TW202241471A - Methods of increasing nkp30-positive lymphocytes in a subject and uses thereof - Google Patents

Abstract

Provided herein are methods of increasing the number of NKp30-positive lymphocytes in a subject in need thereof, and uses of the same.

Description

Method for increasing NKp30 positive lymphocytes in an individual and use thereof

Cross References to Related Applications

This application claims priority to US Provisional Patent Application Serial No. 63/135,559, filed January 8, 2021. The disclosure of this prior application is considered a part of the disclosure of the present application and is incorporated in its entirety into the present application.

The present invention generally relates to methods of administering enucleated erythroid cells to an individual, methods of increasing NKp30 positive lymphocytes in an individual, and methods of treating B7-H6 positive cancer in an individual. sequence listing

This application contains a Sequence Listing which has been filed electronically in an ASCII text file named 47472.0079WO1_ST25.txt. The ASCII text file was created on January 6, 2022 and is 87 bytes in size. The material in the ASCII text archive is incorporated herein by reference in its entirety.

Enucleated erythroid cells engineered to carry or present exogenous proteins to patients in need are being developed as therapeutic agents.

The present invention is concerned with the use of B7 homolog 6 (B7-H6) as a biomarker for the identification of individuals treated with enucleated erythroid cells comprising an exogenous fusion protein Comprising (i) IL-15 or its functional fragment, and (ii) IL-15 receptor α or its functional fragment. The present invention is based, at least in part, on the discovery that administration of enucleated erythroid cells comprising a first exogenous polypeptide on their extracellular surface and a A second exogenous polypeptide on the extracellular surface, the first exogenous polypeptide comprising (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor α or a functional fragment thereof, the two exogenous The sex polypeptide comprises 4-1BBL or a functional fragment thereof. Therefore, determining the B7-H6 positivity of a cancer in an individual is particularly useful in identifying patients who are likely to respond to therapy with these cells. Provided herein are methods of increasing the number of NKp30-positive lymphocytes in an individual previously identified or diagnosed as having a B7-H6-positive cancer, methods of treating an individual previously identified or diagnosed as having a B7-H6-positive cancer, reducing the number of cells previously identified or diagnosed as having a B7-H6-positive cancer. Methods of Amount and/or Proliferation of B7-H6 Positive Cancer Cells in Individuals Diagnosed with B7-H6 Positive Cancer, Killing of B7-H6 Positive Cancer Cells in Individuals Previously Identified or Diagnosed with B7-H6 Positive Cancer Methods, and methods of reducing the volume of solid tumors in an individual previously identified or diagnosed with a B7-H6 positive cancer.

Provided herein is a method of increasing the number of NKp30-positive lymphocytes in an individual in need thereof previously identified or diagnosed with a B7-H6-positive cancer comprising administering to the individual a therapeutically effective amount of a pharmaceutical composition comprising A group of nucleated erythroid cells comprising on their extracellular surface a first exogenous fusion polypeptide comprising (i) IL-15 or a functional fragment thereof , and (ii) IL-15 receptor alpha or a functional fragment thereof.

In some embodiments of any of the methods described herein, the administering results in an increase in the number of NKp30-positive lymphocytes in the individual by at least 5% compared to the number of NKp30-positive lymphocytes in the individual prior to administration. In some embodiments of any of the methods described herein, the administering results in an increase in the number of NKp30-positive lymphocytes in the individual by at least 10% compared to the number of NKp30-positive lymphocytes in the individual prior to administration. In some embodiments of any of the methods described herein, the NKp30-positive lymphocytes are NKp30-positive NK cells.

In some embodiments of any of the methods described herein, the method further results in an increased number of NKp30-positive/NKG2A-negative lymphocytes in the individual. In some embodiments of any of the methods described herein, the administering step results in an increase in the number of NKp30-positive/NKG2A-negative lymphocytes in the individual by at least 5%. In some embodiments of any of the methods described herein, the administering step results in an increase in the number of NKp30-positive/NKG2A-negative lymphocytes in the individual by at least 10%. In some embodiments of any of the methods described herein, the NKp30-positive/NKG2A-negative lymphocytes are NKp30-positive/NKG2A-negative NK cells.

In some embodiments of any of the methods described herein, the administering step results in an increased number of NKp30-positive, CD45-positive, CD56-positive lymphocytes in the individual. In some embodiments of any of the methods described herein, the administering step results in at least a 1.2-fold increase in the percentage of NKp30-positive CD45-positive, CD56-positive lymphocytes in the individual. In some embodiments of any of the methods described herein, the administering step results in at least a 1.5-fold increase in the percentage of NKp30-positive CD45-positive, CD56-positive lymphocytes in the individual. In some embodiments of any of the methods described herein, the administering step results in an at least 2.0-fold increase in the percentage of NKp30-positive CD45-positive, CD56-positive lymphocytes in the individual. In some embodiments of any of the methods described herein, the NKp30-positive, CD45-positive, CD56-positive lymphocytes are NKp30-positive, CD45-positive, CD56-positive NK cells.

In some embodiments of any of the methods described herein, the administering step results in an increased number of NKp30-positive, CD45-positive, CD16-positive, CD56-dim lymphocytes in the individual. In some embodiments of any of the methods described herein, the administering step results in at least a 1.2-fold increase in the percentage of NKp30-positive CD56-dim, CD16-positive, CD45-positive lymphocytes in the individual. In some embodiments of any of the methods described herein, the administering step results in at least a 1.5-fold increase in the percentage of NKp30-positive CD56-dim, CD16-positive, CD45-positive lymphocytes in the individual. In some embodiments of any of the methods described herein, the administering step results in at least a 1.7-fold increase in the percentage of NKp30-positive CD56-dim, CD16-positive, CD45-positive lymphocytes in the individual. In some embodiments of any of the methods described herein, the NKp30 positive, CD45 positive, CD16 positive, CD56-dim lymphocytes are NKp30 positive, CD45 positive, CD16 positive, CD56-dim NK cells.

In some embodiments of any of the methods described herein, the administering step results in an increase in the number of NKp30-positive, CD45-positive, CD16-positive, and CD56-positive lymphocytes in the individual. In some embodiments of any of the methods described herein, the administering step results in at least a 1.5-fold increase in the percentage of NKp30-positive CD56-positive, CD16-positive, CD45-positive lymphocytes in the individual. In some embodiments of any of the methods described herein, the administering step results in at least a 2.0-fold increase in the percentage of NKp30-positive CD56-positive, CD16-positive, CD45-positive lymphocytes in the individual. In some embodiments of any of the methods described herein, the administering step results in at least a 2.5-fold increase in the percentage of NKp30-positive CD56-positive, CD16-positive, CD45-positive lymphocytes in the individual. In some embodiments of any of the methods described herein, the administering step results in at least a 3.0-fold increase in the percentage of NKp30-positive CD56-positive, CD16-positive, CD45-positive lymphocytes in the individual. In some embodiments of any of the methods described herein, the administering step results in at least a 3.5-fold increase in the percentage of NKp30-positive CD56-positive, CD16-positive, CD45-positive lymphocytes in the individual. In some embodiments of any of the methods described herein, the NKp30 positive, CD56 positive, CD16 positive, CD45 positive lymphocytes are NKp30 positive, CD56 positive, CD16 positive, CD45 positive NK cells.

In some embodiments of any of the methods described herein, the administering step does not result in a significant degree of myeloid cytotoxicity in the individual. In some embodiments of any of the methods described herein, the method further comprises identifying or diagnosing the individual as having a B7-H6 positive cancer. In some embodiments of any of the methods described herein, the B7-H6 positive cancer is selected from the group consisting of adrenocortical carcinoma, cholangiocarcinoma, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, esophageal cancer, Eye cancer, head and neck cancer, acute myeloid leukemia, lymphoid neoplasms, diffuse large b-cell lymphoma, kidney cancer, clear cell renal cell carcinoma, papillary renal papillary cell carcinoma of the kidney, chromophobe kidney, liver cancer, lung cancer, lung adenocarcinoma , squamous cell carcinoma of the lung, mesothelioma, ovarian cancer, pancreatic cancer, rectal cancer, skin cancer, stomach cancer, testicular cancer, thymoma, thyroid cancer, uterine cancer, uterine body endometrial cancer, uterine carcinosarcoma, and Uveal melanoma. In some embodiments of any of the methods described herein, the B7-H6 positive cancer is a B7-H6 positive/HLA-E negative cancer.

In some embodiments of any of the methods described herein, the method further comprises administering to the individual an NKG2A inhibitor. In some embodiments of any of the methods described herein, the NKG2A inhibitor is an antagonist antibody that specifically binds to NKG2A.

Also provided herein are methods of treating an individual previously identified or diagnosed as having a B7-H6 positive cancer comprising administering to the individual previously identified or diagnosed as having a B7-H6 positive cancer a therapeutically effective amount of a pharmaceutical composition, the A pharmaceutical composition comprising a population of enucleated erythroid cells comprising on their extracellular surface a first exogenous polypeptide comprising (i) IL-15 or a functional fragment, and (ii) IL-15 receptor alpha or a functional fragment thereof. In some embodiments of any of the methods described herein, the B7-H6 positive cancer is selected from the group consisting of adrenocortical carcinoma, cholangiocarcinoma, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, esophageal cancer, Eye cancer, head and neck cancer, acute myeloid leukemia, lymphoid neoplasms, diffuse large b-cell lymphoma, kidney cancer, clear cell renal cell carcinoma, papillary renal papillary cell carcinoma of the kidney, chromophobe kidney, liver cancer, lung cancer, lung adenocarcinoma , squamous cell carcinoma of the lung, mesothelioma, ovarian cancer, pancreatic cancer, rectal cancer, skin cancer, stomach cancer, testicular cancer, thymoma, thyroid cancer, uterine cancer, uterine body endometrial cancer, uterine carcinosarcoma, and Uveal melanoma. In some embodiments of any of the methods described herein, the B7-H6 positive cancer is a B7-H6 positive/HLA-E negative cancer.

In some embodiments of any of the methods described herein, the administering step does not result in a significant degree of myeloid cytotoxicity in the individual. In some embodiments of any of the methods described herein, the method further comprises diagnosing or identifying the individual as having a B7-H6 positive cancer. In some embodiments of any of the methods described herein, the method further comprises administering to the individual an NKG2A inhibitor. In some embodiments of any of the methods described herein, the NKG2A inhibitor is an antagonist antibody that specifically binds to NKG2A.

In some embodiments of any of the methods described herein, the invention features a method of reducing the number and/or proliferation of B7-H6-positive cancer cells in an individual previously identified or diagnosed with a B7-H6-positive cancer, the method comprising administering a therapeutically effective amount of a pharmaceutical composition, the pharmaceutical composition comprising a group of enucleated erythroid cells, the group of enucleated erythroid cells comprising a first exogenous polypeptide on its extracellular surface, the first exogenous polypeptide The source polypeptide comprises (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof.

In some embodiments of any of the methods described herein, the administering results in a decrease in the number of B7-H6 positive cancer cells in the individual. In some embodiments of any of the methods described herein, the administering results in at least a 5% reduction in the number of B7-H6 positive cancer cells in the individual as compared to the number of B7-H6 positive cancer cells in the individual before the administration. In some embodiments of any of the methods described herein, the administering results in at least a 10% reduction in the number of B7-H6 positive cancer cells in the individual compared to the number of B7-H6 positive cancer cells in the individual before the administration.

In some embodiments of any of the methods described herein, the administering results in a decrease in the number of B7-H6 positive/HLA-E negative cancer cells in the individual. In some embodiments of any of the methods described herein, the administration results in B7-H6 positive/HLA-E negative cancer cells in the individual compared to the number of B7-H6 positive/HLA-E negative cancer cells in the individual prior to administration decrease by at least 5%. In some embodiments of any of the methods described herein, the administration results in B7-H6 positive/HLA-E negative cancer cells in the individual compared to the number of B7-H6 positive/HLA-E negative cancer cells in the individual prior to administration decrease by at least 10%.

In some embodiments of any of the methods described herein, the administering results in decreased proliferation of B7-H6 positive cancer cells in the individual. In some embodiments of any of the methods described herein, the administering results in at least a 5% decrease in the proliferation of B7-H6 positive cancer cells in the individual compared to the proliferation of B7-H6 positive cancer cells in the individual before the administration. In some embodiments of any of the methods described herein, the administering results in at least a 10% decrease in the proliferation of B7-H6 positive cancer cells in the individual compared to the proliferation of B7-H6 positive cancer cells in the individual before the administration.

In some embodiments of any of the methods described herein, the administering results in decreased proliferation of B7-H6 positive/HLA-E negative cancer cells in the individual. In some embodiments of any of the methods described herein, the administration results in the proliferation of B7-H6 positive/HLA-E negative cancer cells in the individual compared to the proliferation of B7-H6 positive/HLA-E negative cancer cells in the individual prior to the administration The hyperplasia is reduced by at least 5%. In some embodiments of any of the methods described herein, the administration results in the proliferation of B7-H6 positive/HLA-E negative cancer cells in the individual compared to the proliferation of B7-H6 positive/HLA-E negative cancer cells in the individual prior to the administration The hyperplasia is reduced by at least 10%.

In some embodiments of any of the methods described herein, the administering step does not result in a significant degree of myeloid cytotoxicity in the individual.

In some embodiments of any of the methods described herein, the method further comprises identifying or diagnosing the individual as having a B7-H6 positive cancer. In some embodiments of any of the methods described herein, the B7-H6 positive cancer is selected from the group consisting of adrenocortical carcinoma, cholangiocarcinoma, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, esophageal cancer, Eye cancer, head and neck cancer, acute myeloid leukemia, lymphoid neoplasms, diffuse large b-cell lymphoma, kidney cancer, renal clear cell carcinoma, renal papillary cell carcinoma, renal chromophobe, liver cancer, lung cancer, lung adenocarcinoma , squamous cell carcinoma of the lung, mesothelioma, ovarian cancer, pancreatic cancer, rectal cancer, skin cancer, stomach cancer, testicular cancer, thymoma, thyroid cancer, uterine cancer, uterine body endometrial cancer, uterine carcinosarcoma, and Uveal melanoma. In some embodiments of any of the methods described herein, the B7-H6 positive cancer is a B7-H6 positive/HLA-E negative cancer.

In some embodiments of any of the methods described herein, the method further comprises administering to the individual an NKG2A inhibitor. In some embodiments of any of the methods described herein, the NKG2A inhibitor is an antagonist antibody that specifically binds to NKG2A.

Also provided herein is a method of inducing the killing of B7-H6 positive cancer cells in an individual previously identified or diagnosed as having a B7-H6 positive cancer comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an enucleated A population of erythroid cells comprising on their extracellular surface an exogenous fusion polypeptide comprising (i) IL-15 or a functional fragment thereof, and (ii) IL -15 receptor alpha or a functional fragment thereof.

In some embodiments of any of the methods described herein, killing comprises necrosis. In some embodiments, killing comprises apoptosis. In some embodiments of any of the methods described herein, the killing is mediated via NK cell-mediated lysis.

In some embodiments of any of the methods described herein, the B7-H6 positive cancer cells are cancer cells selected from the group consisting of adrenocortical carcinoma, cholangiocarcinoma, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer Carcinoma, Esophageal Cancer, Eye Cancer, Head and Neck Cancer, Acute Myeloid Leukemia, Lymphoma, Diffuse Large B-Cell Lymphoma, Kidney Cancer, Kidney Renal Clear Cell Carcinoma, Kidney Renal Papillary Cell Carcinoma, Kidney Chromophobe, Liver Cancer, Lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, mesothelioma, ovarian cancer, pancreatic cancer, rectal cancer, skin cancer, stomach cancer, testicular cancer, thymoma, thyroid cancer, uterine cancer, uterine endometrial cancer, Uterine carcinosarcoma, and uveal melanoma. In some embodiments of any of the methods described herein, the B7-H6 positive cancer cells are B7-H6 positive and HLA-E negative cancer cells.

In some embodiments of any of the methods described herein, the administering step does not result in a significant degree of myeloid cytotoxicity in the individual.

In some embodiments of any of the methods described herein, the individual has previously been identified or diagnosed as having a B7-H6 positive cancer. In some embodiments of any of the methods described herein, the B7-H6 positive cancer is selected from the group consisting of adrenocortical carcinoma, cholangiocarcinoma, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, esophageal cancer, Eye cancer, head and neck cancer, acute myeloid leukemia, lymphoid neoplasms, diffuse large b-cell lymphoma, kidney cancer, clear cell renal cell carcinoma, papillary renal papillary cell carcinoma of the kidney, chromophobe kidney, liver cancer, lung cancer, lung adenocarcinoma , squamous cell carcinoma of the lung, mesothelioma, ovarian cancer, pancreatic cancer, rectal cancer, skin cancer, stomach cancer, testicular cancer, thymoma, thyroid cancer, uterine cancer, uterine body endometrial cancer, uterine carcinosarcoma, and Uveal melanoma. In some embodiments of any of the methods described herein, the B7-H6 positive cancer is a B7-H6 positive/HLA-E negative cancer.

In some embodiments of any of the methods described herein, the method further comprises administering to the individual an NKG2A inhibitor. In some embodiments of any of the methods described herein, the NKG2A inhibitor is an antagonist antibody that specifically binds to NKG2A.

Also provided herein is a method of reducing the volume of a solid tumor in an individual previously identified or diagnosed as having a B7-H6 positive cancer comprising administering a therapeutically effective amount of a population of enucleated erythroid cells, the population of enucleated erythroid cells It comprises a first exogenous fusion polypeptide on its extracellular surface, and the first exogenous fusion polypeptide comprises (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor α or a functional fragment thereof.

In some embodiments of any of the methods described herein, the administering results in at least a 5% reduction in the volume of the solid tumor in the individual as compared to the volume of the solid tumor before the administration. In some embodiments of any of the methods described herein, the administering results in at least a 10% reduction in the volume of the solid tumor in the individual as compared to the volume of the solid tumor prior to the administration. In some embodiments of any of the methods described herein, the administering step does not result in a significant degree of myeloid cytotoxicity in the individual.

In some embodiments of any of the methods described herein, the B7-H6 positive cancer is selected from the group consisting of adrenocortical carcinoma, cholangiocarcinoma, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, esophageal cancer, Eye cancer, head and neck cancer, acute myeloid leukemia, lymphoid neoplasms, diffuse large b-cell lymphoma, kidney cancer, clear cell renal cell carcinoma, papillary renal papillary cell carcinoma of the kidney, chromophobe kidney, liver cancer, lung cancer, lung adenocarcinoma , squamous cell carcinoma of the lung, mesothelioma, ovarian cancer, pancreatic cancer, rectal cancer, skin cancer, stomach cancer, testicular cancer, thymoma, thyroid cancer, uterine cancer, uterine body endometrial cancer, uterine carcinosarcoma, and Uveal melanoma. In some embodiments of any of the methods described herein, the B7-H6 positive cancer is a B7-H6 positive/HLA-E negative cancer.

In some embodiments of any of the methods described herein, the administering step does not result in a significant degree of myeloid cytotoxicity in the individual.

In some embodiments of any of the methods described herein, the method further comprises diagnosing or identifying the individual as having a B7-H6 positive cancer.

In some embodiments of any of the methods described herein, the method further comprises administering to the individual an NKG2A inhibitor. In some embodiments of any of the methods described herein, the NKG2A inhibitor is an antagonist antibody that specifically binds to NKG2A.

In some embodiments of any of the methods described herein, the enucleated erythroid cells comprise at least 1,000 copies of the exogenous fusion polypeptide. In some embodiments of any of the methods described herein, enucleated erythroid cells are produced by a method comprising: introducing a nucleic acid encoding a first exogenous polypeptide into a nucleated erythroid cell precursor; and The nucleated erythroid cell precursor is cultured under conditions that enucleate the nucleated erythroid cell precursor.

In some embodiments of any of the methods described herein, the enucleated erythroid cells further comprise a second exogenous polypeptide comprising 4-1BBL or a functional fragment thereof, wherein the second exogenous polypeptide is present in the enucleated erythroid cells on the outer surface of the cell. In some embodiments of any of the methods described herein, the enucleated erythroid blood cells comprise at least 1,000 copies of the second exogenous polypeptide.

In some embodiments of any of the methods described herein, the enucleated erythroid cells are produced by a method comprising combining a nucleic acid encoding a first exogenous polypeptide comprising 4-1BBL or a functional fragment thereof with a second exogenous polypeptide encoding Introducing the nucleic acid of the sex polypeptide into the nucleated erythroid cell precursor; and culturing the nucleated erythroid cell under conditions sufficient to express the first exogenous polypeptide and the second exogenous polypeptide and enucleate the nucleated erythroid cell precursor Cell precursors.

In some embodiments of any of the methods described herein, the enucleated erythroid cells are not hypotonic cells. In some embodiments of any of the methods described herein, the enucleated erythroid blood cells do not comprise a sortase-transferred feature. In some embodiments of any of the methods described herein, the individual is a human and the enucleated erythroid cells are human cells.

Also provided herein is a kit comprising: a pharmaceutical composition comprising an enucleated erythroid cell comprising a first exosome on an extracellular surface thereof, and instructions for practicing any of the methods described herein. An exogenous polypeptide, the first exogenous polypeptide comprises (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor α or a functional fragment thereof.

In some embodiments of any of the sets described herein, the enucleated erythroid cells comprise at least 1,000 copies of the first exogenous polypeptide.

In some embodiments of any of the kits described herein, enucleated erythroid cells are produced by a method comprising: introducing a nucleic acid encoding a first exogenous polypeptide into a nucleated erythroid cell precursor; The nucleated erythroid cell precursor is incubated with the first exogenous polypeptide and under conditions that enucleate the nucleated erythroid cell precursor.

In some embodiments of any of the kits described herein, the enucleated erythroid cells further comprise a second exogenous polypeptide comprising 4-1BBL or a functional fragment thereof, wherein the second exogenous polypeptide is present in the enucleated erythroid cells on the outer surface of the cell. In some embodiments of any of the sets described herein, the enucleated erythroid cells comprise at least 1,000 copies of the second exogenous polypeptide.

