from 1 X 104 to l X 105 cells/mL. In a specific aspect, said hematopoietic stem or progenitor cells are initially inoculated into said first medium at about 3 X 104 cells/mL.

In certain aspects, said first population of cells are initially inoculated into said second medium from 5 X 104 to 5 X 105 cells/mL. In a specific aspect, said first tion of cells is initially ated into said second medium at about 1 X 105 cells/mL.

In certain aspects said second population of cells is initially ated into said third medium from 1 X 105 to 5 X 106 cells/mL. In certain aspects, said second population of cells is initially inoculated into said third medium from 1 X 105 to l X 106 cells/mL. In a specific , said second population of cells is initially inoculated into said third medium at about 5 X 105 cells/mL. In a more specific aspect, said second population of cells is lly inoculated into said third medium at about 5 X 105 cells/mL in a spinner flask. In a c aspect, said second population of cells is initially inoculated into said third medium at about 3 X 105 cells/mL. In a more specific aspect, said second population of cells is initially inoculated into said third medium at about 3 X 105 cells/mL in a static culture. .2.10. Isolation of Cells Methods of isolating l killer cells are known in the art and can be used to isolate the natural killer cells, e.g., activated NK cells or TSPNK cells (e.g., NK progenitor cells) produced using the three-step process, described herein. NK cells can be isolated or enriched by staining cells from a tissue source, e.g, peripheral blood, with dies to CD56 and CD3, and selecting for CD56+CD3_ cells. NK cells, e.g., activated NK cells or TSPNK cells, can be isolated using a commercially available kit, for example, the NK Cell Isolation Kit nyi Biotec). NK cells, e.g., activated NK cells or TSPNK cells, can also be isolated or enriched by removal of cells other than NK cells in a tion of cells that comprise the NK cells, e.g., activated NK cells or TSPNK cells. For example, NK cells, e.g., activated NK cells or TSPNK cells, may be ed or enriched by depletion of cells displaying non-NK cell markers using, e. g., antibodies to one or more of CD3, CD4, CD14, CD19, CD20, CD36, CD66b, CD123, HLA DR and/or CD23 5a phorin A). Negative isolation can be carried out using a commercially available kit, e. g., the NK Cell Negative Isolation Kit (Dynal h). Cells isolated by these methods may be additionally sorted, e.g., to separate CD16“ and CD16‘ cells.

Cell separation can be accomplished by, e.g., flow cytometry, cence-activated cell sorting (FACS), or, preferably, ic cell sorting using microbeads conjugated with specific antibodies. The cells may be isolated, e.g, using a magnetic activated cell g (MACS) technique, a method for separating particles based on their y to bind magnetic beads (e.g., about 05—100 um diameter) that comprise one or more specific antibodies, e.g, anti- CD56 antibodies. Magnetic cell tion can be performed and automated using, e.g., an AUTOMACSTM Separator (Miltenyi). A variety of useful modifications can be performed on the magnetic microspheres, including covalent addition of antibody that specifically izes a particular cell e molecule or hapten. The beads are then mixed with the cells to allow g. Cells are then passed through a magnetic field to separate out cells having the specific cell surface marker. In one embodiment, these cells can then isolated and re—mixed with magnetic beads coupled to an antibody against additional cell surface markers. The cells are again passed through a magnetic field, isolating cells that bound both the antibodies. Such cells can then be diluted into separate dishes, such as microtiter dishes for clonal isolation.

In some embodiments, the purity of the isolated or enriched natural killer cells can be confirmed by detecting one or more of CD56, CD3 and CD16. .2.11. Preservation of Cells/Perfusate Cells, e. g., NK cells produced using the methods described herein, e.g., activated NK cells or TSPNK cells (e.g., NK progenitor cells) produced using the three-step process described herein, or placental ate cells comprising hematopoietic stem cells or progenitor cells, or placental perfusate, can be preserved, that is, placed under conditions that allow for long-term storage, or under conditions that inhibit cell death by, e.g., apoptosis or necrosis.

Placental ate can be produced by e of a cell collection composition through at least a part of the placenta, e.g., through the placental vasculature. The cell collection composition comprises one or more compounds that act to preserve cells contained within the perfusate. Such a placental cell tion composition can se an apoptosis inhibitor, necrosis inhibitor and/or an oxygen-carrying perfluorocarbon, as described in related US. ation Publication No. 20070190042, the disclosure of which is hereby incorporated by reference in its ty.

In one embodiment, perfusate or a population of placental cells are collected from a ian, e.g, human, post—partum placenta by bringing the perfusate or population of cells into proximity with a cell collection composition comprising an inhibitor of apoptosis and an oxygen-carrying perfluorocarbon, n said inhibitor of apoptosis is present in an amount and for a time sufficient to reduce or prevent apoptosis in the tion of placental cells, e.g., adherent placental cells, for example, placental stem cells or placental multipotent cells, as compared to a population of cells not contacted or brought into proximity with the inhibitor of apoptosis. For example, the placenta can be perfused with the cell collection composition, and placental cells, e.g, total nucleated tal cells, are isolated therefrom. In a specific ment, the tor of apoptosis is a caspase inhibitor. In another specific embodiment, said inhibitor of apoptosis is a INK inhibitor. In a more c embodiment, said INK inhibitor does not modulate differentiation or proliferation of adherent placental cells, e.g., adherent placental stem cells or adherent placental multipotent cells. In another embodiment, the cell collection ition comprises said inhibitor of apoptosis and said oxygen-carrying perfluorocarbon in separate phases. In another embodiment, the cell collection composition comprises said inhibitor of apoptosis and said oxygen—carrying perfluorocarbon in an emulsion.

In another embodiment, the cell collection composition additionally comprises an emulsifier, e. g., lecithin. In another embodiment, said apoptosis inhibitor and said perfluorocarbon are between about 0 °C and about 25 °C at the time of bringing the placental cells into proximity with the cell collection composition. In another more specific embodiment, said sis inhibitor and said perfluorocarbon are between about 2 °C and 10 °C, or between about 2 °C and about 5 °C, at the time of ng the tal cells into proximity with the cell collection composition. In another more specific embodiment, said bringing into proximity is performed during transport of said population of cells. In another more specific embodiment, said bringing into proximity is performed during ng and thawing of said population of cells.

In another embodiment, placental perfusate and/or tal cells can be collected and preserved by bringing the perfusate and/or cells into proximity with an inhibitor of apoptosis and an organ-preserving compound, wherein said inhibitor of apoptosis is t in an amount and for a time sufficient to reduce or prevent apoptosis of the cells, as compared to perfusate or placental cells not contacted or brought into proximity with the inhibitor of apoptosis. In a specific ment, the organ-preserving compound is UW solution (described in US. Patent No. 4,798,824; also known as VIASPANTM; see also Southard el al., Transplantation 49(2):251- 257 (1990) or a solution described in Stern er al., US. Patent No. 5,552,267, the disclosures of which are hereby incorporated by reference in their entireties. In r embodiment, said organ-preserving composition is hydroxyethyl starch, lactobionic acid, raffinose, or a combination thereof. In another embodiment, the placental cell collection composition additionally comprises an oxygen-carrying perfluorocarbon, either in two phases or as an emulsion.

In another ment, placental cells are brought into proximity with a cell collection composition comprising an apoptosis inhibitor and oxygen—carrying perfluorocarbon, organ-preserving compound, or combination thereof, during perfusion. In another embodiment, placental cells are brought into ity with said cell collection compound after collection by perfusion. [003 02] Typically, during placental cell collection, enrichment and isolation, it is preferable to minimize or eliminate cell stress due to hypoxia and mechanical . In r embodiment of the method, therefore, placental ate or a tion of placental cells is exposed to a hypoxic condition during collection, enrichment or isolation for less than six hours during said preservation, wherein a hypoxic condition is a concentration of oxygen that is less than normal blood oxygen tration. In a more specific embodiment, said perfusate or population of placental cells is d to said hypoxic condition for less than two hours during said preservation. In another more c embodiment, said population of tal cells is exposed to said hypoxic condition for less than one hour, or less than thirty minutes, or is not d to a hypoxic condition, during collection, enrichment or isolation. In another specific embodiment, said population of placental cells is not exposed to shear stress during collection, enrichment or isolation.

Cells, e. g., placental perfusate cells, hematopoietic cells, e.g., CD34+ hematopoietic stem cells; NK cells produced using the processes described herein, e.g., activated NK cells or TSPNK cells (e.g., NK progenitor cells), isolated adherent placental cells provided herein can be cryopreserved, e.g., in eservation medium in small containers, e.g., ampoules or septum vials. In specific ments, cells are or have been erved at a concentration of about 1 X 104 — 5 X 108 cells per mL. In specific embodiments, cells are or have been cryopreserved at a tration of about 1 X 106 — 1.5 X 107 cells per mL. In more specific embodiments, cells provided herein are or have been eserved at a concentration of about 1 X 104, 5 X 104, l X 105, 5 x 105, 1 x 10", 5 x 106, 1 x 107, 1.5 x 107 cells per mL. In certain embodiments, NK cells have been cryopreserved before stration. In certain embodiments, NK cells have not been cryopreserved before administration. [003 04] Suitable eservation medium includes, but is not limited to, normal saline, culture medium including, e.g, growth medium, or cell freezing medium, for example commercially available cell freezing medium, e.g., C2695, C2639 or C6039 (Sigma), CryoStor® CS2, CryoStor® CS5 or CryoStor®CSlO (BioLife Solutions). In one embodiment, cryopreservation medium comprises DMSO (dimethylsulfoxide), at a concentration of, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10% (v/v). Cryopreservation medium may comprise additional agents, for e, methylcellulose, dextran, albumin (e.g., human serum albumin), trehalose, and/or glycerol. In certain ments, the cryopreservation medium comprises about 1%- % DMSO, about 25%-75% dextran and/or about 20-60% human serum albumin (HSA). In n embodiments, the cryopreservation medium comprises about l%-lO% DMSO, about %—75% trehalose and/or about 20-60% human HSA. In a specific embodiment, the cryopreservation medium comprises 5% DMSO, 55% dextran and 40% HSA. In a more specific embodiment, the cryopreservation medium comprises 5% DMSO, 55% dextran (10% w/v in normal saline) and 40% HSA. In another specific embodiment, the cryopreservation medium comprises 5% DMSO, 55% trehalose and 40% HSA. In a more specific embodiment, the cryopreservation medium comprises 5% DMSO, 55% ose (10% w/v in normal saline) and 40% HSA. In another specific embodiment, the cryopreservation medium comprises CryoStor® CS5 In another specific embodiment, the eservation medium comprises CryoStor®CSlO. [003 05] Cells can be cryopreserved by any of a variety of methods known in the art, and at any stage of cell culturing, expansion or differentiation. For example, cells provided herein can be cryopreserved right after ion from the origin tissues or organs, e.g., tal perfusate or umbilical cord blood, or during, or after either the first or second step of the methods outlined above. In certain embodiments, the hematopoietic cells, e.g., hematopoietic stem or progenitor cells are cryopreserved within about 1, 5, 10, 15, 20, 30, 45 minutes or within about 1, 2, 4, 6, , 12, 18, 20 or 24 hours after ion from the origin tissues or organs. In certain embodiments, said cells are cryopreserved within 1, 2 or 3 days after ion from the origin tissues or organs. In certain ments, said cells are cryopreserved after being cultured in a first medium as described above, for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 days. In some embodiments, said cells are cryopreserved after being ed in a first medium as described above, for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 days, and in a second medium for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 days as described above. In some embodiments, when TSPNK cells (e.g., NK progenitor cells) are made using a three-step process described herein, said cells are cryopreserved after being cultured in a first medium about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days; and/or after being cultured in a second medium about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or days; and/or after being ed in a third medium about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22,23, 24, or 25 days. In a specific embodiment, NK progenitor cells are made using a three-step process described herein, and said cells are eserved after being ed in a first medium for 9 days, after being cultured in a second medium for 5 days, and after being cultured in a third medium for 7 days.

In one aspect, a population ofNK cells, e.g., activated NK cells, are produced by a process comprising: (a) seeding a population of hematopoietic stem or progenitor cells in a first medium comprising interleukin-15 (IL-15) and, optionally, one or more of stem cell factor (SCF) and interleukin-7 (IL-7), wherein said IL-15 and optional SCF and IL-7 are not comprised within an undefined component of said medium, such that the population expands, and a plurality of hematopoietic stem or itor cells within said population of hematopoietic stem or progenitor cells differentiate into NK cells during said expanding; (b) expanding the cells from step (a) in a second medium comprising interleukin-2 (IL-2), to produce a population of activated NK cells, and (c) cryopreserving the NK cells from step (b) in a cryopreservation WO 09668 medium. In a specific ment, said step (c) further comprises (1) preparing a cell suspension solution; (2) adding cryopreservation medium to the cell suspension solution from step (1) to obtain cryopreserved cell suspension; (3) cooling the cryopreserved cell suspension from step (3) to obtain a cryopreserved sample; and (4) storing the cryopreserved sample below - 80 °C. In certain embodiments, the method includes no intermediary steps between step (a) and (b), and between step (b) and (c), and/or no additional culturing steps prior to step (a).

In another embodiment, the cryopreserving of a population ofNK cells, e.g., activated NK cells or TSPNK cells (e.g., NK progenitor , comprises: (a) expanding a population of hematopoietic stem or progenitor cells in a first medium comprising one or more of stem cell factor (SCF), IL-2, interleukin-7 (IL-7), interleukin-15 (IL-15) and heparin, and wherein said SCF, IL-2, IL-7 and IL-15 are not comprised within an undefined ent of said medium, and wherein a plurality of hematopoietic stem or progenitor cells within said population of hematopoietic stem or progenitor cells differentiate into NK cells during said expanding, (b) expanding the cells from step (a) in a second medium comprising interleukin-2 (IL-2), to produce activated NK cells; and (c) eserving the NK cells from step (b) in a cryopreservation medium. In a specific embodiment, said step (c) further ses (1) ing a cell suspension solution; (2) adding cryopreservation medium to the cell suspension solution from step (1) to obtain cryopreserved cell suspension; (3) cooling the cryopreserved cell suspension from step (3) to obtain a cryopreserved sample, and (4) storing the cryopreserved sample below -80 °C. In n embodiments, the method includes no intermediary steps between step (a) and (b), and between step (b) and (c).

Cells are preferably cooled in a lled-rate freezer, e.g., at about 0.1, 0.3, 0.5, l, or 2 °C/min during cryopreservation. A preferred cryopreservation temperature is about -80 °C to about -180 °C, preferably about -l25 °C to about —l40 °C. Cryopreserved cells can be transferred to liquid nitrogen prior to thawing for use. In some embodiments, for example, once the ampoules have reached about -90 °C, they are transferred to a liquid nitrogen e area. eserved cells preferably are thawed at a temperature of about 25 °C to about 40 °C, preferably to a temperature of about 37 °C. In certain embodiments, the cryopreserved cells are thawed after being cryopreserved for about 1, 2, 4, 6, 10, 12, 18, 20 or 24 hours, or for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 days. In certain ments, the cryopreserved cells are thawed after being cryopreserved for about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27 or 28 months. In certain embodiments, the cryopreserved cells are thawed after being cryopreserved for about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 years.

Suitable thawing medium includes, but is not limited to, normal saline, plasmalyte culture medium including, for example, growth medium, e.g., RPMI medium. In preferred embodiments, the thawing medium comprises one or more of medium supplements (e.g., nutrients, cytokines and/or factors), Medium supplements le for thawing cells provided herein include, for e without limitation, serum such as human serum AB, fetal bovine serum (PBS) or fetal calf serum (FCS), vitamins, human serum albumin (HSA), bovine serum albumin (BSA), amino acids (e.g., L-glutamine), fatty acids (e.g., oleic acid, linoleic acid or palmitic acid), insulin (e.g., recombinant human insulin), transferrin (iron saturated human transferrin), aptoethanol, stem cell factor (SCF), Fms-like-tyrosine kinase 3 ligand (Flt3- L), cytokines such as eukin-2 (IL-2), interleukin-7 (IL-7), interleukin-15 (IL-15), thrombopoietin (Tpo) or heparin. In a specific embodiment, the thawing medium useful in the methods provided herein comprises RPMI. In r specific embodiment, said thawing medium comprises plasmalyte. In another specific embodiment, said thawing medium comprises about 05-20% FBS. In another specific embodiment, said thawing medium comprises about 1, 2, 5, 10, 15 or 20% FBS. In another specific embodiment, said thawing medium comprises about 0.5%-20% HSA. In r c embodiment, said thawing medium comprises about 1, 2.5, 5, 10, 15, or 20% HSA. In a more specific embodiment, said thawing medium comprises RPMI and about 10% FBS. In another more c embodiment, said thawing medium comprises plasmalyte and about 5% HSA.

The cryopreservation methods provided herein can be optimized to allow for long- term storage, or under conditions that inhibit cell death by, e.g., apoptosis or necrosis. In one embodiments, the post-thaw cells comprise greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% of viable cells, as ined by, e.g., automatic cell r or trypan blue method.

In another embodiment, the post-thaw cells comprise about 0.5, 1, 5, 10, 15, 20 or 25% of dead cells. In another embodiment, the post-thaw cells comprise about 0.5, 1, 5, 10, 15, 20 or 25% of early apoptotic cells. In r embodiment, about 0.5, 1, 5, 10, 15 or 20% of post-thaw cells undergo sis after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15,16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 days after being thawed, e.g., as determined by an apoptosis assay (e.g., 3 or AnnV/PI Apoptosis assay kit). In certain ments, the post-thaw cells are re- eserved after being cultured, expanded or differentiated using methods provided herein. .3. Genetically Modified NK Cells In another aspect, NK cells can be genetically modified to enhance target specificity and/or homing specificity.

In some embodiments, the genetically modified NK cells are NK cells that comprise a chimeric n receptor (CAR). CAR is an artificial membrane—bound protein that directs an immune cell (e.g., a T lymphocyte) to an n, and stimulates the immune cell to kill a cell ying the antigen. See, e. g., Eshhar, US. Patent No. 7,741,465; US. Patent Application ation No. 093 842, International Application ation No. 85, and International Application Publication No. . At a m, the CAR comprises an extracellular domain that binds to an antigen, e.g., an antigen on a cell, a transmembrane domain, and an intracellular (cytoplasmic) signaling domain (i.e., intracellular stimulatory domain) that transmits a primary activation signal to an immune cell. All other conditions being satisfied, when the CAR is expressed on the surface of, e.g., a T lymphocyte, for example, a primary T lymphocyte, and the extracellular domain of the CAR binds to an antigen, the intracellular signaling domain its a signal to the T lymphocyte to activate and/or proliferate, and, if the antigen is present on a cell surface, to kill the cell expressing the antigen.

Because some immune cells, e.g., T lymphocytes and NK cells, require two signals, a piimary activation signal and a costimulatory signal, in order to maximally activate, CARs can also optionally comprise a costimulatory domain such that binding of the antigen to the extracellular domain results in transmission of both a primary activation signal and a costimulatory signal.

Adaptive immune responses are initiated in secondary lymphoid organs, including the lymph nodes. B cells and T cells are sequestered in distinct regions of the lymph nodes, termed the “B cell zone,” located in the outer cortex of the lymph node, or les, and the “T cell zone,” which is more diffusely distributed in the area surrounding the follicles (also known as the paracortex) respectively. B cells and T cells express receptors that allow them to home to these respective zones so that they can be exposed to antigen. Intact antigens are present in the B cell zone, whereas in the T cell zone, antigens are presented by antigen-presenting cells, such as dendritic cells. Intact antigens, such as tumor antigens, are also present at the site of the tumor.

In some embodiments, the genetically modified NK cells are NK cells that comprise a homing receptor, which causes a cell comprising said homing receptor to home to a particular anatomical zone, a particular tissue, or a particular type of cell, e.g., B cell zone of the lymph nodes, gastrointestinal tract, or skin.

In certain embodiments, the genetically modified NK cells are NK cells that comprise both a CAR and a homing or as described herein.

Without wishing to be bound by any particular mechanism or theory, it is thought that when the genetically modified cells herein s homing ors that cause a cell expressing said homing receptor to home to a particular zone, they are more likely to be d to native n, where the cells, for example, cells expressing a CAR, are capable of being activated.

The NK cells that comprise a CAR and/or a homing receptor can be generated by any method known in the art. In some embodiments, the NK cells comprising a CAR and/or a homing receptor are first produced as bed in Section 5.2 (e.g., by a two-step process or by a three-step process), and are then engineered to express the CAR and/or the homing receptor by introducing the NK cells to (e.g., by ection) one or more vectors comprising the nucleic acid sequence(s) encoding the CAR and/or the homing receptor. In some embodiments, the cells (e.g., CD34+ hematopoietic stem cells), from whom NK cells can be produced, are first engineered to express a CAR and/or a homing receptor by introducing to the cells (e.g., by transfection) one or more vectors comprising the nucleic acid sequence(s) encoding the CAR and/or the homing receptor, and are then used to derive NK cells comprising the CAR and/or the homing or by any process described in Section 5.2 (e.g, a two-step process or a three-step process). .3.1. General CAR Structure and Intracellular Domain In certain ments, the intracellular domain of the CAR is or comprises an intracellular domain or motif of a protein that is expressed on the surface of immune cells and triggers activation and/or proliferation of said NK cells. Such a domain or motif is able to transmit a primary n-binding signal that is necessary for the activation of a NK cell in response to the n’s binding to the CAR’s extracellular portion. Typically, this domain or motif ses, or is, an ITAM (immunoreceptor tyrosine-based activation motif). ITAM- containing polypeptides suitable for CARs include, for example, the zeta CD3 chain (CD3C) or ITAM-containing portions thereof. In a specific embodiment, the intracellular domain is a CD36; intracellular ing domain. In other specific embodiments, the intracellular domain is from a cyte receptor chain, a 3 complex protein, an Fc receptor subunit or an IL-2 receptor subunit.

In certain embodiments, the CAR additionally comprises one or more mulatory domains or motifs, e.g., as part of the intracellular domain of the ptide. The one or more co-stimulatory domains or motifs can be, or comprise, one or more of a co-stimulatory CD27 polypeptide ce, a co-stimulatory CD28 polypeptide sequence, a co-stimulatory 0X40 (CD134) polypeptide sequence, a co—stimulatory 4-1BB (CD137) polypeptide sequence, a costimulatory ble T-cell costimulatory (ICOS) polypeptide sequence, a co-stimulatory PD-l polypeptide sequence, a co-stimulatory CTLA—4 polypeptide sequence, a co—stimulatory NKp46 polypeptide sequence, a mulatory I\Kp44 polypeptide sequence, a mulatory NKp30 polypeptide sequence, a co—stimulatory 1\KG2D polypeptide sequence, a co—stimulatory DAPlO polypeptide sequence, a co-stimulatory DAP12 polypeptide sequence, or other costimulatory domain or motif.

The transmembrane region can be any transmembrane region that can be incorporated into a functional CAR, typically a embrane region from a CD4 or a CD8 molecule. .3.2. CAR Extracellular Domain The extracellular domain of the polypeptide binds to an antigen of interest. In certain embodiments, the extracellular domain comprises a receptor, or a portion of a receptor, that binds to said antigen. The extracellular domain may be, e.g., a receptor, or a portion of a receptor, that binds to said antigen. In certain embodiments, the extracellular domain comprises, or is, an antibody or an n-binding portion thereof. In specific embodiments, the extracellular domain comprises, or is, a single-chain FV domain. The single-chain FV domain can comprise, for e, a VL linked to VH by a flexible linker, wherein said VL and VH are from an antibody that binds said antigen.

The antigen to which the extracellular domain of the polypeptide binds can be any antigen of interest, e.g., can be an antigen on a tumor cell or an antigen on an infected cell. The tumor cell may be, e.g., a cell in a solid tumor, or a cell of a blood cancer. The n can be any antigen that is expressed on a cell of any tumor or cancer type, e.g., cells of a lymphoma, a lung cancer, a breast cancer, a prostate cancer, an adrenocortical carcinoma, a d carcinoma, a nasopharyngeal carcinoma, a melanoma, e.g., a malignant melanoma, a skin carcinoma, a colorectal carcinoma, a d tumor, a desmoplastic small round cell tumor, an endocrine 2015/068069 tumor, an Ewing a, a peripheral ive neuroectodermal tumor, a solid germ cell tumor, a hepatoblastoma, a neuroblastoma, a non-rhabdomyosarcoma soft tissue a, an osteosarcoma, a retinoblastoma, a rhabdomyosarcoma, a Wilms tumor, a glioblastoma, a myxoma, a fibroma, a lipoma, or the like. In more c embodiments, said lymphoma can be chronic lymphocytic leukemia (small lymphocytic lymphoma), B-cell prolymphocytic leukemia, plasmacytic lymphoma, Waldenstrom macroglobulinemia, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, extranodal marginal zone B cell lymphoma, MALT lymphoma, nodal marginal zone B cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, Burkitt’s lymphoma, T lymphocyte prolymphocytic leukemia, T lymphocyte large granular lymphocytic leukemia, aggressive NK cell leukemia, adult T cyte leukemia/lymphoma, extranodal NK/T lymphocyte lymphoma, nasal type, enteropathy-type T lymphocyte lymphoma, hepatosplenic T lymphocyte lymphoma, blastic NK cell ma, mycosis des, Sezary syndrome, primary cutaneous anaplastic large cell lymphoma, lymphomatoid papulosis, angioimmunoblastic T lymphocyte lymphoma, peripheral T lymphocyte lymphoma (unspecified), stic large cell lymphoma, Hodgkin lymphoma, a non-Hodgkin lymphoma, or multiple myeloma.

In certain ments, the antigen is a tumor-associated antigen (TAA) or a tumor- specific n (TSA). In various specific embodiments, without limitation, the tumor- associated antigen or tumor-specific antigen is Her2, prostate stem cell antigen (PSCA), alpha- fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen-125 (CA-125), CAl9-9, calretinin, MUC-l, epithelial membrane n (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), CD19, CD20, CD34, CD45, CD99, CD117, chromogranin, cytokeratin, desmin, glial fibrillary acidic protein , gross cystic disease fluid protein (GCDFP-lS), HMB-45 antigen, high molecular weight ma-associated antigen (HMW-MAA), protein melan-A (MART-l), myo—Dl, muscle-specific actin (MSA), neurofilament, neuron-specific enolase (NSE), placental alkaline phosphatase, synaptophysis, thyroglobulin, thyroid transcription factor-l, the dimeric form of the pyruvate kinase isoenzyme type M2 (tumor M2-PK), an al ras protein, or an abnormal p53 protein.

In certain embodiments, the TAA or TSA is a cancer/testis (CT) antigen, e.g., BAGE, CAGE, CTAGE, FATE, GAGE, HCA661, HOM-TES-85, MAGEA, MAGEB, MAGEC, NA88, NY-ESO-1,NY-SAR—35, -l, SPANXBl, SPA17, SSX, SYCPl, or TPTE.

In certain other embodiments, the TAA or TSA is a carbohydrate or ganglioside, e.g., fuc-GMl, GM2 (oncofetal antigen—immunogenic-l; OFA-I—l); GD2 (OFA-I-2), GM3, GD3, and the like.