In some embodiments of any of the kits described herein, the enucleated erythroid cells are produced by a method comprising introducing into the nucleated cells a nucleic acid encoding a first exogenous polypeptide and a nucleic acid encoding a second exogenous polypeptide in the erythroid cell precursor; and culturing the nucleated erythroid cell precursor under conditions sufficient to express the first exogenous polypeptide and the second exogenous polypeptide and to enucleate the nucleated erythroid cell precursor.

In some embodiments of any of the kits described herein, the enucleated erythroid cells are not hypotonic cells. In some embodiments of any of the kits described herein, the enucleated erythroid cells do not comprise sortase-transferred features. In some embodiments of any of the kits described herein, the enucleated erythroid cells are human cells.

The term "enucleated erythroid cell engineered" means an enucleated erythroid cell (e.g., human enucleated erythroid cell) comprising one or more (e.g., two, three, four, five or six) exogenous proteins (eg, any combination of the exemplary exogenous proteins described herein or known in the art). For example, an enucleated erythroid engineered can have one or more exogenous proteins present in its cytoplasm. In some examples, engineered enucleated erythroid cells can have one or more exogenous proteins present on their extracellular surface. In some examples, an enucleated erythroid engineered can have (i) one or more exogenous proteins present in its cytoplasm, and (ii) one or more exogenous proteins present on its extracellular surface. source protein. Non-limiting examples of engineered enucleated erythroid cells include click-engaged enucleated erythroid cells, enucleated erythroid cells that have been hypotonically loaded, and enucleated erythroid cells that have been subjected to physical manipulation (e.g., as described herein or known in the art). Any exemplary type of physical manipulation) loaded enucleated erythroid cells. Other non-limiting aspects of engineered enucleated erythroid cells are described herein.

The term "conjugated enucleated erythroid" means an enucleated erythroid engineered to have at least one exogenous protein conjugated to another exogenous protein through catalytic activity of an enzyme and/or peptide sequence, and/or chemical reaction. A protein on the extracellular surface of the engineered enucleated erythroid cell (eg, an endogenous protein or a different exogenous protein of the enucleated erythrocyte).

"Hypotonically-loaded enucleated erythroid cells" means enucleated erythroid cells that have been engineered, at least in part, by adding enucleated erythroid cells or erythroid cells containing one or more exogenous proteins Cellular precursors are produced by exposure to a low ionic strength buffer (eg, any of the exemplary low ionic strength buffers described herein). Non-limiting examples of methods that can be used to generate hypotonically loaded enucleated erythroid cells are described herein. Other methods of generating hypotonically loaded enucleated erythroid cells are known in the art.

The term "enucleated erythroid cell loaded by physical manipulation" means an enucleated erythroid cell, at least a portion of which has been loaded by encoding one or more exogenous proteins, such as any described herein or known in the art. Exemplary exogenous proteins) and/or nucleic acids of exogenous polypeptides are introduced into erythroid cell precursors to generate erythroid cell precursors by physical manipulation. Non-limiting examples of physical manipulations that can be used to introduce nucleic acids encoding one or more exogenous proteins into erythroid cell precursors include electroporation and particle-mediated transfection. Other examples of physical manipulations that can be used to introduce nucleic acids encoding one or more exogenous proteins into erythroid cell precursors are known in the art.

The term "exogenous protein" refers to a protein that is introduced into or onto a cell, or is expressed by a cell by introducing an exogenous nucleic acid encoding the protein into the cell or into a cellular precursor of the cell. In some embodiments, an exogenous protein is a protein encoded by an exogenous nucleic acid introduced into a cell or a cellular precursor of a cell, which nucleic acid is optionally not retained by the cell. In some embodiments, the exogenous protein is a protein bound to the cell surface by chemical or enzymatic means. Non-limiting classes of exogenous proteins include enzymes, interleukins, cytokine receptors, Fc binding molecules, T cell activating ligands, T cell receptors, immunosuppressive molecules, MHC molecules, APC binding molecules, autologous Antigens, allergens, toxins, targeting agents, receptor ligands (eg, receptor agonists or receptor antagonists), and antibodies or antibody fragments.

The term "extracellular surface" when used in the context of exogenous polypeptides means: (1) exogenous polypeptides (e.g., transmembrane proteins, peripheral Membrane proteins, lipid-anchored proteins (such as GPI anchors, N-myristoylated proteins, or S-palmitoylated proteins), or (2) proteins that stably bind to their cognate receptors, wherein the cognate receptors Physically attached to the membrane of the enucleated erythroid cell (eg, a ligand that binds to its cognate receptor, wherein the cognate receptor is physically attached to the membrane of the enucleated erythroid cell). Non-limiting methods for determining the presence of exogenous proteins on the outer surface of enucleated erythroid cells include fluorescence activated cell sorting (FACS), immunohistochemistry, cell fractionation analysis, and Western blotting.

The term "erythroid cell precursor" means a mammalian cell capable of ultimate differentiation/development into an enucleated erythroid cell. In some embodiments, the erythroid cell precursors are cord blood stem cells, CD34 ⁺ cells, hematopoietic stem/cell precursors (HSC, HSPC), colony forming spleen (CFU-S) cells, common myeloid cell precursors (CMP) Cells, blastoid colony forming cells, burst forming unit erythroid/erythroid (BFU-E), megakaryocyte-erythroid precursor (MEP) cells, erythroid colony forming unit or colony forming unit erythrocyte (CFU-E) , induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs), or a combination thereof.

The term "individual" refers to any mammal. In some embodiments, the individual or "individual in need of treatment" can be a primate (e.g., a human, an ape (e.g., a monkey (e.g., a marmoset or a baboon), or an ape (e.g., a gorilla, chimpanzee, orangutans, or gibbons)), rodents (eg, mice, guinea pigs, hamsters, or rats), rabbits, dogs, cats, horses, sheep, cows, pigs, or goats. In some embodiments, the individual or A "subject suitable for treatment" may be a non-human mammal, particularly a mammal, which is commonly used as a model for demonstrating therapeutic efficacy in humans (e.g., mice, pigs, rats, or non-human primates) In some examples, an individual may be previously diagnosed or identified as in need of treatment (e.g., previously diagnosed or identified as having Have B7-H6 positive cancer or have been previously diagnosed or identified as having both B7-H6 positive and HLA-E negative cancer).

The term "adult human subject" refers to a human being 18 years of age or older (e.g., 20 years or older, 25 years or older, 30 years or older, 35 years or older, 40 years or older, 45 years or Over, 55 or over, 60 or over, 65 or over, 70 or over, 75 or over, 80 or over, 85 or over, 90 or over, 95 or over, or 100 or above).

The term "pediatric human subject" means a human under the age of 18 (e.g., 17 and under, 16 and under, 15 and under, 14 and under, 13 and under, 12 and under, 11 and under, 10 and under, 9 and under, 8 and under, 7 and under, 6 and under, 5 and under, 4 and under, 3 and under, 2 and under, or 1 age and under).

As used herein, "treating" means reducing the number, severity, frequency and/or duration of one or more symptoms of a medical disease or condition in a subject.

As used herein, the term "B7-H6" means a B7-H6 polypeptide or B7-H6 mRNA, including wild-type human B7-H6 polypeptide and wild-type human B7-H6 mRNA, and variants thereof (e.g., truncated or mutated forms ). Non-limiting examples of methods of detecting B7-H6 levels are described herein.

As used herein, the term "B7-H6 positive cancer" means a cancer comprising B7-H6 positive cancer cells. Non-limiting examples of B7-H6 positive cancers are described herein.

As used herein, the term "B7-H6 positive cancer cells" means cancer cells with a B7-H6 content (B7-H6 protein or B7-H6 mRNA transcript) higher than a B7-H6 reference content. Non-limiting examples of B7-H6 reference levels are described herein. Non-limiting examples of B7-H6 positive cancer cells are also described herein.

As used herein, the term "HLA-E" refers to human leukocyte antigen-E (HLA-E) polypeptide or HLA-E mRNA, including wild-type human HLA-E polypeptide and wild-type human HLA-E mRNA and variants thereof ( For example, truncated or mutated forms). Non-limiting examples of methods of detecting HLA-E levels are described herein.

As used herein, the term "HLA-E negative cancer" refers to a cancer whose HLA-E content (HLA-E protein or HLA-E mRNA transcript) is lower than a reference level for HLA-E expression. Non-limiting examples of HLA-E reference levels are described herein. Non-limiting examples of HLA-E negative cancers are also described herein.

As used herein, the term "HLA-E-negative cancer cells" means cancers comprising HLA-E-negative cancer cells. Non-limiting examples of cancers that may be HLA-E negative cancers are also described herein.

As used herein, the term "NKp30" means NKp30 polypeptide or NKp30 mRNA, including wild-type human NKp30 polypeptide and wild-type human NKp30 mRNA, and variants (eg, truncated or mutated forms) thereof. The term "NKp30" also includes all known NKp30 protein and NKp30 mRNA isoforms. Non-limiting examples of methods of detecting NKp30 levels are described herein.

As used herein, the term "NKp30 positive lymphocytes" means lymphocytes with NKp30 content (either NKp30 protein or NKp30 mRNA transcript) higher than the NKp30 reference content. Non-limiting examples of reference amounts of NKp30 are described herein.

As used herein, the term "NKG2A" means NK group 2 member A (NKG2A) polypeptide or NKG2A mRNA, including wild-type human NKG2A polypeptide and wild-type human NKG2A mRNA, and variants (eg, truncated or mutated forms) thereof. Non-limiting examples of methods of detecting NKG2A levels are also described herein.

The nkg2a gene encodes two isoforms, NKG2A and NKG2B, the latter lacking the stalk region. As used herein, the term "NKG2A" does not include NKG2B protein or mRNA transcripts.

As used herein, the term "NKG2A-negative lymphocyte" means a lymphocyte whose NKG2A content (either NKG2A protein or NKG2A mRNA) is lower than a reference NKG2A content. Non-limiting examples of reference amounts of NKG2A are described herein.

As used herein, the term "NK cell-mediated cytotoxicity" means that NK cells induce the killing of other cells. In some embodiments, "NK cell-mediated cytotoxicity" is a mechanism used by NK cells to induce the killing of cancer cells.

As used herein, the term "NK cell-mediated lysis" refers to NK cells capable of releasing lysed particles for inducing the killing of other cells. In some embodiments, the lytic particles include at least perforin and granzymes.

As used herein, the term "sortase transfer signature" means an exogenous protein or polypeptide comprising a sequence that can be produced by a sortase reaction. Non-limiting examples of proteins and polypeptides lacking the transfer feature of sortase are described in WO 2017/123646, which is incorporated by reference in its entirety.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other convenient methods and materials known in the art can also be used. The materials, methods and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

Other features and advantages of the present invention will be apparent from the following detailed description and drawings and claims.

Provided herein are methods of increasing the number of NKp30-positive lymphocytes (e.g., NKp30-positive NK cells) in an individual in need thereof, comprising administering to an individual previously identified or diagnosed with a B7-H6-positive cancer a therapeutically effective amount of a drug comprising: A pharmaceutical composition of a group of nucleated erythroid cells comprising on its extracellular surface a first exogenous polypeptide comprising (i) IL-15 or a function thereof fragment, and (ii) IL-15 receptor alpha or a functional fragment thereof. Also provided herein is a method of increasing the number of NKp30-positive lymphocytes (e.g., NKp30-positive NK cells) in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of a pharmaceutical composition comprising a group of enucleated erythroid cells, the denucleated erythroid A population of nuclear erythroid cells comprising on their extracellular surface a first exogenous fusion polypeptide comprising (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor α or a functional fragment thereof; and a second exogenous polypeptide on the extracellular surface thereof, the second exogenous polypeptide comprising 4-1BBL or a functional fragment thereof. Some embodiments of these methods result in an increase in the number of NKp30-positive lymphocytes (e.g., NKp30-positive NK cells) in an individual by at least 1%, by at least 2%, by at least 3%, by at least 4%, by at least 5%, by at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, increased by at least 95%, or increased by at least 100% (eg, compared to the number of NKp30-positive lymphocytes (eg, NKp30-positive NK cells) in the individual prior to administration)). Some embodiments of these methods result in an increase in the number of NKp30-positive lymphocytes (e.g., NKp30-positive NK cells) in an individual from about 1% to about 50%, from about 1% to about 45%, from about 1% to about 40%, about 1% up to about 35% up, about 1% up to about 30% up, about 1% up to about 25% up, about 1% up to about 20% up, about 1% up to about 20% up 15%, about 1% up to about 10% up, about 1% up to about 5% up, about 5% up to about 50% up, about 5% up to about 45% up, about 5% up to about 5% up 40%, from about 5% to about 35%, from about 5% to about 30%, from about 5% to about 25%, from about 5% to about 20%, from about 5% to about 15%, from about 5% to about 10%, from about 10% to about 50%, from about 10% to about 45%, from about 10% to about 40%, from about 10% to about 35%, about 10% higher to about 30% higher, about 10% higher to about 25% higher, about 10% higher to about 20% higher, about 10% higher to about 15% higher, about 15% higher to about 15% higher 50%, from about 15% to about 45%, from about 15% to about 40%, from about 15% to about 35%, from about 15% to about 30%, from about 15% to about 25%, from about 15% to about 20%, from about 20% to about 50%, from about 20% to about 45%, from about 20% to about 40%, from about 20% to about 40% 35%, about 20% up to about 30% up, about 20% up to about 25% up, about 25% up to about 50% up, about 25% up to about 45% up, about 25% up to about 25% up 40%, about 25% higher to about 35% higher, about 25% higher to about 30% higher, about 30% higher to about 50% higher, about 30% higher to about 45% higher, about 30% higher to about 30% higher 40%, about 30% up to about 35% up, about 35% up to about 50% up, about 35% up to about 45% up, about 35% up to about 40% up, about 40% up to about 40% up 50%, an increase of about 40% to an increase of about 45%, or an increase of about 45% to an increase of about 50% (e.g., compared to the number of NKp30-positive lymphocytes (e.g., NKp30-positive NK cells) in the individual prior to administration). In some embodiments of any of the methods described herein, the NKp30-positive lymphocytes are NKp30-positive NK cells.

Some specific examples of these methods result in an increase in the number of NKp30-positive/NKG2A-negative lymphocytes (e.g., NKp30-positive/NKG2A-negative NK cells) in the individual by at least 1%, by at least 2%, by at least 3%, by at least 4%, by at least At least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least At least 35% increase, at least 40% increase, at least 45% increase, at least 50% increase, at least 55% increase, at least 60% increase, at least 65% increase, at least 70% increase, at least 75% increase, at least 80% increase, increase At least 85%, at least 90% increase, at least 95% increase, or at least 100% increase (e.g., compared to the number of NKp30-positive/NKG2A-negative lymphocytes (e.g., NKp30-positive/NKG2A-negative NK cells) in the individual prior to administration ). Some embodiments of these methods result in an increase in the number of NKp30-positive/NKG2A-negative lymphocytes (e.g., NKp30-positive/NKG2A-negative NK cells) in an individual by about 1% to about 50%, about 1% to about 45%, about About 1% up to about 40% up, about 1% up to about 35% up, about 1% up to about 30% up, about 1% up to about 25% up, about 1% up to about 20% up, up about 1% up to about 20% up, up About 1% up to about 15% up, about 1% up to about 10% up, about 1% up to about 5% up, about 5% up to about 50% up, about 5% up to about 45% up, up about 5% up to about 45% up, up About 5% up to about 40% up, about 5% up to about 35% up, about 5% up to about 30% up, about 5% up to about 25% up, about 5% up to about 20% up, up about 5% up to about 20% up, up About 5% to about 15% higher, about 5% higher to about 10% higher, about 10% higher to about 50% higher, about 10% higher to about 45% higher, about 10% higher to about 40% higher, higher About 10% to about 35% higher, about 10% higher to about 30% higher, about 10% higher to about 25% higher, about 10% higher to about 20% higher, about 10% higher to about 15% higher, higher About 15% up to about 50% up, about 15% up to about 45% up, about 15% up to about 40% up, about 15% up to about 35% up, about 15% up to about 30% up, up about 15% up to about 30% up, up From about 15% to about 25%, from about 15% to about 20%, from about 20% to about 50%, from about 20% to about 45%, from about 20% to about 40%, About 20% to about 35% higher, about 20% higher to about 30% higher, about 20% higher to about 25% higher, about 25% higher to about 50% higher, about 25% higher to about 45% higher, higher About 25% up to about 40% up, about 25% up to about 35% up, about 25% up to about 30% up, about 30% up to about 50% up, about 30% up to about 45% up, up about 30% up to about 45% up, up About 30% up to about 40% up, about 30% up to about 35% up, about 35% up to about 50% up, about 35% up to about 45% up, about 35% up to about 40% up, up about 35% up to about 40% up, up About 40% to about 50% increase, about 40% increase to about 45% increase, or about 45% increase to about 50% increase (e.g., compared to NKp30-positive/NKG2A-negative lymphocytes (e.g., NKp30- positive/NKG2A-negative NK cells) compared to the number of). In some embodiments, the NKp30-positive/NKG2A-negative lymphocytes are NKp30-positive/NKG2A-negative NK cells.

Some embodiments of the methods described herein result in an increase in trafficking of NKp30-positive lymphocytes (e.g., NKp30-positive NK cells) in an individual by at least 1%, by at least 2%, by at least 3%, by at least 4%, by at least 5%, At least 6% increase, at least 7% increase, at least 8% increase, at least 9% increase, at least 10% increase, at least 15% increase, at least 20% increase, at least 25% increase, at least 30% increase, at least 35% increase, At least 40% increase, at least 45% increase, at least 50% increase, at least 55% increase, at least 60% increase, at least 65% increase, at least 70% increase, at least 75% increase, at least 80% increase, at least 85% increase, Increased by at least 90%, increased by at least 95%, or increased by at least 100% (eg, compared to trafficking of NKp30-positive lymphocytes (eg, NKp30-positive NK cells) in the subject prior to administration)). Some embodiments of any of the methods described herein result in an increase in trafficking of NKp30-positive lymphocytes (e.g., NKp30-positive NK cells) in an individual by about 1% to about 50%, about 1% to about 45%, about 1% Up to about 40% increase, about 1% increase to about 35% increase, about 1% increase to about 30% increase, about 1% increase to about 25% increase, about 1% increase to about 20% increase, about 1% increase Up to about 15% higher, about 1% higher to about 10% higher, about 1% higher to about 5% higher, about 5% higher to about 50% higher, about 5% higher to about 45% higher, about 5% higher Up to about 40% increase, about 5% increase to about 35% increase, about 5% increase to about 30% increase, about 5% increase to about 25% increase, about 5% increase to about 20% increase, about 5% increase Up to about 15% increase, about 5% increase to about 10% increase, about 10% increase to about 50% increase, about 10% increase to about 45% increase, about 10% increase to about 40% increase, about 10% increase Up to about 35% increase, about 10% increase to about 30% increase, about 10% increase to about 25% increase, about 10% increase to about 20% increase, about 10% increase to about 15% increase, about 15% increase Up to about 50% increase, about 15% increase to about 45% increase, about 15% increase to about 40% increase, about 15% increase to about 35% increase, about 15% increase to about 30% increase, about 15% increase Up to about 25% increase, about 15% increase to about 20% increase, about 20% increase to about 50% increase, about 20% increase to about 45% increase, about 20% increase to about 40% increase, about 20% increase Up to about 35% up, up to about 20% up to about 30% up, up to about 20% up to about 25% up, up to about 25% up to about 50% up, up to about 25% up to about 45% up, up to about 25% up Up to about 40% increase, about 25% increase to about 35% increase, about 25% increase to about 30% increase, about 30% increase to about 50% increase, about 30% increase to about 45% increase, about 30% increase Up to about 40% increase, about 30% increase to about 35% increase, about 35% increase to about 50% increase, about 35% increase to about 45% increase, about 35% increase to about 40% increase, about 40% increase To about 50% increase, about 40% increase to about 45% increase, or about 45% increase to about 50% increase (e.g., relative to trafficking of NKp30-positive lymphocytes (e.g., NKp30-positive NK cells) in the subject prior to administration Compare)).

Some embodiments of the methods described herein result in an increase in trafficking of NKp30-positive/NKG2A-negative lymphocytes (e.g., NKp30-positive/NKG2A-negative NK cells) in an individual by at least 1%, by at least 2%, by at least 3%, by at least 4% , at least 5% increase, at least 6% increase, at least 7% increase, at least 8% increase, at least 9% increase, at least 10% increase, at least 15% increase, at least 20% increase, at least 25% increase, at least 30% increase , at least 35% increase, at least 40% increase, at least 45% increase, at least 50% increase, at least 55% increase, at least 60% increase, at least 65% increase, at least 70% increase, at least 75% increase, at least 80% increase , increase by at least 85%, increase by at least 90%, increase by at least 95%, or increase by at least 100% (e.g., compared to trafficking of NKp30-positive/NKG2A-negative lymphocytes (e.g., NKp30-positive/NKG2A-negative NK cells) in the subject prior to administration compared to)). Some embodiments of any of the methods described herein result in an increase in trafficking of NKp30-positive/NKG2A-negative lymphocytes (e.g., NKp30-positive/NKG2A-negative NK cells) in an individual from about 1% to about 50%, from about 1% to about 45% %, from about 1% to about 40%, from about 1% to about 35%, from about 1% to about 30%, from about 1% to about 25%, from about 1% to about 20% %, about 1% up to about 15% up, about 1% up to about 10% up, about 1% up to about 5% up, about 5% up to about 50% up, about 5% up to about 45% up %, from about 5% to about 40%, from about 5% to about 35%, from about 5% to about 30%, from about 5% to about 25%, from about 5% to about 20% %, from about 5% to about 15%, from about 5% to about 10%, from about 10% to about 50%, from about 10% to about 45%, from about 10% to about 40% %, about 10% higher to about 35% higher, about 10% higher to about 30% higher, about 10% higher to about 25% higher, about 10% higher to about 20% higher, about 10% higher to about 15% higher %, about 15% higher to about 50% higher, about 15% higher to about 45% higher, about 15% higher to about 40% higher, about 15% higher to about 35% higher, about 15% higher to about 30% higher %, about 15% higher to about 25% higher, about 15% higher to about 20% higher, about 20% higher to about 50% higher, about 20% higher to about 45% higher, about 20% higher to about 40% higher %, about 20% higher to about 35% higher, about 20% higher to about 30% higher, about 20% higher to about 25% higher, about 25% higher to about 50% higher, about 25% higher to about 45% higher %, about 25% higher to about 40% higher, about 25% higher to about 35% higher, about 25% higher to about 30% higher, about 30% higher to about 50% higher, about 30% higher to about 45% higher %, about 30% higher to about 40% higher, about 30% higher to about 35% higher, about 35% higher to about 50% higher, about 35% higher to about 45% higher, about 35% higher to about 40% higher %, an increase of about 40% to an increase of about 50%, an increase of about 40% to an increase of about 45%, or an increase of about 45% to an increase of about 50% (e.g., compared to NKp30-positive/NKG2A-negative lymphocytes (e.g., , NKp30-positive/NKG2A-negative NK cells) compared to trafficking))).

Some embodiments of the methods described herein result in increased numbers of NKp30-positive, CD45-positive, CD56-positive lymphocytes (eg, NKp30-positive, CD45-positive, and CD56-positive NK cells) in an individual. Some embodiments of these methods result in at least a 1.1-fold increase, at least a 1.2-fold increase, at least a 1.3-fold increase in the percentage of NKp30-positive CD45-positive, CD56-positive lymphocytes (e.g., NKp30-positive CD45-positive and CD56-positive NK cells) in an individual, at least 1.4 times higher, at least 1.5 times higher, at least 1.6 times higher, at least 1.7 times higher, at least 1.8 times higher, at least 1.9 times higher, at least 2.0 times higher, at least 2.2 times higher, at least 2.4 times higher, at least 2.6 times higher, Increased by at least 2.8 times, increased by at least 3.0 times, increased by at least 3.2 times, increased by at least 3.4 times, increased by at least 3.6 times, increased by at least 3.8 times, increased by at least 4.0 times, increased by at least 4.2 times, increased by at least 4.4 times, increased by at least 4.6 times, An increase of at least 4.8-fold, or an increase of at least 5.0-fold (e.g., compared to the percentage of NKp30-positive CD45-positive, CD56-positive lymphocytes (e.g., NKp30-positive CD45-positive, CD56-positive NK cells) in the individual prior to administration). Some embodiments of any of the methods described herein result in an increase in the percentage of NKp30-positive CD45-positive, CD56-positive lymphocytes (e.g., NKp30-positive CD45-positive and CD56-positive NK cells) in an individual from about 1.1-fold to about 5-fold (e.g., increased From about 1.1 times to about 4.5 times, from about 1.1 times to about 4.0 times, from about 1.1 times to about 3.5 times, from about 1.1 times to about 3.0 times, from about 1.1 times to about 2.5 times, from about 1.1 times to about 3.5 times 2.4 times, about 1.1 times to about 2.2 times, about 1.1 times to about 2.0 times, about 1.1 times to about 1.8 times, about 1.1 times to about 1.6 times, about 1.1 times to about 1.4 times, about 1.1 times to about 1.4 times 1.1 times to about 1.2 times, about 1.2 times to about 5 times, about 1.2 times to about 4.5 times, about 1.2 times to about 4.0 times, about 1.2 times to about 3.5 times, about 1.2 times to about 3.0 times times, about 1.2 times to about 2.5 times, about 1.2 times to about 2.4 times, about 1.2 times to about 2.2 times, about 1.2 times to about 2.0 times, about 1.2 times to about 1.8 times, about 1.2 times 1.6 times, 1.2 to 1.4 times, 1.4 to 5 times, 1.4 to 4.5 times, 1.4 to 4.0 times, 1.4 to 3.5 times , increase by about 1.4 times to about 3.0 times, increase by about 1.4 times to about 2.5 times, increase by about 1.4 times to about 2.4 times, increase by about 1.4 times to about 2.2 times, increase by about 1.4 times to about 2.0 times, increase by about 1.4 times to about 1.8 times, about 1.4 times to about 1.6 times, about 1.6 times to about 5 times, about 1.6 times to about 4.5 times, about 1.6 times to about 4.0 times, about 1.6 times to about 3.5 times, Increased by about 1.6 times to about 3.0 times, increased by about 1.6 times to about 2.5 times, increased by about 1.6 times to about 2.4 times, increased by about 1.6 times to about 2.2 times, increased by about 1.6 times to about 2.0 times, increased by about 1.6 times to About 1.8 times, about 1.8 times to about 5 times, about 1.8 times to about 4.5 times, about 1.8 times to about 4.0 times, about 1.8 times to about 3.5 times, about 1.8 times to about 3.0 times, about 1.8 times to about 3.0 times 1.8 times to about 2.5 times, about 1.8 times to about 2.4 times, about 1.8 times to about 2.2 times, about 1.8 times to about 2.0 times, about 2.0 times to about 5 times, about 2.0 times to about 4.5 times times, about 2.0 times to about 4.0 times, about 2.0 times to about 3.5 times, about 2.0 times to about 3.0 times, about 2.0 times to about 2.5 times, about 2.0 times to about 2.4 times, about 2.0 times times to about 2.2 times, about 2.2 times to about 5 times, about 2.2 times to about 4.5 times, about 2.2 times to about 4.0 times, about 2.2 times to about 3. 5 times, about 2.2 times to about 3.0 times, about 2.2 times to about 2.5 times, about 2.2 times to about 2.4 times, about 2.4 times to about 5 times, about 2.4 times to about 4.5 times, about 2.4 times to about 4.5 times 2.4 times to about 4.0 times, about 2.4 times to about 3.5 times, about 2.4 times to about 3.0 times, about 2.4 times to about 2.5 times, about 2.5 times to about 5 times, about 2.5 times to about 4.5 times times, about 2.5 times to about 4.0 times, about 2.5 times to about 3.5 times, about 2.5 times to about 3.0 times, about 3.0 times to about 5 times, about 3.0 times to about 4.5 times, about 3.0 times 4.0 times, 3.0 to 3.5 times, 3.5 to 5 times, 3.5 to 4.5 times, 3.5 to 4.0 times, 4.0 to 5 times , increased by about 4.0-fold to about 4.5-fold, or increased by about 4.5-fold to about 5-fold) (e.g., compared to NKp30-positive CD45-positive, CD56-positive lymphocytes in individuals before administration (e.g., NKp30-positive CD45-positive, CD56-positive lymphocytes) NK cells) compared to the percentage). In some embodiments, the NKp30-positive, CD45-positive, CD56-positive lymphocytes are NKp30-positive, CD45-positive, CD56-positive NK cells.

Some embodiments of the methods described herein result in increased numbers of NKp30-positive, CD45-positive, CD16-positive, CD56-dim lymphocytes (eg, NKp30-positive, CD45-positive, CD16-positive, CD56-dim NK cells) in an individual. Some specific examples of these methods result in an increase in the percentage of NKp30-positive CD45-positive, CD16-positive, CD56-dim lymphocytes (e.g., NKp30-positive CD45-positive, CD16-positive, CD56-dim NK cells) in an individual by at least 1.1-fold, by at least 1.2 times, at least 1.4 times, at least 1.6 times, at least 1.8 times, at least 2.0 times, at least 2.2 times, at least 2.4 times, at least 2.6 times, at least 2.8 times, at least 3.0 times, at least 3.2-fold, at least 3.4-fold, at least 3.6-fold, at least 3.8-fold, at least 4.0-fold, at least 4.2-fold, at least 4.4-fold, at least 4.6-fold, at least 4.8-fold, or at least 5.0-fold (e.g. , compared to the percentage of NKp30-positive CD45-positive, CD16-positive, CD56-dim lymphocytes (eg, NKp30-positive CD45-positive, CD16-positive, CD56-dim NK cells) in the individual before administration). Some embodiments of any of the methods described herein result in an approximately 1.1-fold increase in the percentage of NKp30-positive CD45-positive, CD16-positive, CD56-dim lymphocytes (e.g., NKp30-positive CD45-positive, CD16-positive, CD56-dim NK cells) in an individual to about 5-fold (e.g., about 1.1-fold to about 4.5-fold, about 1.1-fold to about 4.0-fold, about 1.1-fold to about 3.5-fold, about 1.1-fold to about 3.0-fold, about 1.1-fold to about 2.5 times, about 1.1 times to about 2.4 times, about 1.1 times to about 2.2 times, about 1.1 times to about 2.0 times, about 1.1 times to about 1.8 times, about 1.1 times to about 1.6 times, about 1.1 times 1.4 times, 1.1 to 1.2 times, 1.2 to 5 times, 1.2 to 4.5 times, 1.2 to 4.0 times, 1.2 to 3.5 times , increase by about 1.2 times to about 3.0 times, increase by about 1.2 times to about 2.5 times, increase by about 1.2 times to about 2.4 times, increase by about 1.2 times to about 2.2 times, increase by about 1.2 times to about 2.0 times, increase by about 1.2 times to about 1.8 times, about 1.2 to about 1.6 times, about 1.2 to about 1.4 times, about 1.4 to about 5 times, about 1.4 to about 4.5 times, about 1.4 to about 4.0 times, Increased by about 1.4 times to about 3.5 times, increased by about 1.4 times to about 3.0 times, increased by about 1.4 times to about 2.5 times, increased by about 1.4 times to about 2.4 times, increased by about 1.4 times to about 2.2 times, increased by about 1.4 times to About 2.0 times, about 1.4 times to about 1.8 times, about 1.4 times to about 1.6 times, about 1.6 times to about 5 times, about 1.6 times to about 4.5 times, about 1.6 times to about 4.0 times, about 1.6 times to about 4.0 times About 1.6 times to about 3.5 times, about 1.6 times to about 3.0 times, about 1.6 times to about 2.5 times, about 1.6 times to about 2.4 times, about 1.6 times to about 2.2 times, about 1.6 times to about 2.2 times 2.0 times, about 1.6 times to about 1.8 times, about 1.8 times to about 5 times, about 1.8 times to about 4.5 times, about 1.8 times to about 4.0 times, 1.8 times to about 3.5 times, about 1.8 times 1.8 to 2.5 times, 1.8 to 2.4 times, 1.8 to 2.2 times, 1.8 to 2.0 times, 2.0 to 5 times , increase by about 2.0 times to about 4.5 times, increase by about 2.0 times to about 4.0 times, increase by about 2.0 times to about 3.5 times, increase by about 2.0 times to about 3.0 times, increase by about 2.0 times to about 2.5 times, increase by about 2.0 times to about 2.4 times, about 2.0 times to about 2.2 times, about 2.2 times to about 5 times, about 2.2 times to about 4.5 times, about 2.2 times 4.0 times, 2.2 to 3.5 times, 2.2 to 3.0 times, 2.2 to 2.5 times, 2.2 to 2.4 times, 2.4 to 5 times , by about 2.4 times to about 4.5 times, by about 2.4 times to about 4.0 times, by about 2.4 times to about 3.5 times, by about 2.4 times to about 3.0 times, by about 2.4 times to about 2.5 times, by about 2.5 times to about 5 times, about 2.5 times to about 4.5 times, about 2.5 times to about 4.0 times, about 2.5 times to about 3.5 times, about 2.5 times to about 3.0 times, about 3.0 times to about 5 times, Increased by about 3.0 times to about 4.5 times, increased by about 3.0 times to about 4.0 times, increased by about 3.0 times to about 3.5 times, increased by about 3.5 times to about 5 times, increased by about 3.5 times to about 4.5 times, increased by about 3.5 times to About 4.0-fold, about 4.0-fold to about 5-fold increase, about 4.0-fold to about 4.5-fold increase, or about 4.5-fold to about 5-fold increase) (e.g., compared to NKp30-positive CD45-positive, CD16-positive, CD16-positive, Percentage of CD16-dim lymphocytes (eg, NKp30-positive versus CD45-positive, CD16-positive, CD16-dim NK cells). In some embodiments, the NKp30 positive, CD45 positive, CD16 positive, CD56-dim lymphocytes are NKp30 positive, CD45 positive, CD16 positive, CD56-dim NK cells.

Some embodiments of the methods described herein result in increased numbers of NKp30-positive, CD45-positive, CD16-positive, CD56-positive lymphocytes (eg, NKp30-positive, CD45-positive, CD16-positive, CD56-positive NK cells) in an individual. Some specific examples of these methods result in an increase in the percentage of NKp30-positive CD45-positive, CD16-positive, CD56-positive lymphocytes in an individual by at least 1.1-fold, by at least 1.2-fold, by at least 1.4-fold, by at least 1.6-fold, by at least 1.8-fold, by at least 1.8-fold, At least 2.0 times, at least 2.2 times, at least 2.4 times, at least 2.6 times, at least 2.8 times, at least 3.0 times, at least 3.2 times, at least 3.4 times, at least 3.6 times, at least 3.8 times, At least 4.0-fold increase, at least 4.2-fold increase, at least 4.4-fold increase, at least 4.6-fold increase, at least 4.8-fold increase, or at least 5.0-fold increase (e.g., compared to NKp30-positive CD45-positive, CD16-positive, CD56-positive lymphoid spheres (eg, NKp30-positive CD45-positive, CD16-positive, CD56-positive NK cells) compared to percentages). Some embodiments of any of the methods described herein result in an increase in the percentage of NKp30-positive CD45-positive, CD16-positive, CD56-positive lymphocytes (e.g., NKp30-positive CD45-positive, CD16-positive, CD56-positive NK cells) in an individual from about 1.1-fold to about 5-fold (e.g., about 1.1-fold to about 4.5-fold, about 1.1-fold to about 4.0-fold, about 1.1-fold to about 3.5-fold, about 1.1-fold to about 3.0-fold, about 1.1-fold to about 2.5-fold, Increased by about 1.1 times to about 2.4 times, increased by about 1.1 times to about 2.2 times, increased by about 1.1 times to about 2.0 times, increased by about 1.1 times to about 1.8 times, increased by about 1.1 times to about 1.6 times, increased by about 1.1 times to About 1.4 times, about 1.1 times to about 1.2 times, about 1.2 times to about 5 times, about 1.2 times to about 4.5 times, about 1.2 times to about 4.0 times, about 1.2 times to about 3.5 times, about 1.2 times to about 3.5 times About 1.2 times to about 3.0 times, about 1.2 times to about 2.5 times, about 1.2 times to about 2.4 times, about 1.2 times to about 2.2 times, about 1.2 times to about 2.0 times, about 1.2 times to about 2.0 times 1.8 times, about 1.2 times to about 1.6 times, about 1.2 times to about 1.4 times, about 1.4 times to about 5 times, about 1.4 times to about 4.5 times, about 1.4 times to about 4.0 times, about 1.4 times to about 4.0 times 1.4 times to about 3.5 times, about 1.4 times to about 3.0 times, about 1.4 times to about 2.5 times, about 1.4 times to about 2.4 times, about 1.4 times to about 2.2 times, about 1.4 times to about 2.0 times times, about 1.4 times to about 1.8 times, about 1.4 times to about 1.6 times, about 1.6 times to about 5 times, about 1.6 times to about 4.5 times, about 1.6 times to about 4.0 times, about 1.6 times 1.6 to 3.0 times, 1.6 to 3.0 times, 1.6 to 2.5 times, 1.6 to 2.4 times, 1.6 to 2.2 times, 1.6 to 2.0 times , increase by about 1.6 times to about 1.8 times, increase by about 1.8 times to about 5 times, increase by about 1.8 times to about 4.5 times, increase by about 1.8 times to about 4.0 times, increase by 1.8 times to about 3.5 times, increase by about 1.8 times to About 3.0 times, about 1.8 times to about 2.5 times, about 1.8 times to about 2.4 times, about 1.8 times to about 2.2 times, about 1.8 times to about 2.0 times, about 2.0 times to about 5 times, about 2.0 times to about 5 times About 2.0 times to about 4.5 times, about 2.0 times to about 4.0 times, about 2.0 times to about 3.5 times, about 2.0 times to about 3.0 times, about 2.0 times to about 2.5 times, about 2.0 times to about 2.0 times 2.4 times, about 2.0 times to about 2.2 times, about 2.2 times to about 5 times, about 2.2 times to about 4.5 times, about 2.2 times to about 4. 0 times, about 2.2 times to about 3.5 times, about 2.2 times to about 3.0 times, about 2.2 times to about 2.5 times, about 2.2 times to about 2.4 times, about 2.4 times to about 5 times, about 2.4 times to about 5 times 2.4 times to about 4.5 times, about 2.4 times to about 4.0 times, about 2.4 times to about 3.5 times, about 2.4 times to about 3.0 times, about 2.4 times to about 2.5 times, about 2.5 times to about 5 times times, about 2.5 times to about 4.5 times, about 2.5 times to about 4.0 times, about 2.5 times to about 3.5 times, about 2.5 times to about 3.0 times, about 3.0 times to about 5 times, about 3.0 times 4.5 times, 3.0 to 4.0 times, 3.0 to 3.5 times, 3.5 to 5 times, 3.5 to 4.5 times, 3.5 to 4.0 times , increased by about 4.0-fold to about 5-fold, increased by about 4.0-fold to about 4.5-fold, or increased by about 4.5-fold to about 5-fold) (e.g., compared to NKp30-positive CD45-positive, CD16-positive, CD56-positive lymphoid spheres (eg, NKp30-positive CD45-positive, CD16-positive, CD56-positive NK cells) compared to percentages). In some embodiments, the NKp30-positive, CD56-positive, CD16-positive, CD45-positive lymphocytes are NKp30-positive, CD56-positive, CD16-positive, CD45-positive NK cells.

Some embodiments of the method result in increased numbers or concentrations of circulating NK cells (CD3 ⁻ CD16/CD56 ⁺ cells). In some embodiments, the methods result in at least a 1.1-fold increase, at least a 1.2-fold increase, at least a 1.4-fold increase, at least a 1.6-fold increase, at least a 1.6-fold increase, at least a 1.8-fold increase in the number or concentration of circulating NK cells (CD3 ⁻ CD16/CD56 ⁺ cells) , increase by at least 2.0 times, increase by at least 2.2 times, increase by at least 2.4 times, increase by at least 2.6 times, increase by at least 2.8 times, increase by at least 3.0 times, increase by at least 3.2 times, increase by at least 3.4 times, increase by at least 3.6 times, increase by at least 3.8 times , at least 4.0-fold increase, at least 4.2-fold increase, at least 4.4-fold increase, at least 4.6-fold increase, at least 4.8-fold increase, or at least 5.0-fold increase (e.g., compared to circulating NK cells (CD3 ⁻ CD16/CD56 ⁺ cells) compared to the number or concentration). In some embodiments, the methods result in an increase in the number or concentration of circulating NK cells (CD3 ⁻ CD16/CD56 ⁺ cells) by about 1.1-fold to about 5-fold (e.g., about 1.1-fold to about 4.5-fold, about 1.1-fold to about 4.0 times, about 1.1 to about 3.5 times, about 1.1 to about 3.0 times, about 1.1 to about 2.5 times, about 1.1 to about 2.4 times, about 1.1 to about 2.2 times, Increased by about 1.1 times to about 2.0 times, increased by about 1.1 times to about 1.8 times, increased by about 1.1 times to about 1.6 times, increased by about 1.1 times to about 1.4 times, increased by about 1.1 times to about 1.2 times, increased by about 1.2 times to About 5 times, about 1.2 times to about 4.5 times, about 1.2 times to about 4.0 times, about 1.2 times to about 3.5 times, about 1.2 times to about 3.0 times, about 1.2 times to about 2.5 times, about 1.2 times to about 2.5 times About 1.2 times to about 2.4 times, about 1.2 times to about 2.2 times, about 1.2 times to about 2.0 times, about 1.2 times to about 1.8 times, about 1.2 times to about 1.6 times, about 1.2 times to about 1.2 times 1.4 times, about 1.4 times to about 5 times, about 1.4 times to about 4.5 times, about 1.4 times to about 4.0 times, about 1.4 times to about 3.5 times, about 1.4 times to about 3.0 times, about 1.4 times to about 3.0 times 1.4 times to about 2.5 times, about 1.4 times to about 2.4 times, about 1.4 times to about 2.2 times, about 1.4 times to about 2.0 times, about 1.4 times to about 1.8 times, about 1.4 times to about 1.6 times times, about 1.6 times to about 5 times, about 1.6 times to about 4.5 times, about 1.6 times to about 4.0 times, about 1.6 times to about 3.5 times, about 1.6 times to about 3.0 times, about 1.6 times 1.6 to 2.5 times, 1.6 to 2.4 times, 1.6 to 2.2 times, 1.6 to 2.0 times, 1.6 to 1.8 times, 1.8 to 5 times , increased by about 1.8 times to about 4.5 times, increased by about 1.8 times to about 4.0 times, increased by about 1.8 times to about 3.5 times, increased by about 1.8 times to about 3.0 times, increased by about 1.8 times to about 2.5 times, increased by about 1.8 times to About 2.4 times, about 1.8 times to about 2.2 times, about 1.8 times to about 2.0 times, about 2.0 times to about 5 times, about 2.0 times to about 4.5 times, about 2.0 times to about 4.0 times, about 2.0 times to about 4.0 times About 2.0 times to about 3.5 times, about 2.0 times to about 3.0 times, about 2.0 times to about 2.5 times, about 2.0 times to about 2.4 times, about 2.0 times to about 2.2 times, about 2.2 times to about 2.2 times 5 times, about 2.2 times to about 4.5 times, about 2.2 times to about 4.0 times, about 2.2 times to about 3.5 times, about 2.2 times to about 3.0 times, about 2.2 times to about 2.5 times , increase by about 2.2 times to about 2.4 times, increase by about 2.4 times to about 5 times, increase by about 2.4 times to about 4.5 times, increase by about 2.4 times to about 4.0 times, increase by about 2.4 times to about 3.5 times, increase by about 2.4 times to about 3.0 times, about 2.4 to about 2.5 times, about 2.5 to about 5 times, about 2.5 to about 4.5 times, about 2.5 to about 4.0 times, about 2.5 to about 3.5 times, Increased by about 2.5 times to about 3.0 times, increased by about 3.0 times to about 5 times, increased by about 3.0 times to about 4.5 times, increased by about 3.0 times to about 4.0 times, increased by about 3.0 times to about 3.5 times, increased by about 3.5 times to about 5-fold, about 3.5-fold to about 4.5-fold, about 3.5-fold to about 4.0-fold, about 4.0-fold to about 5-fold, about 4.0-fold to about 4.5-fold, or about 4.5-fold to about 5-fold) (eg, compared to the number or concentration of circulating NK cells (CD3 ⁻ CD16/CD56 ⁺ cells) in the subject prior to administration).