In certain other ments, the TAA or TSA is alpha-actinin-4, Bage—l, BCR- ABL, Bcr-Abl fusion n, beta-catenin, CA 125, CA 15-3 (CA 27.29\BCAA), CA 195, CA 242, CA-50, CAM43, Casp-8, cdc27, cdk4, cdkn2a, CEA, coa-l, dek-can fusion protein, EBNA, EF2, Epstein Barr Virus antigens, ETV6-AML1 fusion protein, HLA—A2, HLA-Al 1, hsp70—2, KIAAO205, Mart2, Mum-l, 2, and 3, neo-PAP, myosin class I, OS-9, pml-RARoc fusion protein, PTPRK, K-ras, N—ras, triosephosphate isomerase, Gage 3,4,5,6,7, GnTV, HerV-K—mel, Lage—l, NA-88, -l/Lage-2, SP17, SSX-2, TRP2-Int2, gp100 (Pmel 17), tyrosinase, TRP-l, TRP- 2, MAGE-l, MAGE-3, RAGE, , GAGE-2, pl5(58), RAGE, , SCP-l, Hom/Mel-40, PRAME, p53, H-Ras, HER-2/neu, E2A-PRL, H4-RET, IGH—IGK, MYL-RAR, human papillomaVirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE—6, p185erbB2, p180erbB-3, c—met, nm—23H1, PSA, —4, CA l9-9, CA 72-4, CAM 17.1, NuMa, K-ras, 13-Catenin, Mum-1, p16, TAGE, PSMA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, 13HCG, BCA225, BTAA, CD68\KP1, CO-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344, MA- 50, MG7-Ag, MOV18, NB\70K, l, RCASl, SDCCAG16, TA-90, TAAL6, TAG72, TLP, TPS, CD19, CD22, CD27, CD30, CD70, GD2 (ganglioside G2), EGFRVIII (epidermal growth factor variant III), sperm protein 17 (Spl7), mesothelin, PAP (prostatic acid phosphatase), prostein, TARP (T cell receptor gamma alternate reading frame protein), Trp-p8, STEAPl ransmembrane epithelial n of the prostate 1), an al ras protein, or an abnormal p53 protein. In another specific embodiment, said tumor-associated antigen or tumor- specific n is in UNB3 (CD61), galactin, K-Ras (V-Ki-rasZ n rat sarcoma Viral oncogene), or Ral-B.

In specific embodiments, the TAA or TSA is CD20, CD123, CLL-l, CD38, CS—l, CD138, RORl, FAP, MUCl, PSCA, EGFRVIII, EPHA2, or GD2. In further specific embodiments, the TAA or TSA is CD123, CLL-l, CD3 8, or CS-l. In a specific embodiment, the extracellular domain of the CAR binds CS-l. In a further specific embodiment, the extracellular domain comprises a single-chain version of elotuzumab and/or an antigen-binding WO 09668 fragment of umab. In a specific embodiment, the extracellular domain of the CAR binds CD20. In a more specific embodiment, the extracellular domain of the CAR is an scFv or antigen-binding fragment thereof binds to CD20.

Other tumor-associated and tumor-specific antigens are known to those in the art.

] Antibodies, and scFvs, that bind to TSAs and TAAs are known in the art, as are nucleotide sequences that encode them.

In certain specific embodiments, the antigen is an antigen not considered to be a TSA or a TAA, but which is nevertheless ated with tumor cells, or damage caused by a tumor.

In specific embodiments, the antigen is a tumor microenvironment-associated antigen (TMAA).

In certain embodiments, for example, the TMAA is, e.g., a growth factor, cytokine or interleukin, e.g., a growth factor, cytokine, or interleukin associated with angiogenesis or vasculogenesis.

Such growth factors, cytokines, or interleukins can include, e.g., vascular elial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), cyte growth factor (HGF), insulin-like growth factor (IGF), or interleukin-8 (IL-8).

Tumors can also create a hypoxic environment local to the tumor. As such, in other specific embodiments, the TMAA is a hypoxia—associated factor, e.g, HIF-lor, HIF-lB, HIF-ZOL, HIP—2B, HIF-30t, or HIF-3B. Tumors can also cause localized damage to normal tissue, causing the release of molecules known as damage associated molecular n molecules (DAMPs; also known as alarmins). In certain other c embodiments, therefore, the TMAA is a DAMP, e. g., a heat shock protein, chromatin—associated protein high mobility group box 1 (HMGB 1), SlOOA8 (MRP8, calgranulin A), SlOOA9 (MRPl4, calgranulin B), serum amyloid A (SAA), or can be a deoxyribonucleic acid, adenosine triphosphate, uric acid, or heparin sulfate. In specific embodiments, the TMAA is VEGF-A, EGF, PDGF, IGF, or bFGF.

In a specific embodiment, in which the cancer a gastrointestinal cancer, for example, liver cancer, stomach cancer, esophageal cancer, gallbladder cancer, colorectal cancer, anal cancer, or pancreatic cancer, the antigen is an antigen specific for or associated with a gastrointestinal cancer. In a specific embodiment, NK cells comprise a intestinal homing receptor and also comprise a CAR with an extracellular domain that binds to an n ated with a intestinal cancer. In a specific embodiment, the extracellular domain of the CAR binds CEA. In other specific embodiments, the extracellular domain of the CAR binds Her2, CA242,MUC1, CA125, or CA19-9.

In a specific embodiment, in which the cancer is a skin cancer, for example, melanoma, squamous cell carcinoma, or basal cell carcinoma, the antigen is an antigen specific for or associated with a skin cancer. In a c embodiment, NK cells comprise a skin homing receptor and also comprise a CAR with an extracellular domain that binds to an antigen associated with a skin cancer. In a specific embodiment, the ellular domain of the CAR binds HMW—MAA. In other specific ments, the ellular domain of the CAR binds Her2, GD2, GD3, CEA, or SPAG9, In certain embodiments, the extracellular domain is joined to said transmembrane domain by a linker, spacer or hinge polypeptide sequence, e.g., a sequence from CD28. .3.3. atory System Homing Receptors In certain ments, the homing receptor causes a cell comprising said homing receptor to home to the circulatory system. Such a receptor is referred to herein as a “circulatory system homing or.” In various ments, the circulatory system homing receptor is a chemotactic receptor. In specific embodiment, the chemotactic receptor is CXCR4, VEGFRZ, or CCR7.

In one embodiment, the homing receptor causes a cell comprising said homing receptor to home to the bone marrow. Such a receptor is referred to herein as a “bone marrow homing receptor.” In specific embodiments, the bone marrow homing receptor is CXCR4, for example, human CXCR4. GenBankTM accession numbers 008540.1 and NM_003467.2 provide exemplary nucleotide sequences for human CXCR4. GenBankTM accession numbers NP_001008540.1 and NP_003458.1 provide exemplary amino acid sequences for human CXCR4. Exemplary nucleotide and amino acid sequences for human homing receptors can be found in Table 1.

In another embodiment, the homing receptor causes a cell comprising said homing receptor to home to a secondary lymphoid organ, e.g, a lymph node. Such a or is referred to herein as a “secondary lymphoid organ homing receptor.” In c embodiments, the secondary lymphoid organ homing or is CCR7, for example, human CCR7. GenBankTM accession numbers NM_001301714.1, NM_001301716.1, NM_001301717.1, NM_001301718.1 and NM_0018383 provide exemplary tide sequences for human CCR7. GenBankTM accession s NP_001288643.1, NP_001288645.1 NP_001288646.1, NP_001288647.1 and NP_001829.1 provide exemplary amino acid sequences for human CCR7. Exemplary nucleotide and amino acid sequences for human homing receptors can be found in Table 1.

In another embodiment, the homing or causes a cell comprising said homing receptor to home to the vascular endothelium. Such a receptor is ed to herein as a “vascular endothelium homing receptor.” In specific embodiments, the vascular endothelium homing receptor is VEGFR2, for example, human VEGFR2. GenBankTM ion number NM_002253.2 provides exemplary nucleotide sequences for human VEGFR2. GenBankTM accession number NP_002244.1 provides exemplary amino acid sequences for human VEGFR2. ary nucleotide and amino acid sequences for human homing receptors can be found in Table 1.

In another embodiment, the homing receptor causes a cell comprising said homing receptor to home to the B cell zone of the lymph nodes, e.g., the follicles of the lymph node.

Such a receptor is referred to herein as a “B cell zone homing receptor.” In specific embodiments, the B cell zone homing or is CXCRS, for e, human CXCRS.

GenBankTM accession numbers NM_001716.4 and NM_032966.2 provide exemplary nucleotide sequences for human CXCRS. GenBankTM accession numbers NP_116743.1 and NP_001707.1 provide exemplary amino acid sequences for human CXCRS. Exemplary nucleotide and amino acid ces for human homing receptors can be found in Table 1.

In some embodiments, the step of ering a NK cell to comprise a circulatory system homing or comprises a step of introducing to the cells one or more vectors comprising the receptor nucleic acid sequence(s), i.e., the nucleic acid sequence (s) encoding the receptor(s). In specific embodiments, the vector comprises the nucleic acid ce for human CXCR4, CCR7, VEGFR2 or CXCRS. In a certain embodiment, the step of engineering a NK cell to comprise a circulatory system homing receptor is performed by any method known to one of skill in the art.

Also bed herein is a method of generating genetically engineered NK cells that home to the circulatory system, comprising a step of engineering a NK cell to se a circulatory system homing receptor, e.g., CXCR4, CCR7, VEGFR2 or CXCRS, wherein said circulatory system homing receptor is expressed by the cell at a sufficient level or sufficient amount to cause the cell to home to the circulatory system. In some embodiments, the step of engineering a NK cell to comprise a circulatory system homing or comprises a step of introducing to the cells one or more vectors comprising the receptor nucleic acid sequence(s), 2015/068069 1'. e., the nucleic acid sequence (s) encoding the receptor(s). In specific embodiments, the vector comprises the c acid sequence for human CXCR4, CCR7, VEGFRZ or CXCRS. In a certain ment, the step of engineering a NK cell to comprise a circulatory system homing receptor is performed by any method known to one of skill in the art. .3.4. Gastrointestinal Homing Receptors In one embodiment, the homing receptor causes a cell comprising said homing receptor to home to the intestinal tract, e.g., gastrointestinal organs, tissues, or cells. Such a receptor that causes a cell to home to the gastrointestinal tract is referred to herein as a “gastrointestinal homing receptor.” In certain embodiments, the gastrointestinal homing or is CCR9 or integrin 0t4B7, for example, human CCR9 or human integrin 0L4B7. kTM accession numbers NM_031200.2 and NM0012563 69.1 provide exemplary nucleotide sequences for human CCR9. GenBankTM accession numbers 477.1 and NP_001243298.1 provide exemplary amino acid sequences for human CCR9. GenBankTM accession numbers NM_000885.4 and NM_000889.2 provide exemplary nucleotide sequences for human (14 and human B7, respectively. GenBankTM ion numbers NP_000876.3 and NP_000880.1 provide exemplary amino acid sequences for human 0L4 and human B7, respectively. Exemplary tide and amino acid sequences for human homing receptors can be found in Table 1. In some embodiments, the I\K cells further comprise a second gastrointestinal homing receptor. In some embodiments, the I\K cells comprise a first gastrointestinal homing receptor, wherein the first gastrointestinal homing receptor is CCR9, and further comprise a second gastrointestinal homing receptor, wherein the second gastrointestinal homing receptor is integrin (1457.

In other specific ments, the NK cells comprise the gastrointestinal-homing receptor CXCR3.

In certain embodiments, the NK cells comprising one or more gastrointestinal homing receptors are expanded, activated, or both expanded and activated in the presence of a Vitamin A metabolite. In specific embodiments, the ion, activation, or both expansion and activation occurs in vivo, in vitro, or ex vivo. In specific embodiments, the Vitamin A metabolite is retinoic acid. In certain embodiments, the NK cells comprising one or more gastrointestinal homing receptors additionally comprise a B cell zone homing receptor. In c embodiments, the B cell zone homing receptor is CXCRS.

] Also described herein are methods of generating cally modified NK cells that home to the intestinal tract, e.g., gastrointestinal organs, skin, or tissue. In certain embodiments, NK cells comprising one or more homing ors that that cause a cell comprising the one or more receptors to home to the intestinal tract, e.g., CCR9 or integrin (1437, are generated by a method comprising a step of engineering a NK cell to express one or more gastrointestinal homing receptors. In some embodiments, the step of engineering a NK cell to comprise one or more gastrointestinal homing ors comprises introducing to the cells one or more vectors comprising a nucleic acid sequence encoding the homing receptor. In specific embodiments, the vector comprises the c acid sequence for human CCR9, the nucleic acid sequence for human integrin (1437, or both.

In certain embodiments, NK cells that home to the gastrointestinal tract are generated by a method comprising a step of treating the cells with a molecule that induces the expression of one or more gastrointestinal homing receptors, e.g, CCR9 or (x487. In specific embodiments, the molecule is Vitamin A.

In n embodiments, the method for generating the genetically modified NK cells that se one or more receptors that that cause a cell comprising the one or more receptors to home to the gastrointestinal tract comprises a step of expanding the cells, which step is carried out in the presence of a vitamin A metabolite. In certain embodiments, the method for generating the genetically modified NK cells that comprise one or more receptors homing to the gastrointestinal tract comprises a step of activating the cells, which step is carried out in the presence of a vitamin A metabolite. In certain embodiments, both the expanding and activating steps are carried out in the presence of a vitamin A metabolite. In certain embodiments the vitamin A metabolite is retinoic acid. In a certain embodiment, the step of ering a NK cell to se a gastrointestinal homing receptor is performed by any method known to one of skill in the art. .3.5. Skin Homing Receptors In one embodiment, the homing receptor causes a cell comprising said homing receptor to home to the skin, e.g., skin , or skin cells. In certain embodiments, the skin homing receptor is CCRlO, CCR8, CCR4, or CLA, for example, human CCRlO, human CCR8, human CCR4, or human CLA. GenBankTM accession s 602.2 and AF215981.1 provide exemplary nucleotide sequences for human CCRlO. GenBankTM accession numbers NP_057686.2 and P460923 provide exemplary amino acid sequences for human CCRlO.

GenBankTM accession numbers NM_0052013 and BC107159.1 provide ary nucleotide sequences for human CCR8. GenBankTM accession s NP_005192.1 and AAIO7 160.1 provide exemplary amino acid ces for human CCR8. GenBankTM accession number NM_005508.4 es an exemplary nucleotide sequence for human CCR4. GenBankTM accession number P5 1679.1 provides an exemplary amino acid sequence for human CCR4.

GenBankTM accession numbers NM_001206609.1 and NM_OO3 006.4 provide exemplary nucleotide sequences for human CLA. GenBankTM accession numbers NP_001193538.1 and NP_002997.2 provide exemplary amino acid ces for human CLA. Exemplary nucleotide and amino acid sequences for human homing receptors can be found in Table 1. In some embodiments, the NK cells further se a second skin homing receptor. In some embodiments, the NK cells comprise a first skin homing receptor, wherein the first skin homing receptor is CCRlO, and further comprise a second skin homing receptor, wherein the second skin homing receptor is CLA. In some embodiments, the NK cells comprise a first skin homing receptor, wherein the first skin homing receptor is CCRlO, and further comprise a second skin homing receptor, wherein the second skin homing receptor is CCR4. In some embodiments, the NK cells comprise a first skin homing receptor, wherein the first skin homing receptor is CCR4, and further comprise a second skin homing receptor, wherein the second skin homing receptor is CLA. In some embodiments, the NK cells r se a third skin homing receptor. In some embodiments, the NK cells comprise a first skin homing receptor, wherein the first skin homing receptor is CCRlO, further comprise a second skin homing receptor, wherein the second skin homing receptor is CCR4, and further comprise a third skin homing receptor, wherein the third skin homing receptor is CLA. In some embodiments, the NK cells comprise a first skin homing receptor, wherein the first skin homing receptor is CCR8, and further comprise a second skin homing receptor, wherein the second skin homing or is CLA, CCR4, or CCRlO. In some embodiments, the NK cells comprise a first skin homing receptor, wherein the first skin homing or is CCR8, further se a second skin homing receptor, wherein the second skin homing receptor is CLA, CCR4, or CCRlO, and further comprise a third skin homing receptor, wherein the third skin homing receptor is distinct from the second skin homing receptor, and is selected from the group consisting of CLA, CCR4, and CCRlO. In some embodiments, the NK cells further comprise a third skin homing or. In some embodiments, the NK cells comprise a first skin homing receptor, wherein the first skin homing receptor is CCRlO, further comprise a second skin homing receptor, wherein the second skin homing receptor is CCR4, further comprise a third skin homing receptor, wherein the third skin homing receptor is CLA, and further compiise a fourth skin homing receptor, wherein the fourth skin homing receptor is CCR8. In certain embodiments, the NK cells comprise one or more skin homing receptors. In other specific embodiments, the NK cells se the skin-homing receptor CCR6.

] In certain embodiments, the NK cells comprising one or more skin homing receptors are expanded, activated, or both expanded and activated in the presence of a Vitamin D metabolite. In specific embodiments, the expansion, activation, or both expansion and activation occurs in vivo, in vitro, or ex vivo. In specific ments, the Vitamin D metabolite is 1,25- dihydroxycholecalciferol (l,25(OH)2D3). In certain embodiments, the NK cells sing one or more skin homing receptors are expanded, activated, or both expanded and activated in the presence of IL-12. In specific ments, the expansion, activation, or both expansion and activation occurs in vivo, in vitro, or ex vivo. In more specific embodiments, the NK cells comprising one or more skin homing receptors are expanded, activated, or both expanded and ted in the presence of a Vitamin D metabolite and IL-12. In specific embodiments, the expansion, activation, or both expansion and activation occurs in vivo, in vitro, or ex vivo. In n embodiments, the NK cells comprising one or more skin homing ors additionally se a B cell zone homing receptor. In specific embodiments, the B cell zone homing receptor is CXCRS.

Also described herein are methods of generating genetically modified NK cells that home to the skin, e.g, skin tissue or cells. In certain embodiments, NK cells that home to the skin are generated by a method comprising a step of engineering the NK cells to comprise a skin homing receptor, e.g., CCR4, CCR8, CCRlO, or CLA. In some embodiments, the step of engineering the NK cells to comprise a skin homing receptor comprises introducing into the cells one or more vectors comprising the receptor nucleic acid sequence(s), 1'. e., the nucleic acid sequence(s) encoding the or(s). In specific embodiments, the vector comprises the nucleic acid sequence for human CCRlO, the nucleic acid sequence for human CLA, or both. In specific embodiments, the vector comprises the c acid sequence for human CCR4, and optionally the nucleic acid ce for human CLA. In specific embodiments, the vector comprises the nucleic acid sequence for human CCR4 and the nucleic acid sequence for human CCRlO, In specific embodiments, the vector comprises the nucleic acid sequence for human CCRlO, the nucleic acid sequence for human CCR4, and the nucleic acid sequence for human CLA. In specific ments, the vector comprises the nucleic acid sequence for human CCR8. In specific embodiments, the vector comprises the nucleic acid sequence for human CCR8, and optionally the nucleic acid sequence for human CLA. In specific embodiments, the vector comprises the nucleic acid sequence for human CCR8 and the nucleic acid sequence for human CCRlO. In specific ments, the vector comprises the nucleic acid sequence for human CCR8, the nucleic acid ce for human CCR4, and the nucleic acid sequence for human CLA. In specific ments, the vector comprises the nucleic acid ce for human CCR8, the nucleic acid sequence for human CCRlO, and the nucleic acid sequence for human CLA. In specific embodiments, the vector ses the nucleic acid sequence for human CCR8, the nucleic acid sequence for human CCR4, and the nucleic acid sequence for human CCRlO. In specific embodiments, the vector ses the nucleic acid sequence for human CCR8, the nucleic acid sequence for human CCR4, the nucleic acid for CCRlO, and the c acid sequence for human CLA.

In certain embodiments, cells, e.g, NK cells, that home to the skin are ted by a method comprising a step of treating the cells, e.g, NK cells, with a molecule that induces, e.g., increases, the expression of one or more skin homing receptors, e.g., CCR4, CCRlO, CCR8, or CLA. In specific ments, the molecule is Vitamin D. In certain embodiments, the induction of expression of skin homing receptors is aided by treating the cells, e.g., NK cells, with IL-12, e.g., contacting the cells with IL-12 in an amount and for a time sufficient to increase expression of one or more of CCR4, CCR8, CCRlO, or CLA by said cells.

] In certain embodiments, the method for generating the NK cells that comprise one or more homing receptors that cause a cell comprising the one or more receptors to home to the skin, comprises a step of ing the cells, which step is carried out in the presence of a vitamin D metabolite and, optionally, IL-12. In certain embodiments, the method for generating the NK cells that comprise one or more receptors that that cause a cell comprising the one or more receptors to home to the gastrointestinal tract, comprises a step of activating the cells, which step is carried out in the presence of a vitamin D metabolite, and, optionally, IL-12. In certain embodiments, both the expanding and activating steps are d out in the presence of a vitamin D metabolite, and, optionally, IL-12. In certain embodiments the vitamin D metabolite is 1,25(OH)2D3. In a certain embodiment, the step of engineering a NK cell to comprise a skin homing receptor is performed by any method known to one of skill in the art.

Table l. Exemplary nucleotide and amino acid sequences for human homing receptors.