Some embodiments of the method result in an increased number or concentration of CD8 positive memory T cells expressing granzyme B (granzyme B positive, CD8 positive, CD45RA negative cells). In some embodiments, the methods result in at least a 1.1-fold increase, at least a 1.2-fold increase, at least a 1.4-fold increase, at least a 1.6-fold increase in the percentage of CD8-positive memory T cells (CD8-positive, CD45RA-negative cells) expressing granzyme B, Increased by at least 1.8 times, increased by at least 2.0 times, increased by at least 2.2 times, increased by at least 2.4 times, increased by at least 2.6 times, increased by at least 2.8 times, increased by at least 3.0 times, increased by at least 3.2 times, increased by at least 3.4 times, increased by at least 3.6 times, At least a 3.8-fold increase, at least a 4.0-fold increase, at least a 4.2-fold increase, at least a 4.4-fold increase, at least a 4.6-fold increase, at least a 4.8-fold increase, or at least a 5.0-fold increase (e.g., compared to a CD8 positive expression of granzyme B in an individual prior to administration The percentages of memory T cells (CD8 positive, CD45RA negative cells) were compared). In some embodiments, the methods result in an increase in the percentage of CD8-positive memory T cells (CD8-positive, CD45RA-negative cells) expressing granzyme B from about 1.1-fold to about 5-fold (e.g., from about 1.1-fold to about 4.5-fold, Increased by about 1.1 times to about 4.0 times, increased by about 1.1 times to about 3.5 times, increased by about 1.1 times to about 3.0 times, increased by about 1.1 times to about 2.5 times, increased by about 1.1 times to about 2.4 times, increased by about 1.1 times to About 2.2 times, about 1.1 to about 2.0 times, about 1.1 to about 1.8 times, about 1.1 to about 1.6 times, about 1.1 to about 1.4 times, about 1.1 to about 1.2 times, about 1.1 to about 1.2 times About 1.2 times to about 5 times, about 1.2 times to about 4.5 times, about 1.2 times to about 4.0 times, about 1.2 times to about 3.5 times, about 1.2 times to about 3.0 times, about 1.2 times to about 4.0 times 2.5 times, about 1.2 times to about 2.4 times, about 1.2 times to about 2.2 times, about 1.2 times to about 2.0 times, about 1.2 times to about 1.8 times, about 1.2 times to about 1.6 times, about 1.2 times to about 1.6 times 1.2 times to about 1.4 times, about 1.4 times to about 5 times, about 1.4 times to about 4.5 times, about 1.4 times to about 4.0 times, about 1.4 times to about 3.5 times, about 1.4 times to about 3.0 times times, about 1.4 times to about 2.5 times, about 1.4 times to about 2.4 times, about 1.4 times to about 2.2 times, about 1.4 times to about 2.0 times, about 1.4 times to about 1.8 times, about 1.4 times 1.6 times to 1.6 times, 1.6 to 5 times, 1.6 to 4.5 times, 1.6 to 4.0 times, 1.6 to 3.5 times, 1.6 to 3.0 times , increase by about 1.6 times to about 2.5 times, increase by about 1.6 times to about 2.4 times, increase by about 1.6 times to about 2.2 times, increase by about 1.6 times to about 2.0 times, increase by about 1.6 times to about 1.8 times, increase by about 1.8 times to about 5 times, about 1.8 times to about 4.5 times, about 1.8 times to about 4.0 times, about 1.8 times to about 3.5 times, about 1.8 times to about 3.0 times, about 1.8 times to about 2.5 times, about 1.8 times to about 2.5 times About 1.8 times to about 2.4 times, about 1.8 times to about 2.2 times, about 1.8 times to about 2.0 times, about 2.0 times to about 5 times, about 2.0 times to about 4.5 times, about 2.0 times to about 2.0 times 4.0 times, about 2.0 times to about 3.5 times, about 2.0 times to about 3.0 times, about 2.0 times to about 2.5 times, about 2.0 times to about 2.4 times, about 2.0 times to about 2.2 times, about 2.0 times to about 2.2 times 2.2 times to about 5 times, about 2.2 times to about 4.5 times, about 2.2 times to about 4.0 times, about 2.2 times to about 3.5 times, about 2.2 times to about 3.0 times, about 2.2 times to about 2.5 times, about 2.2 times to about 2.4 times, about 2.4 times to about 5 times, about 2.4 times to about 4.5 times, about 2.4 times to about 4.0 times, about 2.4 times to about 3.5 times, Increased by about 2.4 times to about 3.0 times, increased by about 2.4 times to about 2.5 times, increased by about 2.5 times to about 5 times, increased by about 2.5 times to about 4.5 times, increased by about 2.5 times to about 4.0 times, increased by about 2.5 times to About 3.5 times, about 2.5 times to about 3.0 times, about 3.0 times to about 5 times, about 3.0 times to about 4.5 times, about 3.0 times to about 4.0 times, about 3.0 times to about 3.5 times, about 3.0 times to about 3.5 times From about 3.5 times to about 5 times, from about 3.5 times to about 4.5 times, from about 3.5 times to about 4.0 times, from about 4.0 times to about 5 times, from about 4.0 times to about 4.5 times, or from about 4.5 times to about 5-fold) (eg, compared to the percentage of CD8-positive memory T cells (CD8-positive, CD45RA-negative cells) expressing granzyme B in the individual before administration).

Provided herein are methods of treating an individual previously identified or diagnosed as having a B7-H6 positive cancer, the method comprising administering to the individual previously identified or diagnosed as having a B7-H6 positive cancer a therapeutically effective amount of a pharmaceutical composition, the method comprising: A pharmaceutical composition comprising a population of enucleated erythroid cells comprising on their extracellular surface a first exogenous polypeptide comprising (i) IL-15 or a functional fragment, and (ii) IL-15 receptor alpha or a functional fragment thereof. Also provided herein is a method of treating an individual with a B7-H6 positive cancer comprising administering to the individual a therapeutically effective amount of a pharmaceutical composition comprising a population of enucleated erythroid cells, the enucleated erythroid cells The group comprises on its extracellular surface a first exogenous fusion polypeptide comprising (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or its a functional fragment; and a second exogenous polypeptide on the extracellular surface thereof, the second exogenous polypeptide comprising 4-1BBL or a functional fragment thereof.

Provided herein are methods of reducing the number and/or proliferation of B7-H6 positive cancer cells in an individual previously identified or diagnosed as having a B7-H6 positive cancer, the methods comprising administering to the individual a therapeutically effective amount of a pharmaceutical composition, the method comprising administering to the individual a therapeutically effective amount of a pharmaceutical composition, the A pharmaceutical composition comprising a population of enucleated erythroid cells comprising on their extracellular surface a first exogenous polypeptide comprising (i) IL-15 or a functional fragment thereof , and (ii) IL-15 receptor alpha or a functional fragment thereof. Also provided herein is a method of reducing the number and/or proliferation of B7-H6 positive cancer cells in an individual previously identified or diagnosed with a B7-H6 positive cancer, the method comprising administering to the individual a therapeutically effective amount of a pharmaceutical composition, The pharmaceutical composition comprises a population of enucleated erythroid cells comprising on their extracellular surface a first exogenous fusion polypeptide comprising (i) IL -15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof; and a second exogenous polypeptide comprising 4-1BBL or a functional fragment thereof, wherein the exogenous polypeptide is located on its extracellular surface .

Some specific examples of these methods result in a reduction in the number of B7-H6 positive cancer cells in the individual by at least 1%, by at least 2%, by at least 3%, by at least 4%, by at least 5%, by at least 6%, by at least 7% %, at least 8% reduction, at least 9% reduction, at least 10% reduction, at least 15% reduction, at least 20% reduction, at least 25% reduction, at least 30% reduction, at least 35% reduction, at least 40% reduction, at least 45% reduction %, at least 50% reduction, at least 55% reduction, at least 60% reduction, at least 65% reduction, at least 70% reduction, at least 75% reduction, at least 80% reduction, at least 85% reduction, at least 90% reduction, at least 95% reduction %, or at least a 99% reduction (e.g., compared to the number of B7-H6 positive cancer cells in the individual prior to administration). Some specific examples of these methods result in about 1% to about 99% reduction, about 1% to about 90% reduction, about 1% to about 80% reduction, about 1% to about 80% reduction, about 1% reduction in the number of B7-H6 positive cancer cells in an individual % to about 70% reduction, about 1% to about 60% reduction, about 1% to about 50% reduction, about 1% to about 40% reduction, about 1% to about 30% reduction, about 1% reduction % to about 20% reduction, about 1% to about 10% reduction, about 5% to about 99% reduction, about 5% to about 90% reduction, about 5% to about 80% reduction, about 5% reduction % to about 70% reduction, about 5% to about 60% reduction, about 5% to about 50% reduction, about 5% to about 40% reduction, about 5% to about 30% reduction, about 5% reduction % to about 20% reduction, about 5% to about 10% reduction, about 10% to about 99% reduction, about 10% to about 90% reduction, about 10% to about 80% reduction, about 10% reduction % to about 70% reduction, about 10% to about 60% reduction, about 10% to about 50% reduction, about 10% to about 40% reduction, about 10% to about 30% reduction, about 10% reduction % to about 20% reduction, about 20% reduction to about 99% reduction, about 20% reduction to about 90% reduction, about 20% reduction to about 80% reduction, about 20% reduction to about 70% reduction, about 20% reduction % to about 60% reduction, about 20% to about 50% reduction, about 20% to about 40% reduction, about 20% to about 30% reduction, about 30% to about 99% reduction, about 30% reduction % to about 90% reduction, about 30% to about 80% reduction, about 30% to about 70% reduction, about 30% to about 60% reduction, about 30% to about 50% reduction, about 30% reduction % to about 40% reduction, about 40% reduction to about 99% reduction, about 40% reduction to about 90% reduction, about 40% reduction to about 80% reduction, about 40% reduction to about 70% reduction, about 40% reduction % to about 60% reduction, about 40% to about 50% reduction, about 50% to about 99% reduction, about 50% to about 90% reduction, about 50% to about 80% reduction, about 50% reduction % to about 70% reduction, about 50% reduction to about 60% reduction, about 60% reduction to about 99% reduction, about 60% reduction to about 90% reduction, about 60% reduction to about 80% reduction, about 60% reduction % to about 70% reduction, about 70% reduction to about 99% reduction, about 70% reduction to about 90% reduction, about 70% reduction to about 80% reduction, about 80% reduction to about 99% reduction, about 80% reduction % to about 90% reduction, or about 90% reduction to about 99% reduction (eg, compared to the number of B7-H6 positive cancer cells in the subject prior to administration).

Some specific examples of these methods result in at least a 1% reduction, at least a 2% reduction, at least a 3% reduction, at least a 4% reduction, at least a 5% reduction, at least a 6% reduction in the number of B7-H6 positive/HLA-E negative cancer cells in an individual %, at least 7% reduction, at least 8% reduction, at least 9% reduction, at least 10% reduction, at least 15% reduction, at least 20% reduction, at least 25% reduction, at least 30% reduction, at least 35% reduction, at least 40% reduction %, at least 45% reduction, at least 50% reduction, at least 55% reduction, at least 60% reduction, at least 65% reduction, at least 70% reduction, at least 75% reduction, at least 80% reduction, at least 85% reduction, at least 90% reduction %, a reduction of at least 95%, or a reduction of at least 99% (eg, compared to the number of B7-H6 positive/HLA-E negative cancer cells in an individual prior to administration). Some specific examples of these methods result in about 1% to about 99% reduction, about 1% to about 90% reduction, about 1% to about 80% reduction in the number of B7-H6 positive/HLA-E negative cancer cells in an individual %, from about 1% to about 70%, from about 1% to about 60%, from about 1% to about 50%, from about 1% to about 40%, from about 1% to about 30% %, from about 1% to about 20%, from about 1% to about 10%, from about 5% to about 99%, from about 5% to about 90%, from about 5% to about 80% %, from about 5% to about 70%, from about 5% to about 60%, from about 5% to about 50%, from about 5% to about 40%, from about 5% to about 30% %, from about 5% to about 20%, from about 5% to about 10%, from about 10% to about 99%, from about 10% to about 90%, from about 10% to about 80% %, from about 10% to about 70%, from about 10% to about 60%, from about 10% to about 50%, from about 10% to about 40%, from about 10% to about 30% %, from about 10% to about 20%, from about 20% to about 99%, from about 20% to about 90%, from about 20% to about 80%, from about 20% to about 70% %, from about 20% to about 60%, from about 20% to about 50%, from about 20% to about 40%, from about 20% to about 30%, from about 30% to about 99% %, from about 30% to about 90%, from about 30% to about 80%, from about 30% to about 70%, from about 30% to about 60%, from about 30% to about 50% %, from about 30% to about 40%, from about 40% to about 99%, from about 40% to about 90%, from about 40% to about 80%, from about 40% to about 70% %, from about 40% to about 60%, from about 40% to about 50%, from about 50% to about 99%, from about 50% to about 90%, from about 50% to about 80% %, from about 50% to about 70%, from about 50% to about 60%, from about 60% to about 99%, from about 60% to about 90%, from about 60% to about 80% %, from about 60% to about 70%, from about 70% to about 99%, from about 70% to about 90%, from about 70% to about 80%, from about 80% to about 99% %, about 80% reduction to about 90% reduction, or about 90% reduction to about 99% reduction (e.g., compared to the number of B7-H6 positive/HLA-negative cancer cells in an individual prior to administration).

Some embodiments of these methods result in a reduction of at least 1%, a reduction of at least 2%, a reduction of at least 3%, a reduction of at least 4%, a reduction of at least 5%, a reduction of at least 6%, a reduction of at least 7% in the individual %, at least 8% reduction, at least 9% reduction, at least 10% reduction, at least 15% reduction, at least 20% reduction, at least 25% reduction, at least 30% reduction, at least 35% reduction, at least 40% reduction, at least 45% reduction %, at least 50% reduction, at least 55% reduction, at least 60% reduction, at least 65% reduction, at least 70% reduction, at least 75% reduction, at least 80% reduction, at least 85% reduction, at least 90% reduction, at least 95% reduction %, or a reduction of at least 99% (eg, compared to the proliferation of B7-H6 positive cancer cells in an individual prior to administration). Some embodiments of these methods result in about 1% to about 99% reduction, about 1% to about 90% reduction, about 1% to about 80% reduction, about 1% to about 80% reduction, about 1% reduction in the proliferation of B7-H6 positive cancer cells in an individual. % to about 70% reduction, about 1% to about 60% reduction, about 1% to about 50% reduction, about 1% to about 40% reduction, about 1% to about 30% reduction, about 1% reduction % to about 20% reduction, about 1% to about 10% reduction, about 5% to about 99% reduction, about 5% to about 90% reduction, about 5% to about 80% reduction, about 5% reduction % to about 70% reduction, about 5% to about 60% reduction, about 5% to about 50% reduction, about 5% to about 40% reduction, about 5% to about 30% reduction, about 5% reduction % to about 20% reduction, about 5% to about 10% reduction, about 10% to about 99% reduction, about 10% to about 90% reduction, about 10% to about 80% reduction, about 10% reduction % to about 70% reduction, about 10% to about 60% reduction, about 10% to about 50% reduction, about 10% to about 40% reduction, about 10% to about 30% reduction, about 10% reduction % to about 20% reduction, about 20% reduction to about 99% reduction, about 20% reduction to about 90% reduction, about 20% reduction to about 80% reduction, about 20% reduction to about 70% reduction, about 20% reduction % to about 60% reduction, about 20% to about 50% reduction, about 20% to about 40% reduction, about 20% to about 30% reduction, about 30% to about 99% reduction, about 30% reduction % to about 90% reduction, about 30% to about 80% reduction, about 30% to about 70% reduction, about 30% to about 60% reduction, about 30% to about 50% reduction, about 30% reduction % to about 40% reduction, about 40% reduction to about 99% reduction, about 40% reduction to about 90% reduction, about 40% reduction to about 80% reduction, about 40% reduction to about 70% reduction, about 40% reduction % to about 60% reduction, about 40% to about 50% reduction, about 50% to about 99% reduction, about 50% to about 90% reduction, about 50% to about 80% reduction, about 50% reduction % to about 70% reduction, about 50% reduction to about 60% reduction, about 60% reduction to about 99% reduction, about 60% reduction to about 90% reduction, about 60% reduction to about 80% reduction, about 60% reduction % to about 70% reduction, about 70% reduction to about 99% reduction, about 70% reduction to about 90% reduction, about 70% reduction to about 80% reduction, about 80% reduction to about 99% reduction, about 80% reduction % to about 90% reduction, or about 90% reduction to about 99% reduction (eg, compared to the proliferation of B7-H6 positive cancer cells in an individual prior to administration).

Some specific examples of these methods result in at least a 1% reduction, at least a 2% reduction, at least a 3% reduction, at least a 4% reduction, at least a 5% reduction, at least a 6% reduction in the proliferation of B7-H6 positive/HLA-E negative cancer cells in the individual %, at least 7% reduction, at least 8% reduction, at least 9% reduction, at least 10% reduction, at least 15% reduction, at least 20% reduction, at least 25% reduction, at least 30% reduction, at least 35% reduction, at least 40% reduction %, at least 45% reduction, at least 50% reduction, at least 55% reduction, at least 60% reduction, at least 65% reduction, at least 70% reduction, at least 75% reduction, at least 80% reduction, at least 85% reduction, at least 90% reduction %, reduced by at least 95%, or reduced by at least 99% (e.g., compared to proliferation of B7-H6 positive/HLA-E negative cancer cells in an individual prior to administration). Some embodiments of these methods result in about 1% to about 99% reduction, about 1% to about 90% reduction, about 1% to about 80% reduction in proliferation of B7-H6 positive and HLA-E negative cancer cells in an individual %, from about 1% to about 70%, from about 1% to about 60%, from about 1% to about 50%, from about 1% to about 40%, from about 1% to about 30% %, from about 1% to about 20%, from about 1% to about 10%, from about 5% to about 99%, from about 5% to about 90%, from about 5% to about 80% %, from about 5% to about 70%, from about 5% to about 60%, from about 5% to about 50%, from about 5% to about 40%, from about 5% to about 30% %, from about 5% to about 20%, from about 5% to about 10%, from about 10% to about 99%, from about 10% to about 90%, from about 10% to about 80% %, from about 10% to about 70%, from about 10% to about 60%, from about 10% to about 50%, from about 10% to about 40%, from about 10% to about 30% %, from about 10% to about 20%, from about 20% to about 99%, from about 20% to about 90%, from about 20% to about 80%, from about 20% to about 70% %, from about 20% to about 60%, from about 20% to about 50%, from about 20% to about 40%, from about 20% to about 30%, from about 30% to about 99% %, from about 30% to about 90%, from about 30% to about 80%, from about 30% to about 70%, from about 30% to about 60%, from about 30% to about 50% %, from about 30% to about 40%, from about 40% to about 99%, from about 40% to about 90%, from about 40% to about 80%, from about 40% to about 70% %, from about 40% to about 60%, from about 40% to about 50%, from about 50% to about 99%, from about 50% to about 90%, from about 50% to about 80% %, from about 50% to about 70%, from about 50% to about 60%, from about 60% to about 99%, from about 60% to about 90%, from about 60% to about 80% %, from about 60% to about 70%, from about 70% to about 99%, from about 70% to about 90%, from about 70% to about 80%, from about 80% to about 99% %, about 80% reduction to about 90% reduction, or about 90% reduction to about 99% reduction (e.g., compared to the proliferation of B7-H6 positive and HLA-E negative cancer cells in an individual prior to administration).

Provided herein is a method of inducing the killing of B7-H6-positive cancer cells in an individual previously identified or diagnosed as having a B7-H6-positive cancer, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an enucleated A population of erythroid cells comprising on their extracellular surface a first exogenous polypeptide comprising (i) IL-15 or a functional fragment thereof, and (ii) ) IL-15 receptor alpha or a functional fragment thereof. Also provided herein is a method of killing B7-H6 positive cancer cells in an individual previously identified or diagnosed as having a B7-H6 positive cancer, the method comprising administering to the individual a therapeutically effective amount of a pharmaceutical composition comprising A population of enucleated erythroid cells comprising on their extracellular surface a first exogenous fusion polypeptide comprising (i) IL-15 or a functional fragment thereof , and (ii) IL-15 receptor alpha or a functional fragment thereof; and a second exogenous polypeptide comprising 4-1BBL or a functional fragment thereof, wherein the exogenous polypeptide is present on the extracellular surface thereof.

Provided herein is a method of reducing the volume of a solid tumor in an individual previously identified or diagnosed as having a B7-H6 positive cancer, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising a population of enucleated erythroid cells , the group of enucleated erythroid cells includes on its extracellular surface a first exogenous polypeptide comprising (i) IL-15 or a functional fragment thereof, and (ii) an IL-15 receptor body α or its functional fragments. Also provided herein is a method of reducing the volume of a solid tumor in an individual previously identified or diagnosed as having a B7-H6 positive cancer, the method comprising administering to the individual a therapeutically effective amount of a pharmaceutical composition comprising enucleated erythrocytes A group of enucleated erythroid cells comprising a first exogenous fusion polypeptide on its extracellular surface, the first exogenous fusion polypeptide comprising (i) IL-15 or a functional fragment thereof, and (ii ) IL-15 receptor alpha or a functional fragment thereof; and a second exogenous polypeptide comprising 4-1BBL or a functional fragment thereof, wherein the exogenous polypeptide is on an extracellular surface thereof.

Some specific examples of these methods result in at least a 1% reduction, at least a 2% reduction, at least a 3% reduction, at least a 4% reduction, at least a 5% reduction, at least a 6% reduction, at least a 7% reduction, at least an 8% reduction in the solid tumor volume in the individual %, at least 9% reduction, at least 10% reduction, at least 15% reduction, at least 20% reduction, at least 25% reduction, at least 30% reduction, at least 35% reduction, at least 40% reduction, at least 45% reduction, at least 50% reduction %, at least 55% reduction, at least 60% reduction, at least 65% reduction, at least 70% reduction, at least 75% reduction, at least 80% reduction, at least 85% reduction, at least 90% reduction, at least 95% reduction, or at least 99% (e.g. compared to solid tumor volume in the individual prior to administration).

Some embodiments of these methods result in a solid tumor volume reduction of about 1% to about 99%, about 1% to about 90%, about 1% to about 80%, about 1% to about 70% reduction in volume of a solid tumor in an individual %, from about 1% to about 60%, from about 1% to about 50%, from about 1% to about 40%, from about 1% to about 30%, from about 1% to about 20% %, from about 1% to about 10%, from about 5% to about 99%, from about 5% to about 90%, from about 5% to about 80%, from about 5% to about 70% %, from about 5% to about 60%, from about 5% to about 50%, from about 5% to about 40%, from about 5% to about 30%, from about 5% to about 20% %, from about 5% to about 10%, from about 10% to about 99%, from about 10% to about 90%, from about 10% to about 80%, from about 10% to about 70% %, from about 10% to about 60%, from about 10% to about 50%, from about 10% to about 40%, from about 10% to about 30%, from about 10% to about 20% %, from about 20% to about 99%, from about 20% to about 90%, from about 20% to about 80%, from about 20% to about 70%, from about 20% to about 60% %, from about 20% to about 50%, from about 20% to about 40%, from about 20% to about 30%, from about 30% to about 99%, from about 30% to about 90% %, from about 30% to about 80%, from about 30% to about 70%, from about 30% to about 60%, from about 30% to about 50%, from about 30% to about 40% %, from about 40% to about 99%, from about 40% to about 90%, from about 40% to about 80%, from about 40% to about 70%, from about 40% to about 60% %, from about 40% to about 50%, from about 50% to about 99%, from about 50% to about 90%, from about 50% to about 80%, from about 50% to about 70% %, from about 50% to about 60%, from about 60% to about 99%, from about 60% to about 90%, from about 60% to about 80%, from about 60% to about 70% %, from about 70% to about 99%, from about 70% to about 90%, from about 70% to about 80%, from about 80% to about 99%, from about 80% to about 90% %, or about 90% to about 99% reduction (e.g., compared to the solid tumor volume before administration).

In some embodiments of any of the methods described herein, the individual is an adult individual. In some embodiments of any of the methods described herein, the individual is a pediatric human individual.

Non-limiting aspects of these compositions and methods are described below. As those in the art appreciate, the exemplary aspects listed below can be used in any combination and combined with other aspects known in the art. NKp30

As used herein, NKp30 polypeptide refers to the amino acid sequence encoded by the natural cytotoxicity triggering receptor 3 (NCR3) gene. Exemplary NKp30 polypeptides include, but are not limited to, the amino acid sequences corresponding to NCBI reference sequences: NP_001138938.1, NP_001138939.1, and NP_667341.1.

In some embodiments, the NKp30 polypeptide comprises a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical to a sequence % identity) amino acid sequence: MAWMLLLILIMVHPGSCA LWVSQPPEIRTLEGSSAFLPCSFNASQGRLAIGSVTWFRDEVVPGKEVRNGTPEFRGRLAPLASSRFLHDHQAELHIRDVRGHDASIYVCRVEVLGLGVGTGNGTRLVVEKEHPQLGAGTVLLLRAGFYAVSFLSVAVGSTVYYQGKYAKSTLSGFPQL: (with sequence number) or not. The underlined part of the above SEQ ID NO: 1 represents the message peptide.

In some embodiments, the NKp30 polypeptide consists of a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) % identical) encoded by the nucleic acid sequence: ATGGCCTGGATGCTGTTGCTCATCTTGATCATGGTCCATCCAGGATCCTGTGCTCTCTGGGTGTCCCAGCCCCCTGAGATTCGTACCCTGGAAGGATCCTCTGCCTTCCTGCCCTGCTCCTTCAATGCCAGCCAAGGGAGACTGGCCATTGGCTCCGTCACGTGGTTCCGAGATGAGGTGGTTCCAGGGAAGGAGGTGAGGAATGGAACCCCAGAGTTCAGGGGCCGCCTGGCCCCACTTGCTTCTTCCCGTTTCCTCCATGACCACCAGGCTGAGCTGCACATCCGGGACGTGCGAGGCCATGACGCCAGCATCTACGTGTGCAGAGTGGAGGTGCTGGGCCTTGGTGTCGGGACAGGGAATGGGACTCGGCTGGTGGTGGAGAAAGAACATCCTCAGCTAGGGGCTGGTACAGTCCTCCTCCTTCGGGCTGGATTCTATGCTGTCAGCTTTCTCTCTGTGGCCGTGGGCAGCACCGTCTATTACCAGGGCAAATATGCCAAATCTACTCTCTCCGGATTCCCCCAACTCTGA (SEQ ID NO: 2)。

In some embodiments, the NKp30 polypeptide comprises a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical to a sequence % identity) Amino acid sequence: MAWMLLLILIMVHPGSCAL WVSQPPEIRTLEGSSAFLPCSFNASQGRLAIGSVTWFRDEVVPGKEVRNGTPEFRGRLAPLASSRFLHDHQAELHIRDVRGHDASIYVCRVEVLGLGVGTGNGTRLVVEKEHPQLGAGTVLLLRAGFYAVSFLSVAVGSTVYYQGKCHQNOSECHSSDGPRG IDEPEP sequence number has. The underlined part of the above SEQ ID NO: 3 represents the message peptide.

In some embodiments, the NKp30 polypeptide consists of a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) % identical) encoded by the nucleic acid sequence: ATGGCCTGGATGCTGTTGCTCATCTTGATCATGGTCCATCCAGGATCCTGTGCTCTCTGGGTGTCCCAGCCCCCTGAGATTCGTACCCTGGAAGGATCCTCTGCCTTCCTGCCCTGCTCCTTCAATGCCAGCCAAGGGAGACTGGCCATTGGCTCCGTCACGTGGTTCCGAGATGAGGTGGTTCCAGGGAAGGAGGTGAGGAATGGAACCCCAGAGTTCAGGGGCCGCCTGGCCCCACTTGCTTCTTCCCGTTTCCTCCATGACCACCAGGCTGAGCTGCACATCCGGGACGTGCGAGGCCATGACGCCAGCATCTACGTGTGCAGAGTGGAGGTGCTGGGCCTTGGTGTCGGGACAGGGAATGGGACTCGGCTGGTGGTGGAGAAAGAACATCCTCAGCTAGGGGCTGGTACAGTCCTCCTCCTTCGGGCTGGATTCTATGCTGTCAGCTTTCTCTCTGTGGCCGTGGGCAGCACCGTCTATTACCAGGGCAAATGCCACTGTCACATGGGAACACACTGCCACTCCTCAGATGGGCCCCGAGGAGTGATTCCAGAGCCCAGATGTCCCTAG (SEQ ID NO: 4)。

In some embodiments, the NKp30 polypeptide comprises a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical to a sequence %一致)的胺基酸序列： MAWMLLLILIMVHPGSCAL WVSQPPEIRTLEGSSAFLPCSFNASQGRLAIGSVTWFRDEVVPGKEVRNGTPEFRGRLAPLASSRFLHDHQAELHIRDVRGHDASIYVCRVEVLGLGVGTGNGTRLVVEKEHPQLGAGTVLLLRAGFYAVSFLSVAVGSTVYYQGKCLTWKGPRRQLPAVVPAPLPPPCGSSAHLLPPVPGG (SEQ ID NO: 5) (具有或不具有訊號序列)。 The underlined part of the above SEQ ID NO: 5 represents the message peptide.

In some embodiments, the NKp30 polypeptide consists of a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) % identical) encoded by the nucleic acid sequence: ATGGCCTGGATGCTGTTGCTCATCTTGATCATGGTCCATCCAGGATCCTGTGCTCTCTGGGTGTCCCAGCCCCCTGAGATTCGTACCCTGGAAGGATCCTCTGCCTTCCTGCCCTGCTCCTTCAATGCCAGCCAAGGGAGACTGGCCATTGGCTCCGTCACGTGGTTCCGAGATGAGGTGGTTCCAGGGAAGGAGGTGAGGAATGGAACCCCAGAGTTCAGGGGCCGCCTGGCCCCACTTGCTTCTTCCCGTTTCCTCCATGACCACCAGGCTGAGCTGCACATCCGGGACGTGCGAGGCCATGACGCCAGCATCTACGTGTGCAGAGTGGAGGTGCTGGGCCTTGGTGTCGGGACAGGGAATGGGACTCGGCTGGTGGTGGAGAAAGAACATCCTCAGCTAGGGGCTGGTACAGTCCTCCTCCTTCGGGCTGGATTCTATGCTGTCAGCTTTCTCTCTGTGGCCGTGGGCAGCACCGTCTATTACCAGGGCAAATGTCTGACCTGGAAAGGTCCAAGAAGGCAGCTGCCGGCTGTGGTCCCAGCGCCCCTCCCACCACCATGTGGGAGCTCAGCACATCTGCTTCCCCCAGTCCCAGGAGGCTGA (SEQ ID NO: 6)。

In some embodiments, the methods comprise detecting the number of NKp30 positive lymphocytes in the individual. Any suitable sample containing NKp30 positive lymphocytes can be obtained from an individual. In some embodiments, whole blood samples, plasma samples, serum samples, samples containing cell fractions (PBMC) of whole blood samples, tissue samples, biopsy samples, and laser capture cleaved biological samples ( For example, tumor or tissue samples) to determine the number of NKp30 positive lymphocytes. In some embodiments, the number of NKp30-positive lymphocytes is detected before, simultaneously with, or after administration of a therapeutically effective amount of a pharmaceutical composition comprising a population of enucleated erythroid cells (e.g., any of the exemplary enucleated erythroid cells). In some embodiments, NKp30 positive cells can be assayed using any antibody or antigen-binding fragment thereof that specifically binds to NKp30 (eg, Beckman Coulter, clone Z25). In some embodiments, NKp30 protein expression can be assessed using fluorescence-assisted cell sorting (FACS) (e.g., using compensation beads (e.g., Bangs beads)) or by other immunological-based methods, including But not limited to Western blot analysis, ELISA, tissue array analysis, in situ hybridization, and immunofluorescence using, for example, antibodies or antigen-binding fragments thereof that specifically bind to NKp30. In some embodiments, NKp30 positive cells can be determined by measuring NKp30 mRNA. Non-limiting methods of quantifying RNA include: qRT-PCR, RNA sequencing, fluorescence in situ hybridization combined with flow cytometry, microfluidic capillary electrophoresis, and in situ hybridization.

NKp30-positive lymphocytes are determined by comparing the NKp30 content in lymphocytes to a reference content (e.g., NKp30 content in control non-activated or resting lymphocytes, or NKp30 content in cells that are not lymphocytes) as described herein . In some embodiments, the reference level of NKp30 can be one transcript per million transcripts. B7-H6

As used herein, B7-H6 polypeptide refers to the amino acid sequence encoded by the natural killer cell cytotoxicity receptor 3 ligand 1 (NCR3LG1) gene. Exemplary B7-H6 polypeptides include, but are not limited to, the amino acid sequence corresponding to NCBI Reference Sequence: NP_001189368.1. B7-H6, a member of the B7 immunoreceptor family, is the cell surface ligand of NKp30.

In some embodiments, the B7-H6 polypeptide can comprise a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical) ，或100%一致)的序列： MTWRAAASTCAALLILLWALTTEG DLKVEMMAGGTQITPLNDNVTIFCNIFYSQPLNITSMGITWFWKSLTFDKEVKVFEFFGDHQEAFRPGAIVSPWRLKSGDASLRLPGIQLEEAGEYRCEVVVTPLKAQGTVQLEVVASPASRLLLDQVGMKENEDKYMCESSGFYPEAINITWEKQTQKFPHPIEISEDVITGPTIKNMDGTFNVTSCLKLNSSQEDPGTVYQCVVRHASLHTPLRSNFTLTAARHSLSETEKTDNFSIHWWPISFIGVGLVLLIVLIPWKKICNKSSSAYTPLKCILKHWNSFDTQTLKKEHLIFFCTRAWPSYQLQDGEAWPPEGSVNINTIQQLDVFCRQEGKWSEVPYVQAFFALRDNPDLCQCCRIDPALLTVTSGKSIDDNSTKSEKQTPREHSDAVPDAPILPVSPIWEPPPATTSTTPVLSSQPPTLLLPLQ (SEQ ID NO: 7) (具有或不具有訊號序列)。 The underlined part of the above SEQ ID NO: 7 represents the message peptide.

In some embodiments, the B7-H6 polypeptide consists of a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) nucleic acid sequence encoded by: ATGACGTGGAGGGCTGCCGCCTCCACGTGCGCGGCGCTCCTGATTCTGCTGTGGGCGCTGACGACCGAAGGTGATCTGAAAGTAGAGATGATGGCAGGGGGGACTCAGATCACACCCCTGAATGACAATGTCACCATATTCTGCAATATCTTTTATTCCCAACCCCTCAACATCACGTCTATGGGTATCACCTGGTTTTGGAAGAGTCTGACGTTTGACAAAGAAGTCAAAGTCTTTGAATTTTTTGGAGATCACCAAGAGGCATTCCGACCTGGAGCCATTGTGTCTCCATGGAGGCTGAAGAGTGGGGACGCCTCACTGCGGCTGCCTGGAATCCAGCTGGAGGAAGCAGGAGAGTACCGATGTGAGGTGGTGGTCACCCCTCTGAAGGCACAGGGAACAGTCCAGCTTGAAGTTGTGGCTTCCCCAGCCAGCAGATTGTTGCTGGATCAAGTGGGCATGAAAGAGAATGAAGACAAATATATGTGTGAGTCAAGTGGGTTCTACCCAGAGGCTATTAATATAACATGGGAGAAGCAGACCCAGAAGTTTCCCCATCCCATAGAGATTTCTGAGGATGTCATCACTGGTCCCACCATCAAGAATATGGATGGCACATTTAATGTCACTAGCTGCTTGAAGCTGAACTCCTCTCAGGAAGACCCTGGGACTGTCTACCAGTGTGTGGTACGGCATGCGTCCTTGCATACCCCCTTGAGGAGCAACTTTACCCTGACTGCTGCTCGGCACAGTCTTTCTGAAACTGAGAAGACAGATAATTTTTCCATTCATTGGTGGCCTATTTCATTCATTGGTGTTGGACTGGTTTTATTAATTGTTTTGATTCCTTGGAAAAAGATATGTAACAAATCATCTTCAGCCTATACTCCTCTCAAGTGCATTCTGAAACACTGGAACTCCTTTGACACTCAGACTCTGAAGAAAGAGCACCTCATATTCTTTTGCACTCGGGCATGGCCGTCTTACCAGCTGCAGGATG GGGAGGCTTGGCCTCCTGAGGGAAGTGTTAATATTAATACTATTCAACAACTAGATGTTTTCTGCAGACAGGAGGGCAAATGGTCCGAGGTTCCTTATGTGCAAGCCTTCTTTGCCTTGCGAGACAACCCAGATCTTTGTCAGTGTTGTAGAATTGACCCTGCTCTCCTAACAGTTACATCAGGCAAGTCCATAGATGATAATTCCACAAAGTCTGAGAAACAAACCCCTAGGGAACACTCGGATGCAGTTCCGGATGCCCCAATCCTTCCTGTCTCCCCTATCTGGGAACCTCCTCCAGCCACAACATCAACAACTCCAGTTCTATCCTCCCAACCCCCAACTTTACTGTTACCCCTACAGTAA (SEQ ID NO: 8)。

In some embodiments, the methods comprise detecting the presence of B7-H6 positive cancer cells in the individual. Any suitable sample containing B7-H6 positive cancer cells can be obtained from an individual. In some embodiments, whole blood samples, plasma samples, serum samples, samples containing cell fractions (PBMC) of whole blood samples, tissue samples, biopsy samples, and laser capture cleaved biological samples ( For example, a tumor or tissue sample) to determine the number of B7-H6 positive cancer cells. In some embodiments, B7-H6 positive cancer cells are detected before, concurrently with, or after administration of the enucleated erythroid cells. In some embodiments, any antibody or antigen-binding fragment thereof that specifically binds to B7-H6 (eg, B7-H6 (Abcam)) can be used to detect B7-H6 positive cancer cells. B7-H6 protein expression can be assessed using fluorescence-assisted cell sorting (FACS) or by other immunological-based methods including, but not limited to, Western blotting, ELISA, and immunofluorescence. In some embodiments, B7-H6 positive cells can be determined by measuring B7-H6 mRNA. Non-limiting methods of quantifying RNA include: qRT-PCR, RNA sequencing, fluorescence in situ hybridization combined with flow cytometry, microfluidic capillary electrophoresis, and in situ hybridization.

Exemplary B7-H6 reference levels that can be used to identify B7-H6 positive cancer cells or B7-H6 positive cancers can be B7-H6 levels in non-cancerous cells, B7-H6 median levels in multiple tumor biopsy samples, higher than One standard deviation of the median B7-H6 content in multiple tumor biopsy samples of the same type of cancer or multiple different cancers, higher than the median value of B7-H6 in multiple tumor biopsy samples of the same type of cancer or multiple different cancers One-quarter standard deviation of the B7-H6 median content in multiple tumor biopsy samples of the same type of cancer or multiple different cancers, one-half standard deviation of the median content of B7-H6 in multiple tumor biopsy samples, Three-quarters of the standard deviation of the median B7-H6 content in multiple tumor biopsy samples from a different cancer, higher than the median B7-H6 content in multiple tumor biopsy samples from the same type of cancer or multiple different cancers One and one-half standard deviations above, or two standard deviations above the median B7-H6 level in multiple tumor biopsy samples of the same type of cancer or multiple different cancers.

In some embodiments, the reference level of B7-H6 is the level of B7-H6 in normal (non-cancerous) tissue of the same type. In some embodiments, the reference level of B7-H6 is the level of B7-H6 in healthy tissues adjacent to solid tumors. In some embodiments, the reference level is a level that is 50% higher than the level of B7-H6 in adjacent or corresponding healthy tissue. NK2A

As used herein, NKG2A polypeptide refers to the amino acid sequence encoded by the killer lectin-like receptor C1 (KLRCl) gene. NKG2A forms a complex with KLRD1/CD94. Exemplary KLRCl polypeptides include, but are not limited to, the amino acid sequences corresponding to NCBI reference sequences: NP_001291377.1, NP_002250.2, and NP_998823.1.

In some embodiments, the NKG2A polypeptide comprises a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) % consistent) sequence: MDNQGVIYSDLNLPPNPKRQQRKPKGNKNSILATEQEITYAELNLQKASQDFQGNDKTYHCKDLPSAPEKLIVGILGIICLILMASVVTIVVIPSTLIQRHNNSSLNTRTQKARHCGHCPEEWITYSNSCYYIGKERRTWEESLLACTSKNSSLLSIDNEEEMKFLSIISPSSWIGVFRNSSHHPWVTMNGLAFKHEIKDSDNAELNCAVLQVNRLKSAQCGSSIIYHCKHKL (SEQ ID NO: 9)。

In some embodiments, the NKG2A polypeptide consists of a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) % identical) encoded by the nucleic acid sequence: ATGGATAACCAAGGAGTAATCTACTCAGACCTGAATCTGCCCCCAAACCCAAAGAGGCAGCAACGAAAACCTAAAGGCAATAAAAACTCCATTTTAGCAACTGAACAGGAAATAACCTATGCGGAATTAAACCTTCAAAAAGCTTCTCAGGATTTTCAAGGGAATGACAAAACCTATCACTGCAAAGATTTACCATCAGCTCCAGAGAAGCTCATTGTTGGGATCCTGGGAATTATCTGTCTTATCTTAATGGCCTCTGTGGTAACGATAGTTGTTATTCCCTCTACATTAATACAGAGGCACAACAATTCTTCCCTGAATACAAGAACTCAGAAAGCACGTCATTGTGGCCATTGTCCTGAGGAGTGGATTACATATTCCAACAGTTGTTACTACATTGGTAAGGAAAGAAGAACTTGGGAAGAGAGTTTGCTGGCCTGTACTTCGAAGAACTCCAGTCTGCTTTCTATAGATAATGAAGAAGAAATGAAATTTCTGTCCATCATTTCACCATCCTCATGGATTGGTGTGTTTCGTAACAGCAGTCATCATCCATGGGTGACAATGAATGGTTTGGCTTTCAAACATGAGATAAAAGACTCAGATAATGCTGAACTTAACTGTGCAGTGCTACAAGTAAATCGACTTAAATCAGCCCAGTGTGGATCTTCAATAATATATCATTGTAAGCATAAGCTTTAG (SEQ ID NO: 10)。

In some embodiments, the NKG2A polypeptide can comprise a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% consistent) sequence: MDNQGVIYSDLNLPPNPKRQQRKPKGNKSSILATEQEITYAELNLQKASQDFQGNDKTYHCKDLPSAPEKLIVGILGIICLILMASVVTIVVIPSTLIQRHNNSSLNTRTQKARHCGHCPEEWITYSNSCYYIGKERRTWEESLLACTSKNSSLLSIDNEEEMKFLSIISPSSWIGVFRNSSHHPWVTMNGLAFKHEIKDSDNAELNCAVLQVNRLKSAQCGSSIIYHCKHKL (SEQ ID NO: 11)。

In some embodiments, the NKG2A polypeptide consists of a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) % identical) encoded by the nucleic acid sequence: ATGGATAACCAAGGAGTAATCTACTCAGACCTGAATCTGCCCCCAAACCCAAAGAGGCAGCAACGAAAACCTAAAGGCAATAAAAACTCCATTTTAGCAACTGAACAGGAAATAACCTATGCGGAATTAAACCTTCAAAAAGCTTCTCAGGATTTTCAAGGGAATGACAAAACCTATCACTGCAAAGATTTACCATCAGCTCCAGAGAAGCTCATTGTTGGGATCCTGGGAATTATCTGTCTTATCTTAATGGCCTCTGTGGTAACGATAGTTGTTATTCCCTCTACATTAATACAGAGGCACAACAATTCTTCCCTGAATACAAGAACTCAGAAAGCACGTCATTGTGGCCATTGTCCTGAGGAGTGGATTACATATTCCAACAGTTGTTACTACATTGGTAAGGAAAGAAGAACTTGGGAAGAGAGTTTGCTGGCCTGTACTTCGAAGAACTCCAGTCTGCTTTCTATAGATAATGAAGAAGAAATGAAATTTCTGTCCATCATTTCACCATCCTCATGGATTGGTGTGTTTCGTAACAGCAGTCATCATCCATGGGTGACAATGAATGGTTTGGCTTTCAAACATGAGATAAAAGACTCAGATAATGCTGAACTTAACTGTGCAGTGCTACAAGTAAATCGACTTAAATCAGCCCAGTGTGGATCTTCAATAATATATCATTGTAAGCATAAGCTTTAG(SEQ ID NO: 12)。

In some embodiments, the methods comprise detecting NKG2A levels in lymphocytes in the individual. Any suitable sample containing NKp30-positive/NKG2A-negative lymphocytes can be obtained from an individual. In some embodiments, whole blood samples, plasma samples, serum samples, samples containing cell fractions (PBMC) of whole blood samples, tissue samples, biopsy samples, and laser capture cleaved biological samples ( For example, tumor or tissue samples) to determine the number of NKp30-positive/NKG2A-negative lymphocytes. In some embodiments, the level of NKG2A in lymphocytes is detected before, concurrently with, or after administration of the therapeutic enucleated erythroid cells. In some embodiments, NKG2A-negative lymphocytes can be assayed using an antibody or antigen-binding fragment thereof that specifically binds to NKG2A (eg, Beckman Coulter, clone Z199.1). In some embodiments, NKG2A levels in lymphocytes can be assessed using fluorescence-assisted cell sorting. NKG2A levels in lymphocytes can also be assessed by other immunological-based methods, including but not limited to Western blotting, ELISA, and immunofluorescence. In some embodiments, NKG2A positive cells can be determined by measuring NKG2A mRNA. Non-limiting methods of quantifying RNA include: qRT-PCR, RNA sequencing, fluorescence in situ hybridization combined with flow cytometry, microfluidic capillary electrophoresis, and in situ hybridization.