GenBank Accession Sequence Number and Description N14 001008540.1 tttttt2tCt tCCCtC;agt gggngggca gaggagttag ccaagatgtg aCtttgaaaC CthagCgtC tcagtgccct ctaa acaaagaatt :tgtaattgg ttctaccaaa Exemplary nucleic gaagga ;ata m :gaag;CaC aaaa gatggggagg agagttgtag gattCtacat acid sequence taattC2Ctt l9 :gCCC2tag cccactaCtt cagaa2ttCC :gaagaaagC aagCCtgaat encoding human tggttt2tta mattgc:tta aaaatttttt gggt :aatgctth tgaattggaa CXCR4 isoform a gtgaatgtCC m :tCCt2th CtCttttgca gatatacaCt :CagataaCt acaCCgagga aa:gggtha l9gggaC2atg athcatgaa ggaaCCCtgt :2CCgtgaag aaaathtaa tt:caa:aaa m th ccaccatcta thca:catc ::Cttaactg gcattgtggg caatggattg m :CatCCtgg tcatgggtta ccagaagaaa C;gagaagca tgangacaa gtacaggCtg ;Cag tggCCgaCC; CC;Ct;tgtC a :CanCttC gggC ag:tga:gcc (Q :ggcaaact ggtactttgg gaact2CCta gcag tccatgtcat CtacacagtC 951) 0 0 fl. 0 :aca gcagtgtCC; ca:CC;ggCC :;CatcagtC tggaCCgCta CC;ggccatC l9 :Ccancca ccaacagtca gaggccaagg aagCtgttgg aggt gg:cta:gtt DLO0LO ('1' 0 :gga tCCCthCC: CC:gC:gaCt gaCt ttgc caantcagt E)aggcagatg acagatata; CtgtgaCCgC :;Ctacccca atgaCttgtg gg;ggt;gtg W ttC agcacatca; gg;tggCCtt a;CCthCtg gtattgtcat Cth Wattgcatta tcatctccaa gC:gtcacaC ggCC accagaagcg CCtC magaccacag tcatCtha; CC:ggCtttC tgtt tta C :acat;ggg m :Cagcatcg aCtCCttca; CC:CCtggaa :CatcaagC aagggtgtga 9 ::tgagaac caca agtggatttc ca:CaCCgag CCCtagCtt tCttccaCtg t :gtCtgaaC cccatCCtCt athtttCC; tggagccaaa :2taaaaCCt Ctgcccagca CgcathaCC tCtgtgagca gagggtccag CC;CaagatC C;thcaaag gaaagCgagg tggacattca tCtgtttcca Ctgagtctga gtCttcaagt ::tcactcca gCtaacacag a :gtaaaaga Ctttttttta tanataaa aaCttttttt :aagttacaC atttt:Caga tataaaagaC tgaccaatat tgtacagtt ttatthttg :2ggattttt gtCttgtg tCtttagttt ttgtgaagtt taattgact atttatataa a:tttttttg tttca:at a :gtgtgtCt aggcaggaCC tgtggccaag ttCttagttg C:gtatgtCt agga C :gtagaaaa gggaaCtgaa cattccagag Cgtgtagtga a;Cantaaa gCtagaaa;g a :Ccccagct gtttatgcat agataatCtC tccattCCCg tggaacgttt ttcctgttct taagantga tttthtgta gaagatggca Cttataacca aagcccaaag tggta:agaa a :gCtggttt ttcagttttC aggagtgggt tgatttcagC aCCtacagtg tacag;Ct tattaagttg ttaataaaag tacatgttaa acttaaaaaa aaaaaaaaaa aa DEW 0034672 aacttcagtt tgttggCth ggcagcaggt agcaaagtga CgCCgagggC Ctgag;gC ccac Cgcatctgga gaaccagcgg ttaccatgga ggggatcagt atatacac EXGHHflaU/nudsm cagataaCta ggaa atgggthag gggaCtatga thcatgaag gaaCCCtg acid sequence tCCgtgaaga aaathtaat ttcaataaaa tCttCCthC caccatCtaC tccatcatCt encoding human tCttaaCtgg cattgtgggc aatggattgg tcatCCtggt catgggttac cagaagaaac CXCR4 isoform b tgagaagcat gangacaag tacaggCth aCCtgtcagt ggCCgaCCtC gtca tcanCttCC ggca gttgathCg tggcaaaCtg gtaCtttggg aaCttCCtat gcaaggcagt ccatgtcatc tacacagtca acctctacag cagtgtCCtC gcct tcatcagtCt CtaC Ctggccath tCCanccaC caacagtcag aggccaagga GenBank Accession Number and Sequence Description agctgttggc ggtg gtctatgttg gcgtctggat ccctgccc acta :tcccgactt catctttgcc aacgtcagtg aggcagatga cagatata tgtgaccgc; :ctaccccaa tgacttgtgg gtggttgtgt tccagtttca gcacatca gttggcctta :cctgcctgg tattgtcatc ctgtcctgct attgcattat catctccaag cac: gcca ccagaagcgc aaggccctca agaccacagt catcctca;c Ctggctttc; :cgcctgttg gctgccttac :acattggga tcagcatcga thcttca;c ctcctggaaa :catcaagca agggtgtgag :ttgagaaca ctgtgcacaa gtggatttcc atcaccgagg ccctagcttt cttccactgt aacc ccatcctcta tgctttcc:t ggagccaaa; :taaaacctc tgcccagcac gcactcacct ctgtgagcag agggtccagc ctcaagatcc :ctccaaagg aggt ggacattcat ctgtttccac tgagtctgag agt :tcactccag ctaacacaga :gtaaaagac ttttttttat acgataaa:a acttttttt aagttacaca tttttcagat ataaaagact tatt gtacagtt;t tattgcttg tttg tcttgtgttt Ctttagtttt tgtgaagttt aattgact:a tttatataaa :tttttttgt ttcatattga :gtgtgtcta ggcaggacct aagt tcttagttgc :gtatgtctc gtggtaggac :gtagaaaag ggaactgaac attccagagc gtgtagtgaa :cacgtaaag ctagaaatga :Ccccagctg tttatgcata gataatctct ccattcccgt tttt tcctgttctt aagacgtgat tttgctgtag aagatggcac ttataaccaa agcccaaagt gaaa :gctggtttt tcagttttca ggagtgggtt gatttcagca gtgt acagtcttgt attaagttgt taataaaagt acatgttaaa cttaaaaaaa aaaaaaaaaa a NP_001008540.1 msiplpllqi ytsdnyteem gsgdydsmke pcfreenanf tiys iifltgivgn glvilvmqu kklrsmtdky rlhlsvadll fvitlpfwav davanwyfgn flckavhviy Exemplary amino tvnlyssvli lafisldryl aivhatnsqr ekvv yvngipall ltipdfifan acid sequence for vseaddryic drfypndlwv vqufqhimv glilpgivil scyciiiskl shskghqkrk human CXCR4 alkttvilil affacwlpyy igisidsfil leiikqgcef entvhkwisi tealaffhcc isoform a lnpilyaflg akfktsaqha gssl kilskgkrgg eses ssfhss NP_003458.1 megisiytsd nyteemgsgd ydsmkepcfr eenanfnkif lptiysiifl tgivgnglvi lvmqukklr smtdkyrlhl svadllfvit dava flc< avhviytvnl Exemplary amino yssvlilafi sldrylaivh atnsqrprkl yvgv wipallltip dfifanvsea acid sequence for ddryicdrfy pndlwvvqu fqhimvglil scyc iiisklshs< ghqkrkalkt human CXCR4 tvililaffa cwlpyyigis idsfilleii kqgcefentv hkwisiteal affhcclnpi isoform b lyaflgakfk tsaqhaltsv srgsslkils kgkrgghssv ssfn SS 301714.1 cacttcctcc ccagacaggg gtagtgcgag gccgggcaca gccttcc gtggtt2tac cgcccagaga gcgtcatgga cctgggtatg cctgtgtcaa gatgagg cggacgatta ary nucleic ca:cggagac aacaccacag tggactacac tttgt :cgag tctttgtgc ccaagaagga acid sequence cg;gcggaac gcct ggttcctccc tatca :gtac tccatca;t gtttcg;ggg ng human cc:actgggc ctgg tcgtgttgac ctata :ctat ttcaagaggc tcaagaccat CCR'7 isoform b gaccgatacc tacctgctca acctggcggt ggcagacatc ctcttcc tgacccztcc Ct;ctgggcc tacagcgcgg ccaagtcctg ggtct :ngt gtccact gcaagc;cat ct:tgccatc tacaagatga gcttcttcag tggca :gctc ctacttc : gcatcagcat tgaccgctac gtggccatcg tccaggctgt ctcagctcac cgccaccgtg cccgcg:cct tc;catcagc aagctgtcct gtgtgggcat ctgga :acta gccacag:gc tctcca;ccc agagctcctg tacagtgacc tccagaggag cagcagtgag caagcga:gc gatgctctct ca:cacagag catgtggagg cctttatcac catccaggtg gcccaga:gg tgatcggctt tc;ggtcccc Ctgctggcca tgagcttctg ttacc :tgtc atcatccgca ccctgc;cca GenBank Accession Number and Sequence Description ggcacgcaac tttgagcgca acaaggccat caaggtgatc atcgctgtgg tcgtggtctt catagtcttc cagctgccct gggt ggtcctggcc cagacggtgg ccaacttcaa catcaccagt agcacctgtg agctcagtaa gcaactcaac atcgcctacg acgtcaccta cagcctggcc cgct gctgcgtcaa ccctttcttg tacgccttca tcggcgtcaa gttccgcaac gatctcttca agctcttcaa ggacctgggc tgcctcagcc aggagcagct ccggcagtgg tcttcctgtc tccg gcgctcctcc atgagtgtgg aggccgagac caccaccacc ttctccccat ctct tctgcctgga ctagagggac ctctcccagg gtccctgggg tggggatagg gagcagatgc aatgactcag gacatccccc cgccaaaagc tgctcaggga aaagcagctc tcccctcaga gtgcaagccc Ctgctccaga agatagcttc accccaa:cc cagctacctc aaccaatgcc aaaaaaagac agggctgata agctaacacc acaa cactgggaaa cagaggctat tgtcccctaa accaaaaact gaaagtgaaa aaac tgttcccacc tgctggagtg aaggggccaa ggagggtgag tgcaaggggc gzgg cctgaagagt cctctgaatg aaccttctgg cctcccacag actcaaatgc tcagaccagc tcttccgaaa accaggcctt aaga atag tggggagact tcttggC2tg aaaa gcggacatca gctggtcaaa caaactctct gaacccctcc thcatcgtt ttcttcactg tcctccaagc cagcgggaat ggcagctgcc acgccgccct aaaagcacac tcatcccctc acttgccgcg tcgccctccc aggctctcaa caggggagag tgtggtg;tt cctgcaggcc aggccagctg cctccgcgtg atcaaagcca cactctgggc tccagag:gg ggatgacatg gctc ttggctccac tgggatggga ggagaggaca agggaaa;gt caggggcggg gagggtgaca gtggccgccc aaggcccacg ttct ttgttct2tg tcacagggac tgaaaacctc tcctcatgtt Ctgctttcga ttcgttaaga gagcaacatt ttacccacac acagataaag ttttcccttg aggaaacaac agctttaaaa gaaaaagaaa aaaaaagtct ttggtaaatg gcaaaaaaaa aaaaaaaaaa aaaaaaa N14_001301716J ctctagatga gtcagtggag ggcggg:gga gcgttgaacc agtg tggttgggcg taaacgtgga cttaaactca ggagctaagg ggtaattcag tgaaaaaggg gaatgagcgg ExenqflaU’HUdem tggggagctc tgttgcaaca ggg;ccaatc gcagcaggac tacaaatgcc Cgagcgcagg acid sequence ctgggaacga ggggacagcg gctgcc:gtc cccagaatag aaaatgcagc taggaagccc encoding human tc:ttgagtg gacagcggag gac2ggactg ccaggccaag catcaggggc ctca CCR7 isoform c gtta gagcccctga gga2ttagga ggaagggaaa ccaatgaaaa gcgtgc;ggt precursor gg:ggctctc cttgtcattt tccagg:atg cctgtgtcaa gatgagg:ca cggacgatta ca:cggagac aacaccacag tggactacac tttgt:cgag tgct ccaagaagga Cg;gcggaac gcct ggt;cc;ccc tatca;gtac a2tt gtttcg;ggg cc:actgggc aatgggctgg tcg:gt:gac ctata;ctat ttcaagaggc tcaagaccat gaccgatacc tacctgctca cggt ggcagacatc ctcttcc:cc ztcc Ct;ctgggcc tacagcgcgg ccaagtcctg ggtct2ngt gtccact2tt gcaagc;cat ct:tgccatc tacaagatga gct:ct:cag gctc c:tt gcatcagcat tgaccgctac atcg tccaggctgt ctcagctcac cgccaccgtg cccgcgzcct tc;catcagc aagctgtcct gtg;gggcat ctgga2acta gccacag;gc tctcca;ccc agagctcctg tacagtgacc tccagaggag cagcagtgag caagcga:gc gatgctctct ca:cacagag catgtggagg cct:tatcac catccaggtg gcccaga:gg tgatcggctt tc;ggtcccc gcca tgagcttctg ttacc;tgtc atcatccgca ccctgc;cca ggcacgcaac tttgagcgca acaaggccat caagg;gatc atcgctg:gg tcgtgg;ctt ca:agtcttc cagctgccct acaatggggt ggtcc:ggcc g:gg ccaact:caa ca;caccagt agcacctgtg agctcagtaa gcaac;caac atcgcctacg acgtcaccta ggcc tgcgtccgct gctgcgtcaa ccctt2cttg tacgcct;ca tcggcg;caa gt:ccgcaac ttca agctcttcaa ggacc:gggc tgcctcagcc aggagcagct ccggcagtgg tcttcctgtc ggcacatccg gcgctcctcc atgagtg:gg aggccgagac GenBank Accession Nnnflmrand Sequence Description caccaccacc ttctccccat aggcgactct tctgcctgga ctagagggac ctctcccagg gtccctgggg tggggatagg gagcagatgc aatgactcag gacatccccc cgccaaaagc tgctcaggga aaagcagctc tcccctcaga gtgcaagccc caga agatagcttc accccaa2cc cagctacctc aaccaatgcc aaaaaaagac agggctgata agctaacacc agacagacaa cactgggaaa ctat tgtcccctaa accaaaaact gaaagtgaaa gtccagaaac tgttcccacc tgctggagtg aaggggccaa ggagggtgag tgcaaggggc gtgggag:gg cctgaagagt cctctgaatg aaccttctgg cctcccacag actcaaatgc tcagaccagc tcttccgaaa cctt atctccaaga ccagagatag tggggagact tcttggC2tg gtgaggaaaa gcggacatca gctggtcaaa caaactctct gaacccctcc thcatcgtt ttcttcactg tcctccaagc cagcgggaat ggcagctgcc acgccgccct aaaagcacac tcatcccctc acttgccgcg tcgccctccc tcaa caggggagag tgtggtg;tt cctgcaggcc gctg cctccgcgtg atcaaagcca cactctgggc gzgg ggatgacatg cactcagctc ccac tgggatggga ggagaggaca agggaaa;gt caggggcggg gagggtgaca gtggccgccc aaggcccacg agcttgttct ttgttct2tg tcacagggac tgaaaacctc tcctcatgtt Ctgctttcga ttcgttaaga gagcaacatt ttacccacac acagataaag ttttcccttg aggaaacaac agctttaaaa gaaaaagaaa aaaaaagtct ttggtaaatg gcaaaaaaaa aaaaaaaaaa aaaaaaa 1301717J thtagatga gtcagtggag ggcgggtgga gcgttgaacc gtgaagagtg tggttgggcg :gga cttaaactca ggagctaagg gggaaaccaa tgaaaagcgt gctggtgg Exemplary nucleic gctctccttg tcattttcca cctg tgtcaagatg agg :cacgga Cgattaca acid sequence aaca ccacagtgga ctacactttg ttcgagtctt tgtgctccaa gaaggacg encoding human ngaac:tta aagcctggtt cctccctatc atgtactcca tca :ttgttt cgtgggcc CCR7 isoform c Ctgggcaatg ggctggtcgt gttgacctat ttca agaggctcaa gaccatgacc precursor gatacc:acc tgctcaacct ggcggtggca gaca :cctct tcc :cctgac ccttccct :aca gcgcggccaa gtcctgggtc ttcggtgtcc act :ttgcaa gctcatct gccatc;aca gctt Cttcagtggc atgc :Cctac ttc :ttgcat cagcattgac cgctacgtgg ccatcgtcca ggctgtctca gctcaccgcc cccg cgtccttc 2C atcagcaagc tgtcctgtgt gggcatctgg atac :agcca cag :gctctc catcccagag thctg;aca gtgacctcca gaggagcagc agtgagcaag cga :gcgatg thtctca acagagcatg tggaggcctt tatcaccatc cagg :ggccc aga :ggtgat cggctttc :9 gtccccctgc tggccatgag Cttctgttac cttg :Catca ccct gctccaggca Cgcaac;ttg agcgcaacaa ggccatcaag gtga :Catcg Ctg t ggtcttca :a gtcttccagc tgccctacaa tggggtggtc ctggcccaga C99 :ggccaa cttcaaca :C accagtagca agct cagtaagcaa ctcaacatcg cctacgacgt cacctacagc Ctggcc;gcg tccgctgctg Cgtcaaccct ttct :gtacg cct :catcgg Cgtcaagt 2C cgcaacgatc tcttcaagct cttcaaggac ctgggctgcc tcagccagga gcagctccgg cagtgg:ctt cctgtcggca catccggcgc tcctccatga gtg :ggaggc cgagaccacc accacc;tct ccccataggc gactcttctg cctggactag ctct cccagggtcc :ggg gagc agatgcaatg actcaggaca tccccccgcc aaaagctgct cagggaaaag cagctctccc tgc aagcccctgc tccagaagat agcttcaccc cagc tacctcaacc aatgccaaaa aaagacaggg Ctgataagct agac agacaacact gggaaacaga ggctattgtc ccctaaacca aaaactgaaa gtgaaagtcc agaaactgtt cccacctgct ggagtgaagg ggccaaggag ggtgagtgca aggggcgtgg cctg aagagtcctc tgaatgaacc ttctggcctc ccacagactc aaatgctcag accagctctt ccgaaaacca ggccttatct ccaagaccag agatagtggg gagacttctt ggcttggtga ggaaaagcgg acatcagctg gtcaaacaaa ctctctgaac ccctccctcc atcgttttct tcactgtcct ccaagccagc gggaatggca gctgccacgc Cgccctaaaa GenBank Accession Number and Sequence Description gcacactcat CCCthactt gCCgcgthC CthccaggC tthaacagg ggagagtgtg gtgtttCCtg caggccaggC cagCthCtC CgCg;gatca aagccacaCt :gggthca gagtggggat gacatgcact cagCtCttgg thcaCtggg atgggaggag ggacaaggg aaatgtcagg ggcggggagg gtgacagtgg CCgcccaagg cccacgagct gttctttgt tCtttgtcaC agggaCtgaa aaCCtCtCCt catg;tCth th :aagagagc aacattttac ccacacacag ataaagtttt CCCt;gagga aacaacagCt :aaaagaaa aagaaaaaaa aag:ctttgg taaatggcaa aaaaaaaaaa aaaaaaaaaa aa pflw:0013017181 aggagaaggt aaca can catt;CCtgg CgC;attgag :tggagCtg ccaagggCCt gccztcactt gtggcathC agttaCtgaC tthcagtgg ccaggCCCt ExenqflaU7HUd€m Ctgg gaCCtgaggg tan ggaagagggC taC;gCCgCC gta acid ce gggaaaccaa tgaaaagcgt gC;ggtgg gCtC;CCttg tca2tttcca gtathCtg encoding human tgtcaagatg aggzcacgga Cgattaca ggagacaaca ccacagtgga :acaCtttg CCR7 m c t :CgagtCtt tgthtccaa gaaggan ngaaC2tta ggtt CtCCCtatC precursor a ca tca2ttgttt Cg;gggCC Ctgggcaatg ggC;ggtht a :Ctatttca agaggctcaa gaccatgaCC gataCC:aCC th:CaaCCt gacatCCtCt tCC;CCtgaC CC;tCCCt;C tgggCC2aca caa t :ngtgtCC aCt;ttgcaa gC;CatCt;t gccatC2aca aga;gagCtt OLQLQLQOOLQQLQO :tgaCCtatgcggtggca:CCtgggtC:tcagtggc a :gctCCtaC ttC:ttgcat cagcattgac Cgctantgg cca:cgtcca ggctgtctca gthaCCgCC aCCgthCCg Cg;CCttC;C atcagcaagC tgtCCtgtgt gggcatCtgg a :aCtagcca cag;gCtCtC ca;Cccagag CtCCtg;aca gtgaCthca gaggagcagc agtgagcaag Cga:gCgatg Ctthtca:C acagagcatg cctt catc CaggtggCCC aga:ggtgat ngCtttC;g gtCCCCCth tggccatgag CttCtgttaC C :tgtcatca tCCgcaCCCt ggca CgcaaC2ttg agCgcaacaa ggccatcaag 9 :gatcatcg ctg:ggtcgt gg:cttca:a gtCttccagC thCCtacaa ggtc C aga ng:ggccaa Ct;Caaca:C accagtagca CCtgtgagCt cagtaagcaa C :Caacath CCtanant caCCtacagC CtggCC;gCg tCCgCthtg CgtcaaCCCt t :Cttgtan tcgg Cg:Caagt:C Cgcaacgatc tCttcaagCt Cttcaaggac C :gggCthC tcagccagga gcagCtCng cagtgg;Ctt CCtgthgca CgC tCthcatga gtg;ggaggC CgagaccaCC accaCC2tCt aggC gaCtCttCtg CCtggaCtag agggaCCtCt cccagggtCC Ctgggg:ggg gatagggagc agatgcaatg athaggaca tCCCCCCgCC aaaagCtht cagggaaaag tCCC tgc aagCCCCth tccagaagat agCttcaCCC caa;CccagC taCthaaCC aatgccaaaa aaagacaggg ctgataagct aacaccagac agacaacact caga ggctattgtc CCCtaaacca aaaaCtgaaa gtgaaagtCC agaaaCtgtt cccaCCtht ggagtgaagg ggccaaggag ggtgagtgca aggggCgtgg gag;ggCCtg aagagtCCtC tgaatgaaCC ttctggCCtC ccacagactc aaatgctcag accagCtCtt CCgaaaacca ggccttatct ccaagaccag agatagtggg gagacttCtt ggC:tggtga ggaaaagng acatcagctg gtcaaacaaa gaaC CCCtCCCtCC athttttCt tcaCtgtCCt ccaagccagc gggaatggca gCtgccach Cgccctaaaa gcacathat CCCthaCtt gccgcgtcgc CthccaggC tthaacagg ggagagtgtg gtg:ttCCtg caggccaggC cagCthCtC CgCgtgatca aagccacact hca ggat caCt cagCtCttgg thcactggg atgggaggag aggacaaggg aaa;gtcagg agg gtgacagtgg CCgcccaagg cccanagCt tgttCtttgt tCt:tgtcaC agggactgaa aaCCtCtCCt catgttCth ttthatth ttaagagagC aacattttac ccacacacag ataaagtttt CCCttgagga aacaacagct ttaaaagaaa aagaaaaaaa aagtctttgg taaatggcaa aaaaaaaaaa aaaaaaaaaa aaa GenBank Accession Number and Sequence Description N14 0018383 cacttcctcc ccagacaggg gtagtgcgag gccgggcaca gccttcctgt gtggttttac Cgcccagaga gcg;catgga cctggggaaa ccaatgaaaa tggt ggtggctctc aU7HUd€m cttgtcattt tccaggtatg cctgtgtcaa gatgagg;ca ngacgatta catcggagac acid sequence acag tggactacac tttgttcgag tctttgtgct ccaagaagga cgtgcggaac ng human :ttaaagcct ggt;cctccc tatcatgtac tccatca2tt gtttcgtggg cctactgggc CCR7 isoform a aatgggctgg tcg;gttgac Ctatatctat ttcaagaggc tcaagaccat gaccgatacc precursor :acctgctca acczggcggt ggcagaca:c thttcc:cc tgacccttcc ggcc :acagcgcgg ccaagtcctg ggtcttcggt gtccact2tt gcaagctcat catc :acaagatga gct;cttcag tggcatgc;c ctacttc;tt gcatcagcat tgaccgctac gtggccatcg tccaggctgt ctcagctcac cgccaccgtg cccgcgtcct tctcatcagc aagctgtcct gtg;gggcat Ctggatacza gccacag;gc tctccatccc agagctcctg :acagtgacc tccagaggag cagcagtgag caagcga;gc gatgctctct catcacagag catgtggagg cctztatcac catccaggzg gcccaga:gg gctt tctggtcccc Ctgctggcca tgagcttctg ttaccttg atcatccgca ccctgctcca caac :ttgagcgca acaaggccat caaggtga;c atcgctg;gg tcgtggtctt catagtcttc cagctgccct acaatggggt ggtcctggcc cagacgg:gg ccaacttcaa catcaccagt agcacctgtg agctcagtaa gcaactcaac atcgcctacg acgtcaccta cagcctggcc :gcgtccgct gctgcgtcaa ccctttctzg tacgcct2ca tcggcgtcaa gttccgcaac gatctcttca agctcttcaa ggacctgggc tgcctcagcc aggagcagct gtgg :cttcctgtc ggcacatccg gcgctcctcc atgagtg;gg agac cacc :tctccccat aggcgactct tctgcctgga ctagagggac thtcccagg gtccctgggg :ggggatagg gagcagatgc aatgactcag gacatccccc aagc tgctcaggga aaagcagctc tcccctcaga gtgcaagccc ctgctccaga agatagcttc accccaa2cc cagctacctc aaccaatgcc agac agggctgata agctaacacc agacagacaa cactgggaaa cagaggctat tgtcccctaa accaaaaact gaaa aaac tgttcccacc tgctggagtg aaggggccaa ggagggtgag tgcaaggggc gtgggag;gg cctgaagagt cctctgaatg ctgg cctcccacag actcaaatgc tcagaccagc tcttccgaaa cctt atctccaaga ccagagatag tggggagact tcttggc:tg gtgaggaaaa gcggacatca gctggtcaaa caaactctct gaacccctcc ctccatcgtt ttcttcactg tcctccaagc cagcgggaat ggcagctgcc acgccgccct aaaagcacac tcatcccctc acttgccgcg tcgccctccc aggctctcaa caggggagag tgtggtg:tt cctgcaggcc aggccagctg cctccgcgtg atcaaagcca gggc tccagag;gg catg cactcagctc ttggctccac tgggatggga ggagaggaca agggaaa;gt caggggcggg gagggtgaca gtggccgccc aaggcccacg agc ttgttct2tg tcacagggac tgaaaacctc tcctcatgtt ctgctttcga gagcaacatt ttacccacac acagataaag ttttcccttg aggaaacaac gaaaaagaaa aaaaaagtct ttggtaaatg gcaaaaaaaa aaaaaaaaaa NP 0012886431 mysiicfvgl lgnglvvlty iyfkrlktmt dtyllnlava waysaakswv fgvhfcklif aiykmsffsg mllllcisid ryvaivqavs isklscvgiw ao’anfino sipe qrss seqamrcsli tehveafiti ' vpllamsfcy acid sequence for lviirtllqa rnfernkaik viiavvvvfi qulpyngvv ' tsstcelskq human CCR7 lniaydvtys cvnp flyafigka rndlfklfkd lgclsqeqlr hirr isoform b ssmsveaett ttfsp NP 0012886451 mksvlvvall Vifqvclch igdn ttvdytlfes vrnf kawflpimys iicfvgllgn glvvltyiyf krlktmtdty llnlavadil fway saakswvfgv ExenqflaU’aHflHO hfcklifaiy kmsffsgmll llcisidryv aivqavsahr hrarvllisk lscvgiwila WO 09668 GenBank Accession Number and Sequence Description acid sequence for tvlsipelly sdlqrssseq amrcsliteh qmvigflvpl lamsfcylvi human CCR7 irtllqarnf ernkaikvii avvvvfiqu tvanfnitss tcelskqlni m 0 aydvtyslac vrccvnpfly afigkarnd lsqeqquws scrhirrssm precursor sveaettttf Sp NP_001288646.1 mksvlvvall Vifqvclch evtddyigdn ttvdy21fes lcskkdvrnf kawflpimys iicfvgllgn glvvltyiyf krlktmtdty llnlavadil flltlpfway saakswvfgv Exemplary amino hfcklifaiy kmsffsgmll dryv sahr lisk lscvgiwila acid sequence for tvlsipelly sdlqrssseq amrcsliteh veafi2iqva qmvigflvpl lamsfcylvi human CCR7 irtllqarnf ernkaikvii avvvvfiqu lpyngvvlaq itss tcelskqlni isoform c aydvtyslac vrccvnpfly rnd lfklf<dlgc lsqeqquws scrhirrssm precursor sveaettttf SP NP_001288647.1 mksvlvvall vifqvclch evtddyigdn 1fes lcskkdvrnf kawflpimys llgn yiyf krlktmtdty llnlavadil flltlpfway saakswvfgv Exemplary amino hfcklifaiy kmsffsgmll llcisidryv aivqavsahr hrarvllisk lscvgiwila acid sequence for tvlsipelly sdlqrssseq amrcsliteh iqva qmvigflvpl lamsfcylvi human CCR7 irtllqarnf ernkaikvii avvvvfiqu vlaq tvanfnitss tcelskqlni isoform c slac vrccvnpfly afigkarnd dlgc lsqeqquws scrhirrssm precursor sveaettttf Sp NP_001829. 1 mdlgkpmksv lvvallvifq vclchevtd dyigdnttvd ytlfeslcsk kdvrnfkawf lpimysiicf vgllgnglvv ltyiyfkrlk tmtdtyllnl avadilfllt lpfwaysaak Exemplary amino hfck lifaiykmsf fsgmllllci sidryvaivq avsahrhrar vllisklscv acid sequence for giwilatvls ipellysdlq rssseqamrc slitehveaf itiqvaqmvi gflvpllams human CCR7 fcylviirtl lqarnfernk aikviiavvv vfiqulpyn gvvlaqtvan fnitsstcel isoform a skqlniaydv tyslacvrcc vnpflyafig karndlfkl fkdlgclsqe qquwsscrh precursor irrssmsvea ettttfsp NM_002253 .2 actgagtccc gggaccccgg gagagcggtc aatgtgtggt gttt cctctgcctg Cgccgggcat cacttgcgcg ccgcagaaag tccgtctggc agcctggata tcctctccta Exemplary nucleic ccggcacccg cagacgcccc tgcagccgcg gtcggcgccc gggctcccta gccctgtgcg acid sequence gtc Ctgcgc;gcg gggtgccgcg agttccacct ccgcgcctcc ttctctagac encoding human aggcgctggg agaaagaacc ggctcccgag ttctgggcat ttcgcccggc tcgaggtgca VEGFRZ ggatgcagag caaggtgctg Ctggccgtcg ccctgtggct ctgcgtggag acccgggccg precursor cctctgtggg tttgcczag gtttctcttg atctgcccag gctcagcata caaaaagaca tacttacaat taaggc:aa acaactcttc aaattacttg caggggacag agggacttgg tttg gcccaa:aa cagagtggca gtgagcaaag ggtggaggtg actgagtgca gcgatggcct Cttctg;aag acactcacaa ttccaaaagt gatcggaaat gacactggag cctacaagtg cttctaccgg gaaactgact tggcctcggt catttatgtc tatgttcaag attacagatc tccatt:at: gcttctgtta gtgaccaaca tggagtcgtg tacattactg agaacaaaaa caaaac;gtg gtgattccat gtctcgggtc aaat thaacgtgt cactttgtgc aagataccca agat ttgttcctga tggtaacaga atttcctggg acagcaagaa :ac: attcccagct acatgatcag ctatgctggc atggtcttct caaa ;ga; gaaagttacc agtctattat gtacatagtt gtcgttgtag ggtataggat ttatga;gtg gttctgagtc cgtctcatgg aattgaacta tctgttggag aaaagcttgt Cttaaa:tg: acagcaagaa taaa gatt gacttcaact gggaataccc ttcttcgaag catcagcata ttgt aaaccgagac Ctaaaaaccc GenBank Accession Number and Sequence ption agtctgggag tgagatgaag aaatttttga gcaccttaac tatagatggt gtaacccgga gtgaccaagg attg :acacc t9 :gcagcat ccagtgggct gatgaccaag aagaacagca catttgtcag ggtccatgaa tttg ttgcttttgg aagtggcatg gaatctctgg tggaagccac ggtgggggag cg :gtcagaa tccctgcgaa gtaccttggt tacccacccc cagaaataaa atgg :ataaa aa ac cccttgagtc caatcacaca attaaagcgg ggcatgtact gacgattatg gaagtgagtg aaagagacac aggaaattac actgtcatcc ttaccaatcc catt :caaag gagaagcaga gccatgtggt ctctctggtt gtgtatgtcc agat tggtgagaaa tc :ctaatct ctcctgtgga :tcctaccag tacggcacca thaaacgct gaca :gtacg gtctatgcca ttcctccccc gcatcacatc cactggtatt ggcagttgga gtgc gccaacgagc ccagccaagc :gtctcagtg acaaacccat acccttgtga agaa :ggaga ag :gtggagg acttccaggg aggaaataaa attgaagtta ataaaaatca atttgctcta at :gaaggaa aaaacaaaac :gtaagtacc cttgttatcc aagcggcaaa tgtg :cagct ttgtacaaat gtgaagcggt caacaaagtc gggagaggag agaggg2gat ctcc :tccac gtgaccaggg gtcctgaaat :actttgcaa cctgacatgc agcccactga gcaggagagc gtgtctttgt ggtgcactgc agacagatct acgtttgaga catg gtacaagctt 99cccacagc ctctgccaat ccatgtggga gagttgccca cacctg:ttg caagaacttg gatactcttt ggaaattgaa :gccaccatg aata gcacaaatga cattttgatc atggagctta agaatgcatc Cttgcaggac caaggagact atgtctgcct tgctcaagac aggaagacca agaaaagaca :tgcgtggtc aggcagctca cagtcc:aga ggca cccacgatca caggaaacct ggagaatcag acgacaagta ttggggaaag catcgaagtc tcatgcacgg ggaa :Ccccctcca cagatcatgt ggtttaaaga taatgagacc Cttgtagaag actcaggcat :g:attgaag gatgggaacc ggaacc:cac tatccgcaga gtgaggaagg aggacgaagg cc;ctacacc tgccaggcat gcagtg;tct tggctgtgca aaagtggagg catttttcat aa;agaaggt gcccaggaaa agacgaactt ggaaatcatt attctagtag gcacggcggt ga:tgccatg tggc tacttc:tgt catcatccta cggaccgtta agcgggccaa tggaggggaa ctgaagacag ;gtc catcgtcatg gatccagatg aactcccatt ggatgaacat tgtgaacgac tgccttatga tgccagcaaa tgggaattcc ccagagaccg gc:gaagcta ggtaagcctc ttggccgtgg tgcctttggc caagtgattg aagcaga2gc ct;tggaatt acag caacttgcag gacagtagca gtcaaaatgt tgaaagaagg agcaacacac agtgagcatc gagctctcat gtctgaactc ctca ttcatat:gg tcaccatctc aatg:ggtca accttctagg tgcctgtacc aagccaggag ggccactcat tgtg tgca aatttggaaa cact agga gaaa tgaatttgtc ccctacaaga ccaaaggggc acgattccgt caagggaaag actacgt:gg agcaatccc: gtggatctga aacggcgctt ggacagcatc accagtagcc agagctcagc cagctctgga tttgzggagg agaagtccct cagtgatgta gagg aagctcc;ga agatctgta; aaggacttcc tgga gcatctcatc tg :tacagct tccaagtggc :aagggcatg gagt:cttgg catcgcgaaa gtgtatccac agggacctgg cggcacgaaa :atcctctta tcggagaaga acgtggttaa tgac tt :ggcttgg cccggga2at :tataaaga; ccagattatg tcagaaaagg agatgctcgc ctccctttga tggc cccagaaaca attt:tgaca gagtgtacac aatccagagt gacgtctggt g:gt :ttgctgtgg gaaa2atttt ccttaggtgc ttctccatat cc :ggggtaa agattga2ga agaattttg; aggcgattga aagaaggaac tagaatgagg gcccctgatt atactacacc agaaatgtac cagaccatgc tggactgctg gcacggggag cccagtcaga gacccacgtt :tcagagttg gtggaacatt tgggaaatct Cttgcaagct aa :gctcagc aggatggcaa agactacat; gttcttccga tatcagagac tttgagcatg gaagaggatt ctggactctc :ctgcctacc tcacctgttt tgga ggaggaggaa gtatgtgacc tcca :tatgacaac ggaa tcagtcagta tctgcagaac ag aa agagccggcc :gtgagtgta aaaacatttg GenBank Accession Nnnflmrand Sequence Description aaga:atCCC gttagaagaa ccagaagtaa aagtaa:CCC agatgacaac cagacggaca gtggzatggt tCtththa gaagagCtga ;gga aaCC aaattatCtC catC;tttgg tggaatggtg aaaa gcagggagtc tgtggcatCt gaaggthaa accagacaag ngCtaccag tCngatatC aCtCCga:ga caCC aCCgtgtaCt ccag:gagga agcagaactt ttaaagCtga :agaga;;gg agtgcaaaCC ggtagcacag cccagattCt ccagCCtgaC thgggacca caCtgagCtC tCCtCCtgtt taaaaggaag catccacaCC cccaactcct ggacatcaca :gagagg:gc thtcagatt ttcaagtgtt gttC2ttcca ccagcaggaa gtagCCgcat :tgatt;;Ca aaC agaaaaagga CCthgaCtg cagggagcca gtCttCtagg catatCC;gg aagaggCttg tgacccaaga atgtgtCtgt tCCC agtgttgaCC :gatCC:Ctt tttcattcat ttaaaaagca ttta2Cath CCCCthth gggtctcaCC atgggt22ag aacaaagan ttcaagaaat ggccccatCC tcaaagaagt agcagtaCCt ggggagC2ga caCttctgta aaaCtagaag ataaaccagg caatgtaagt gtthaggtg :tgaaga:gg gaaggat:tg cagggCtgag tCta:Ccaag aggCtttgtt taggantgg gtcccaagCC aagCCttaag tgtggaa2tC ggat;gatag aaaggaagac taanttaCC :thtttgga gagtactgga gCCtgcaaat gcat:gtgtt thtCtggtg gaggtgggca :ggggtc:gt tctgaaa:gt aaagggt:ca gangggttt ttag aaggtthgt gttCtthag ttgggctaaa gtagagt2Cg t :gthtgtt tCtgaCtCCt aa;gagagtt CCttccagaC Cgttan;gt CtCCtggcca agccccagga aggaaatgat ctgg CtCCttg:Ct cccaggc:ga :Ccttta::c agaataccac aaagaaagga gCtC aaggCtCCCt gCCg;gt:ga agagttC;ga C :gcacaaaC cagCt2Ctgg tt;CttCtgg aatgaataCC tha2atCtg :CCtgatg a tga gaCtgaath gggaggttca atgtgaagct gtgtgtggtg :Caaagt- aggaaggatt ttaCCCtttt gt;CttCCCC Ctgtccccaa cccaCtC2Ca CCCCgcaa Catcagtatt ttagt2attt ggCCtCtaCt ccagtaaaCC tgat;gggtt :gttcaC C :gaatgatt attagccaga aatt attttatagc ccaaattata acatCta tattatttag aCttt2aaca ta;agagCta tttCtaCtga tttt;gCCCt :gttCtg t :tttttcaa aaaagaaaat gtgttttttg tttggtacca gaaa :gCtgggaaC aatgaCtata agaca:gcta tggcacatat atttatagtc tgtt:atgta gaaacaaa taatatatta aagCC2tata ta2aatgaaC tttgtaCtat tcacattttg :atcagta a :gtagcata acaaaggtca taathtttC agcaattgat gtca2tttat acat tgaaaaactt gaaggaatCC Ct:tgcaagg ttgcattact gtacccatca :ttctaaaat ggaagagggg gtggC;gggC acagtggCCg acaCCtaaaa acccagcaCt :tggggggCC aaggtgggag gathCttga gcccaggagt ccag tCtggccaaC atggtcagat tccatctcaa agaaaaaagg taaaaataaa ataaaatgga gaagaaggaa :caga NP_002244. 1 mqskvllava lwlcvetraa svglpsvsld qkdi ltikantth rdld wlwpnnqsgs eqrvevtecs dglfcktlti dtga kafyretdl asviyvyqu Exemplary nucleic yrspfiasvs dqhgvvyite nknktvvipC lgsisnlnvs lcarypekrf vpdgnriswd acid sequence skkgftipsy mfo eakindesyq simyivvvvg yriydvvlsp shgielsvge ng human tart elnvgidfnw eypsskhqhk lktq sgsemkkfls tltidgvtrs VEGFRZ dqglytcaas sglmtkknst fvrvhekpfv afgsgmeslv eatvgervri pakylgyppp eikwykngip ikag hvltimevse rdtgnytvil tnpiskekqs hvvslvvyvp pqigekslis pvd8yqygtt qtltctvyai ppphhihwyw qleeecanep sqavsvtnpy pceewrsved fqggnkievn kanaliegk nktvstlviq aanvsalykc eavnkvgrge rvisfhvtrg peithpqu vslw Ctadrstfen gpqp lpihvgelpt pvcknldtlw klnatmfsns melk quy vclaqdrktk krhcvvrqlt vlervaptit gnlenqttsi gesievscta sgnpppqimw fkdnetlved sgivlkdgnr vrke deglythaC svlgcakvea ffiiegaqek tnleiiilvg taviamffwl 2015/068069 GenBank Accession Nnnflmrand Sequence Description llviilrtvk ranggelktg ylsivmdpde lpldehcerl pydaskwefp rdrlklgkpl grgafgqvie adafgidkta tcrtvavkml kegathsehr almselkili highhlnvvn llgactkpgg plmvivefck fgnlstylrs pykt kgarfrqgkd yvgaipvdlk rrldsitssq ssassgfvee kslsdveeee apedlykdfl tlehlicysf qvakgmefla srkcihrdla arnillsekn vvkicdfgla rdiykdpdyv lplk wmapetifdr vytiqsdes fgvllweifs lgaSPypgvk ideefcrrlk egtrmrapdy qtml dcwhgepsqr ptfselvehl naqq dgkdyivlpi setlsmeeds glslptspvs cmeeeevcdp kfhydntagi sqqunskrk srpvsvktfe dipleepevk vipddnqtds gmvlaseelk tledrtklsp sfggmvpsks resvasegsn qtsqusgyh tvys seeaellkli eiqutgsta qilqusgtt lssppv NM_001716.4 aaaaaaaaaa agtgatgagt gcag gtcgcggccc tactgcctca ggagacgatg cgcagc:cat ttgcttaaat ttgcagctga cggctgccac ctctctagag gcacctggcg Exemplary nucleic gggagcctct caacataaga cagtgaccag tctggtgact cacagccggc acagccatga acid sequence cgct ggaa atggacctcg agaacctgga ggacctgttc tgggaactgg ng human acagat:gga caactataac gacacctccc :ggaaaa ctgc cctgccacag CXCR5 aggggcccct catggcctcc ttcaaggccg :cgtgcc cgtggcctac agcc :catct tcctcc;ggg cgtgatcggc aacgtcctgg :ggtgat cctggagcgg caccggcaga cacgcagttc cacggagacc ttcctgttcc :ggccgt ggccgacctc ctgc :ggtct tcatct:gcc ctttgccgtg gccgagggct :gggctg ggtcctgggg acct :cctct gcaaaactgt gattgccctg cacaaagtca act :ctactg cagcagcctg ctcc :ggcct gcatcgccgt ggaccgctac ctggccattg tccacgccgt ccatgcctac cgccaccgcc gcctcc:ctc catccacatc acctgtggga cca :ctggct ggtgggcttc ctcc :tgcct tgccagagat tctcttcgcc aaagtcagcc atca caacaactcc ctgccacgtt gcacct:ctc ccaagagaac caagcagaaa cgcatgcctg gttcacctcc cgat :cctct accatg:ggc gggattcctg atgc tgg :gatggg ctggtgctac gtgggggtag ggtt gcgccaggcc cagcggcgcc ctcagcggca gaaggcagtc aggg :ggcca :gac aagcatcttc ttcctctgct ggtcacccta ccacatcgtc atct :cctgg acaccc:ggc gaggctgaag gccgtggaca atacctgcaa gctgaatggc tctc :ccccg tggcca2cac catgtgtgag ttcctgggcc tggcccactg ctgcctcaac ccca :gctct acactt:cgc cggcgtgaag ttccgcagtg acc :gtcgcg gacg aagc :gggct gtaccggccc cctg tgccagctct tccctagctg gcgcaggagc agtc :ctctg agtcagagaa ctct ctcaccacgt tccc agtgtcccct ttta :tgctg cttttcct;g gggcaggcag tgatgctgga tgc :ccttcc aacaggagct 9993 :cctaa gggctcaccg tggctaagag tgtcctagga gta :cctcat ttggggtagc tagaggaacc aaccccca;t tctagaacat ccctgccagc tct :ctgccg gccctggggc taggctggag gagc ggaaagcagc tcaaaggcac ggct gtccttaccc atctgcaccc ccctgggc:g agagaacctc acgcacctcc catcctaatc atccaatgct caagaaacaa cttctactzc tgcccttgcc aacggagagc gcc :gcccct cccagaacac actccatcag cttaggggct gctgacctcc acagcttccc ctc :ctcctc ctgcccacct gtcaaacaaa gccagaagct gagcaccagg ggatgagtgg agg :taaggc tgaggaaagg ccagctggca gcagagtg;g gccttcggac aactcagtcc ctaaaaacac agacattctg ccaggccccc gcag tcatcttgac caagcaggaa actg gttgagttca ggtagctgcc cctggctc:g accgaaacag cgctgggtcc accccatgtc accggatcct tctg caggcagggc tgactctagg tgcccttgga ggccagccag tgacctgagg aagcgtgaag gccgagaagc gaaa cccgacagag ggaagaaaag agctttcttc ccgaacccca aggagggaga tggatcaatc aaacccggcg gtcccctccg ccaggcgaga tggggtgggg tggagaactc ctagggtggc tgggtccagg ggatgggagg ttgtgggcat tgatggggaa GenBank Accession Number and Sequence Description ggaggctggc ttgtcccctc ctcactccct tcccataagc tatagacccg aggaaactca gagtcggaac aggt ggactggaag gggcccgtgg gagtcatctc aaccatcccc tccgtggcat caccttaggc agggaagtgt aagaaacaca cagg gaagtcccca ggccccagga agccgtgccc tgcccccgtg aggatgtcac tcagatggaa ccgcaggaag Ctgctccgtg cttgtttgct gggt gtgggaggcc Cgtccggcag ttctgggtgc tccctaccac ctccccagcc agg tggggagtca cctg cccttgtccc actcaagcca agcagccaag :ccttggga ggccccactg gggaaataac ggct cacgtgagag tgtcttcacg caggacaac gaggaagccc taagacgtcc cttttttctc tgagtatctc ctcgcaagc: ggtaatcga tgggggagtc tgaagcagat gcaaagaggc ctgg attttgaat: :ctttttaa taaaaaggca cctataaaac aggtcaatac agtacaggca gcacagagac cccggaaca agcctaaaaa ttgtttcaaa ataaaaacca agaagatgtc ttcacatat- :aaaaaaaa aaaaaaaaa NM_032966.2 ccactctaag gaatgcggtc cctttgacag gcgaaaaact gaagttggaa aagt ttca aaattgaaa: t;gaaacttg acatttggtc agtgggccc; atgtaggaaa Exemplary nucleic aaacctccaa gagagctagg g:tcctctca gagaggaaag acaggtcct: aggtcctcac acid sequence cctcccgtct ccttgccct: gcagttctgg gaactggaca gat :ggacaa ctataacgac encoding human acctccctgg tggaaaatca tctctgccct gccacagagg ggcccctca; cttc CXCRS aaggccgtgt tcgtgcccg: ggcctacagc ctcatcttcc tcc :gggcg : gatcggcaac gtcctggtgc tggtgatcc: ggagcggcac cggcagacac gcagttccac ggagaccttc Ctgttccacc tggccgtggc Cgacctcctg ttca tct :gccct; tgccgtggcc gagggctctg tgggctggg: cctggggacc ttcctctgca aaactgtga: tgccctgcac aaagtcaact tctactgcag cagcctgctc ctggcctgca tcgccgtgga ccgctacctg gccattgtcc tcca tgcctaccgc caccgccgcc tcc :ctcca; ccacatcacc tgtgggacca tctggctgg: gggcttcctc cttgccttgc cagagattc: cttcgccaaa gtcagccaag gccatcacaa caactccctg ccacgttgca cct :ctccca agagaaccaa gcagaaacgc atgcctggt; cacctcccga ttcctctacc atg 399C999 attcctgctg ctgg tgatgggctg gtgctacgtg gtgc acaggttgcg ccaggcccag cggcgccctc agcggcagaa ggcagtcagg gtggccatcc tgg :gacaag cttc thtgctggt caccctacca catcgtcatc ttcctggaca CCC :ggcgag ggcc aata cctgcaagct gaatggctct ctccccgtgg cca :caccat gtgtgagttc ctgggcctgg cccactgctg cccc atgctctaca ctt :cgccgg cgtgaagttc gacc ggct cctgacgaag ctgggctgta ccggccctgc thcctgtgc ttcc ctagctggcg caggagcagt ctctctgagt cagagaatgc cacctctctc accacgttct aggtcccagt gtcccctttt attgctgctt gggg gtga tgctggatgc tccttccaac aggagctggg atcctaaggg thaccgtgg Ctaagagtgt cctaggagta tcctcatttg gggtagctag aggaaccaac ccccatttct agaacatccc tgccagctct tctgccggcc Ctggggctag gctggagccc agggagcgga aagcagctca aaggcacagt gaaggctgtc Cttacccatc tgcacccccc tgggctgaga gaacctcacg ccat cctaatcatc caatgctcaa gaaacaactt ctacttctgc ccttgccaac ggagagcgcc tgcccctccc agaacacact gctt aggggctgct gacctccaca gcttcccctc tctcctcctg cccacctgtc aaacaaagcc agaagctgag caccagggga tgagtggagg ttaaggctga ggaaaggcca gctggcagca gagtgtggcc ttcggacaac tcagtcccta aaaacacaga cattctgcca ggcccccaag cctgcagtca tcttgaccaa gcaggaagct ggtt gagttcaggt agctgcccct ggctctgacc gaaacagcgc tgggtccacc ccatgtcacc ggatcctggg tggtctgcag gcagggctga ctctaggtgc ccttggaggc cagccagtga cctgaggaag cgtgaaggcc gagaagcaag aaagaaaccc ggga agaaaagagc tttcttcccg aaccccaagg agggagatgg atcaatcaaa GenBank Accession and Sequence Description ggtc ccctccgcca ggcgagatgg ggtggggtgg agaactccta gggtggctgg m :ccagggga tgggaggttg tgggcattga tggggaagga 99Ctggcttg tcccctcctc mctcccttcc cataagctat agacccgagg agag tcggaacgga gaaaggtgga 0 :ggaagggg cccgtgggag tcatctcaac catcccctcc gtggcatcac cttaggcagg maagtgtaag aaacacactg ggaa aggc CCCaggaagc Cgtgccctgc Occcgtgagg atgtcactca gatggaaccg caggaagctg thcgtgctt tcac 0 :ggggtgtg ggaggcccgt ccggcagttc tgggtgctcc ctaccacctc cccagccttt matcaggtgg ggagtcaggg acccctgccc ttgtcccact caagccaagc agccaagctc 0 :tgggaggc cccactgggg aaataacagc tgtggctcac gtgagagtgt cttcacggca Qgacaacgag ctaa gacgtccctt ttttctctga gta :thctc tggg (* aatcgatgg gggagtctga agcagatgca aagaggcaag aggCtggatt tttc fl taa aaaggcacct ataaaacagg tcaatacagt acaggcagca cagagacccc cggaacaagc ctaaaaattg tttcaaaata aaaaccaaga aga :gtcttc acatattgta aaaaaaaaaa aaaaaa NP_116743.1 masfkavap vayslifllg Vignvlvlvi lerhrqtrss tet Elfhlav adlllvfilp svgw vlgtflcktv ialqunfyc ciav dry.Zaivhav rlls Exemplary amino ihitcgtiwl vgfllalpei ghh nnslprctfs qenqaethaw yhva acid sequence for gfllpmlvmg wcyvgvvhrl rqaqrrpqrq kavrvailvt sif Elcwspy hivifldtla human CXCRS rlkavdntck lngslpvait mceflglahc clnpmlytfa gvk frsdlsr ctgp precursor aslcqlfpsw esen atslttf NP_00 1707. l mnypltlemd lenledlfwe ldrldnyndt slvenhlcpa tegplmasfk avapvaysl ifllgvignv lvlvilerhr qtrsstetfl fhlavadlll vfi lpfavae gsvnglgtf Exemplary amino alhk slll aciavdryla ivhavhayrh rrl lsihitc gfll acid sequence for alpeilfakv sqghhnnslp rctfsqenqa ethawftsrf lyhvagfllp mlvmgwcyvg human CXCRS vvhrquaqr rpqquavrv ailvtsiffl cwspyhivif ldt larlkav dntcklngsl precursor pvaitmcefl glahcclnpm sdlsrlltkl gctgpaslcq lfpswrrssl sesenatslt tf NM_031200.2 gcttcctttc tcgtg::gtt atcgggtagc tgcctgctca gaacccacaa agcctgcccc tcatcccagg cagagagcaa cccagctc:t tccccagaca gctg gtggtgcctg Exemplary