Exemplary NKG2A reference levels that can be used to identify NKG2A negative lymphocytes are known in the art. For example, the reference level may be the level of NKG2A in activated or proliferating lymphocytes.

In some embodiments, the methods comprise modulating the activity of an NKG2A polypeptide. In some embodiments, the method further comprises administering to the individual an NKG2A inhibitor (eg, an antagonist antibody). For example, the method can comprise administering to the individual monalizumab (see Andre et al., Cell 175(7):1731-1743 before, concurrently with, or after administration of any of the enucleated erythroid cells described herein ; 2018) or dasatinib (Chang et al., Front. Immunol. 9:3152l; 2019). In some embodiments, the method further comprises administering S095029 to the individual before, concurrently with, or after administering any of the enucleated erythroid cells described herein. HLA-E

As used herein, an HLA-E polypeptide refers to the amino acid sequence encoded by the major histocompatibility complex class I E (HLA-E) gene. Exemplary HLA-E polypeptides include, but are not limited to, the amino acid sequence corresponding to NCBI Reference Sequence: NP_005507.3.

In some embodiments, the HLA-E polypeptide comprises at least 70% identity (e.g., at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 99% identity) with the following sequence, or 100% identical) amino acid sequence: MVDGTLLLLLSEALALTQTWAGSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRFDNDAASPRMVPRAPWMEQEGSEYWDRETRSARDTAQIFRVNLRTLRGYYNQSEAGSHTLQWMHGCELGPDGRFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDASEAEHQRAYLEDTCVEWLHKYLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQQDGEGHTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPVTLRWKPASQPTIPIVGIIAGLVLLGSVVSGAVVAAVIWRKKSSGGKGGSYSKAEWSDSAQGSESHSL (SEQ ID NO: 13)。

In some embodiments, the HLA-E polypeptide consists of a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical to, or 100% identical) nucleic acid sequence encoded by: ATGGTAGATGGAACCCTCCTTTTACTCCTCTCGGAGGCCCTGGCCCTTACCCAGACCTGGGCGGGCTCCCACTCCTTGAAGTATTTCCACACTTCCGTGTCCCGGCCCGGCCGCGGGGAGCCCCGCTTCATCTCTGTGGGCTACGTGGACGACACCCAGTTCGTGCGCTTCGACAACGACGCCGCGAGTCCGAGGATGGTGCCGCGGGCGCCGTGGATGGAGCAGGAGGGGTCAGAGTATTGGGACCGGGAGACACGGAGCGCCAGGGACACCGCACAGATTTTCCGAGTGAATCTGCGGACGCTGCGCGGCTACTACAATCAGAGCGAGGCCGGGTCTCACACCCTGCAGTGGATGCATGGCTGCGAGCTGGGGCCCGACGGGCGCTTCCTCCGCGGGTATGAACAGTTCGCCTACGACGGCAAGGATTATCTCACCCTGAATGAGGACCTGCGCTCCTGGACCGCGGTGGACACGGCGGCTCAGATCTCCGAGCAAAAGTCAAATGATGCCTCTGAGGCGGAGCACCAGAGAGCCTACCTGGAAGACACATGCGTGGAGTGGCTCCACAAATACCTGGAGAAGGGGAAGGAGACGCTGCTTCACCTGGAGCCCCCAAAGACACACGTGACTCACCACCCCATCTCTGACCATGAGGCCACCCTGAGGTGCTGGGCCCTGGGCTTCTACCCTGCGGAGATCACACTGACCTGGCAGCAGGATGGGGAGGGCCATACCCAGGACACGGAGCTCGTGGAGACCAGGCCTGCAGGGGATGGAACCTTCCAGAAGTGGGCAGCTGTGGTGGTGCCTTCTGGAGAGGAGCAGAGATACACGTGCCATGTGCAGCATGAGGGGCTACCCGAGCCCGTCACCCTGAGATGGAAGCCGGCTTCCCAGCCCACCATCCCCATCGTGGGCATCATTGCTGGCCTGGTTCTCCTTGGATCTGTGGTCTCTGGAGCTGTGGTTGCTGCTGTGATATGGAGGAAGAAGAGCT CAGGTGGAAAAGGAGGGAGCTACTCTAAGGCTGAGTGGAGCGACAGTGCCCAGGGGTCTGAGTCTCACAGCTTGTAA (SEQ ID NO: 14).

In some embodiments, the methods comprise detecting HLA-E levels in cancer cells or in individual cancer cells. Any suitable sample containing HLA-E cancer cells can be obtained from an individual. In some embodiments, whole blood, plasma, cellular fractions of whole blood (PBMC), tissue samples, biopsy samples, and laser capture microdissected tumor samples or biopsy samples can be obtained and evaluated to identify HLA- E content in individual cancer cells. In some embodiments, the level of HLA-E in cancer cells is detected before, simultaneously or after administration of enucleated erythroid cells. In some embodiments, HLA-E negative cancer cells can be assayed using antibodies or antigen-binding fragments thereof that specifically bind to HLA-E (eg, Beckman Coulter, clone Z199.1). In some embodiments, the HLA-E content in cancer cells can be determined using fluorescence-assisted cell sorting. HLA-E levels in cancer cells can also be assessed by other immunological-based methods, including but not limited to Western blotting, ELISA, and immunofluorescence. In some embodiments, HLA-E negative cancer cells can be determined by measuring HLA-E mRNA. Non-limiting methods of quantifying RNA include: qRT-PCR, RNA sequencing, fluorescence in situ hybridization combined with flow cytometry, microfluidic capillary electrophoresis, and in situ hybridization. In some embodiments, the HLA-E negative cancer cells are cancer cells that have lost heterozygosity at the HLA-E locus.

An exemplary HLA-E reference level that can be used to identify HLA-E negative cancer cells can be the level of HLA-E present in non-cancer cells or cancer cells that have not lost heterozygosity at the HLA-E locus. Exemplary levels of HLA-E can be determined using the method described in Seliger et al., Oncotarget 7(41):67360-67372, 2016. In some embodiments, the reference level of B7-H6 is the level of B7-H6 in healthy tissues adjacent to solid tumors. In some embodiments, the reference level is a level that is 50% less than the level of HLA-E in adjacent tissue or corresponding healthy tissue. first exogenous polypeptide

In some embodiments, the invention features enucleated erythroid blood cells comprising on their extracellular surface a first exogenous polypeptide comprising (i) interleukin 15 (IL-15) or a functional fragment thereof, and (ii) interleukin-15 receptor alpha (IL-15RA) or a functional fragment thereof.

In some embodiments, the IL-15 comprises an immature form of wild-type human IL-15. In some embodiments, IL-15 comprises at least 70% identity (e.g., at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 99% identity, or 100% identical) amino acid sequence: MRISKPHLRSISIQCYLCLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 15), or a functional fragment thereof. In some embodiments, IL-15 comprises the sequence of SEQ ID NO:15.

In some embodiments, IL-15 comprises a mature form of wild-type human IL-15 or a functional fragment thereof. In some embodiments, IL-15 comprises at least 70% identity (e.g., at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 99% identity, or 100% identical) amino acid sequence: NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 16), or a functional fragment thereof. In some embodiments, IL-15 comprises the sequence of SEQ ID NO:16.

In some embodiments, IL-15 consists of a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) encoded by the nucleic acid sequence: AACTGGGTGAACGTTATTAGTGACCTTAAAAAGATCGAAGATTTGATACAGTCAATGCACATAGACGCGACGCTTTATACAGAATCTGATGTACATCCTTCATGCAAGGTTACTGCTATGAAGTGTTTTCTTCTCGAACTCCAAGTAATAAGTCTTGAGAGCGGAGATGCGAGCATTCATGACACCGTTGAGAATCTTATTATATTGGCTAACAACTCTCTGTCCAGCAATGGTAATGTGACAGAAAGCGGGTGTAAGGAGTGCGAGGAACTCGAGGAGAAGAACATCAAAGAGTTCTTGCAGTCTTTCGTCCATATTGTCCAGATGTTCATAAATACTAGC (SEQ ID NO: 17)。

In some embodiments, the IL-15 polypeptide consists of a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical to, or 100% identical) nucleic acid sequence encoded by: AACTGGGTAAACGTCATAAGCGACCTCAAAAAGATCGAGGACCTTATACAGTCTATGCACATAGATGCGACACTTTACACGGAATCAGACGTGCACCCGTCCTGCAAAGTCACAGCCATGAAGTGCTTTCTTCTCGAACTGCAGGTAATTTCTCTCGAATCAGGTGACGCATCTATCCACGACACAGTTGAAAATCTTATTATCCTGGCTAATAACTCCCTTAGCTCAAACGGCAATGTCACCGAGAGTGGATGTAAAGAATGTGAGGAACTTGAAGAGAAAAACATAAAGGAATTCTTGCAGAGTTTCGTTCATATTGTGCAAATGTTCATCAATACTAGT (SEQ ID NO: 18)。

In some embodiments, the functional fragment of IL-15 comprises at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 or 160 amino acids. In some embodiments, the functional fragment of IL-15 comprises less than 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 or 160 amino acids. In some embodiments, the functional fragment of IL-15 retains at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% of the ability of wild-type human IL-15 polypeptide to bind IL-15RA polypeptide %, 90%, 95%, 98% or 99%, as measured by assays well known in the art, such as ELISA and surface plasmon resonance (SPR) binding assays, or co-immunoprecipitation. In some embodiments, the functional fragment of the IL-15 polypeptide retains at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%, as measured by assays well known in the art, such as electromobility assays, ELISAs, and other immunoassays. In some embodiments, the functional fragment of the IL-15 polypeptide can be an IL-15 receptor binding fragment of the IL-15 polypeptide.

In some embodiments, interleukin-15 receptor alpha (IL-15RA) or a functional fragment thereof can comprise an immature form of wild-type human IL-15RA. In some embodiments, IL-15RA can comprise at least 70% identity (e.g., at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 99% identity) with the following sequence, or 100% identical) sequence: MAPRRARGCRTLGLPALLLLLLLRPPATRGITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL (SEQ ID NO：19)或其功能片段。 In some embodiments, the IL-15RA polypeptide comprises the sequence of SEQ ID NO:19.

In some embodiments, the IL-15RA comprises a mature form of wild-type human IL-15RA. In some embodiments, IL-15RA comprises at least 70% identity (e.g., at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 99% identity, or 100% identical) amino acid sequence: ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL (SEQ ID NO: 20)或其功能片段。 In some embodiments, IL-15RA comprises the sequence of SEQ ID NO:20.

In some embodiments, IL-15RA comprises an extracellular portion of an IL-15RA polypeptide. For example, an IL-15RA polypeptide may lack the transmembrane domain of wild-type IL-15RA, and optionally lack the intracellular domain of wild-type IL-15RA. In some embodiments, the IL-15RA comprises an immature form of extracellular wild-type human IL-15RA. In some embodiments, the IL-15RA polypeptide can comprise a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical , or 100% identical) sequence: MAPRRARGCRTLGLPALLLLLLLLRPPATRGITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTT: (SEQ ID 2NO), or a functional fragment thereof In some embodiments, the IL-15RA polypeptide comprises the sequence of SEQ ID NO:21.

In some embodiments, the IL-15RA comprises a mature form of extracellular wild-type human IL-15RA. In some embodiments, IL-15RA can comprise at least 70% identity (e.g., at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 99% identity) with the following sequence, or 100% identical) sequence: ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTT (SEQ ID NO: 22) or a functional fragment thereof. In some embodiments, the IL-15RA polypeptide comprises the sequence of SEQ ID NO:22.

In some embodiments, the IL-15RA consists of a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) encoded by the nucleic acid sequence: ATCACATGCCCACCGCCCATGTCTGTTGAACACGCAGACATTTGGGTTAAAAGTTACTCACTTTACTCACGCGAGAGATATATATGCAACAGCGGCTTCAAGCGCAAAGCAGGCACTAGTAGTCTTACAGAGTGCGTGCTCAATAAAGCTACAAATGTAGCTCATTGGACTACTCCTAGTCTCAAATGCATTCGGGACCCCGCGCTTGTGCACCAGAGACCTGCGCCGCCGTCCACAGTGACGACAGCTGGTGTAACCCCCCAACCTGAATCCCTTAGTCCGTCTGGTAAAGAACCGGCGGCGTCTTCACCTTCCAGCAATAATACTGCGGCGACAACAGCCGCGATAGTTCCTGGATCCCAACTCATGCCGTCAAAGTCTCCTTCAACGGGAACGACAGAGATCTCTTCACATGAAAGTTCTCATGGAACACCGAGCCAAACTACGGCAAAGAACTGGGAACTGACTGCCTCAGCAAGCCACCAGCCGCCAGGGGTGTACCCGCAAGGGCACTCAGATACTACT (SEQ ID NO: 23)。

In some embodiments, IL-15RA or a functional fragment thereof includes a "sushi domain" in exon 2 of the receptor extracellular domain (Wei et al., J Immunol. 2001; 167:277-282). In some embodiments, the IL-15RA or functional fragment thereof comprising the sushi domain of wild-type human IL-15RA comprises at least 70% identity (e.g., at least 75% identity, at least 80% identity, at least 85% identity, Amino acid sequence of at least 90% identity, at least 95% identity, at least 99% identity, or 100% identity): ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLECVLNKATNVAHWTTPSLKCIR (SEQ ID NO: 24) or a functional fragment thereof. In some embodiments, IL-15RA or a functional fragment thereof includes the sushi domain of wild-type human IL-15RA, which includes the sequence of SEQ ID NO: 24.

In some embodiments, IL-15RA comprises a sushi domain of wild-type human IL15RA or a functional fragment thereof and additionally at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,30,35,40,45,50,60,70,80,90 or 100 amino acids. In some embodiments, IL-15RA or a functional fragment thereof comprises the sushi domain of wild-type human IL-15RA and an additional 13 amino acids of wild-type human IL-15RA. In some embodiments, IL-15RA comprises at least 70% identity (e.g., at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 99% identity, or 100% identical) amino acid sequence: ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPS (SEQ ID NO: 25) or a functional fragment thereof. In some embodiments, the IL-15RA polypeptide comprises the sequence of SEQ ID NO:25.

In some embodiments, the IL-15RA consists of a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) encoded by the nucleic acid sequence: ATCACCTGCCCGCCTCCCATGAGCGTGGAACACGCGGACATTTGGGTTAAGAGCTACAGTCTTTACAGCCGGGAGCGCTATATCTGCAACTCAGGGTTTAAGCGGAAAGCAGGGACATCAAGTTTGACAGAATGTGTGTTGAACAAGGCTACAAATGTTGCTCACTGGACCACGCCATCTTTGAAGTGTATCCGAGATCCCGCGCTTNOGTCCATCAGCGCCCAGCCC6.

In some embodiments, the functional fragment of IL-15RA comprises at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 amino acids. In some embodiments, the functional fragment of IL-15RA comprises at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 amino acids, and contains the IL-15RA sushi domain. In some embodiments, the IL-15RA fragment or variant retains at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% of the ability of wild-type human IL-15RA to bind IL-15 , 90%, 95%, 98% or 99%, as measured by assays well known in the art, such as ELISA, surface plasmon resonance (SPR) binding assay, or co-immunoprecipitation. In some embodiments, the IL-15RA variant or fragment retains at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%, as measured by assays well known in the art, such as electromobility assays, ELISAs, and other immunoassays. In some embodiments, the functional fragment of the IL-15RA polypeptide includes one or both of the sushi domain and the transmembrane domain of the IL-15RA polypeptide. In some embodiments, the functional fragment of the IL-15RA polypeptide includes a sushi domain. In some embodiments, the functional fragment of the IL-15RA polypeptide includes a transmembrane domain.

In some embodiments, the first exogenous polypeptide further includes a signal peptide. In some embodiments, the first exogenous polypeptide includes a signal peptide including the amino acid sequence listed in Table 3. In some embodiments, the first exogenous polypeptide includes a signal peptide that includes a GPA signal peptide. In some embodiments, the first exogenous polypeptide comprises a signal peptide having the amino acid sequence of SEQ ID NO:27. In some embodiments, the first exogenous polypeptide includes a leader (signal) sequence that includes the amino acid sequence of SEQ ID NO: 27, an IL-15 polypeptide, and an IL-15RA polypeptide. In some embodiments, the first exogenous polypeptide comprises a leader (signal) sequence comprising the amino acid sequence of SEQ ID NO: 27, a mature human IL-15 polypeptide comprising the amino acid sequence of SEQ ID NO: 16, and An IL-15RA polypeptide comprising the amino acid sequence of SEQ ID NO:22. In some embodiments, the mature human IL-15 polypeptide and the IL-15RA polypeptide are linked by a flexible linker having the amino acid sequence of SEQ ID NO:29.

In some embodiments, one or more linkers are disposed between IL-15 or a functional fragment thereof and IL-15RA or a functional fragment thereof. Any linker provided herein can be used. In some embodiments, the linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 in length peptides of one or more amino acids. In some embodiments, the length of the linker is sufficient to preserve the ability of IL-15 to bind to IL-15RA. In other embodiments, the length of the linker is sufficient to preserve the ability of the IL-15/IL-15RA complex to bind to the βγ IL-15 receptor complex and act as an agonist mediating IL-15 signaling. In some embodiments, the linker includes the amino acid sequences listed in Table 2. In some embodiments, the linker comprises the amino acid sequence (GGGGS) _n (SEQ ID NO: 30), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, the linker consists of (GGGGS) _n linker (SEQ ID NO: 30), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, the linker includes the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 29).

Other suitable linkers known to those skilled in the art can be used to link IL-15 and IL-15RA polypeptides. In some embodiments, the linker can be 5 to 25 amino acids, 5 to 20 amino acids, 10 to 25 amino acids, or 10 to 20 amino acids in length. In some embodiments, the linker can be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids in length. In some embodiments, linkers are non-immunogenic.

In some embodiments, the first exogenous polypeptide includes an IL-15 polypeptide or a functional fragment thereof, and an extracellular region of an IL-15Ra polypeptide. In some embodiments, the first exogenous polypeptide includes the amino acid sequence of SEQ ID NO:1. In some embodiments, the first exogenous polypeptide comprises a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical) consistent, or 100% consistent) sequences: NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGGSGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTT (SEQ ID NO: 31)。

In some embodiments, the first exogenous polypeptide consists of a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical) Consistent, or 100% identical) nucleic acid sequence encoded by: AACTGGGTGAACGTTATTAGTGACCTTAAAAAGATCGAAGATTTGATACAGTCAATGCACATAGACGCGACGCTTTATACAGAATCTGATGTACATCCTTCATGCAAGGTTACTGCTATGAAGTGTTTTCTTCTCGAACTCCAAGTAATAAGTCTTGAGAGCGGAGATGCGAGCATTCATGACACCGTTGAGAATCTTATTATATTGGCTAACAACTCTCTGTCCAGCAATGGTAATGTGACAGAAAGCGGGTGTAAGGAGTGCGAGGAACTCGAGGAGAAGAACATCAAAGAGTTCTTGCAGTCTTTCGTCCATATTGTCCAGATGTTCATAAATACTAGCGGGGGAGGTGGCTCTGGTGGAGGCGGGAGTGGCGGGGGCGGCTCAATCACATGCCCACCGCCCATGTCTGTTGAACACGCAGACATTTGGGTTAAAAGTTACTCACTTTACTCACGCGAGAGATATATATGCAACAGCGGCTTCAAGCGCAAAGCAGGCACTAGTAGTCTTACAGAGTGCGTGCTCAATAAAGCTACAAATGTAGCTCATTGGACTACTCCTAGTCTCAAATGCATTCGGGACCCCGCGCTTGTGCACCAGAGACCTGCGCCGCCGTCCACAGTGACGACAGCTGGTGTAACCCCCCAACCTGAATCCCTTAGTCCGTCTGGTAAAGAACCGGCGGCGTCTTCACCTTCCAGCAATAATACTGCGGCGACAACAGCCGCGATAGTTCCTGGATCCCAACTCATGCCGTCAAAGTCTCCTTCAACGGGAACGACAGAGATCTCTTCACATGAAAGTTCTCATGGAACACCGAGCCAAACTACGGCAAAGAACTGGGAACTGACTGCCTCAGCAAGCCACCAGCCGCCAGGGGTGTACCCGCAAGGGCACTCAGATACTACT (SEQ ID NO: 32)。

In some embodiments, the first exogenous polypeptide includes an IL-15 polypeptide and a sushi domain of an IL-15RA polypeptide. In some embodiments, the first exogenous polypeptide includes the amino acid sequence of SEQ ID NO:2. In some embodiments, the exogenous polypeptide comprises a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical , or 100% identical) sequence: NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGGSGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAPPSLTECVLNKATNVAHWTTPSHLK.

In some embodiments, the first exogenous polypeptide is composed of a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical) % identical, or 100% identical) nucleic acid sequence encoded by: AACTGGGTAAACGTCATAAGCGACCTCAAAAAGATCGAGGACCTTATACAGTCTATGCACATAGATGCGACACTTTACACGGAATCAGACGTGCACCCGTCCTGCAAAGTCACAGCCATGAAGTGCTTTCTTCTCGAACTGCAGGTAATTTCTCTCGAATCAGGTGACGCATCTATCCACGACACAGTTGAAAATCTTATTATCCTGGCTAATAACTCCCTTAGCTCAAACGGCAATGTCACCGAGAGTGGATGTAAAGAATGTGAGGAACTTGAAGAGAAAAACATAAAGGAATTCTTGCAGAGTTTCGTTCATATTGTGCAAATGTTCATCAATACTAGTGGCGGGGGGGGAAGCGGTGGTGGAGGGAGCGGGGGTGGTGGATCCATCACCTGCCCGCCTCCCATGAGCGTGGAACACGCGGACATTTGGGTTAAGAGCTACAGTCTTTACAGCCGGGAGCGCTATATCTGCAACTCAGGGTTTAAGCGGAAAGCAGGGACATCAAGTTTGACAGAATGTGTGTTGAACAAGGCTACAAATGTTGCTCACTGGACCACGCCATCTTTGAAGTGTATCCGAGATCCCGCGCTTGTCCATCAGCGCCCAGCGCCTCCCTCC (SEQ ID NO: 34)。

In some embodiments, the first exogenous polypeptide includes an IL-15 polypeptide or a functional fragment thereof, and an IL-15RA polypeptide or a functional fragment thereof (eg, an IL-15 binding fragment). Any IL-15 polypeptide described herein can be combined with any IL-15RA polypeptide described herein to form a first exogenous polypeptide. In some embodiments, the IL-15 polypeptide or its functional fragment and the extracellular part of the IL-15RA polypeptide or its functional fragment exist in a complex. In some embodiments, the IL-15 polypeptide is present as a fusion polypeptide (eg, a first exogenous fusion polypeptide) with the extracellular portion of the IL-15RA polypeptide. In some embodiments, the IL-15 polypeptide is linked to the extracellular portion of the IL-15RA polypeptide via a linker. In some embodiments, the IL-15 polypeptide and the IL-15RA polypeptide are present in a complex. Components of the IL-15/IL-15RA complex can be fused directly, using non-covalent linkages or covalent linkages (eg, combining amino acid sequences via peptide bonds).

In some embodiments, the first exogenous polypeptide, IL-15 polypeptide, IL-15RA polypeptide, or IL-15/IL-15RA complex is not released from erythroid cells (eg, enucleated erythroid cells). In some embodiments, the IL-15 polypeptide, IL-15RA polypeptide, or IL-15/IL-15RA complex or fusion polypeptide is attached to an erythroid membrane (eg, the membrane of an enucleated erythroid). In some embodiments, the first exogenous polypeptide further includes a polypeptide sequence (eg, a transmembrane region) that anchors the polypeptide to the erythroid cell membrane (referred to herein as an anchor or transmembrane domain). In some embodiments, the polypeptide sequence that anchors the first exogenous polypeptide to the erythroid membrane is heterologous to another polypeptide of the first exogenous polypeptide. In some embodiments, the polypeptide sequence that anchors the polypeptide to the erythroid membrane is heterologous to the IL-15 polypeptide and/or IL-15RA polypeptide. In some embodiments, the polypeptide sequence that anchors the polypeptide to the erythroid membrane is a GPA sequence.

In some embodiments, other polypeptides for anchoring the first exogenous polypeptide to the erythroid membrane (e.g., the membrane of an enucleated erythroid) are well known to those skilled in the art and are contemplated to include IL-15, IL-15RA, or the exogenous polypeptide of IL-15/IL-15RA fusion. Non-limiting examples include small integral membrane protein 1 (SMIM1), transferrin receptor, Fas ligand (FasL), Kell, and Band 3.

In some embodiments, the anchoring or transmembrane domain can comprise a type 1 membrane protein or a transmembrane portion thereof. For example, in some embodiments of the first exogenous polypeptide, the anchor or transmembrane domain comprises a Type I membrane protein or a transmembrane portion thereof selected from the group consisting of: glycophorin A (GPA); blood group Glycoprotein B (GPB); basigin (also known as CD147); CD44; CD58 (also known as LFA3); intercellular adhesion molecule 4 (ICAM4); basal cell adhesion molecule (BCAM); CR1; CD99; protein (ERMAP); junctional adhesion molecule A (JAM-A); neuroplastin (NPTN); AMIGO2; and DS cell adhesion molecule-like 1 (DSCAML1). In some embodiments, the anchoring or transmembrane domain comprises or consists of a type 2 membrane protein or a transmembrane portion thereof. For example, in some embodiments of the first exogenous polypeptide, the anchor or transmembrane domain comprises a type 2 membrane protein or a transmembrane portion thereof selected from the group consisting of small integral membrane protein 1 (SMIM1), Transferrin receptor (CD71); Fas ligand (FasL) transmembrane; and Kell. In some embodiments of the first exogenous polypeptide, the anchor is a GPI-linked membrane protein. In some embodiments of the first exogenous polypeptide, the GPI-linked membrane protein anchor is selected from the group consisting of: CD59; CD55; and Semaphorin 7A (SEMA7A).

In some embodiments, the anchoring or transmembrane domain can comprise small integral membrane protein 1 (SMIM1) or a transmembrane portion thereof. In some embodiments, the anchoring or transmembrane domain comprises glycophorin A (GPA), or a fragment thereof (eg, the transmembrane portion thereof). In some embodiments, the anchor or transmembrane domain includes the amino acid sequences provided in Table 1.

In some embodiments, the first exogenous polypeptide includes an IL-15 polypeptide or a functional fragment thereof and the transmembrane region of wild-type human IL-15RA. In some embodiments, the first exogenous polypeptide includes an IL-15 polypeptide or functional fragment thereof and a transmembrane region (eg, any of the exemplary transmembrane regions or transmembrane domains described herein).

In some embodiments, a linker is disposed between the anchor or transmembrane domain and the IL-15 polypeptide, IL-15RA polypeptide or IL-15/IL-15RA polypeptide. Suitable linkers include, but are not limited to, any of the linker amino acid sequences provided in Table 2. In some embodiments, the linker between the anchor or transmembrane domain (eg, GPA) and the IL-15 polypeptide, IL-15RA polypeptide or IL-15/IL-15RA fusion polypeptide comprises or consists of an HA linker . In some embodiments, the linker comprises or consists of the amino acid sequence of SEQ ID NO: 33.

In some embodiments, the first exogenous polypeptide can further include an anchor. In some embodiments, the first exogenous polypeptide may comprise the amino acid sequence of SEQ ID NO: 36 (which is encoded by the nucleic acid sequence of SEQ ID NO: 37), an interleukin-15 (IL-15) polypeptide and an interleukin-15 (IL-15) polypeptide. The extracellular portion of the interleukin 15 receptor alpha (IL-15RA) polypeptide. In some embodiments, the first exogenous polypeptide can comprise an anchor comprising the amino acid sequence of SEQ ID NO: 36, a mature human IL-15 comprising the amino acid sequence of SEQ ID NO: 16, and a mature human extracellular IL-15RA (which comprises the amino acid sequence of SEQ ID NO: 22), whereby the mature human IL-15 amino acid sequence and the mature human extracellular IL-15 RA amino acid sequence are defined by comprising the amino acid sequence of SEQ ID NO: 29 flexible linker connections.

In some specific examples, the exogenous fusion polypeptide comprises: a signal peptide (eg, GPA signal peptide) comprising the amino acid sequence of SEQ ID NO: 27, a mature human IL-15 comprising the amino acid sequence of SEQ ID NO: 16 , a flexible linker comprising the amino acid sequence of SEQ ID NO:29 (for example, linking mature human IL-15 and mature human extracellular IL-15RA), a mature human extracellular IL comprising the amino acid SEQ ID NO:22 - 15RA, a linker comprising the amino acid sequence of SEQ ID NO: 35, and an anchor comprising the amino acid sequence of SEQ ID NO: 36. In some embodiments, the exogenous fusion polypeptide comprises (for example, from N-terminal to C-terminal): mature human IL-15 comprising the amino acid sequence of SEQ ID NO: 16, comprising the amino acid sequence of SEQ ID NO: 29 A flexible linker (for example, connecting mature human IL-15 and mature human extracellular IL-15RA), comprising the amino acid sequence of SEQ ID NO: 22 mature human extracellular IL-15RA comprising the amino acid sequence of SEQ ID The linker of NO:35, and the anchor including the amino acid sequence of SEQ ID NO:36. In some embodiments, the exogenous fusion polypeptide includes the sequence of SEQ ID NO:46.

In some embodiments, the first exogenous polypeptide comprises a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical) Consistent, or 100% identical) amino acid sequence: MYGKIIFVLLLSEIVSISANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGSGGSGGYPYDVPDYAGGGSGGGSLSTTEVAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRAHEVSEISVRTVYPPEEETGERVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDTDVPLSSVEIENPETSDQ (SEQ ID NO: 38)或其功能片段。

In some embodiments, the first exogenous polypeptide consists of a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical) Consistent, or 100% identical) nucleic acid sequence encoded by: ATGTATGGAAAAATAATCTTTGTATTACTATTGTCAGAAATTGTGAGCATATCAGCAAACTGGGTAAATGTGATTTCAGACCTGAAAAAAATCGAAGACCTTATTCAATCCATGCACATCGACGCGACACTTTATACTGAATCAGACGTACACCCGTCCTGTAAGGTTACTGCGATGAAGTGCTTTCTGTTGGAATTGCAAGTGATCTCCCTCGAATCAGGGGATGCATCCATTCATGATACCGTCGAGAATTTGATCATTCTGGCAAATAACTCCCTCAGTAGTAACGGGAATGTGACCGAGTCTGGGTGTAAGGAGTGCGAAGAGTTGGAGGAAAAGAATATCAAAGAATTCCTTCAGTCCTTTGTTCACATCGTGCAAATGTTTATTAATACATCTGGAGGATCTGGCGGGTCTGGAGGCTACCCCTATGACGTGCCCGACTATGCCGGCGGAGGGTCTGGAGGCGGTTCCTTAAGTACCACTGAGGTGGCAATGCACACTTCAACCTCTTCTTCAGTCACAAAGAGTTACATCTCATCACAGACAAATGATACGCACAAACGGGACACATATGCAGCCACTCCTAGAGCTCATGAAGTTTCAGAAATTTCTGTTAGAACTGTTTACCCTCCAGAAGAGGAAACCGGAGAAAGGGTACAACTTGCCCATCATTTCTCTGAACCAGAGATAACACTCATTATTTTTGGGGTGATGGCTGGTGTTATTGGAACGATCCTCTTAATTTCTTACGGTATTCGCCGACTGATAAAGAAAAGCCCATCTGATGTAAAACCTCTCCCCTCACCTGACACAGACGTGCCTTTAAGTTCTGTTGAAATAGAAAATCCAGAGACAAGTGATCAA (SEQ ID NO: 39)。

In some embodiments, the first exogenous polypeptide comprises a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical) Consistent, or 100% identical) amino acid sequence: MYGKIIFVLLLSEIVSISANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGGSGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTGGSGGSGGYPYDVPDYAGGGSGGGSLSTTEVAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRAHEVSEISVRTVYPPEEETGERVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDTDVPLSSVEIENPETSDQ (SEQ ID NO: 40)或其功能片段。

In some embodiments, the first exogenous polypeptide consists of a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical) Consistent, or 100% identical) nucleic acid sequence encoded by: ATGTATGGAAAAATAATCTTTGTATTACTATTGTCAGAAATTGTGAGCATATCAGCAAACTGGGTGAACGTTATTAGTGACCTTAAAAAGATCGAAGATTTGATACAGTCAATGCACATAGACGCGACGCTTTATACAGAATCTGATGTACATCCTTCATGCAAGGTTACTGCTATGAAGTGTTTTCTTCTCGAACTCCAAGTAATAAGTCTTGAGAGCGGAGATGCGAGCATTCATGACACCGTTGAGAATCTTATTATATTGGCTAACAACTCTCTGTCCAGCAATGGTAATGTGACAGAAAGCGGGTGTAAGGAGTGCGAGGAACTCGAGGAGAAGAACATCAAAGAGTTCTTGCAGTCTTTCGTCCATATTGTCCAGATGTTCATAAATACTAGCGGGGGAGGTGGCTCTGGTGGAGGCGGGAGTGGCGGGGGCGGCTCAATCACATGCCCACCGCCCATGTCTGTTGAACACGCAGACATTTGGGTTAAAAGTTACTCACTTTACTCACGCGAGAGATATATATGCAACAGCGGCTTCAAGCGCAAAGCAGGCACTAGTAGTCTTACAGAGTGCGTGCTCAATAAAGCTACAAATGTAGCTCATTGGACTACTCCTAGTCTCAAATGCATTCGGGACCCCGCGCTTGTGCACCAGAGACCTGCGCCGCCGTCCACAGTGACGACAGCTGGTGTAACCCCCCAACCTGAATCCCTTAGTCCGTCTGGTAAAGAACCGGCGGCGTCTTCACCTTCCAGCAATAATACTGCGGCGACAACAGCCGCGATAGTTCCTGGATCCCAACTCATGCCGTCAAAGTCTCCTTCAACGGGAACGACAGAGATCTCTTCACATGAAAGTTCTCATGGAACACCGAGCCAAACTACGGCAAAGAACTGGGAACTGACTGCCTCAGCAAGCCACCAGCCGCCAGGGGTGTACCCGCAAGGGCACTCAGATACTACTGGAGGATCTGGCGGGTCTGGAGGCTACCCCT ATGACGTGCCCGACTATGCCGGCGGAGGGTCTGGAGGCGGTTCCTTAAGTACCACTGAGGTGGCAATGCACACTTCAACCTCTTCTTCAGTCACAAAGAGTTACATCTCATCACAGACAAATGATACGCACAAACGGGACACATATGCAGCCACTCCTAGAGCTCATGAAGTTTCAGAAATTTCTGTTAGAACTGTTTACCCTCCAGAAGAGGAAACCGGAGAAAGGGTACAACTTGCCCATCATTTCTCTGAACCAGAGATAACACTCATTATTTTTGGGGTGATGGCTGGTGTTATTGGAACGATCCTCTTAATTTCTTACGGTATTCGCCGACTGATAAAGAAAAGCCCATCTGATGTAAAACCTCTCCCCTCACCTGACACAGACGTGCCTTTAAGTTCTGTTGAAATAGAAAATCCAGAGACAAGTGATCAA (SEQ ID NO: 41)。

In some embodiments, the first exogenous polypeptide comprises a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical) Consistent, or 100% identical) amino acid sequence: MYGKIIFVLLLSEIVSISANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGGSGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSGGSGGSGGYPYDVPDYAGGGSGGGSLSTTEVAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRAHEVSEISVRTVYPPEEETGERVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDTDVPLSSVEIENPETSDQ (SEQ ID NO: 42)或其功能片段。

In some embodiments, the first exogenous polypeptide consists of a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical) Consistent, or 100% identical) nucleic acid sequence encoded by: ATGTATGGAAAAATAATCTTTGTATTACTATTGTCAGAAATTGTGAGCATATCAGCAAACTGGGTAAACGTCATAAGCGACCTCAAAAAGATCGAGGACCTTATACAGTCTATGCACATAGATGCGACACTTTACACGGAATCAGACGTGCACCCGTCCTGCAAAGTCACAGCCATGAAGTGCTTTCTTCTCGAACTGCAGGTAATTTCTCTCGAATCAGGTGACGCATCTATCCACGACACAGTTGAAAATCTTATTATCCTGGCTAATAACTCCCTTAGCTCAAACGGCAATGTCACCGAGAGTGGATGTAAAGAATGTGAGGAACTTGAAGAGAAAAACATAAAGGAATTCTTGCAGAGTTTCGTTCATATTGTGCAAATGTTCATCAATACTAGTGGCGGGGGGGGAAGCGGTGGTGGAGGGAGCGGGGGTGGTGGATCCATCACCTGCCCGCCTCCCATGAGCGTGGAACACGCGGACATTTGGGTTAAGAGCTACAGTCTTTACAGCCGGGAGCGCTATATCTGCAACTCAGGGTTTAAGCGGAAAGCAGGGACATCAAGTTTGACAGAATGTGTGTTGAACAAGGCTACAAATGTTGCTCACTGGACCACGCCATCTTTGAAGTGTATCCGAGATCCCGCGCTTGTCCATCAGCGCCCAGCGCCTCCCTCCGGAGGATCTGGCGGGTCTGGAGGCTACCCCTATGACGTGCCCGACTATGCCGGCGGAGGGTCTGGAGGCGGTTCCTTAAGTACCACTGAGGTGGCAATGCACACTTCAACCTCTTCTTCAGTCACAAAGAGTTACATCTCATCACAGACAAATGATACGCACAAACGGGACACATATGCAGCCACTCCTAGAGCTCATGAAGTTTCAGAAATTTCTGTTAGAACTGTTTACCCTCCAGAAGAGGAAACCGGAGAAAGGGTACAACTTGCCCATCATTTCTCTGAACCAGAGATAACACTCATTATTTTTGGGGTGATGGCTG GTGTTATTGGAACGATCCTCTTAATTTCTTACGGTATTCGCCGACTGATAAAGAAAAGCCCATCTGATGTAAAACCTCTCCCCTCACCTGACACAGACGTGCCTTTAAGTTCTGTTGAAATAGAAAATCCAGGACAAGTGATCAA (SEQ ID NO: 43).

In some embodiments, the first exogenous polypeptide comprises a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical) Consistent, or 100% identical) amino acid sequence: MYGKIIFVLLLSEIVSISANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGSGGSGGGGGSGGGSGGGSGGGSLSTTEVAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRAHEVSEISVRTVYPPEEETGERVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDTDVPLSSVEIENPETSDQ (SEQ ID NO: 44)或其功能片段。

In some embodiments, the first exogenous polypeptide consists of a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical) Consistent, or 100% identical) nucleic acid sequence encoded by: ATGTATGGAAAAATAATCTTTGTATTACTATTGTCAGAAATTGTGAGCATATCAGCAAACTGGGTAAATGTGATTTCAGACCTGAAAAAAATCGAAGACCTTATTCAATCCATGCACATCGACGCGACACTTTATACTGAATCAGACGTACACCCGTCCTGTAAGGTTACTGCGATGAAGTGCTTTCTGTTGGAATTGCAAGTGATCTCCCTCGAATCAGGGGATGCATCCATTCATGATACCGTCGAGAATTTGATCATTCTGGCAAATAACTCCCTCAGTAGTAACGGGAATGTGACCGAGTCTGGGTGTAAGGAGTGCGAAGAGTTGGAGGAAAAGAATATCAAAGAATTCCTTCAGTCCTTTGTTCACATCGTGCAAATGTTTATTAATACATCTGGAGGATCTGGCGGGTCTGGAGGCGGCGGCGGCAGCGGCGGCGGCAGCGGCGGAGGGTCTGGAGGCGGTTCCTTAAGTACCACTGAGGTGGCAATGCACACTTCAACCTCTTCTTCAGTCACAAAGAGTTACATCTCATCACAGACAAATGATACGCACAAACGGGACACATATGCAGCCACTCCTAGAGCTCATGAAGTTTCAGAAATTTCTGTTAGAACTGTTTACCCTCCAGAAGAGGAAACCGGAGAAAGGGTACAACTTGCCCATCATTTCTCTGAACCAGAGATAACACTCATTATTTTTGGGGTGATGGCTGGTGTTATTGGAACGATCCTCTTAATTTCTTACGGTATTCGCCGACTGATAAAGAAAAGCCCATCTGATGTAAAACCTCTCCCCTCACCTGACACAGACGTGCCTTTAAGTTCTGTTGAAATAGAAAATCCAGAGACAAGTGATCAA (SEQ ID NO: 45)。

In some embodiments, the first exogenous polypeptide comprises a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical) Consistent, or 100% identical) amino acid sequence: MYGKIIFVLLLSEIVSISANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGGSGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTGGSGGSGGGGGSGGGSGGGSGGGSLSTTEVAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRAHEVSEISVRTVYPPEEETGERVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDTDVPLSSVEIENPETSDQ (SEQ ID NO: 46)或其功能片段。

In some embodiments, the first exogenous polypeptide consists of a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical) Consistent, or 100% identical) nucleic acid sequence encoded by: ATGTATGGAAAAATAATCTTTGTATTACTATTGTCAGAAATTGTGAGCATATCAGCAAACTGGGTGAACGTTATTAGTGACCTTAAAAAGATCGAAGATTTGATACAGTCAATGCACATAGACGCGACGCTTTATACAGAATCTGATGTACATCCTTCATGCAAGGTTACTGCTATGAAGTGTTTTCTTCTCGAACTCCAAGTAATAAGTCTTGAGAGCGGAGATGCGAGCATTCATGACACCGTTGAGAATCTTATTATATTGGCTAACAACTCTCTGTCCAGCAATGGTAATGTGACAGAAAGCGGGTGTAAGGAGTGCGAGGAACTCGAGGAGAAGAACATCAAAGAGTTCTTGCAGTCTTTCGTCCATATTGTCCAGATGTTCATAAATACTAGCGGGGGAGGTGGCTCTGGTGGAGGCGGGAGTGGCGGGGGCGGCTCAATCACATGCCCACCGCCCATGTCTGTTGAACACGCAGACATTTGGGTTAAAAGTTACTCACTTTACTCACGCGAGAGATATATATGCAACAGCGGCTTCAAGCGCAAAGCAGGCACTAGTAGTCTTACAGAGTGCGTGCTCAATAAAGCTACAAATGTAGCTCATTGGACTACTCCTAGTCTCAAATGCATTCGGGACCCCGCGCTTGTGCACCAGAGACCTGCGCCGCCGTCCACAGTGACGACAGCTGGTGTAACCCCCCAACCTGAATCCCTTAGTCCGTCTGGTAAAGAACCGGCGGCGTCTTCACCTTCCAGCAATAATACTGCGGCGACAACAGCCGCGATAGTTCCTGGATCCCAACTCATGCCGTCAAAGTCTCCTTCAACGGGAACGACAGAGATCTCTTCACATGAAAGTTCTCATGGAACACCGAGCCAAACTACGGCAAAGAACTGGGAACTGACTGCCTCAGCAAGCCACCAGCCGCCAGGGGTGTACCCGCAAGGGCACTCAGATACTACTGGAGGATCTGGCGGGTCTGGAGGCGGCGGCG GCAGCGGCGGCGGCAGCGGCGGAGGGTCTGGAGGCGGTTCCTTAAGTACCACTGAGGTGGCAATGCACACTTCAACCTCTTCTTCAGTCACAAAGAGTTACATCTCATCACAGACAAATGATACGCACAAACGGGACACATATGCAGCCACTCCTAGAGCTCATGAAGTTTCAGAAATTTCTGTTAGAACTGTTTACCCTCCAGAAGAGGAAACCGGAGAAAGGGTACAACTTGCCCATCATTTCTCTGAACCAGAGATAACACTCATTATTTTTGGGGTGATGGCTGGTGTTATTGGAACGATCCTCTTAATTTCTTACGGTATTCGCCGACTGATAAAGAAAAGCCCATCTGATGTAAAACCTCTCCCCTCACCTGACACAGACGTGCCTTTAAGTTCTGTTGAAATAGAAAATCCAGAGACAAGTGATCAA (SEQ ID NO: 47)。

In some embodiments, the exogenous fusion polypeptide includes the amino acid sequence of SEQ ID NO:38. In some embodiments, the exogenous fusion polypeptide includes the amino acid sequence of SEQ ID NO:40. In some embodiments, the exogenous fusion polypeptide includes the amino acid sequence of SEQ ID NO:42. In some embodiments, the exogenous fusion polypeptide includes the amino acid sequence of SEQ ID NO:44. In some embodiments, the exogenous fusion polypeptide includes the amino acid sequence of SEQ ID NO:46.