nucleic 0 :gtcccagg gagag;;gca tcgccctcca cagagcaggc :tgcatctga Ctgacccacc acid sequence m :gacaccca cagac;:cac aagcccta;t cctaacatgg ctgatgacta tggctctgaa ng human fi' ccacatct cca:ggaaga Ctacgttaac ttcaacztca ctgacttcta ctgtgagaaa CCR9 mm0m9)wm :ca ggcag;;tgc gagccatt ctcccaccct :g;actggc; Cgtgttcatc m :gggtgcc: tgggcaacag tc;tgtta;c cttgtc;act gg;actgcac aagagtgaag mccatgaccg aca:g::cct tt:gaatt:g gcaattgctg acc :cctct tcttgtcact 0 :tccct;c; 2tgc tgctgctgac aagt :CCagacct catgtgcaag m :ggtcaaca gca:g;acaa ga;gaact;c tacagc;gtg :g; :gctga catgtgcatc mgcgtggaca ggtaca:tgc caztgcccag gccatgagag cacatacttg gagggagaaa mggcttt;g; acagcaaaat gg;ttgct;t accatc;ggg :ggcagc tgctctctgc m :cccagaaa tct;a;acag ccaaatcaag gaggaa2ccg :tgcta ctgcaccatg m :ttacccta gcgatgagag caccaaac:g aagtcagctg :gaccc gaaggtcatt O 2C; tcc;tccctt Cg;ggtca;g gcttgc;gct ataccatca cattcacacc 0 :gatacaag ccaagaagtc ttccaagcac aaagccctaa :gacca cactgtcctg accgtct:tg :gtctca ctac aactgcattt :g :ggtgca gaccattgac gcctatgcca tgt;catctc caactgtgcc gtttccacca aca :tgaca Ctgcttccag WO 09668 GenBank Accession and Sequence Description gtcacccaga ccatcgcctt cttccacagt tgcctgaacc ctgttctcta tgtttttg'g agat tccgccggga tctcgtgaaa accctgaaga acttgggttg catcagccag gcccagtggg tttcatttac aaggagagag ggaagcttga agctgtcgtc tatgttgc gagacaacct caggagcact ctccctctga ggggtcttct tgca tggttctt ggaagaaatg agaaatacag aaacagtttc cccactgatg ggaccagaga gagtgaaaga gaaaagaaaa ctcagaaagg gatgaatctg aactatatga ttac2tgtag tcagaatt;g ccaaagcaaa tatttcaaaa :caactgact agtgcaggag gctg:tgatt tgac tgtgatgccc ctca aaggaggact aaggaccggc actg;ggagc gc tgccactcgc cggagcatca atgccgctgc ctctggagga gccc2tggat tttc:cca gaac ttctgtggct :cagttctca :gctgcctct tccaaaaggg gacacagaag cactggctgc tgctacagac Cgcaaaagca gaaag;;tcg tgaaaatgtc catc:ttggg aaattttcta ccctgctctt gata acccazgcca ggtc;tatag attcctga;c tagaaccttt ccaggcaatc :Cagacctaa :ttcc::ctg ttctccttgt tctg:tctgg gccagtgaag gtccttgttc :gattttgaa acgatc;gca ggtc2tgcca gtgaacccct ggacaactga ccac tcca aagtc;g;tg gcttccaatc catt;ctg tcctgctgga ggt:ttaacc :agacaagga ::at tcct:ggtat ggtgacag tctctccatg gcag ggagattata acagc;gggt tcgcaggagc cagccttggc cctgttgtag gct;gttctg :tgagtggca cttgc ;;gg g;ccaccgtc tgtc;gctcc Ctagaaaatg ggc:ggttct :ttggccctc :tctt:c:ga gcccacttt attc:gagga atacagtgag cagatatggg cagcagccag gtagggcaaa gggtgaagc gcaggccttg Ctggaaggct att;acttcc atgcttctcc :tttc;;act :atagtggc aaca;tttaa ttaa cttagagatt aaaa aaataag:aa ggaattcac ctttgcatct tttgtgtctt tct:a;catg atttggcaaa atgca2cacc :tgaaaata tttcacatat tggaaaagtg Ctt;t;aatg tgtatatgaa gcattaa2ta :tgtcactt ccct gtctcaatat tttaagtgtg tgcaattaaa gatcaaa:ag :acatt N}4001256369_1 gcttcctttc tcg;g;tgtt atcgggtagc 2ca gaacccacaa agcc;gcccc cagg cagagagcaa cccagctctt tccccagaca C:gagagctg gtgg:gcctg ExenqflaU’HUdem ctgtcccagg gagag;tgca tcgccctcca aggc t:gcatctga ctgacccacc acid sequence atgacaccca cagac;tcac atctcctcca ggccccgctc cagatcacct tccc;cgctg encoding human gcccaggaat ccatc:cctt ccaggacctt agcccaggac taacacaagc ccta:tccta CCR9 acatggctga tgactatggc tctgaatcca catct:ccat ggaagactac gttaacttca ac;tcactga Cttctactgt gagaaaaaca atg;caggca gtttgcgagc ctcc caccct;g;a ctggc;cgtg ttcatcg;gg gtgcc;tggg caacagtc;t gtta:ccttg gg:a Ctgcacaaga gtgaagacca tgaccgacat gttccttt:g aatt:ggcaa ttgctgacc cctct2tctt gtcactc;tc cct;c;gggc cattgctgct gctgaccagt gacct2catg tgcaagg;gg gcat gtacaaga aact:ctaca gctgazcatg tgcatcagcg tggacaggta cattgcca:t gcccaggcca tacttggagg gagaaaaggc ttt;g;acag caaaatgg;t tgct2tacca ggcagctgct ctctgca;cc tctt ccaa atcaaggagg tgctazctgc accatgg:tt accctagcga tgagagcacc aaac:gaagt gaccc;gaag gtcattc;gg ggt;c;tcct tcccttcgzg gtca;ggctt catca2catt cacaccc;ga ccaa ttcc aagcacaaag gaccazcact gtcctgaccg tct:tgtctt gtctcagt:t ccctacaact ggtgcagacc attgacgcct atgccatgtt catctccaac gttt ccaccaaca tgaca2ctgc ttccagg;ca cccagaccat cgccttct cacagttgcc tgaaccctg tctctatgtt tttgtgggtg tccg ccgggatc:c gtgaaaaccc tgaagaac; gggttgcatc agccaggccc agtgggtttc atttacaagg agagagggaa GenBank Accession Number and Sequence Description gcttgaagct fi'Ofi'mmfi'fi'fl'fi'OLQLQ :gccagtga:cgtctatg ttgctggaga caacctcagg agcactctcc :ctgagggg tcttctctga gtgcatggt tcttttggaa gaaatgagaa atacagaaac gtttcccca ctgatgggac agagagagt gaaagagaaa agaaaactca gaaagggatg atctgaact atatgattac :gtagtcag aatttgccaa agcaaatatt tcaaaatcaa :gactagtg caggaggctg :gattggct Cttgactgtg atgcccgcaa ttctcaaagg ggactaagg accggcactg ggagcaccc :ggctttgcc actcgccgga atgc gctgcctct ggaggagccc :ggattttc cact gtgaacttct gtggcttcag gct gcctcttcca aaggggaca cagaagcact ggctgctgct acagaccgca SD 9)LO0 SD LQ SD 9) DJ SDSJJOLQWOOLQOFl'le—Q :cacctttg:2tcgtgaa atgtccatc aaat tttctaccct gctcttgagc :gataaccc :gccaggtc :atagattc taga acctttccag gcaatctcag O ‘1. Sb 9) :tc ;;ctgttct cttgttctg :tctgggcca gtgaaggtcc ttgttctgat O :gcaggtc ggac aactgaccac aagg :gttggctt ccaatccatt :Ctgtgtcct gctggaggtt taga Q :tattcct ggtg acagtgtctc tccatggcct gagcagggag :gggttcgc aggagccagc Cttggccctg ttgtaggctt gttctgttga :ggcac ::tgggtcc accgtctgtc ctag aaaatgggct tttg ccctc: gcc cactttattc atac caga tatgggcagc gccagg gcaaagggg tgaagcgcag gccttgctgg aaggctattt atgc WQJKQLQQJOOFT'gOSDWOSDOSDSDO:ctcc; ::actctat agtggcaaca :tttaaaagc ttttaactta aggc tgaaaaaaat agtaatgga attcaccttt gcatcttttg tgtctttctt atcatgattt ggcaaaa;gc aaaatatttc tgga aaagtgcttt ttaatgtgta tatgaagcat taattacttg tctt :accctgtct caatatttta agtgtgtgca attaaagatc aaatagatac att NP_112477.1 mtptdftspi pnmaddygse dyvn fnftdfycek nnvrqfashf lpplywlvfi vgalgnslvi lvywyctrvk tmtdmfllnl aiadllflvt lpfwaiaaad qwqutfmck Exemplary amino vvnsmykmnf yscvllimci svdryiaiaq amrahtwrek rllyskmvcf tiwvlaaalc acid ce for ipeilysqik eesgiaictm stkl ksavltlkvi lgfflpfvvm accytiiiht human CCR9 liqakkssch kalkvtitvl tvalsquy ncillvqtid snca vstnidich precursor vtqtiafqu yva gerfrrdlvk tlknlgcisq aqusftrre gslklssmll ettsgalsl NP_001243298.1 l maddygses ; ssmedyvnfn ftdfyceknn vrqfashflp plywlvfivg algnslvilv 61 ywyctrvk;m nlai adllflvtlp fwaiaaadqw qutfmckvv nsmykmnfys Exemplary amino 121 cvllimcisv dryiaiaqam rahtwrekrl lyskmvcfti wvlaaalcip eilysqikee acid sequence for 181 sgiaictmvy psdestklks avltlkvilg fflpfvvmac cytiiihtli qakksskhka human CCR9 241 lkvtitv1;v fvlsquync illvqtiday amfisncavs tnidichvt qtiaffhscl precursor 301 npvlyvage rfrrdlvktl knlgcisqaq wvsftrregs lklssmllet tsgalsl NM_000885.4 ataacgtc tgtcactaaa atgttcccca 9999CCtth gcgagtcttt ttgtttggtt ttttgttt:t aatctgtggc tcttgataat ttatctagtg gttgcctaca cctgaaaaac Exemplary nucleic aagacacagt gtttaactat caacgaaaga actggacggc ccgc agtcccactc acid sequence cccgagtt;g tggctggcat ttgggccacg ccgggctggg cggtcacagc gaggggcgcg encoding human cagtttgggg tcacacagct ccgcttctag gccccaacca ccgttaaaag gggaagcccg tgccccatca ctct tgctgagccc agagccatcc Cgcgctctgc gggctgggag gcccgggcca ggacgcgagt cagc cgaggttccc cagcgccccc tgcagccgcg cgtaggcaga gacggagccc ggccctgcgc ctccgcacca cgcccgggac cccacccagc ggcccgtacc cggagaagca gcgcgagcac ccgaagctcc cggctggcgg cagaaaccgg 2015/068069 GenBank Accession Number and Sequence Description gagtggggcc gggcgagtgc gcggcatccc aggccggccc gaacgctccg cccgcggtgg gccgacttcc cctcctcttc cctctctcct tcctttagcc Cgctggcgcc ggacacgctg Cgcctcatct cttggggcgt tcttccccgt tggccaaccg tcgcatcccg tgcaactttg gggtagtggc cgtttagtgt tgaatgttcc ccaccgagag cgcatggctt gggaagcgag gcgcgaaccc ggcccccgaa gggccgccgt ccgggagacg gtgatgctgt tgctgtgcct gggggtcccg accggccgcc cctacaacgt ggacactgag agcgcgctgc tttaccaggg cccccacaac acgctgttcg gctactcggt Cgtgctgcac gggg cgaaccgatg gctcctagtg ggtgcgccca Ctgccaactg gctcgccaac gcttcagtga tcaatcccgg ggcgatttac agga tcggaaagaa tcccggccag acgtgcgaac agctccagct ccct aatggagaac cttgtggaaa gacttg:ttg gaagagagag acaatcagtg gttgggggtc acac;ttcca gacagccagg agaaaa;gga tccatcgtga cttgtgggca tagatggaaa aata2atttt agaa tgaaaa2aag thcccactg gtggttgcta tggagtgccc cctgatttac gaacagaact gagtaaaaga atagctccgt aaga ttatgtgaaa aaa;;tggag aaaattttgc atcatg;caa gctggaatat ccagttttta cacaaaggat ttaa2tgtga tgggggcccc aggatcatct tactggactg gctctctttt caat ataactacaa ataaatacaa ggcttt:tta gacaaacaaa atcaagtaaa atttggaagt tat;;aggat attcagtcgg agctgg;cat :tcggagcc agcatactac Cgaagtagtc ggaggagctc thaacatga gcagat;ggt aggcatata tattcagcat tgatgaaaaa aata tct:acatga aatgaaaggt aaaagcttg gatcgtactt tggagcttct gctg tggacctcaa tgcaga;ggc :ctcagatc tgctcgtggg agcacccatg cagagcacca tcagagagga aggaagagtg :tgtgtaca tcaactctgg gca gtaa:gaatg caa:ggaaac aaacctcgtt aca ctgc aagatttggg :Ctatag ttaatcttgg cgacat;gac gct ttgaagatgt tgctatcgga caag aagatgactt gcaagg;gct :ttatattt acaatggccg tgcagatggg atc:cgtcaa cct:ctcaca gagaat:gaa gacttcaga aatc gttaagtatg tttggacagt Cta2atcagg acaaat;gat cagataata atggctatgt agatgtagca gctt ttcggtctga ttctgc;gtc :gctaagga ctgt agtaattgt gacgcttctt accc tgagtcagta OWWLQLQQJQJLQWFT'QJSDFT'atagaacga aatt:gactg tgttgaaaa ggatggcctt Ctg;gtgcat agatctaaca tct cata2aaggg caaggaagt ccaggttaca tgtt ttataacatg agtttggatg tgaacagaaa ggcagagtc ccaccaagat tctatttctc ttctaatgga acttctgacg tgat:acagg aagcatacag gtgtccagca gagaagctaa ctgtagaaca catcaagcat ttatgcggaa agatgtgcgg gacatcctca ccccaattca gattgaagct gcttaccacc ttgg;cc;ca tgtcatcag: aaacgaagta cagaggaatt cccaccactt cagccaattc ttcagcagaa gaaagaaaaa atga aaaaaacaat aaact:tgca aggttttgtg ccca:gaaaa ttgttc;gc gatttacagg caaa gattgggttt ttgaagcccc atgaaaa2aa aacata;ct ggga agac attga:gttg aatgtgtcct tgtt:aa;gc tggaga:ga gcatatgaaa Cgactctaca tgtcaaacta cccgtgggtc ttta2ttcat taagat2tta gagctggaag agaagcaaat aaactgtgaa gtcacagata actc;ggcgt ggtacaact: gactgcagta ttggctatat atatg;agat catctctcaa ggataga2at tagctt:ctc ctggatgtga gctcactcag cagagcggaa gaggacctca gtatcacagt gcath2aCC tgtgaaaatg aagaggaaat ggacaatcta agca gagtgac;gt acc: ttaaaatatg aggttaagct gactg;tcat gggtttgtaa acccaac2tc atttgtgta: ggatcaaatg atgaaaatga gcctgaaacg tgcatggtgg agaaaatgaa Cttaac;ttc catgttatca acactggcaa tagta;ggct cccaatgtta gtgtggaaat aatggtacca aattctttta gcccccaaac tgataagctg ttcaacattt tggatgtcca gactac2ac: ggagaatgcc actttgaaaa ttatcaaaga gtgtgtgcat tagagcagca aaagag;gca atgcagacct tgaaaggcat agtccggttc ttgtccaaga Ctgataagag GenBank Accession Number and ce Description gtac tgcataaaag ctgatccaca ttgtttaaat ttcttgtgta KQQJQJS‘DFV'SDKQLQLQWWQSD :tttgggaa aatggaaagt gaag ccagtgttca actg gaaggccggc atccatttt agaaatggat gagacttcag cactcaag;t tgaaataaga gcaacaggtt :ccagagcc aaatccaaga gtaattgaac taaacaagga tgagaatgtt gcgcatgttc actggaagg actacatcat caaagaccca at;t caccatagtg attatttcaa :agcttgct acttggactt attgtacttc tgttgatc;c atatgttatg tggaaggctg cttctttaa aagacaatac aaatctatcc tacaagaaga aaacagaaga gacagttgga :tatatcaa cagtaaaagc aatgatgatt aaggacttct ttcaaattga gagaatggaa acagactca ggttgtagta aagaaattta aaagacaczg tt:acaagaa aaaatgaatt gac :tcttt2act catgatcttg tgacatat:a tg:cttcatg caaggggaaa caa :gattactct ttgagataga agaactgcaa aggtaataat acagccaaag :aatctctc agcttt2aaa gaga aacactaaag ca;tcaattt attcaagaaa gtaagccct :atc ttgaaatgaa agtataaczg ag:taaatta agaa :cttagact :gaaatacta cttaccatat gtgcttgcct cagtaaaatg ac:g ggtgggcaga ggttca2ttc aaatacatct ttgatactzg ttcaaaatat taaa aatataattt :ttagagagc tgttcccaaa ttttctaacg ag:ggaccat :atcactt:a aagccc:tta :ttataatac atttcctacg ggctgtgt;c caacaaccat :ttttttcag cagac:atga :agt attataggcc aaactggcaa ac;tcagact gaacatgtac actgg;;tga :gaa attacttctg gataatta:t tt:ttataat :atggatt:c accatc:ttc :ata tatacatgtg :ttttatg;a gg:atatatt :accattc ; cctatc;att Cttcctataa cacaccttta :Caagcatac ccaggagtaa :Cttcaaa;c ;;ttg;;ata :tctgaaaca aaagattgtg agtgttgcac tt:acctgat acacgctga: ::agaaaata cagaaaccat acctcactaa :aactttaaa atcaaagctg :gcaaagac: agggggccta :acttcatat gtattatgta aaaa tattgactat cacacaacza ;;tCC;;gga :gtaattctt tgttaccctt :acaagtata ag:gttacct :acatggaaa aaca aaattcataa attc ataaatt:ag gata c:gattcaa: ;;gta:acag :gaatataaa tgagacgaca gcaaaat2tt catgaaatgt aaaatat;;; :atag;;tgt :catactata tgaggttcta :t::aaa:ga ggat ::taaaaaa: ::ctt:aaat acaatcattt ttgtaatatt :a;;tta:gc tta:gatcta gataattgca gaata:ca;t :tatctgact Ctgccttcat aagagagctg tggccgaatt :2gaaca;c; g;tataggga gtgatcaaat tagaaggcaa :ggaaaaa caa:tctggg aagatt:c: ::ata:gaag :ccctgccac gcca :cc:aat:ga tgaaagttat :gttcacag gcctgcag;g atggtgagga atgttctgag at;;gcgaag gca2ttgagt gtgaaa aagcacaaaa cctcc:gaac ccagagtgtg :a;acacagg aataaacttt :gacat :gtat;tt:a aaaaactttg tatcgttata aaaaggczag tca:tctttc ctaggatcat agatgaaaaa tcaagccccg at;;agaact gtc2tctcca :aaggaaa;t tggt tctttcctac :cagaac:ac tcagaaacaa atct gagcacagtg aaagcagagt ac:atgg:tg tccaacacag :acaagggga ttac atattgggct aga2tttgcc cag;tcaaaa :atcaact ctttg;tact tgtatcatga at;:taaaac actt taagaagaca gggatggg attct2tttt ggcaggtagg Cta:ataact atg:gatttt gaaatttaac :gctctggat tagggagcag tgaatcaagg cagacttatg aaa;ctgtat tatatttgta acagaata ggaaa;ttaa cataattgat gagctcaaat cctgaaaaat gaaagaatcc aaattatt agaat2atct aggttaaata ttgatgtatt atgatggttg caaagttttt :tgtgtgtcc aataaacaca ttgtaaaaaa aa 26 N14 0008892 aaatc2tccc caccctgggg actt cctcctctgc cgtctcccag atcagtacac aaaggctgct gcca gaggaaggac tgctctgcac gcacctatgt ggaaactaaa GenBank Accession Number and ce ption Exenqflanlnudcm gcccagagag tgac ttgccccaca gccagtgagt gactgcagca gcaccagaat addsequcnce Ctggtctgtt tcctgtttgg acc actacggctt gggatctcgg gcatggtggc encmflnglnnnan tttgccaatg gtccttgttt tgctgctggt cctgagcaga ggtgagagtg aattggacgc fi7 caagatccca tccacagggg atgccacaga atggcggaat cctcacctgt ccatgctggg ccag ccagccccct cctgccagaa gtgcatcctc tcacacccca gctgtgcatg gtgcaagcaa ctgaacttca ccgcgtcggg agaggcggag gcgcggcgct gcgcccgacg agaggagctg ctggctcgag gctgcccgct ggaggagctg gaggagcccc gcggccagca ggagg:gctg caggaccagc cgctcagcca gggcgcccgc ggagagggtg ccacccagct ggcgccgcag cgggtccggg tcacgctgcg gcctggggag ccccagcagc tccaggtccg Cttcc:tcgt gctgagggat acccggtgga cctgtactac cttatggacc tgagctactc ggac gacctggaac gcgtgcgcca gctcgggcac gctctgctgg tccggctgca ggaag;cacc cattctgtgc gcattggttt tggttccttt aaaa cggtgctgcc Ctttg:gagc acagtaccct ccaaactgcg ccacccctgc cccacccggc tggagcgctg ccagtcacca ttcagctttc accatgtgct gtccctgacg ggggacgcac aagccttcga gcgggaggzg gggcgccaga gtgtgtccgg caatctggac tcgcctgaag g :ggcttcga tc:g caggctgcac tctgccagga tggc tggagaaatg tgtcccggct gctgg:gt;c acttcagacg acacattcca tacagctggg gacgggaagt tgggcggcat tttca;gccc agtgatgggc actgccactt ggacagcaat ggcctctaca g cac agagt:tgac tacccttctg tgggtcaggt agcccaggcc ctctctgcag tcca ct;t gctgtcacca gtgccgcact gcctgtctac caggagctga g;aaactgat tcctaagtct gcagttgggg agctgagtga ggactccagc aacgtggtac agctcatcat ggatgcttat aatagcctgt Cttccaccgt gacccttgaa tcac tccctcctgg ggtccaca;t tcttacgaat cccagtgtga gggtcctgag gagg g tga ggatcgagga cagtgcaacc acgtccgaat caaccagacg gtgactttct ctct ccaagccacc cactgcctcc cagagcccca gagg thcgggccc t :ggcttctc gczg attgtggagt tgcacacgct gtgtgactgt aattgcagtg acacccagcc ccaggctccc cactgcagtg atggccaggg acaa tgtggtgtat gcagctgtgc ccctggccgc ctaggtcggc tctgtgagtg ctctgtggca gagctgtcct ccccagacct tggg tgccgggctc ccaatggcac agggcccctg tgcagtggaa agggtcactg tcaatgtgga Cgctgcagct gcagtggaca gagctctggg catctgtgcg agtgtgacga tgccagctgt gagcgacatg agggcatcct ctgcggaggc tttggtcgct gtgg agtatgtcac tg:catgcca accgcacggg cagagcatgc gaa;gcagtg gggacatgga cagttgcatc ag;cccgagg gagggctctg cagtgggcat ggacgctgca aatgcaaccg ctgccagtgc ttggacggct actatggtgc tctatgcgac caa;gcccag gctgcaagac cgag agacaccggg actgtgcaga gtgtggggcc ttcaggactg gcccactggc caccaactgc ag;acagctt gtgcccatac caatgtgacc cccctatctt ggatgatggc tggtgcaaag agcggaccct ggacaaccag tcttggtgga ggatgacgcc agaggcacgg tcgtgctcag agtgagaccc gagcagacca cacgcaggcc at;gtgctgg tagg gggcatcgtg tggggctggt cctggcttac cggctctcgg tggaaatcta tgaccgccgg gctttgagaa ggagcagcaa caactcaact ggaagcagga cagtaatcct a gtgccatcac gaccaccatc aa;cctcgct ttcaagaggc agacagtccc actctctgaa ggagggaggg acacttaccc aaggctcttc tccttggagg acagtgggaa ctggagggtg agaggaaggg tgggtctgta agaccttggt aggggactaa ttcactggcg agg:gcggcc accaccctac ttcattttca gagtgacacc caagagggct gcttcccatg cctgcaacct tgcatccatc tgggctaccc cacccaagta tacaataaag tcttacctca aaaa aaaaaaaa NP 0008763 1 mawearrepg prraavretv mlllclgvpt grpynvdtes hnt lfgysvvlhs 2015/068069 GenBank Nunflmrand Sequence Description hganrwllvg aptanwlana svinpgaiyr crigknpgqt ceqlqlgspn gepcgktcle ary amino erdnqwlgvt lsrquengs ivtcghrwkn enkl ptggcygvpp skri acid sequence for apcyqdyvkk fgenfascqa gissfytkdl ivmgapgssy wtgslfvyni afld human (14 kqnqkagsy aghf rsqhttevvg gapqheqigk ayifsideke lnilhemkgk precursor klgsyfgasv cavdlnadgf sdllvgapmq grvf vyinsgsgav mnametnlvg sdkyaarfge sivnlgdidn dgfedvaiga pqeddlqgai yiyngradgi sstfsqrieg lqiskslsmf gqsisgqida dnngyvdvav gafrsdsavl lrtrpvvivd aslshpesvn rtkfdcveng wpsvcidltl kevp gyivlfynms ldvnrkaesp prfyfssngt sdvitgsiqv ssreancrth qafmrkdvrd iltpiqieaa yhlgphvisk rsteefpplq piqukkekd imkktinfar fcahencsad lquakigfl kphenktyla vgsmktlmln vslfnagdda yettlhvklp vglyfikile leekqincev tdnsgvvqld csigyiyvdh lsridisfll dvsslsraee dlsitvhatc eneeemdnlk hsrvtvaipl kyevkltvhg fvnptsfvyg sndenepetc mvekmnltfh Vintgnsmap nvsveimvpn sfqutdklf nildvqtttg echfenyqrv caleqqksam qtlkgivrfl sktdkrllyc clnf lcnfgkmesg iqle grpsilemde tsalkfeira tgfpepnprv ielnkdenva hvlleglhhq tivi issslllgli Vlllisyvmw kagffquyk silqeenrrd sksn dd NP_000880.1 mvalpmvlvl llvlsrgese ldakipstgd atewrnphls mlgscqpaps chcilshps cawckqlnft asgeaearrc arreellarg cpleeleepr gqqevlqdqp lsqgargega Exemplary amino tqlapqrvrv tlrpgepqql quflraegy pvdlyylmdl sysmkddler vrqlghallv acid sequence for rlqevthsvr igfgsfvdkt vlpfvstvps klrhpcptrl ercqspfsfh daq human B7 grqs vsgnldspeg qaal cqeqigwrnv srllvftsdd tfhtagdgkl ggifmpsdgh Chldsnglys rstefdypsv gqvaqalsaa avts aalpquels klipksavge lsedssnvvq limdaynsls stvtlehssl syes qcegpekreg kaedrgqcnh vrinqtvtfw vslqathclp ephllrlral ivel htlcdcncsd tqpqaphcsd gqghlchvc scapgrlgrl elss pdlesgcrap ngtgplcsgk ghcchrcsc sgqssghlce cddascerhe gilcggfgrc chvchchan rtgracecsg dmdscispeg glcsghgrck cnrcqcldgy ygalcquPg cktpcerhrd caecgafrtg platncstac ahtnvtlala pilddgwcke rtldnqlfff lveddargtv vlrvrpqekg adhtqaivlg cvggivavgl glvlayrlsv eiydrreysr fekeqqqlnw kqunplyks aitttinprf qeadsptl NM_016602.2 agagatgggg acggaggcca cagagcaggt ttcctggggc cattactctg gggatgaaga ggacgcatac tcggctgagc cactgccgga gC :ttgctac aaggccgatg tccaggcctt Exemplary nucleic cagccgggcc ttccaaccca gtgtctccct gaccgtggct gcgctgggtc tggccggcaa acid sequence tggcctggtc ctggccaccc acctggcagc ccgacgcgca tcgc ccacctctgc encoding human ccacctgctc cagctggccc tggccgacct ct :gctggcc ctgc ccttcgcggc CCRlO ggct Cttcagggct ggagtctggg aagtgccacc tgccgcacca tctctggcct ctactcggcc tccttccacg ccggcttcct ct :cctggcc tgtatcagcg ccgaccgcta cgtggccatc gcgcgagcgc tcccagccgg gccgcggccc tccactcccg gccgcgcaca Cttggtctcc gtcatcgtgt tgtc ac :gctcctg gcgctgcctg tctt cagccaggat gggcagcggg aaggccaacg acgctgtcgc ctcatcttcc ccgagggcct cacgcagacg gtgaaggggg Cgagcgccgt ggcgcaggtg gccctgggct tcgcgctgcc gctgggcgtc gcct gctacgcgct to :gggccgc acgctgctgg ccgccagggg gcccgagcgc cggcgtgcgc tgcgcgtcgt 99 :ggctctg gtggcggcct tcgtggtgct gcagctgccc tacagcctcg ccctgctgct ggatactgcc gatctactgg ctgcgcgcga gcggagctgc cctgccagca aacgcaagga t9 tg Ctggtgacca gcggcttggc GenBank Accession Nnnflmrand Sequence Description cctcgcccgc tgtggcctca ttct ctacgccttc ctgggcctgc gcttccgcca ggacctgcgg aggctgctac ggag ctca gggcctcaac cccgccgcgg ctgcccccgc cggccccgcc tttcttcctg ctcagctccc acggagaccc acagtctctc ctgggacaac tagggctgcg aatctagagg 3999990399 gtcg tgggaaaggg gagtaggtgg gggaacactg agaaagaggc agggacctaa agggactacc tctgtgcctt gccacattaa attgataaca tggaaatgag atgcaaccca acaa AF215981.1 agagatgggg acggaggcca cagagcaggt ttcctggggc cattactctg gggatgaaga ggacgcatac tcggctgagc cactgccgga gC :ttgctac aaggccgatg tccaggcctt Exemplary nucleic cagccgggcc ttccaaccca gtgtctccct gaccgtggct gcgctgggtc tggccggcaa acid sequence tggcctggtc ctggccaccc acctggcagc ccgacgcgca gcgcgctcgc ccacctctgc encoding human gctc cagctggccc tggccgacct ct cc Ctgactctgc ccttcgcggc CCRIO ggct cttcagggct ggagtctggg cacc tgccgcacca tctctggcct Ctactcggcc tccttccacg ccggcttcct ct cc agcg ccgaccgcta Cgtggccatc gcgcgagcgc tcccagccgg gccgcggccc cccg gccgcgcaca Cttggtctcc gtcatcgtgt ggctgctgtc ac :gctcctg gcgctgcctg cgctgctctt cagccaggat gggcagcggg aaggccaacg tcgc thatcttcc ccgagggcct cacgcagacg gtgaaggggg Cgagcgccgt ggcgcaggtg gccctgggct tcgcgctgcc gctgggcgtc atggtagcct gctacgcgct tc :gggccgc acgctgctgg ccgccagggg gcccgagcgc cggcgtgcgc tgcgcgtcgt 99 :ggctctg gtggcggcct tcgtggtgct gcagctgccc tacagcctcg ccctgctgct ggatactgcc ctgg gcga gcggagctgc cctgccagca aacgcaagga t9 :cgcactg acca gcggcttggc cctcgcccgc tgtggcctca atcccgttct ctacgccttc Ctgggcctgc gcttccgcca ggacctgcgg aggctgctac ggggtgggag ctcgccctca gggcctcaac cccgccgcgg Ctgcccccgc cggccccgcc tttcttcctg ctcagctccc acggagaccc acagtctctc Ctgggacaac tagggctgcg aatctagagg agggggcagg ctgagggtcg tgggaaaggg gagtaggtgg gggaacactg agaaagaggc agggacctaa agggactacc tctgtgcctt gccacattaa attgataaca tggaaatgaa aaaaaaaaaa aaaa NP_057686.2 mgteatequ wghysgdeed aysaeplpel cykadvqafs rafqpsvslt vaalglagng lvlathlaar raarsptsah adll laltlpfaaa galqgwslgs sgly ary amino sasfhagflf lacisadryv aiaralpagp rpstpgrahl vsvivwllsl llalpallfs acid sequence for ngqregqrr crlifpeglt qtvkgasava qvalgfalpl gvmvacyall grtllaargp human CCRIO errralrvvv alvaafvvlq lpyslallld tadllaarer scpaskrkdv allvtsglal precursor arcglnpvly aflglrfrqd lrrllrggsc psgpqprrgc sscs aptethslsw P460923 mgteatequ wghysgdeed aysaeplpel cykadvqafs rafqpsvslt vaalglagng laar raarsptsah llqlaladll laltlpfaaa galqgwslgs atcrtisgly Exemplary amino sasfhagflf lacisadryv aiaralpagp rpstpgrahl vsvivwllsl llalpallfs acid ce for ngqregqrr crlifpeglt qtvkgasava qvalgfalpl gvmvacyall grtllaargp human CCRIO errralrvvv vvlq lpyslallld tadllaarer scpaskrkdv allvtsglal arcglnpvly aflglrfrqd lrrllrggsc psgpqprrgc prrprlsscs aptethslsw NM_005201.3 tttgtagtgg acct ccagagaggc tcat tgagctgcac tcacatgagg atacagactt tgtgaagaag gaattggcaa cactgaaacc acaa aggctgtcac Exemplary nucleic taaggtcccg Ctgccttgat ggattataca cttgacctca gtgtgacaac agtgaccgac GenBank Accession Nnnflmrand Sequence Description acid sequence tactactacc ctgatatct ctcaagcccc tgtgatgcgg aac:tattca gacaaatggc encoding human aagt :gc ZCC ttgctgtct ttattgcc;c ctgtttgtat :Cagtcttct gggaaacagc CCR8 Ctgg :ca :CC :Ccttg gg;ctgcaag aagctgagga gca2cacaga tgtatacctc ttgaacc 399 cc:gcttt tcct :cccctttca gacctac :at Ctgc :ggacc tgggactg atgtgcaaag :gg;gtctgg Cttttat :ac attggct :Ct gt;tttca accctcatga gtg;ggacag gtacctggct gttg :ccatg cc:aaagg aggacgatca gga:gggcac aacgctg :gc Ctggcag :at ca2tatggct accatcccat :gc;agtgtt ttaccaagtg gcctctgaag acagtgttat tcattt :aca agac tttgaag 199 aaga :ct :ca aa:gaaca:t ttaggc :tgt :ga:cccatt caccatc :tt atgt :Ctgct cc;gcaccag ctgaagaggt gtcaaaacca caacaagacc aaggcca :Ca ca;tgtgg;c attgca :ctt :ac;tttctg ggtccca :tc aacg 399 :tc ttccttgcac agtatgcaca :ct:ggatgg atgtagcata agccaacagc caccca:g;c atca :ttcctttac tcactgc :gt gtgaaccctg tt;tgt;ggg gagaag :tca agaaacacct ctcagaaata tttcagaaaa caac tacctaggaa gacaaatgcc tagggagagc tgtgaaaagt ccagcagcac tcc:cccgtt cctccagcgt agactacatt ttgtgaggat :gaagac ;aaa aaacat :ttc ttgaatggca tgctagtagc agtgagcaaa ggtgtgggtg tgaaaggttt ccaaaaaaag ttcagcatga aggatgccat atatgttgtt gccaacactt ggaacacaat atagttgtgc atgcctggca caacatcaag attg tgttta;tga caagtggtaa Ctttaaagga atgc caagtgaaaa atgt t BCH07159J Ctttgtgaag aaggaattgg caacac;gaa ctgt cactaaggtc cctt ga :ggattat gacc aacagtgacc gactactact Exemplary nucleic accctgatat ct :thaagc ccctgtgatg tcagacaaat ggcaagt :gc acid sequence :Ccttgctgt Ct :ttattgc 0 :Cctg;ttg aaac agcctgg :ca encoding human :cctgg :Cct t9 399 :ctgc magaagctga agatgtatac ctcttgaacc CCRS :ggccc :gtc tgacc :gctt fi' :tgtc2tct tcagacctac :atctgc 399 accagtgggt gt :tgggact 0 :aatg;gca tggcttttat :acattggct :ctacagcag ca :gt :tttc m :caccctca caggtacctg g ICC atgccg :gta tgccc :aaag m :gaggacga cacaacgctg :gcctggcag :atggc :aac cgcca :tatg mCtaccatcc gttttaccaa gtggcctctg aagatggtgt tc :acagtgt r* attcattt; gactttgaag :ggaaga :ct :caccaactt caaaa :gaac m :tttaggc: attcaccatc :ttatgt :ct gctaca :taa aa ZCC :gcac 0agctgaaga ccacaacaag accaaggcca :caggt :ggt gC :ca :tgtg (Q :cattgca; ctgggtccca :tcaacg 399 :tcttt :cct cacttccttg 0acagtatgc tggatgtagc caac agctgactta tgccacccat m :cacagaaa tactcactgc :gtgtgaacc ctgtta :cta tgctt :tgtt ggggagaag; tcaagaaaca cctctcagaa caga aaagttgcag ccaaa :cttc aactacctag gaagacaaat ggag agctgtgaaa agtcatcatc Ctgccagcag cactcctccc ccag Cgtagactac attttgtgag gatcaa :gaa gactaaatat aaaaaacat; ttcttgaatg gcatgctagt agcagtgagc aaaggtgtgg gtgtgaaagg tttccaaaaa aagttcagca atgc cgtgtgtgtt gttgccaaca Cttggaacac gatgactggg gacgtggttg tgcatgcctg gcacaacatc aagcctgtga ttgtgtttat tgt; gaacaagtgg tggctttgga ggattctgta tgccaagtga aaggggagat gtctgacctc cttcatatag GenBank Accession and Sequence ption NP_005192.1 mdytldlsvt tvtdyyypdi fsspcdaeli llav fycllfvfsl lgnslvilvl vvckklrsit dvyllnlals dllfvfsfpf qtyylldqu fgtvmckvvs gfyyigfyss Exemplary amino mffitlmsvd rylavvhavy irmg ttlclavwlt aimatipllv fyqvasedgv acid sequence for lq0ysfynqq tlkwkiftnf kmnilgllip ftifmfcyik ilhqlkrcqn hnktkairlv human CCR8 livviasllf vafnvvlfl tslhsmhild gcsisqqlty athvteiisf thccvnpviy afvgekfkkh lseiquscs grqm presceksss cqqhssrsss vdyil AAIO7 160. l mdytldlsvt tvtdyyypdi fsspcdaeli qtngklllav fycllfvfsl lgnslvilvl vvckklrsit lals dllfvfsfpf qtyylldqu fgtvmckvvs gfyyigfyss Exemplary amino mffitlmsvd havy alkvrtirmg ttlclavwlt pllv fyqvasedgv acid sequence for qustynqq tlkwkiftnf kmnilgllip ftifmfcyik ilhqlkrcqn hnktkairlv human CCR8 sllf vafnvvlfl tslhsmhild gcsisqqlty a;hvteiisf thccvnpviy precursor afvgekfkkh lseiquscs qifnylgrqm presceksss cqqhssrsss deil NM_005508.4 gctcacagga agccacgcac ccttgaaagg caccgggtcc :Cttagca cgtgcttcct gagcaagcct ggcattgcct cacagacctt cctcagagcc ctttcagaa aagcaagctg Exemplary nucleic Cttctggttg ggcccagacc tgccttgagg agcctgtaga :taaaaaa gaaccccacg acid sequence gatatagcag acaccaccct Cgatgaaagc agca LQLQOFT'QJWOLQSDLQLQW :tatactga:tactatc gtatgaaagt encoding human atccccaagc cttgcaccaa agaaggcatc aaggcatttg ggagctct cctgccccca CCR4 Ctgtattcct ttgt atttggtctg cttggaaatt :gtggtgg tctggtcczg ttcaaataca agcggctcag gtccatgact gatgtgtacc gctcaacc tgccatctcg gatc:gctct tcgtgttttc cctccctttt tggggctact aga ccagtggg:t tttgggctag gcaa gatgatttcc tggatgtact LOLO fl. LOLOLO0 fl. :acagtggc atattctttg tcatgctcat gagcattgat ctgg aattgtgca gcggtgt tcct:gaggg caaggacctt gacttatggg gtcatcacca :ttggctac :ggtcag gctg:gttcg cctcccttcc tctg ttcagcactt cat acctactgca aaaccaagta aac tccacgacgt ggaaggttct agctccc gaaa:caaca ttctcggatt gg:gatcccc ttagggatca tgctgttttg :actcca atca:cagga ccttgcagca :tgtaaaaat gagaagaaga acaaggcggt aagatga tttgccgtgg tggtcctctt gttc tggacacctt acaacatagt ctcttcc gagaccctgg tggagctaga ag;ccttcag gactgcacct ttgaaagata :tggactat gcca:ccagg ccacagaaac :c;ggctttt gttcactgct atcc atcatctac ttttttctgg gggagaaatt :Cgcaagtac atcc;acagc tcttcaaaac :gcaggggc cttt:tgtgc tctgccaata ctgtgggctc ctccaaattt actctgctga acccccagc tcatcttaca Cgcagtccac ca;ggatcat gatc:ccatg atgctctgta aaaaatgaa atggtgaaat gcagagtcaa :gaactttcc acat;cagag Cttacttaaa SDLQOQOOLQLQOOLQQIOFT‘ ttt agtaagagat tcctgagcca gtgtcaggag gaaggcttac acccacagtg gaaagacagc ttctcatcct gcaggcagct :tztctctcc cactagacaa gtccagcctg gcaagggttc acctgggctg cctt CC :cacacca ggct;gcctg caggcatgag tcagtctgat gagaactctg agcagtgctt gaatgaagtt gtaggtaata ttgcaaggca aagactattc ccttctaacc gatg ggzttctcca gagggaattg cagagtactg ggag taaatcgcta ccttttgctg tggcaaatgg gccc;ct P5 1679.1 mnptdiadtt ldesiysnyy lyesipkpct kegi<afgel flpplyslvf vfgllgnsvv ykrl rsmtdvylln laisdllfvf slpfwgyyaa dqufglglc kmiswmylvg Exemplary amino fysgiffvml msidrylaiv havfslrart ltygvitsla twsvavfasl pgflfstcyt acid sequence for ernhtycktk yslnsttwkv lssleinilg lviplgimlf cysmiirth hcknekknka human CCR4 vkmifavvvl flgfwtpyni vlfletlvel evlqdctfer yldyaiqate tlafvhccln WO 09668 GenBank Accession Number and Sequence Description pmcumor lgek qlfk tcrglfvlcq ycgllqiysa dtpsssytqs tmdhdlhdal N14 6091 aatcatccga gaaccttgga gggtggacag tttt acagatgaga aaactgaggc ttgaagggga gaagcagctg cctctggcgg catggcttct ggctgcagga tgcccatgga ExenqflaU’HUdem gttcgtggtg accctaggcc tgtgtctcgg cttcctttgc tgaacttgaa caggaagatg acid ce gcagtggggg ccagtggtct agaaggagat aagatggctg gtgccatgcc tctgcaactc ng human ctcctgttgc tgatcctact gggccctggc aacagcttgc agctgtggga cacctgggca gatgaagccg cctt gggtcccctg cttgcccggg accggagaca ggccaccgaa tatgagtacc tagattatga tttcctgcca gaaacggagc ctccagaaat gctgaggaac agcactgaca ccactcctct gactgggcct ggaacccctg agtctaccac tgtggagcct gctgcaaggc ctgg cctggatgca ggaggggcag tcacagagct gaccacggag ctggccaaca tggggaacct gtccacggat tcagcagcta taca gaccactcaa ccagcagcca cggaggcaca gaccactcaa ccagtgccca cggaggcaca gaccactcca ctggcagcca cagaggcaca gacaactcga ctgacggcca cggaggcaca gaccactcca ctggcagcca cagaggcaca gaccactcca ccagcagcca cggaagcaca gaccactcaa cccacaggcc tggaggcaca gaccactgca ccagcagcca tggaggcaca gaccactgca ccagcagcca caca gaccactcca ccagcagcca tggaggcaca gaccactcaa accacagcca tggaggcaca gaccactgca ccagaagcca cggaggcaca gaccactcaa gcca cggaggcaca gaccactcca ctggcagcca tggaggccct gtccacagaa cccagtgcca cagaggccct gtccatggaa acca aaagaggtct gttcataccc ttttctgtgt cctctgttac tcacaagggc attcccatgg cagccagcaa tttgtccgtc aactacccag cccc agaccacatc tctgtgaagc tgct ggccatccta atcttggcgc tggtggccac tatcttcttc gtgtgcactg tggtgctggc cctc tcccgcaagg gccacatgta ccccgtgcgt tccc ccaccgagat ggtctgcatc tcatccctgt tgcctgatgg ggg:gagggg ccctctgcca cagccaatgg gggcctgtcc aaggccaaga gcccgggcct gacgccagag gagg accgtgaggg ggatgacctc accctgcaca gcttcctccc ttagctcact ctgccatctg ttttggcaag accccacctc cacgggctct cctgggccac ccc:gagtgc ccagacccca ttccacagct ctgggcttcc tcggagaccc ctggggatgg gga;cttcag ggaaggaact accc aaacaggaca agagcagcct ggggccaagc agacgggcaa gtggagccac thtttcctc cctccgcgga tgaagcccag ccacatttca gccgaggtcc aaggcaggag gccatttact tgagacagat tctctcct;t ttcctgtccc cca;cttctc tgggtccctc taacatctcc catggctctc cccgcttc;c ctggtcactg gag;ctcctc cccatgtacc caaggaagat cccc catcccacac gcactgcact gccattgtct tttggttgcc atggtcacca aacaggaagt ggacattc;a agggaggagt actgaagagt gacggacttc tgaggctgtt tcctgctgct cctctgac;t ggggcagctt ggg;cttctt gggcacctct Ctgggaaaac ccagggtgag gttcagcczg tgagggctgg gatgggtttc gtgggcccaa gggcagacct ttctttggga Ctgtgtggac caaggagctt cca2ctagtg acaagtgacc cccagctatc gcctcttgcc ttcccctg ttcc agggtggact ctgtcttgtt cactgcagta tcccaactgc aggtccag ataa ata:gtgatg gacaaacgat agcggaatcc ttcaaggttt caaggctg tccttcaggc agccttcccg gaattctcca tccctcagtg caggatgggg gctggtcc agctgtctgc cctcagcccc tggcccccca ggaagcctct ttcatgggct gttaggttga cttcagtttt gcc:cttgga gggg tcttgtacat ccttgggtga ccaggaaaag ttcaggctat ggggggccaa agggagggct gccccttccc caccagtgac GenBank Accession Number and ce Description cactttattc cacttcctcc attacccagt tttggcccac tggt cccccccaaa acca atatccctct aaacatcaat ctatcctcct gttaaagaaa aaaaaaaa N14 0030064 acacacagcc attgggggtt gctcggatcc gggactgccg cagggggtgc cacagcagtg cctggcagcg tgggctggga ccttgtcact aaagcagaga agccacttct tctgggccca ExenqflaU7HUd€m Cgaggcagct gtcccatgct Ctgctgagca cggtggtgcc atgcctctgc aactcctcct acid sequence gttgctgatc ctactgggcc ctggcaacag cttgcagctg tgggacacct gggcagatga encoding human gaaa gccttgggtc ccctgcttgc ccgggaccgg agacaggcca ccgaatatga agat ttcc tgccagaaac ggagcctcca gaaatgctga ggaacagcac tgacaccact cctctgactg ggcctggaac ccctgagtct gtgg agcctgctgc aaggcgttct actggcctgg atgcaggagg ggcagtcaca acca cggagctggc gggg aacctgtcca ngattcagc agctatggag atacagacca ctcaaccagc ggag gcacagacca ctcaaccagt gcccacggag gcacagacca ctccactggc agccacagag gcacagacaa ctcgactgac ggccacggag acca ctccactggc agccacagag gcacagacca ctccaccagc agccacggaa gcacagacca ctcaacccac aggcctggag gcacagacca ctgcaccagc ggag gcacagacca ctgcaccagc agccatggaa gcacagacca ctccaccagc agccatggag gcacagacca ctcaaaccac agccatggag gcacagacca ctgcaccaga agccacggag gcacagacca ctcaacccac agccacggag gcacagacca ctccactggc agccatggag gccctgtcca cagaacccag tgccacagag gccctgtcca ctac taccaaaaga ggtctgttca tacccttttc tgtgtcctct gttactcaca agggcattcc catggcagcc agcaatttgt ccgtcaacta cccagtgggg gccccagacc acatctctgt gaagcagtgc ctgctggcca tcctaatctt ggcgctggtg gccactatct :Cttcgtgtg cactgtggtg Ctggcggtcc gcctctcccg caagggccac cccg :gcgtaatta ctcccccacc gagatggtct gcatctcatc cctgttgcct gatgggggtg aggggccctc tgccacagcc aatgggggcc tgtccaaggc caagagcccg ggcctgacgc cagagcccag ggaggaccgt gagggggatg ccct gcacagcttc thccttagc :Cactctgcc atctgttttg gcaagacccc acctccacgg gctctcctgg gccacccctg agtgcccaga ccccattcca cagctctggg cttcctcgga gacccctggg gatc :tcagggaag gaactctggc cacccaaaca ggacaagagc agcctggggc caagcagacg ggcaagtgga gccacctctt tcctccctcc gaag caca tttcagccga ggtccaaggc aggaggccat ttacttgaga ctct cctttttcct gtcccccatc :tctctgggt aaca tctcccatgg ctctccccgc ttctcctggt cactggagtc :Cctccccat aagg aagatggagc atcc cacacgcact gcactgccat :gtcttttgg ttgccatggt caccaaacag gaagtggaca ttctaaggga ggagtactga agagtgacgg acttctgagg ctgtttcctg ctgctcctct gacttggggc agcttgggtc :tcttgggca cctctctggg aaaacccagg gtgaggttca gcctgtgagg gctgggatgg gtttcgtggg cccaagggca gacctttctt tgggactgtg tggaccaagg agcttccatc :agtgacaag tgacccccag ctatcgcctc ttgccttccc Ctgtggccac tttccagggt ggactctgtc ttgttcactg cagtatccca actgcaggtc cagtgcaggc aataaatatg :gatggacaa acgatagcgg aatccttcaa ggtttcaagg ctgtctcctt caggcagcct :cccggaatt ctccatccct cagtgcagga tgggggctgg tcctcagctg tctgccctca gcccctggcc ccccaggaag cctctttcat gggctgttag gttgacttca gttttgcctc :tggacaaca gggggtcttg tacatccttg cagg aaaagttcag gctatggggg gccaaaggga gggctgcccc ttccccacca gtgaccactt tattccactt ttac ccagttttgg cccacagagt ttggtccccc ccaaacctcg gaccaatatc aaca tatc ctcctgttaa agaaaaaaaa aaa NP 0011935381 1 mavgasgleg dkmagamplq lllllillgp gnslqlwdtw adeaekalgp llardrrqat Number and Sequence Description. . eyeyldydfl peteppemlr nstdttpltg pgtpesttve paarrstgld aggavteltt Exenqflaqzannno elanmgnlst dsaameiqtt qpaateaqtt qpvpteaqtt plaateaqtt rltateaqtt addsequencefbr plaateaqtt ppaateaqtt qptgleaqtt apaameaqtt aqtt ppaameaqtt hunuHICLA. qttameaqtt apeateaqtt qptateaqtt plaamealst epsatealsm epttkrglfi pecumo1’ 1‘ pfsvssvthk gipmaasnls vnypvgapdh isvchllai lilalvatif lavr lsrkghmypv rnysptemvc issllpdgge gpsatanggl gltp epredregdd ltlhsflp 42 NP 0029972 mplqllllli llgpgnslql wdtwadeaek algpllardr rqateyeyld ydflpetepp emlrnstdtt pltgpgtpes ttvepaarrs tgldaggavt elttelanmg nlstdsaame Exenqflao’anfino iqttqpaate aqttqpvpte aqttplaate aqttrltate aqttplaate aqttppaate uencefbr aqttQPtg1e a ttag Paame aqtta aameP aqttPPaame aqttqttame aqttaPeate hu CLA. aqttqptate aqttplaame alstepsate alsmepttkr glfipfsvss Vthkgipmaa pmcumor snlsvnypvg apdhisvch llaililalv atiffvctvv lavrlsrkgh yspt emvcissllp dggegpsata ngglskaksp redr egddltlhsf 1p .3.6. Polynucleotide for Generating CAR and/or Homing Receptor Described herein are polynucleotide sequences (326., nucleic acid sequences) that encode the chimeric receptors and homing receptors. The polynucleotides may be contained within any polynucleotide vector suitable for the transformation of immune cells, e.g., NK cells.

For example, NK cells may be transformed using synthetic vectors, lentiviral or retroviral vectors, autonomously replicating ds, a virus (e.g., a retrovirus, lentivirus, adenovirus, or herpes virus), or the like, containing polynucleotides ng the first and second polypeptides (e.g., chimeric receptors). Lentiviral vectors suitable for ormation ofNK cells include, but are not d to, e.g, the lentiviral vectors described in US. Patent Nos. 5,994,136; 6,165,782; 6,428,953, 7,083,981; and 299, the disclosures of which are hereby incorporated by reference in their entireties. HIV vectors le for transformation ofNK cells include, but are not limited to, e.g., the vectors bed in US. Patent No. 5,665,577, the disclosure of which is hereby incorporated by reference in its entirety. [003 53] Nucleic acids useful in the production of the polypeptides descn'bed herein, e.g., within a NK cell, include DNA, RNA, or nucleic acid analogs. Nucleic acid analogs can be modified at the base , sugar moiety, or ate backbone, and can include deoxyuridine substitution for deoxythymidine, 5-methyl-2'—deoxycytidine or 5-bromo-2'—deoxycytidine substitution for deoxycytidine. Modifications of the sugar moiety can include modification of the 2' hydroxyl of the ribose sugar to form 2'—O-methyl or 2'—O-allyl sugars. The ibose phosphate ne can be d to produce morpholino nucleic acids, in which each base moiety is linked to a six membered, lino ring, or peptide c acids, in which the deoxyphosphate backbone is replaced by a pseudopeptide backbone and the four bases are retained. See, for example, Summerton and Weller (1997) Antisense Nucleic Acid Drug Dev. 7: 187-195, and Hyrup et al. (1996) Bioorgan. Med. Chain. 4:5-23. In addition, the deoxyphosphate backbone can be replaced with, for example, a phosphorothioate or orodithioate backbone, a phosphoroamidite, or an alkyl phosphotriester backbone. [003 54] A nucleic acid encoding a polypeptide described herein may be uced into host cells as part of a vector, such as, e.g., an expression vector. In addition, a polypeptide described herein may be produced by transfecting a host cell with a nucleic acid encoding such a polypeptide, and such nucleic acid may be part of a vector. In a c embodiment, the vector is an expression vector that is capable of directing the expression of a nucleic acid encoding a polypeptide described herein. Non-limiting examples of expression vectors include, but are not limited to, ds and viral vectors, such as replication defective retroviruses, adenoviruses, adeno—associated viruses, Newcastle e virus, ia virus and baculoviruses. Standard molecular biology techniques may be used to introduce a nucleic acid ng a polypeptide bed herein into an expression vector.