In some embodiments, the first exogenous polypeptide is as described in US Patent Application Publication No. 2019/0298769, which is incorporated herein by reference in its entirety. second exogenous polypeptide

In some embodiments, the enucleated erythroid cells can further include exogenous polypeptides on their extracellular surfaces. In some embodiments, the enucleated erythroid cell includes a first exogenous polypeptide on its extracellular surface and a second exogenous polypeptide on its extracellular surface, the first exogenous polypeptide including (i) interleukin IL-15 or a functional fragment thereof, and (ii) interleukin 15 receptor alpha (IL-15RA) or a functional fragment thereof, and the second exogenous polypeptide includes 4-1BBL polypeptide or a functional fragment thereof.

As used herein, 4-1BBL polypeptide refers to the amino acid sequence encoded by the tumor necrosis factor superfamily member 9 (TNFSF9 or CD137L) gene. 4-1BBL is the ligand for 4-1BB (also known as tumor necrosis factor receptor superfamily, member 9 (TNFRSF9) or CD137), a member of a family of receptors found on the surface of cells in the immune system. See Alderson et al., 1994, Eur. J. Immunol. 24:2219-2227.

In some embodiments, 4-1BBL is in its native trimeric form.

In some embodiments, 4-1BBL comprises at least 70% identity (e.g., at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 99% identity, or 100% identical) amino acid sequence: ACPWAVSGARASPPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPPEIPAGL or a functional fragment thereof (ID:PRSETVLGLFRVTPPEIPAGL) In some embodiments, 4-1BBL comprises the sequence of SEQ ID NO:48.

In some embodiments, 4-1BBL consists of a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) encoded by the nucleic acid sequence: GCCTGCCCCTGGGCCGTGTCCGGGGCTCGCGCCTCGCCCGGCTCCGCGGCCAGCCCGAGACTCCGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA (SEQ ID NO: 49)。

In some embodiments, the second exogenous polypeptide comprises a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical) Consistent, or 100% identical) amino acid sequence: MYGKIIFVLLLSEIVSISAACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSEGGSGGSGGGPEDEPGSGSGGGSGGGSLSTTEVAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRAHEVSEISVRTVYPPEEETGERVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDTDVPLSSVEIENPETSDQ (SEQ ID NO: 50)。 In some embodiments, the 4-1BBL polypeptide comprises the sequence of SEQ ID NO:50.

In some embodiments, the second exogenous polypeptide consists of a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical) Consistent, or 100% identical) nucleic acid sequence encoded by: ATGTATGGAAAAATAATCTTTGTATTACTATTGTCAGAAATTGTGAGCATATCAGCAGCCTGCCCCTGGGCCGTGTCCGGGGCTCGCGCCTCGCCCGGCTCCGCGGCCAGCCCGAGACTCCGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAAGGAGGATCTGGCGGGTCTGGAGGCGGCCCCGAGGACGAGCCCGGCAGCGGCAGCGGCGGAGGGTCTGGAGGCGGTTCCTTAAGTACCACTGAGGTGGCAATGCACACTTCAACCTCTTCTTCAGTCACAAAGAGTTACATCTCATCACAGACAAATGATACGCACAAACGGGACACATATGCAGCCACTCCTAGAGCTCATGAAGTTTCAGAAATTTCTGTTAGAACTGTTTACCCTCCAGAAGAGGAAACCGGAGAAAGGGTACAACTTGCCCATCATTTCTCTGAACCAGAGATAACACTCATTATTTTTGGGGTGATGGCTGGTG TTATTGGAACGATCCTCTTAATTTCTTACGGTATTCGCCGACTGATAAAGAAAAGCCCATCTGATGTAAAACCTCTCCCCTCACCTGACACAGACGTGCCTTTAAGTTCTGTTGAAATAGAAAATCCAGGACAAGTGATCAA (SEQ ID NO: 51).

In some embodiments, the exogenous polypeptide further includes a leader (signal) sequence. In some embodiments, 4-1BBL or a functional fragment thereof is fused to a leader (signal) sequence. Non-limiting examples of leader (signal) sequences include the amino acid sequences listed in Table 3. In some embodiments, the leader (signal) sequence includes a GPA signal peptide. In some embodiments, the leader (signal) sequence includes the amino acid sequence of SEQ ID NO:27. In some specific examples, the exogenous polypeptide includes 4-1BBL or a functional fragment thereof, and a leader (signal) sequence of amino acid sequence SEQ ID NO:27. In some embodiments, the exogenous polypeptide comprises a leader (signal) sequence comprising the amino acid sequence of SEQ ID NO:27, and 4-1BBL having the amino acid sequence of SEQ ID NO:48.

In some embodiments, the second exogenous polypeptide is attached to the erythroid cell membrane (eg, the membrane of an enucleated erythroid cell). In some embodiments, the second exogenous polypeptide is attached to the extracellular surface of the enucleated erythroid blood cell. In some embodiments, the second exogenous polypeptide further comprises an anchoring or transmembrane domain that anchors the second exogenous polypeptide to an erythroid membrane (eg, an enucleated erythroid membrane). In some embodiments, the anchoring or transmembrane domain is heterologous to the second exogenous polypeptide (eg, 4-1BBL). In some embodiments, the anchoring or transmembrane domain comprises an endogenous erythrocyte transmembrane protein or a fragment or transmembrane portion thereof. In certain embodiments, the anchor or transmembrane domain comprises GPA or a transmembrane portion thereof. In some embodiments, the anchoring or transmembrane domain comprises small integral membrane protein 1 (SMIM1), transferrin receptor, Fas ligand (FasL), Kell band 3, or a transmembrane portion thereof (e.g., transmembrane domain). In some embodiments, the second exogenous polypeptide can comprise any of the anchoring or transmembrane domains described herein. In some embodiments, the second exogenous polypeptide comprises an anchoring or transmembrane domain listed in Table 1.

In some embodiments, the second exogenous polypeptide includes one or more linkers (eg, any of the exemplary linkers described herein). For example, the second exogenous polypeptide can include one or more linkers provided in Table 2. In some embodiments, the linker is arranged between 4-1BBL or its functional fragment and the anchoring domain or transmembrane domain in the second exogenous polypeptide.

In some embodiments, the second exogenous polypeptide can further include a signal peptide (eg, any of the exemplary signal peptides described herein). For example, the second exogenous polypeptide can include a signal peptide provided in Table 3.

In some embodiments, the exogenous polypeptide includes a signal peptide, 4-1BBL or a functional fragment thereof, and an anchor. In some embodiments, the exogenous polypeptides include signal peptides, 4-1BBL or functional fragments thereof, linkers, and anchors. In some embodiments, the exogenous polypeptide comprises (for example, from N-terminus to C-terminus) a signal peptide comprising the amino acid sequence of SEQ ID NO: 27, a 4-1BBL comprising the amino acid sequence of SEQ ID NO: 48, or Its functional fragment, a linker comprising the amino acid sequence of SEQ ID NO: 52 (for example, placed between 4-1BBL or its functional fragment and the anchor), and an anchor comprising the amino acid sequence of SEQ ID NO: 36 . In some embodiments, the exogenous polypeptide comprises 4-1BBL or a functional fragment thereof, a linker and an anchor. In some embodiments, the exogenous polypeptide comprises (for example, from N-terminal to C-terminal) 4-1BBL comprising the amino acid sequence of SEQ ID NO: 48 or a functional fragment thereof, comprising the amino acid sequence of SEQ ID NO: 52 A linker (eg, placed between 4-1BBL or a functional fragment thereof and the anchor), and an anchor comprising the amino acid sequence of SEQ ID NO:36. In some embodiments, the exogenous polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 50.

In some embodiments, the first and/or second exogenous polypeptide can have a post-translational modification characteristic of a eukaryotic cell (eg, a mammalian cell, eg, a human cell). In some embodiments, one or more (eg, 2, 3, 4, 5 or more) exogenous proteins are glycosylated, phosphorylated, or both. In vitro detection of glycoproteins is usually accomplished on SDS-PAGE gels and Western blotting using a modified Periodic acid-Schiff (PAS) method. Cellular localization of glycoproteins can be accomplished using lectin fluorescent conjugates known in the art. Phosphorylation can be assessed by Western blotting using phospho-specific antibodies.

Post-translational modifications also include conjugation to hydrophobic groups (e.g., myristylation, palmitoylation, prenylation, polyprenylation, or glycosylphosphatidylinositolation), conjugation to cofactors (e.g., , lipidation, flavin moieties (eg, FMN or FAD), heme C attachment, phosphopanthiolation or retinylidene Schiff base formation), dibenzamide formation, ethanolamine phosphoglycerol attachment, putrescine Hypusine formation, acylation (eg O-acylation, N-acylation or S-acylation), formylation, acetylation, alkylation (eg methylation or ethylation), Amidation, butyrylation, γ-carboxylation, malonylation, hydroxylation, iodination, nucleotide addition (such as ADP-ribosylation), oxidation, phosphate (O-linkage) or amine groups Phosphate (N-linked) formation, (eg, phosphorylation or adenylation), propionylation, pyroglutamate formation, S-glutathionylation, S-nitrosylation, succinylation, sulfate ylation, ISGylation, sumoylation, ubiquitination, neddylation, or chemical modification of amino acids (such as citrullination, desamidation, elimination, or carbamidylation), disulfide bonds Formation, racemization (eg of proline, serine, alanine or methionine). In particular examples, glycosylation includes adding a sugar group to arginine, asparagine, cysteine, hydroxylysine, serine, threonine, tyrosine, or tryptophan to produce glycoprotein. In particular examples, glycosylation comprises, for example, O-linked glycosylation or N-linked glycosylation.

In some embodiments, the first and second exogenous polypeptides are fusion proteins, eg, are fusions with an endogenous hemoglobin protein or a fragment thereof (eg, a transmembrane protein, such as GPA or a transmembrane fragment thereof). In some embodiments, one or more exogenous polypeptides are fused to a dimerization- or multimerization-promoting domain, eg, to a second fused exogenous polypeptide optionally comprising a dimerization domain. In some embodiments, the dimerization domain comprises a portion of an antibody molecule, eg, an Fc domain or a CH3 domain. In some embodiments, the first and second dimerization domains comprise intrapore knob mutations (eg, T366Y knob and Y407T pore) to promote heterodimerization. anchor/transmembrane domain

In some embodiments, the transmembrane domain comprises or consists of a transmembrane domain of a type 1 membrane protein. In some embodiments, the type 1 membrane protein is selected from the group consisting of: glycophorin A (GPA); glycophorin B (GPB); basigin (also known as CD147); CD44; CD58 (also known as LFA3 ); intercellular adhesion molecule 4 (ICAM4); basal cell adhesion molecule (BCAM); CR1; CD99; and DS cell adhesion molecule-like 1 (DSCAML1). In some embodiments, the transmembrane domain comprises or consists of a transmembrane domain of a type 2 membrane protein. In some embodiments, the type 2 membrane protein is selected from the group consisting of small integral membrane protein 1 (SMIM1), transferrin receptor (CD71); Fas ligand (FasL) transmembrane; and Kell. In some embodiments, the polypeptide sequence that anchors the exogenous polypeptide to the enucleated erythrocyte membrane comprises, consists of, or is derived from (eg, a fragment of) a GPI-linked membrane protein. In some embodiments, the GPI-linked membrane protein is selected from CD59; CD55; and SEMA7A (SEMA7A).

In certain embodiments, the transmembrane domain comprises GPA or a transmembrane portion thereof. Without being bound by theory, GPA is preferred in certain embodiments because it has a cytoplasmic domain that interacts with the reticulocyte cytoskeleton, which has the ability to retain GPA during cell differentiation and maturation. effect. In some embodiments, the transmembrane domain comprises small integral membrane protein 1 (SMIM1) or a transmembrane portion thereof. In some embodiments, the anchor is selected from the amino acid sequences listed in Table 1. Table 1. Anchoring sequence SEQ ID NO: sequence name sequence description amino acid sequence 54 GPA full-length GPA MYGKIIFVLLLSAIVSISALSTTEVAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRAHEVSEISVRTVYPPEEETGERVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDDVPLSSVEIEENPETSDQ 36 GPA Fragment of GPA containing the transmembrane domain LSTTEVAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRAHEVSEISVRTVYPPEEETGE RVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDTDVPLSS VEIENPETSDQ 55 SMIM1 SMIM1 MQPQESHVHYSRWEDGSRDGVSLGAVSSTEEASRCRRISQRLCTGKLGIAMKVLGGVALFWIIFILGYLTGYYVHKCK

In some embodiments where the enucleated erythroid cells include first and second exogenous polypeptides, the first exogenous polypeptide (such as any of the exogenous fusion polypeptides described herein) or the second exogenous polypeptide (such as any of the exogenous fusion polypeptides described herein) Any exogenous polypeptide described above) comprises endogenous red blood cell protein or its fragment (such as transmembrane protein, such as GPA or its transmembrane fragment). In some embodiments, the exogenous fusion polypeptide or one or more exogenous polypeptides (e.g., any of the exogenous polypeptides described herein) are fused to a domain that promotes dimerization or multimerization, e.g., to an optional A second fusion exogenous polypeptide comprising a dimerization domain is fused. In some embodiments, the dimerization domain comprises a portion of an antibody molecule, eg, an Fc domain or a CH3 domain. In some embodiments, the first and second dimerization domains comprise intrapore knob mutations (eg, T366Y knob and Y407T pore) to promote heterodimerization. Linker

In some embodiments where the enucleated erythroid cells include first and second exogenous polypeptides, the first exogenous fusion polypeptide (such as any fusion polypeptide described herein) and/or the second exogenous polypeptide (such as any fusion polypeptide described herein) Any of the exemplary polypeptides described above) may include one or more linkers. For example, a linker can be placed between a cytokine polypeptide sequence (eg, IL-15 or a functional fragment thereof) and a transmembrane domain sequence, or between IL-15 or a functional fragment thereof and IL-15RA or a functional fragment thereof. In another example, a linker can be placed between the 4-1BBL polypeptide, a functional fragment thereof, and the transmembrane domain sequence.

In some embodiments, the linker comprises or consists of the following: the length is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20 or more amino acids. In some embodiments, the linker comprises about 5 to about 25 amino acids in length, about 5 to about 20 amino acids in length, about 10 to about 25 amino acids in length, or about 10 to about 25 amino acids in length. or consisting of 20 amino acids. In some embodiments, linkers useful in the present invention comprise or consist of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids in length. In a preferred embodiment, the linker is non-immunogenic.

In some embodiments, the linker is selected from the amino acid sequences shown in Table 2. Table 2. Linker sequences SEQ ID NO. sequence description amino acid sequence 56 G4S linker GGGGS 29 (G4S) ₃ linker GGGGSGGGGSGGGGS 57 Linker-HA-Linker GGSGGSGGYPYDVPDYAGGGSGGGS 35 Linker GGSGGSGGGGGSGGGSGGGSGGGS 52 Linker GGSGGSGGGPEDEPGSGSGGGSGGGS 58 Linker GSGSGSGSGSSEEDEDEDEDGSGSGSGSGS 59 Linker GGGGSGGGGSGGGGSGGGGS 60 Linker GSGSGSGSEDGSGSGSGS 61 Linker GSGSGSGSGSGSGSGSGSGS 62 Linker GCGGSGGGGSGGGGS 63 Linker SGRGGGGSGGGGSGGGGSGGGGSSPA 64 Linker GGGGSGGGGSGGGGSGGGGSGGGG 65 Snorkel linker SGRGASSGSSGSGSQKKPRYEIRWKVVVISAILALVVLTVISLIILIMLWGSGMQSPA

In some embodiments, the linker comprises the amino acid sequence (GGGGS) _n (SEQ ID NO: 30), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, the linker consists of (GGGGS) _n linker (SEQ ID NO: 30), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, the linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 29). In some embodiments, the linker consists of the amino acid sequence of SEQ ID NO:29. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:57. In some embodiments, the linker consists of the amino acid sequence of SEQ ID NO:57. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:35. In some embodiments, the linker consists of the amino acid sequence of SEQ ID NO:35. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:52. In some embodiments, the linker consists of the amino acid sequence of SEQ ID NO:52. Other suitable linkers that can be used with exogenous fusion polypeptides or exogenous polypeptides (such as any of the exogenous polypeptides described herein) are known in the art. preamble (signal) sequence

In some embodiments where the enucleated erythroid cells include first and second exogenous polypeptides, the first exogenous fusion polypeptide or exogenous polypeptide (such as any of the exemplary exogenous polypeptides described herein) comprises a leader (signal )sequence. In some embodiments, the leader sequence is selected from the sequences listed in Table 3. Table 3. Leader (Signal) Sequence SEQ ID NO. sequence description amino acid sequence 27 GPA signal peptide MYGKIIFVLLLSEIVSISA 66 Ig heavy chain V region 3 message peptide MGWSCIILFLVATATGVHS 67 light chain leader MRVPAQLLGLLLLWLPGARC Exemplary Enucleated Erythroid Cells

In some embodiments, the enucleated erythroid cells comprise a combination of a first exogenous fusion polypeptide comprising IL-15 or a fragment thereof linked to IL-15 receptor alpha (IL-15Rα) The extracellular portion of IL-15 receptor alpha (IL-15Rα) or fragments thereof (e.g., IL-15Rα sushi binding domain), the extracellular portion of the IL-15 receptor alpha (IL-15Rα) or fragments thereof are linked to transmembrane proteins (e.g., GPA or its transmembrane fragment); and a second exogenous polypeptide comprising 4-1BBL or a fragment thereof linked to a transmembrane protein (e.g., GPA or a transmembrane fragment thereof); for example, as described in U.S. Patent Application Publication No. 2019/0298769, incorporated herein by reference).

In some embodiments, the enucleated erythroid cells described herein (eg, human enucleated erythroid cells) are negative for (i.e., do not include) one or more minor blood group antigens, such as Le(a ^- b ^- ) (Lewis antigen system (Lewis antigen system)), Fy(a ^- b ^- ) (Duffy system), JK(a ^- b ^- ) (Kidd system), M ^- N ^- (MNS system), K ^- k ^- (Kell system), Lu(a ^- b ^- ) (Lutheran system) and H antigen negative (Bombay phenotype) or any combination thereof. In some embodiments, the enucleated erythroid cells are also type O and/or Rh ⁻ . Minor blood types are described, for example, in Agarwal et al., “Blood group phenotype frequencies in blood donors from a tertiary care hospital in north India,” Blood Res. 48(1):51-54, 2013, and Mitra et al., "Blood groups systems," Indian J. Anaesth. 58(5):524-528, 2014, the description of which is incorporated herein by reference.

In some embodiments, the enucleated erythroid cells described herein (e.g., human enucleated erythroid cells) exhibit a protein Isolated, uncultured, enucleated erythroid cells have essentially the same osmotic membrane fragility. In some embodiments, the population of enucleated erythroid cells has an osmotic fragility of less than 50% cell lysis at 0.3%, 0.35%, 0.4%, 0.45%, or 0.5% NaCl. In some embodiments, osmotic fragility is determined using the method described in Example 59 of WO 2015/073587, the description of which is incorporated herein by reference.

In some embodiments, the enucleated erythroid cells (eg, human enucleated erythroid cells) have approximately the same diameter or volume as wild-type, unprocessed enucleated erythroid cells. In some embodiments, the population of enucleated erythroid cells (e.g., human enucleated erythroid cells) has a mean diameter of about 4, 5, 6, 7, 8, 9, 10, 11, or 12 microns, or about 4.0 to about 12.0 microns , about 4.0 to about 11.5 microns, about 4.0 to about 11.0 microns, about 4.0 to about 10.5 microns, about 4.0 to about 10 