An expression vector comprises a nucleic acid encoding a polypeptide described herein in a form le for expression of the nucleic acid in a host cell or non-human subject.

In a specific embodiment, an expression vector includes one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operably linked to the nucleic acid to be sed. Within an expression vector, "operably linked" is intended to mean that a nucleic acid of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleic acid (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). Regulatory sequences include promoters, enhancers and other expression control elements (e.g., enylation signals). Regulatory sequences include those which direct constitutive expression of a nucleic acid in many types of host cells, those which direct expression of the nucleic acid only in certain host cells (e.g., tissue- specific regulatory sequences), and those which direct the expression of the nucleic acid upon stimulation with a particular agent (e.g., inducible regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such s as, e. g., the choice of the host cell to be transformed, the level of expression of protein desired, etc.

An expression vector can be introduced into host cells via conventional transformation or ection techniques. Such techniques include, but are not limited to, calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, and electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al,, 1989, Molecular Cloning - A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, New York, and other tory manuals. In certain embodiments, a host cell is ently transfected with an expression vector containing a nucleic acid encoding a polypeptide described herein. In other embodiments, a host cell is stably transfected with an expression vector ning a nucleic acid encoding a polypeptide bed herein.

] Cells containing any of the polynucleotide may be selected using one or more selectable s. .4. Methods of Treating Hematological Disorders or Solid Tumors Provided herein are methods of treating a hematological disorder or a solid tumor using NK cells or genetically modified NK cells (e.g., NK cells comprising a CAR and/or a homing receptor) as described above. .4.1. NK Combination Therapies [003 59] In one aspect, provided herein are methods of treating a hematological disorder or a solid tumor in a subject in need thereof, sing: (a) administering to said subject an isolated population of natural killer (NK) cells or a pharmaceutical composition thereof, or an isolated population of genetically modified NK cells (e.g., NK cells comprising a CAR and/or a homing receptor) or a pharmaceutical composition thereof, and (b) administering to said subject a second agent or a pharmaceutical composition thereof. The second agent can be any pharmaceutically acceptable agent that can be used to treat the logical er or the solid tumor, and includes, but is not limited to, an antibody (e.g., a monoclonal dy), a bispecific killer cell engager (BiKE), an anti-inflammatory agent, an immunomodulatory agent (e.g., an odulatory compound as described in section 5.2.7.1), a cytotoxic agent, a cancer vaccine, a chemotherapeutic agent, an HDAC inhibitor, or an siRNA. .4.1.1. NK Combinations with Antibodies In certain embodiments, the second agent is an antibody or antigen—binding fragment thereof.

As used herein, the terms “antibody” and “immunoglobulin” and “Ig” are terms of art and can be used interchangeably herein and refer to a molecule with an antigen binding site that specifically binds an antigen. [003 62] Antibodies can include, for example, monoclonal antibodies, recombinantly produced antibodies, monospeciflc antibodies, multispecific antibodies (including bispeciflc antibodies), human antibodies, humanized dies, such as composite human antibodies or deimmunized antibodies, murine antibodies (e.g., mouse or rat antibodies), chimeric antibodies, synthetic antibodies, and tetrameric antibodies sing two heavy chain and two light chain les.

In specific ments, antibodies can include, but are not limited to an antibody light chain monomer, an antibody heavy chain monomer, an antibody light chain dimer, an antibody heavy chain dimer, an antibody light chain- antibody heavy chain pair, intrabodies, conjugate antibodies, single domain antibodies, and monovalent antibodies. In a specific embodiment, antibodies can include antigen—binding fragments or epitope binding fragments such as, but not limited to, single chain antibodies or single-chain Fvs (scFv) (e.g., including monospeciflc, bispecific, eta), camelized antibodies, affybodies, Fab fragments, F(ab’) fragments, F(ab’)2 fragments, and disulfide-linked Fvs (dev). In specific embodiments, dies described herein refer to monoclonal antibodies.

Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA or IgY), any class, (e.g., IgGl, Ing, IgG3, IgG4, IgA1 or IgAz), or any ss (e.g., Inga or Ingb) of globulin le. In certain embodiments, antibodies descnbed herein are IgG antibodies, or a class (e.g., human IgG1, Ing, or IgG4) or subclass thereof. In certain embodiments, antibodies described herein are IgG2 antibodies (e.g., human IgG2) or a ss thereof (e.g., human IgG;a or human Ingb, or a e thereof). In certain embodiments, antibodies described herein are IgG1 antibodies (e.g., human IgG) or a ss thereof. In certain embodiments, IgG1 antibodies described herein comprise one or more amino acid substitutions and/or deletions in the constant . [003 64] As used herein, the term “monoclonal antibody” is a well known term of art that refers to an antibody obtained from a population of homogenous or substantially homogeneous 2015/068069 antibodies. The term “monoclonal” is not d to any particular method for making the antibody. Generally, a population of monoclonal antibodies can be ted by cells, a population of cells, or a cell line. In specific embodiments, a “monoclonal antibody,” as used herein, is an antibody produced by a single cell or cell line wherein the antibody immunospecifically binds to an epitope as determined, e.g., by ELISA or other antigen-binding or competitive binding assay known in the art. In particular embodiments, a monoclonal antibody can be a chimeric antibody or a humanized antibody. In certain embodiments, a onal antibody is a monovalent antibody or alent (e.g., bivalent) antibody.

In specific embodiments, the antibody or antigen—binding fragment thereof specifically binds to a tumor-associated antigen (TAA), which is bed in Section 5.3.2. In a further specific embodiment, the antibody or antigen-binding fragment thereof binds to CS—l. In a more specific embodiment, the antibody or antigen-binding fragment thereof is elotuzumab, or an antigen-binding fragment thereof. In a further specific embodiment, the antibody or antigen- binding fragment thereof binds to CD20.

In specific embodiments, the antibody or antigen-binding fragment thereof specifically binds to a tumor microenvironment-associated antigen (TMAA), which is bed in Section 5.3.2. [003 67] In specific embodiments, the antibody or antigen-binding fragment thereof specifically binds to and antagonizes the activity of an immune checkpoint protein. In more specific embodiments, the immune checkpoint n is CTLA-4, PD-l, PD-Ll, PD-L2, or LAG-3. In more specific embodiments, the immune checkpoint-related protein is BTLA, KIR, TIM-3, A2aR, B7-H3, or B7-H4. In other specific embodiments, the dy or antigen- g fragment thereof specifically binds to and antagonizes the activity of a costimulatory signaling n. In more specific embodiments, the costimulatory signaling protein is ICOS, CD28, 4-1BB, 0X40, CD27, or CD40. .4.1.2. NK Combinations with Bispecific Killer Cell Engagers In certain embodiments, the second agent is a bispecific killer cell engager (BiKE). [003 69] BiKEs are reagents that contain two single chain variable fragments (scFvs) and specifically engage both target cells (e.g., tumor cells or infected cells) and NK cells to mediate target cell g. They are used to colocalize target cells (e.g, tumor cells or infected cells) with NK cells, and thereby triggering NK—cell mediated antibody—dependent cellular cytotoxicity (ADCC). BiKEs can be generated by any method known in the art, for e, as described in Gleanson, M. K, et a1., Mol Cancer Ther, 11: 2674-2684 (2012); Vallera, D. A., et al., Cancer Biother Radiopharm, 28: 274-282 , Wiernik, A., et al., Clin Cancer Res, 19: 3844-3 855 ; Reiners, K. S., et a1., Mol Ther, 21: 895-903 (2013); Singer, H., et al., J Immunother, 33: 599-608 (2010), or Gleason, M. K, et al., Blood, 123: 3016—3026 (2014). One scFv ofBiKE specifically binds to an antigen on the surface of target cells (e.g., tumor cells or infected cells), and the other scFv specifically binds to a receptor (e.g., an Fc receptor, such as CD16) on NK cells.

In specific embodiments, the BiKE comprises a first scFv that specifically binds to a TAA, which is described in Section 5.3.2. In further specific embodiments, the BiKE comprises a second scFv that specifically binds to CD16. .4.1.3. NK Combinations with Other Anti Cancer Agents Other anticancer agents that can be administered as the second agent are well-known in the art and e anti-inflammatory , immumodulatory agents, cytotoxic agents, cancer vaccines, chemotherapeutics, HDAC tors, and siRNAs. Specific anticancer agents that may be administered to an dual having cancer, e.g, an individual having tumor cells, in addition to the NK cells produced using the methods described herein and optionally perfusate, perfusate cells, natural killer cells other than NK cells produced using the methods described herein include, but are not d to: acivicin, aclarubicin; acodazole hydrochloride, acronine, adozelesin; adriamycin, adrucil, aldesleukin, altretamine, ambomycin, ametantrone acetate; amsacrine; anastrozole; anthramycin; asparaginase (e.g., from Erwinia chrysan; Erwinaze), asperlin, avastin (bevacizumab), azacitidine, azetepa, azotomycin, stat, benzodepa; bicalutamide, bisantrene hydrochloride; bisnafide dimesylate; sin; bleomycin sulfate; brequinar sodium, bropirimine, busulfan, cactinomycin, calusterone; caracemide, carbetimer, latin; carmustine; carubicin hydrochloride, carzelesin, cedefingol; celecoxib (COX—2 inhibitor), CC—l22; CC-486 (oral azacididine), Cerubidine; chlorambucil; cirolemycin, cisplatin, cladribine, crisnatol mesylate, cyclophosphamide; cytarabine, dacarbazine; dactinomycin; daunorubicin hydrochloride; decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate, diaziquone, docetaxel, doxorubicin, bicin hydrochloride, ifene; ifene citrate; dromostanolone propionate; duazomycin; edatrexate; eflomithine hydrochloride, elsamitrucin, Elspar, enloplatin; enpromate, epipropidine, epirubicin hloride; erbulozole; esorubicin hydrochloride; estramustine; estramustine phosphate sodium; etanidazole; etoposide; etoposide phosphate; hos; etoprine; fadrozole hloride; fazarabine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil; flurocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride; hydroxyurea; Idamycin; idarubicin hydrochloride; ifosfamide; ilmofosine; iproplatin; irinotecan; irinotecan hydrochloride; tide acetate; lenalidomide; letrozole; leuprolide acetate; liarozole hloride; lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprocol; maytansine; mechlorethamine hydrochloride; rol acetate; melengestrol acetate; melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; enolic acid; nocodazole; nogalamycin; ormaplatin; an; paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride; plicamycin; plomestane; pomalidomide; porf1mer sodium; porf1romycin; prednimustine; procarbazine hydrochloride; Proleukin; Purinethol; puromycin; puromycin hydrochloride; pyrazofurin; Rheumatrex; riboprine; safmgol; safingol hydrochloride; semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermanium hloride; spiromustine; spiroplatin; onigrin; streptozocin; nur; Tabloid; mycin; tecogalan sodium; taxotere; tegafur; teloxantrone hydrochloride; temoporf1n; teniposide; teroxirone; testolactone; omide; thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; Toposar; toremifene e; trestolone acetate; Trexall; triciribine phosphate; trimetrexate; trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracil mustard; uredepa; vapreotide; verteporfln; Vinblastine sulfate; Vincristine sulfate; Vindesine; Vindesine sulfate; Vinepidine sulfate; Vinglycinate sulfate; rosine e; Vinorelbine tartrate; Vinrosidine sulfate; idine e; vorozole; atin; zinostatin; and zorubicin hydrochloride. [003 72] Other anti-cancer drugs include; but are not limited to: 20-epi-l;25 oxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti-dorsalizing morphogenetic protein-1; antiandrogen; prostatic carcinoma; antiestrogen; antineoplaston; nse oligonucleotides; aphidicolin 2015/068069 glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine; tane; stine; axinastatin l; tatin 2; axinastatin 3; azasetron; azatoxin; osine; in III derivatives; balanol; batimastat; BCR/ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactam derivatives; beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor; tamide; bisantrene; bisaziridinylspermine; bisnafide; bistratene A; sin; breflate; bropirimine; budotitane; buthionine sulfoximine; calcipotriol; calphostin C; camptosar (also called ; irinotecan) camptothecin derivatives; capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinase inhibitors ; castanospermine; cecropin B; elix; chlorlns; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine; clomifene analogues; mazole; collismycin A; collismycin B; combretastatin A4; combretastatin analogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8; cryptophycin A tives; curacin A; cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor; cytostatin; dacliXimab; decitabine; dehydrodidenmin B; deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone; didemnin B; didox; diethylnorspermine; dihydro-S—azacytidine; dihydrotaxol; 9-; dioxamycin; diphenyl spiromustine; docetaxel; docosanol; dolasetron; doxifluridine; doxorubicin; droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab; eflornithine; elemene; emitefur; epirubicin; epristeride; estramustine analogue; estrogen agonists; estrogen antagonists; etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine; inide; fllgrastim; finasteride; flavopiridol; flezelastine; fluasterone; fludarabine (e.g; Fludara); fluorodaunorunicin hloride; forfenimex; forrnestane; fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid; idarubicin; ene; idramantone; ilmofosine; ilomastat; imatinib (e.g.; GLEEVEC®); imiquimod; immunostimulant peptides; insulin-like growth factor-1 or inhibitor; interferon agonists; interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol; 4-; iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia inhibiting factor; leukocyte alpha interferon; lide + estrogen + progesterone; leuprorelin; levamisole; liarozole; linear polyamine analogue; lipophilic disaccharide peptide; lipophilic platinum compounds; lissoclinamide 7; lobaplatin; cine; lometrexol; lonidamine; ntrone; bine; lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides; maitansine; tatin A; marimastat; masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone; meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone; miltefosine; mirimostim; mitoguazone; mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growth factor-saporin; ntrone; mofarotene; molgramostim; anti-EGFR antibody (e.g; Erbitux (cetuximab)); anti-CD19 antibody; anti-CD20 antibody (e.g.; rituximab); anti-disialoganglioside (GD2) dy (e.g.; monoclonal antibody 3F8 or chl4>l8); anti-ErbBZ antibody (e.g.; herceptin); human chon'onic gonadotrophin; monophosphoryl lipid A+myobacterium cell wall sk; mopidamol; mustard ncer agent; mycaperoxide B; mycobacterial cell wall extract; orone; N— acetyldinaline; N—substituted ides; nafarelin; nagrestip; naloxone+pentazocine; napavin; naphterpin; nartograstim; atin; nemorubicin; onic acid; nilutamide; cin; nitric oxide modulators; nitroxide antioxidant; nitrullyn; oblimersen (GENASENSE®); O6- benzylguanine; octreotide; okicenone; oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral ne inducer; ormaplatin; osaterone; oxaliplatin (e.g.; Floxatin); oxaunomycin; paclitaxel; paclitaxel analogues; paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin; pentrozole; perflubron; perfosfamide; perillyl alcohol; inomycin; phenylacetate; phosphatase tors; picibanil; pilocarpine hydrochloride; pirarubicin; exim; placetin A; in B; plasminogen activator inhibitor; platinum complex; platinum compounds; platinum-triamine complex; porfimer sodium; porfiromycin; prednisone; propyl bis—acridone; prostaglandin J2; proteasome inhibitors; protein A—based immune modulator; n kinase C inhibitor; protein kinase C inhibitors; microalgal; protein tyrosine phosphatase inhibitors; puiine nucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists; raltitrexed; ramosetron; ras famesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re 186 nate; rhizoxin; ribozymes; RII retinamide; kine; romurtide; imex; rubiginone Bl; ruboxyl; safingol; saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescence derived inhibitor 1; sense oligonucleotides; signal transduction inhibitors; sizofiran; sobuzoxane; sodium borocaptate; sodium phenylacetate; solverol; somatomedin binding protein; sonermin; sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin l; squalamine; stipiamide; stromelysin inhibitors; sulfinosine; superactive tive intestinal peptide nist; sta; suramin; swainsonine; tallimustine; tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium; rase inhibitors; temoporfin; side; tetrachlorodecaoxide; tetrazomine; thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocene bichloride; topsentin; toremifene; ation inhibitors; tretinoin; triacetyluridine; triciribine; trimetrexate; triptorelin; etron; turosteride; tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenital sinus-derived growth inhibitory factor; urokinase receptor antagonists; vapreotide; variolin B; Vectibix (panitumumab)velaresol; veramine; verdins; verteporfin; Vinorelbine; Vinxaltine; VitaXin; vorozole; Welcovorin (leucovorin); Xeloda (capecitabine); zanoterone; zeniplatin; zilascorb; and atin stimalamer.

In a specific embodiment, the anticancer agent that is administered as the second agent is thalidomide, lenalidomide, pomalidomide, CC-122; idine; decitabine or CC-486 (oral azacididine). In a more specific embodiment; the anticancer agent that is administered as the second agent is lenalidomide or pomalidomide. In a specific embodiment; the anticancer agent that is administered as the second agent is an odulatory compound (e.g.; an immunmodulatory compound as described in section 5.2.7.1). In a specific embodiment; the anticancer agent that is administered as the second agent is psin. .4.2. Treatments Using Genetically Modified NK Cells In another aspect, provided herein are methods of treating a hematological disorder or a solid tumor in a t in need thereof; comprising administering to said t an isolated population ofNK cells or a ceutical composition thereof; wherein the NK cells are genetically d (e.g.; comprising a chimeric antigen receptor (CAR) and/or a homing receptor; wherein said CAR comprises an extracellular domain; a transmembrane domain; an intracellular stimulatory ; and ally a co-stimulatory domain).

The cally modified NK cells (e.g.; NK cells comprising a CAR and/or a homing receptor) are described in Section 5.3. .4.3. Hematological Disorders and Solid Tumors [003 76] In specific embodiments, the hematological disorder is a hematological hyperproliferative er. In specific embodiments, the hematological disorder is a hematological cancer, e.g., a ia or a lymphoma. In more specific embodiments, the hematological cancer is an acute leukemia, e.g., acute T cell leukemia, acute myelogenous leukemia (AML), acute promyelocytic leukemia, acute myeloblastic leukemia, acute megakaryoblastic leukemia, precursor B acute blastic leukemia, precursor T acute lymphoblastic leukemia, Burkitt’s leukemia (Burkitt’s lymphoma), or acute biphenotypic leukemia, a chronic leukemia, e.g., chronic myeloid lymphoma, chronic myelogenous leukemia (CML), chronic monocytic leukemia, chronic lymphocytic leukemia (CLL)/Small lymphocytic ma, or B-cell phocytic leukemia; hairy cell lymphoma; T-cell prolymphocytic leukemia, or a ma, e.g., histiocytic lymphoma, plasmacytic lymphoma (e.g., Waldenstrom macroglobulinemia), splenic marginal zone ma, plasma cell neoplasm (e.g., plasma cell myeloma, plasmacytoma, a monoclonal globulin deposition disease, or a heavy chain disease), extranodal marginal zone B cell lymphoma (MALT lymphoma), nodal marginal zone B cell lymphoma (NMZL), follicular lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, y effusion lymphoma, T cell large granular lymphocytic leukemia, aggressive NK cell ia, adult T cell leukemia/lymphoma, extranodal NK/T cell lymphoma, nasal type, enteropathy-type T cell lymphoma, hepatosplenic T cell lymphoma, blastic NK cell lymphoma, s fungoides (Sezary syndrome), a primary ous CD3 O-positive T cell lymphoproliferative disorder (e.g., primary cutaneous anaplastic large cell lymphoma or lymphomatoid papulosis), angioimmunoblastic T cell lymphoma, peripheral T cell lymphoma, unspecified, stic large cell lymphoma, a Hodgkin’s lymphoma or a nodular lymphocytepredominant Hodgkin’s lymphoma. In another specific embodiment, the hematological cancer is acute myelogenous leukemia (AML). In another specific embodiment, the logical cancer is chronic lymphocytic leukemia (CLL). In another c embodiment, the logical cancer is multiple myeloma or myelodysplastic syndrome.

] The solid tumor can be, but is not limited to, e.g, a carcinoma, such as an adenocarcinoma, an adrenocortical carcinoma, a colon adenocarcinoma, a colorectal adenocarcinoma, a colorectal carcinoma, a ductal cell carcinoma, a lung carcinoma, a thyroid 2015/068069 carcinoma, a nasopharyngeal carcinoma, a melanoma (e.g., a malignant melanoma), a non- melanoma skin carcinoma, or an unspecified carcinoma; a d tumor; a desmoplastic small round cell tumor, an endocrine tumor, an Ewing sarcoma, a germ cell tumor (e.g., testicular cancer, ovarian cancer, carcinoma, endoderrnal sinus tumor, germinoma, etc); a hepatosblastoma, a hepatocellular oma; a neuroblastoma, a non-rhabdomyosarcoma soft tissue sarcoma, an arcoma, a retinoblastoma; a rhabdomyosarcoma; or a Wilms tumor. In another embodiment, the solid tumor is pancreatic cancer or breast cancer, In other embodiments, the solid tumor is an acoustic neuroma; an astrocytoma (e.g., a grade I pilocytic astrocytoma, a grade II low-grade astrocytoma; a grade III anaplastic astrocytoma; or a grade IV glioblastoma multiforme), a ma, a craniopharyngioma, a glioma (e.g., a brain stem glioma; an ependymoma; a mixed glioma; an optic nerve glioma; or a subependymoma); a glioblastoma, a medulloblastoma, a meningioma, a metastatic brain tumor, an oligodendroglioma; a pineoblastoma; a pituitary tumor; a primitive neuroectoderrnal tumor; or a schwannoma. In another embodiment, the solid tumor is prostate .

In certain embodiments, the individual having a hematological cancer or a solid tumor, e.g., an individual having a deficiency of natural killer cells, is an individual that has received a bone marrow transplant before said stering. In certain embodiments, the bone marrow lant was in treatment of said hematological cancer or said solid tumor. In certain other embodiments, the bone marrow transplant was in treatment of a condition other than said hematological cancer or said solid tumor. In certain ments, the individual received an immunosuppressant in addition to said bone marrow transplant. In certain embodiments, the individual who has had a bone marrow transplant exhibits one or more symptoms of graft- versus—host disease (GVHD) at the time of said administration. In certain other embodiments, the individual who has had a bone marrow transplant is administered said cells before a symptom of graft-versus-host disease (GVHD) has manifested.

In certain specific ments, the individual having a hematological cancer or solid tumor has ed at least one dose of a TNFOL inhibitor, e.g, ETANERCEPT® (Enbrel), prior to said administering. In specific embodiments, said individual received said dose of a TNFOL tor within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months of diagnosis of said hematological cancer or said solid tumor. In a specific embodiment, the individual who has received a dose of a TNFOL inhibitor exhibits acute myeloid leukemia. In a more specific embodiment, the individual who has received a dose of a TNFOL inhibitor and exhibits acute myeloid leukemia further exhibits deletion of the long arm of chromosome 5 in blood cells. In r embodiment, the individual having a hematological cancer or solid tumor, for example, a blood cancer, exhibits a Philadelphia chromosome.

In certain other embodiments, a hematological cancer or a solid tumor, in said individual is refractory to one or more anticancer drugs. In a specific embodiment, the hematological cancer or solid tumor is refractory to GLEEVEC® (imatinib mesylate).

In certain embodiments, a hematological cancer or a solid tumor, in said individual responds to at least one anticancer drug; in this ment, placental perfusate, isolated placental perfusate cells, isolated natural killer cells, e.g., placental natural killer cells, e.g., placenta-derived intermediate l killer cells, isolated combined natural killer cells, or activated NK, or TSPNK cells described herein, and/or combinations thereof, and optionally an immunomodulatory compound (e.g., an immunmodulatory compound as described in section .2.7.1), are added as adjunct treatments or as a combination therapy with said anticancer drug.

In certain other embodiments, the individual having a hematological cancer or a solid tumor, has been treated with at least one anticancer drug, and has relapsed, prior to said administering. In certain ments, the individual to be treated has a tory cancer. In one embodiment, the cancer treatment method with the cells described herein protects against (e.g., ts or delays) relapse of cancer. In one embodiment, the cancer treatment method described herein results in remission of the cancer for 1 month or more, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or more, 1 year or more, 2 years or more, 3 years or more, or 4 years or more.

In certain embodiments, NK cells are isolated from a tumor lesion, e.g., are tumor- infiltrating cytes; such NK cells are expected to be specific for a tumor-associated antigen (TAA) or a tumor microenvironment—associated antigen (TMAA).

In one embodiment, provided herein is a method of treating an individual having multiple a, comprising administering to the individual (1) lenalidomide or pomalidomide and (2) CAR NK cells, wherein said CAR NK cells are effective to treat multiple myeloma in said individual. In a specific embodiment, said CAR NK cells are cord blood NK cells, or NK cells produced from cord blood hematopoietic cells, e.g., hematopoietic stem cells. In r embodiment, said CAR NK cells have been ed by a two or stage method bed herein for producing NK cells. In r embodiment, said lenalidomide or pomalidomide, and CAR NK cells are administered separately from each other. In certain specific embodiments of the method of treating an individual with multiple myeloma, said CAR NK cells comprise a CAR extracellular domain, which ellular domain is a CS—l binding domain. In c embodiments, the CS—l binding domain comprises an scFv or antigen-binding fragment of an antibody that binds CS-l. In certain specific embodiments, the CS-l g domain comprises a single-chain n of elotuzumab and/or an antigen-binding nt of elotuzumab.

In one embodiment, provided herein is a method of treating an dual having multiple myeloma, comprising administering to the individual (1) lenalidomide or pomalidomide, (2) umab; and (3) CAR NK cells, wherein said CAR NK cells are effective to treat multiple myeloma in said individual. In a c embodiment, said CAR NK cells are cord blood NK cells, or NK cells produced from cord blood hematopoietic cells, e.g, hematopoietic stem cells. In another embodiment, said CAR NK cells have been produced by a two or three-stage method described herein for producing NK cells. In another ment, said lenalidomide or pomalidomide, elotuzumab, and/or CAR NK cells are administered separately from each other. In certain specific embodiments of the method of treating an individual with multiple myeloma, said CAR NK cells comprise a CAR extracellular domain, which extracellular domain is a CS-l binding domain. In specific embodiments, the CS-l binding domain comprises an scFv or antigen-binding fragment of an dy that binds CS-l.

In another embodiment, provided herein is a method of treating an individual having a blood cancer (e.g., Burkitt’s lymphoma), comprising administering to the individual (1) romidepsin and (2) CAR NK cells, wherein said CAR NK cells are effective to treat the blood cancer (e.g., Burkitt’s lymphoma) in said individual. In certain specific embodiments of the method of treating an individual with blood cancer (e.g., Burkitt’s ma), said CAR NK cells se a CAR extracellular domain, which extracellular domain is a CD20 binding domain. In c embodiments, the CD20 binding domain comprises an scFv or antigen- binding fragment of an dy that binds CD20. .5. Methods of Treating Infectious Diseases Provided herein are methods of treating an infectious disease using NK cells or genetically modified NK cells (e.g., NK cells comprising a CAR and/or a homing receptor) as described above. .5.1. Treatment of Infectious Diseases Using NK Combination Therapies ] In another aspect, provided herein are methods of treating an infectious disease in a subject in need thereof, comprising: (a) administering to said subject an isolated population of natural killer (NK) cells or a pharmaceutical ition thereof, or an isolated population of genetically d NK cells (e.g., NK cells comprising a CAR and/or a homing receptor) or a ceutical composition thereof; and (b) administering to said subject a second agent or a pharmaceutical composition thereof. The second agent can be any pharmaceutically acceptable agent that can be used to treat the infectious disease, and includes, but is not limited to, an antibody (e.g, a monoclonal antibody), a bispecific killer cell engager (BiKE), or an antiviral agent. 1. Antibodies that Binds to an Immune oint Protein In certain ments, the second agent is an antibody or antigen—binding fragment thereof (see Section 5.4.1.1 for description of antibodies). In specific embodiments, the antibody specifically binds to and antagonizes activity of an immune oint protein, immune checkpoint-related protein, or costimulatory signaling protein as described in Section 5.4.1.1. .5.1.2. Bispecific Killer Cell Engager In certain embodiments, the second agent is a BiKE, as described in Section 5.4.1.2. .5.1.3. Antiviral Agent [003 90] In certain embodiments, the second agent is an antiviral agent, which includes, but is not limited to: mod, podofilox, podophyllin, interferon alpha (IFNOL), reticolos, nonoxynol-9, acyclovir, famciclovir, valaciclovir, ganciclovir, cidofovir, amantadine, rimantadine, ribavirin; zanamavir and oseltaumavir; protease inhibitors such as indinavir, nelfinavir, ritonavir, or saquinavir, side reverse transcriptase inhibitors such as didanosine, lamivudine, stavudine, zalcitabine, or zidovudine, or non-nucleoside reverse transcriptase inhibitors such as nevirapine, or efavirenz. .5.2. Treatment of ious Diseases Using Genetically Modified NK Cells In another , provided herein are s of treating an infectious disease in a subject in need thereof, comprising administering to said t an isolated population ofNK cells or a pharmaceutical ition thereof, wherein the NK cells are genetically modified (e.g., comprising a chimeric antigen receptor (CAR) and/or a homing receptor comprise a chimeric antigen receptor (CAR), wherein said CAR comprises an extracellular domain, a transmembrane domain, an intracellular stimulatory domain, and optionally a co-stimulatory Genetically modified NK cells (e.g., NK cells comprising a CAR and/or a homing receptor) are described in Section 5.3. .5.3. Infectious Disease In certain ments, the infectious disease is an infection caused by a Virus, a bacterium, a fungus, or a helminth. In specific embodiments, the infectious disease is a Viral infection.

] In specific embodiments, the Viral ion is an infection by a Virus of the Adenoviridae, Picornaviridae, Herpesviridae, Hepadnaviridae, Flaviviridae, Retroviridae, Orthomyxoviridae, Paramyxoviridae, Papilommaviridae, Rhabdoviridae, or TogaViridae .

In more specific embodiments, said Virus is human immunodeficiency Virus (HIV).coxsackieVirus, hepatitis A Virus (HAV), poliovirus, Epstein-Barr Virus (EBV), herpes simplex type 1 (HSVl), herpes x type 2 (HSV2), human cytomegalovirus (CMV), human virus type 8 (HHV8), herpes zoster Virus (varicella zoster Virus (VZV) or shingles Virus), hepatitis B Virus (HBV), hepatitis C Virus (HCV), hepatitis D Virus (HDV), tis E Virus (HBV), influenza Virus (e.g., influenza A Virus, influenza B virus, influenza C Virus, or thogotovirus), measles Virus, mumps Virus, parainfluenza Virus, papillomavirus, rabies Virus, or rubella virus.

] In other more specific embodiments, said Virus is adenovirus species A, serotype 12, 18, or 31; adenoVirus species B, pe 3, 7, 11, 14, 16, 34, 35, or 50; irus species C, serotype 1, 2, 5, or 6; species D, serotype 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 33, 36, 37, 38, 39, 42, 43, 44, 45, 46, 47, 48, 49, or 51, s B, serotype 4, or species F, serotype 40 or 41. [003 96] In certain other more specific embodiments, the Virus is Apoi Virus (APOIV), Aroa Virus (AROAV), bagaza Virus (BAGV), Banzi Virus (BANV), Bouboui Virus (BOUV), Cacipacore Virus (CPCV), Carey Island Virus (CIV), Cowbone Ridge Virus (CRV), Dengue Virus (DENV), Edge Hill Virus (EHV), Gadgets Gully Virus (GGYV), Ilheus Virus (ILHV), Israel turkey meningoencephalomyelitis Virus (ITV), Japanese encephalitis Virus (JEV), Jugra Virus (IUGV), Jutiapa virus (JUTV), kadam virus (KADV), Kedougou virus (KEDV), Kokobera virus (KOKV), Koutango virus (KOUV), Kyasanur Forest disease virus (KFDV), Langat virus (LGTV), Meaban virus (MEAV), Modoc virus (MODV), Montana myotis leukoencephalitis virus (MMLV), Murray Valley encephalitis virus (MVEV), Ntaya virus (NTAV), Omsk hemorrhagic fever virus (OHFV), Powassan virus (POWV), Rio Bravo virus (RBV), Royal Farm virus (RFV), Saboya virus (SABV), St. Louis encephalitis virus (SLEV), Sal Vieja virus (SVV), San Perlita virus (SPV), Saumarez Reef virus (SREV), Sepik virus (SEPV), Tembusu virus (TMUV), tick-borne encephalitis virus (TBEV), Tyuleniy virus (TYUV), Uganda S virus (UGSV), Usutu virus (USUV), Wesselsbron virus ), West Nile virus (WNV), Yaounde virus (YAOV), Yellow fever virus (YFV), Yokose virus (YOKV), or Zika virus (ZIKV). [003 97] In other embodiments, the NK cells are stered to the subject having a viral infection as part of an ral therapy regimen that includes one or more other antiviral agents.

Specific antiviral agents that may be administered to an individual having a viral infection include, but are not limited to: imiquimod, podofilox, yllin, interferon alpha (IFNoc), los, nonoxynol-9, acyclovir, famciclovir, valaciclovir, ganciclovir, cidofovir; dine, adine; ribavirin; zanamavir and oseltaumavir; protease inhibitors such as indinavir, nelfinavir, ritonavir, or saquinavir, nucleoside reverse transcriptase inhibitors such as didanosine, lamivudine, stavudine, zalcitabine, or zidovudine; and non-nucleoside reverse transcriptase inhibitors such as pine, or efavirenz. .6. Administration [003 98] The NK cells, the genetically modified NK cells, or the second agent as described herein, may be administered to an individual, e.g., an individual having tumor cells or infected cells, by any lly-acceptable route known in the art suitable to the administration of live cells or the second agent. In various embodiments, the cells may be ally ted, injected, infused, e.g., by way of a catheter or syringe, or otherwise administered directly or indirectly to the site in need thereof. In s embodiments, the second agent may be injected, infused, e.g., by way of a catheter or syringe, or otherwise administered directly or indirectly to the site in need f. In one embodiment, the cells or the second agent are administered to an individual intravenously. In another embodiment, the cells or the second agent are administered to the individual at the site of a tumor, e.g., a solid tumor, or an infection. In a specific embodiment in which the individual has a tumor or an infection at more than one site, the cells or the second agent are administered to at least two, or all, tumor/infection sites. In certain other embodiments, the cells or the second agent, or compositions thereof, are administered orally, nasally, intraarterially, parenterally, ophthalmically, uscularly, subcutaneously, intraperitoneally, intracerebrally, intraventricularly, intracerebroventricularly, intrathecally, intracisternally, intraspinally and/or perispinally. In specific embodiments, the cells or the second agent, or compositions thereof, are stered by ion, infusion, intravenous (IV) administration, intrafemoral administration, or intratumor administration. In n specific embodiments, the cells or the second agent are delivered via intracranial or intravertebral needles and/or catheters with or without pump devices.

In specific embodiments, the step of administering to said subject an ed population ofNK cells or a ceutical composition thereof is by ion. In specific embodiments, the injection ofNK cells is local injection. In more specific embodiments, the local ion is directly into a solid tumor (e.g., a sarcoma). In specific embodiments, stration ofNK cells is by injection by syringe. In specific embodiments, administration of NK cells by injection is aided by laparoscopy, endoscopy, ultrasound, computed tomography, magnetic resonance, or ogy.

The NK cells, the genetically modified NK cells, or the second agent, can be administered to an individual in a composition, e.g, a matrix, hydrogel, scaffold, or the like.

In one embodiment, the cells are seeded onto a natural matrix, e.g., a placental biomaterial such as an amniotic membrane material. Such an amniotic membrane material can be, e.g, amniotic membrane dissected ly from a mammalian ta; fixed or heat-treated amniotic membrane, substantially dry (i.e., <20% H20) amniotic membrane, chorionic membrane, substantially dry chorionic membrane, substantially dry amniotic and chorionic membrane, and the like. red tal biomaterials on which placental stem cells can be seeded are described in Hariri, US. Application Publication No. 2004/0048796, the disclosure of which is hereby incorporated by reference in its entirety.

In another embodiment, the cells are suspended in a hydrogel solution suitable for, e. g., injection. Suitable hydrogels for such compositions include self-assembling peptides, such as RAD16. In one embodiment, a hydrogel solution comprising the cells can be allowed to harden, for instance in a mold, to form a matrix having cells dispersed n for implantation.

The cells in such a matrix can also be cultured so that the cells are mitotically expanded prior to 2015/068069 implantation. The hydrogel can be, for example, an organic polymer (natural or tic) that is cross-linked via nt, ionic, or hydrogen bonds to create a three—dimensional open-lattice structure that entraps water molecules to form a gel. el-forming materials include polysaccharides such as alginate and salts thereof, peptides, polyphosphazines, and polyacrylates, which are crosslinked ionically, or block polymers such as polyethylene oxide- polypropylene glycol block copolymers which are inked by temperature or pH, respectively. In some ments, the el or matrix is biodegradable.

In some embodiments, the formulation used in the present invention comprises an in silu polymelizable gel (see, e.g, US. Patent Application Publication 2002/0022676; Anseth el al., J. Control Release, ): 199-209 (2002), Wang el al., Biomaterials, 24(22):3969-80 (2003).

In some embodiments, the polymers are at least partially soluble in aqueous solutions, such as water, buffered salt solutions, or aqueous alcohol ons, that have charged side groups, or a monovalent ionic salt thereof. Examples of polymers having acidic side groups that can be reacted with cations are poly(phosphazenes), poly(acrylic acids), poly(methacrylic acids), mers of acrylic acid and methacrylic acid, poly(vinyl acetate), and sulfonated polymers, such as sulfonated yrene. Copolymers having acidic side groups formed by reaction of acrylic or methacrylic acid and Vinyl ether monomers or polymers can also be used. Examples of acidic groups are carboxylic acid groups, sulfonic acid groups, halogenated (preferably fluorinated) alcohol groups, phenolic OH groups, and acidic OH groups.

The cells can be seeded onto a three-dimensional ork or scaffold and implanted in viva. Such a ork can be implanted in ation with any one or more growth factors, cells, drugs or other components that stimulate tissue formation or otherwise enhance or improve the practice of the methods described herein.

Examples of scaffolds that can be used in the present invention include nonwoven mats, porous foams, or self assembling es. Nonwoven mats can be formed using fibers comprised of a synthetic absorbable copolymer of glycolic and lactic acids (e.g., PGA/PLA) (VICRYL, Ethicon, Inc., Somerville, N.J.). Foams, composed of, e.g., poly(e- caprolactone)/poly(glycolic acid) (PCL/PGA) copolymer, formed by ses such as freeze- drying, or lyophilization (see, e.g., US. Pat. No. 6,355,699), can also be used as scaffolds.

The cells can also be seeded onto, or contacted with, a physiologically-acceptable ceramic material including, but not limited to, mono-, di-, tri-, tri-, beta-tri-, and tetra- calcium phosphate, hydroxyapatite, fluoroapatites, calcium sulfates, calcium fluorides, calcium oxides, calcium carbonates, magnesium calcium phosphates, biologically active s such as BIOGLASS®, and mixtures thereof. Porous biocompatible ceramic materials currently commercially available include SURGIBONE® dica Corp, Canada), ENDOBON® (Merck erial France, France), CEROS® (Mathys, AG, Bettlach, Switzerland), and mineralized collagen bone grafting products such as HEALOSTM (DePuy, Inc, Raynham, MA) and VITOSS®, RHAKOSSTM, and CORTOSS® (Orthovita, n, Pa.). The framework can be a mixture, blend or composite of natural and/or synthetic materials.

In r embodiment, cells can be seeded onto, or contacted with, a felt, which can be, e.g, composed of a lament yarn made from a bioabsorbable material such as PGA, PLA, PCL copolymers or blends, or hyaluronic acid.

The cells can, in another embodiment, be seeded onto foam lds that may be composite structures. Such foam scaffolds can be molded into a useful shape, such as that of a portion of a specific structure in the body to be ed, replaced or augmented. In some embodiments, the framework is d, e.g., with 0.1M acetic acid followed by incubation in polylysine, PBS, and/or collagen, prior to inoculation of the cells described herein in order to enhance cell attachment al surfaces of a matrix may be modified to improve the attachment or growth of cells and differentiation of tissue, such as by plasma-coating the matrix, or addition of one or more proteins (e.g., ens, elastic , reticular fibers), glycoproteins, glycosaminoglycans (e.g., heparin sulfate, chondroitinsulfate, oitinsulfate, dermatan sulfate, keratin sulfate, etc), a cellular , and/or other materials such as, but not limited to, gelatin, alginates, agar, agarose, and plant gums, and the like.

] In some embodiments, the scaffold comprises, or is treated with, materials that render it non-thrombogenic. These treatments and materials may also promote and sustain endothelial growth, migration, and extracellular matrix deposition. Examples of these materials and treatments include but are not limited to natural materials such as basement membrane proteins such as n and Type IV collagen, synthetic materials such as EPTFE, and segmented polyurethaneurea silicones, such as PURSPANTM (The Polymer Technology Group, Inc., Berkeley, Calif). The scaffold can also comprise anti-thrombotic agents such as heparin, the scaffolds can also be treated to alter the surface charge (e.g., coating with plasma) prior to seeding with placental stem cells.

In specific embodiments, the NK cells, the genetically modified NK cells, or the second agent is administered with a pharmaceutical carrier. The pharmaceutical carrier can be any known in the art. In specific embodiments, the NK cells or the genetically d NK cells are fucosylated on the cell surface.

Determination of the number ofNK cells or genetically modified NK cells (e.g., NK cells sing a CAR and/or a homing receptor), or the amount of the second agent can be performed ndently. Such determination can be based on the condition of the subject and can be made by the physician.

In certain embodiments, the NK cells, the genetically modified NK cells, or the second agent, is used, e.g., stered to an individual, in any amount or number that results in a detectable therapeutic benefit to the individual, e.g, an effective , wherein the individual has a viral ion, cancer, or tumor cells, for example, an individual having tumor cells, a solid tumor or a blood cancer, e.g, a cancer patient. Cells can be administered to such an individual by absolute numbers of cells, e.g., said individual can be administered at about, at least about, or at most about, 1x 105, 5 x106,5 x106,1x107,5 x107,1x108,5 x108,1 X 109, 5 X 109, 1 X 1010, 5 X 1010, or 1 X 1011 cells. In other embodiments, cells can be administered to such an dual by ve numbers of cells, e.g., said individual can be administered at about, at least about, or at most about, 1 X 105, 5 X 105, 1 X 106, 5 X 106, 1 X 107, x107,1x108,5 x108,1x109,5 x109,1x101°,5 x 1010, or 1 x 1011 cells. In other embodiments, cells can be administered to such an individual by relative numbers of cells, e.g, said individual can be administered at about, at least about, or at most about, 1 X 105, 5 X 105, 1 X 106, 5 X 106, 1 X 107, 5 X 107, 1 X 108, or 5 X 108 cells. Cells can be administered to such an individual according to an imate ratio between a number ofNK cells or genetically modified NK cells and optionally placental perfusate cells, and a number of tumor/infected cells in said individual (e.g., an estimated number). For eXample, NK cells or the genetically modified NK cells can be administered to said individual in a ratio of about, at least about or at most about 1:1, 1:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1 or 100:1 to the number oftumor/infected cells in the individual. The number of tumor/infected cells in such an individual can be estimated, e.g., by counting the number of tumor/infected cells in a sample of tissue from the individual, e.g., blood sample, biopsy, or the like. In specific embodiments, e.g., for solid , said counting is performed in combination with imaging of the tumor or tumors to obtain an imate tumor volume.

In a specific embodiment, NK cells (or genetically modified NK cells) are supplemented with placental perfusate cells or placental perfusate. In a specific embodiment, about 1 x 104, 5 x104,1x105,5 x105,1x106,5 x106,1x107,5 x107,1x108,5 x108 or more NK cells (or genetically modified NK cells) per milliliter, or 1 x 104, 5 x 104, 1 x 105, 5 x 105, 1 x ", 5 x106,1x107,5 x107,1x108,5 x103,1x109,5 x109,1x101°,5 x 1010, 1x 1011 or more NK cells (or genetically modified NK cells) per milliliter, are supplemented with about, or at least about, 1 x 104, 5 x104,1x105,5 x105,1x10"’,5 x106, 1x107,5 x107,1x108,5 x108 or more isolated placental perfusate cells per milliliter, or 1 X 104, 5 X 104, 1 X 105, 5 X 105, 1 X 106, 5 x106,1x107,5 x107,1x108,5 x108,1x109,5 x109,1x1010,5 x 1010, 1x 1011 or more isolated placental perfusate cells per milliliter. In other more specific embodiments, about 1x 104, 5 x104,1x105,5 x 105, 1x 106, 5 X106,1X107,5 X107,1X108,5 x108 or moreNK cells (or genetically modified NK cells) per milliliter, or 1 x 104, 5 x 104, 1 x 105, 5 x 105, 1 x 106, 5 X106,1X107,5 x108,5 x109,5 x109,1x1010,5 x 1010, 1 x 1011 or more NK cells (or genetically modified NK cells) per milliliter are supplemented with about, or at least about, 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, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 mL of perfusate, or about 1 unit of perfusate.

In another c embodiment, NK cells (or genetically modified NK cells) are supplemented with nt placental cells, e.g., adherent placental stem cells or multipotent cells, e. g., CD34‘, CD10+, CD105+, CD200Jr tissue e plastic-adherent tal cells. In specific embodiments, the NK cells are supplemented with about 1 X 104, 5 X 104, 1 X 105, 5 X 105, 1 x 10", 5 x 106, 1 x 107, 5 x 107, 1 x 103, 5 x 108 or more adherent placental stem cells per milliliter, or 1 x 104, 5 x104,1x105,5 x 105, 1x 10", 5 x106, 1x107,5 x107,1x108,5 x108,1 X 109, 5 X 109, 1 X 1010, 5 X 1010, 1 X 1011 or more adherent tal cells, e.g., adherent placental stem cells or multipotent cells.

In another specific embodiment, NK cells (or genetically modified NK cells) are supplemented with conditioned medium, e.g, medium conditioned by CD34‘, CD10+, CD105+, CD200+ tissue culture plastic-adherent placental cells, e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.1, 0.8, 2015/068069 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mL of stem cell-conditioned culture medium per unit of perfusate, or per 104, 105, 106, 107, 108, 109, 1010, or 1011NK cells (or genetically d NK cells). In certain embodiments, the tissue culture plastic-adherent placental cells are the multipotent nt tal cells described in US. Patent No. 7,468,276 and US. Patent ation Publication No. 2007/0275362, the disclosures of which are incorporated herein by reference in their ties. In another specific embodiment, the method additionally comprises bringing the tumor cells into proximity with, or administering to the individual, an immunomodulatory compound (e.g., an immunmodulatory compound as described in section 5.2.7.1) or thalidomide.

In another specific embodiment, NK cells (or genetically modified NK cells) are supplemented with placental perfusate cells, the perfusate cells are brought into proximity with interleukin-2 (IL-2) for a period of time prior to said bringing into proximity. In certain embodiments, said period oftime is about, at least, or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 or 48 hours prior to said bringing into proximity.

The NK cells, the genetically modified NK cells, or the second agent can be administered once (i.e., in single dose) to an individual having a viral infection, a hematological disorder, or a solid tumor during a course of therapy; or can be administered multiple times (1'. e., in multiple , e.g., once every 1, 2, 3, 4, 5, 6, 7, 8, 9,10,11,12,13,14,15,16,l7,18,l9, , 21, 22 or 23 hours, or once every 1, 2, 3, 4, 5, 6 or 7 days, or once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 24, 36 or more weeks during therapy. In embodiments n both NK cells (or genetically modified NK cells) and a second agent are used, the second agent and the NK cells (or genetically modified NK cells), can be administered to the individual together, e. g., in the same formulation, separately, e.g., in separate formulations, at approximately the same time; or can be administered separately, e.g., on different dosing schedules or at different times of the day. The second agent can be administered before, after, or at the same time as the NK cells (or genetically modified NK . NK cells (or genetically d NK cells) or a second agent can be administered without regard to whether the NK cells (or genetically modified NK cells) or the second agent have been administered to the individual in the past. .7. Patients ] The patient referred to in this disclosure, can be, but is not limited to, a human or non— human vertebrate such as a wild, domestic or farm animal. In certain ments, the patient is 2015/068069 a mammal, e.g., a human, a cow, a dog, a cat, a goat, a horse, a sheep, a pig, a rat, or a mouse. In one embodiment, the patient is a human patient. .8. Kits Provided herein is a pharmaceutical pack or kit comprising one or more containers filled with a composition sing NK cells or genetically modified NK cells (e.g., NK cells sing a CAR and/or a homing receptor) described above, and one or more containers filled with a composition comprising a second agent described above. Also provided herein is a pharmaceutical pack or kit comprising one or more containers filled with a composition comprising NK cells comprising a CAR and/or a homing receptor described above. ally associated with such container(s) can be a notice in the form ibed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

] The kits encompassed herein can be used in accordance with the methods of treating as provided herein, e.g., methods of treating a hematological cancer, a solid tumor, or a viral infection 6. EXAMPLE 6.1. Example 1: Antibody-Dependent Cellular Cytotoxicity (ADCC) Using Rituximab ] The Example presented herein demonstrates that co-administration ofNK cells (here, PiNK cells) and an dy c for a cell surface antigen (in this case, CD20), e. g., a tumor- associated antigen increases NK antibody-dependent cell-mediated cytotoxicity (ADCC) of the NK cells.

The experiments presented herein e an D20 antibody, Iituximab, and Daudi cells (Cat#: CCL-213, ATCC), which are high expressers of CD20, Daudi cells were harvested and labeled with PKH26 (Cat#: PKH26GL—1KT et al., , Sigma-Aldrich) (Ferlazzo, G., J Immunol, 172: 1455—1462 (2004); Lehmann, D., et al., Stem Cells Dev, 21: 2926-2938 (2012)), whose lipophilic aliphatic residue inserts into cell plasma membrane. The cells were washed and incubated with rituximab (and human IgG as an isotype control) at different concentrations as indicated in Fig. 1 for 1h at room temperature. After washing three times, 104 target cells were placed in 96-well U-bottom tissue culture plates and incubated with cultured NK cells at various effector-target (EzT) ratios (50: 1, 20: 1, 10:1 and 2.5: 1) in 200 ul RPMI 1640 supplemented with 10% PB S. The Cultures were incubated for 4h at 37°C in 5% C02. After tion, cells were harvested and TO-PRO-3 (Catalog # T3605, Invitrogen), a ne- impermeable DNA stain, was added to es to 0.25uM final tration followed by FACS analysis using BD FACSCanto II. Cytotoxicity (“% cytotoxicity” in Fig. 1) is expressed as percentage of dead cells (PKH26+TO-PRO-3+) within the total PKH26+ target tumor cells, subtracted by spontaneous cell death.

Incubating Daudi cells with rituximab increases the cytotoxicity of (PiNK) cells compared to human IgG controls, thereby indicating enhanced cytolytic activity of PiNK cells when accompanied by co-administration of the anti-CD20 antibody (Fig. 1). 6.2. Example 2: Cytotoxicity of Three-Stage NK cells Against Multiple Myeloma ype characterization ofMM cell lines and primary MM samples. Primary multiple myeloma (MM) cells (Tissue Solution, donor IDs: 85, l\/11\/I293) or W tumor cell lines: RPMI8226 (ATCC, Cat# 5) and OPM2 (DSMZ, Cat# ACC-50) cells (1 x 106 each) were used for this assay. Cells were stained with D-Ll APC gend, Cat# 329708), anti-CSl PE-Cy7 gend, Cat# 331816) and 7-AAD (BD Bioscience, Cat# 559925) according to the manufacturer’ s protocol. Data were acquired on BD LSRFortessa (BD Biosciences) and analyzed using FLOWJO® software (Tree Star). Data were sed as % positive cells gated under 7-AAD- single cells. Setting of the % positive gate was done using unstained sample as control.

Results. The expression of PD—Ll and CS—l on the MM cells lines is shown in Figure 2. The left-most peak in the panels of Figure 2 indicates the control, whereas the right- most peak indicates the sample. The tage of cells positive for PD-Ll was as follows: 71.6% W285, 70.7% MM293, 66.2% OPM-2, and 94.4% RPMI8226. The percentage of cells positive for CS-1 was as follows: 31.8% MM285, 58.8% MM293, 93.4% OPM-2, and 29.5% RPMI8226. 24-hour Cytotoxicity assay of three-stage NK cells against MM cell lines and primary MM samples. OPM2 cells were labeled with 10 uM PKH26 fluorescent dye (Sigma- Aldrich, Cat# PKH26—GL) prior to co-culture with three-stage NK cells from five different donors at an effector to target (EzT) ratio of 3:1 (3 x 105 and 1 x 105 three-stage NK and OPM2 cells, tively) in 1 mL of RPMIl640 supplemented with 10% FBS and antibiotics (Basal medium), or the experimetnal conditions: IL-lS (5 ng/mL) rogen, Cat# PHC9153), IL-2 (200 IU/mL) (Invitrogen, Cat# PHC0023), anti-PD-Ll (10ng/mL) (Affymetrix, Cat# 16 82); anti-IgG(10ng/mL) (Affymetrix, Cat# 4-82); REVLIMID® (lenalidomide; luM), or DMSO (0.1%) in 48-well plates. Target cells alone were plated as controls, After incubation for 24 hours at 37° C and 5% C02, cells were harvested, followed by staining with 1 uM TO-PRO-3 to identify the dead cells. The number of viable target cells +TO-PRO-3') in each sample was quantified by flow cytometry using ng beads following the protocol ed by the manufacturer (Invitrogen, Cat# C3 6950). Counting beads were introduced in this assay in order to account for any ial proliferation of tumor cells during the prolonged 24 hour culture.

Briefly, the number of viable target cells in each sample was calculated as follows: (% PKH26+TO-PRO-3' live targets) / (% counting beads) X (assigned bead count of the counting bead lot). Percent survival (% survival) in s t cells with co-cultures of three-stage NK cells) was calculated by dividing the absolute number of viable, , target cells remaining in co-cultures with three-stage NK cells after 24 hours with the absolute number of viable, , target cells remaining in culture of target cells alone. Percent cytotoxicity at 24 hours reported was calculated as: 100 - % survival. s were depicted as mean in standard deviation of the mean.

Results. Three-stage NK cells displayed cytotoxic activity against different MM cell lines. The three-stage NK cells exerted 20-60% specific lysis against four primary MM samples at an E:T ratio of 3:1 (Figure 3). Varying susceptibility of MIVI targets from different donors to NK killing was observed. In addition, initial assessment the cytotoxicity of three-stage NK cells against OPM2 indicated an enhancement of tic activity by addition of the cytokines, immunomodulatory compounds, and monoclonal antibodies utilized in these experiments (Figure EQUIVALENTS The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, s modifications of the invention in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

WO 09668 All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

Claims (66)

What is claimed is: 1.
1. l. A method of treating a cancer in a subject in need thereof, comprising: (a) stering to said subject an isolated tion of natural killer (NK) cells or a pharmaceutical composition thereof; and (b) administering to said subject a second agent or a pharmaceutical composition thereof, wherein said second agent can be used to treat said cancer.
2. 2. The method of claim 1, wherein the second agent is an antibody or antigen binding fragment thereof that specifically binds to a tumor-associated antigen (TAA).
3. 3. The method of claim 2, wherein the dy is a monoclonal antibody.
4. 4. The method of claim 2 or 3, n the TAA is selected from the group consisting of CD123, CLL-l, CD38, CS-l, CD138, RORl, FAP, MUC1, PSCA, EGFRVIII, EPHA2, and GD2.
5. 5. The method of claim 1, wherein the second agent is an antibody or antigen binding fragment thereof that specifically binds to a tumor microenVironment-associated antigen (TMAA).
6. 6. The method of claim 5, wherein the antibody is a monoclonal antibody.
7. 7. The method of claim 5 or 6, wherein the TMAA is selected from the group consisting of VEGF-A, EGF, PDGF, IGF, and bFGF.
8. 8. The method of claim 1, wherein the second agent is an antibody or antigen binding nt thereof that specifically binds to and antagonizes the activity of an immune checkpoint protein.
9. 9. The method of claim 8, n the antibody is a monoclonal antibody.
10. 10. The method of claim 8 or 9, wherein the immune checkpoint protein is selected from the group ting of CTLA-4, PD-l, PD-Ll, PD-L2, and LAG-3.
11. 11. The method of claim 1, wherein the second agent is a bispecific killer cell r (BiKE).
12. 12. The method of claim 11, wherein the BiKE comprises a first single chain variable fragment (scFV) that specifically binds to a TAA.
13. 13. The method of claim 12, wherein the TAA is selected from the group consisting of CD123, CLL-l, CD38, CS-l, CD138, RORl, FAP, MUC1, PSCA, EGFRVIII, EPHA2, and GD2. WO 09668
14. 14. The method of any one of claims 11-13, wherein the BiKE comprises a second scFv that specifically binds to CD16
15. 15. The method of claim 1, wherein the second agent is an anti-inflammatory agent.
16. l6. The method of claim 1, wherein the second agent is an immunomodulatory agent.
17. 17. The method of claim 1, wherein the second agent is a cytotoxic agent.
18. 18. The method of claim 1, n the second agent is a cancer vaccine.
19. 19. The method of claim 1, n the second agent is a chemotherapeutic.
20. 20. The method of claim 1, wherein the second agent is an HDAC inhibitor.
21. 21‘ The method of claim 1, wherein the second agent is an siRNA.
22. 22. The method of any one of claims l-2l, wherein the isolated population ofNK cells or a pharmaceutical composition thereof is administered before the second agent or a pharmaceutical ition thereof.
23. 23. The method of any one of claims l-21, wherein the isolated population ofNK cells or a pharmaceutical composition thereof is administered after the second agent or a pharmaceutical composition thereof.
24. 24. The method of any one of claims l-21, n the isolated population ofNK cells or a pharmaceutical composition thereof is administered at the same time as the second agent or a pharmaceutical composition thereof.
25. 25. The method of any one of claims l-24, wherein the step of administering to said subject an isolated population ofNK cells or a pharmaceutical composition f is by injection, infusion, intravenous (IV) administration, intrafemoral administration, or intratumor stration.
26. 26. The method of any one of claims l-25, wherein the step of administering to said subject an isolated population ofNK cells or a pharmaceutical composition thereof is performed with a devise, a matrix, or a scaffold.
27. 27. The method of any one of claims 1-26, wherein the step of administering to said subject a second agent or a pharmaceutical composition f is by injection, infusion, intravenous (IV) administration, intrafemoral stration, or intratumor administration
28. 28. The method of any one of claims 1-27, wherein the step of administering to said subject a second agent or a ceutical composition f is performed with a devise, a matrix, or a scaffold.
29. 29. The method of any one of claims 1—28, wherein the NK cells are fucosylated on the cell surface.
30. 30. The method of any one of claims 1-29, wherein the isolated population ofNK cells or a pharmaceutical composition thereof is stered in a single dose.
31. 31. The method of any one of claims 1-29, wherein the isolated population ofNK cells or a ceutical ition thereof is administered in multiple doses.
32. 32. The method of any one of claims 1-31, wherein the second agent or a pharmaceutical composition thereof is administered in a single dose.
33. 33. The method of any one of claims 1-31, wherein the second agent or a pharmaceutical composition thereof is administered in multiple doses.
34. 34. A method of treating a cancer in a t in need thereof, sing administering to said subject an ed population ofNK cells or a pharmaceutical composition thereof, wherein the NK cells comprise a chimeric antigen receptor (CAR), wherein said CAR comprises an extracellular domain, a transmembrane domain, an intracellular stimulatory domain, and optionally a co-stimulatory domain.
35. 35. The method of claim 34, wherein the CAR comprises an extracellular domain, a transmembrane domain, an intracellular stimulatory , and a co-stimulatory domain.
36. 36. The method of claim 34 or 35, wherein the NK cells sing the CAR are derived from CD34+ hematopoietic stem cells (HSCs) that are engineered to express the CAR.
37. 37. The method of any one of claims 34-3 6, wherein the extracellular domain is an antigen binding domain.
38. 38. The method of claim 37, wherein the antigen binding domain is an scFV domain.
39. 39. The method of claim 37 or 38, wherein the antigen binding domain specifically binds to a TAA,
40. 40. The method of claim 39, wherein the TAA is selected from the group consisting of CD123, CLL-l, CD38, and CS-l.
41. 41. The method of any one of claims 34-40, wherein the intracellular atory domain is a CD3 zeta signaling domain.
42. 42. The method of any one of claims 34-41, wherein the mulatory domain comprises the intracellular domain of CD28, 4—1BB, PD—l, 0X40, CTLA-4, NKp46, NKp44, NKp30, DAPlO or DAPlZ.
43. 43. A method of treating a cancer in a subject in need thereof, comprising administering to said subject an isolated population ofNK cells or a pharmaceutical composition thereof, wherein the NK cells comprise a homing receptor.
44. 44‘ The method of claim 43, wherein the NK cells comprising the homing receptor are derived from CD34+ hematopoietic stem cells (HSCs) that are engineered to express the homing receptor.
45. 45. The method of claim 43 or 44, wherein the homing receptor is a chemotactic receptor,
46. 46. The method of claim 45, wherein the chemotactic receptor is selected from the group consisting of CXCR4, VEGFRZ, and CCR7.
47. 47. A method of treating a cancer in a subject in need thereof, comprising administering to said t an isolated population of Natural Killer (NK) cells or a pharmaceutical ition thereof, wherein the NK cells comprise a chimeric antigen receptor (CAR) and a homing or, wherein said CAR comprises an extracellular domain, a embrane domain, an intracellular stimulatory , and optionally a co- stimulatory domain.
48. 48. The method of claim 47, wherein the CAR comprises an ellular domain, a transmembrane domain, an intracellular stimulatory domain, and a co-stimulatory domain.
49. 49. The method of claim 47 or 48, wherein the NK cells comprising the CAR and the homing receptor are derived from CD34+ hematopoietic stem cells (HSCs) that are engineered to express the CAR.
50. 50. The method of any one of claims 47—49, wherein the NK cells comprising the CAR and the homing receptor are derived from CD34+ hematopoietic stem cells (HSCs) that are engineered to s the homing receptor.
51. 51. The method of any one of claims 47-50, wherein the extracellular domain is an antigen binding domain.
52. 52. The method of claim 51, wherein the antigen binding domain is an scFV domain.
53. 53. The method of claim 51 or 52, wherein the n binding domain cally binds to a TAA.
54. 54. The method of claim 53, wherein the TAA is selected from the group consisting of CD123, CLL-l, CD38, and CS-l.
55. 55. The method of any one of claims 47-54, n the ellular stimulatory domain is a CD3 zeta signaling domain.
56. 56. The method of any one of claims 47—55, wherein the mulatory domain comprises the intracellular domain of CD28, 4-1BB, PD—l, 0X40, CTLA-4, NKp46, NKp44, NKp30, DAPIO or DAP12.
57. 57. The method of any one of claims 47—56, wherein the homing receptor is a chemotactic receptor,
58. 58. The method of claim 57, n the chemotactic receptor is selected from the group consisting of CXCR4, VEGFR2, and CCR7.
59. 59. The method of any one of claims 34—5 8, wherein the step of administering to said subject an isolated population ofNK cells or a pharmaceutical composition thereof is by injection, infusion, intravenous (IV) administration, intrafemoral administration, or intratumor administration.
60. 60. The method of any one of claims 34-59, wherein the step of administering to said t an isolated population ofNK cells or a pharmaceutical composition thereof is performed with a devise, a matrix, or a scaffold.
61. 61. The method of any one of claims 34-60, wherein the NK cells are fucosylated on the cell surface.
62. 62. The method of any one of claims 34-61, wherein the isolated population ofNK cells or a pharmaceutical composition thereof is administered in a single dose.
63. 63. The method of any one of claims 34-61, wherein the isolated population ofNK cells or a pharmaceutical ition thereof is administered in multiple doses.
64. 64. The method of any one of claims 1-63, wherein the cancer is a hematological cancer.
65. 65. The method of any one of claims 1-63, wherein the cancer is a solid tumor.
66. 66. A method of treating a Viral infection in a subject in need thereof, comprising: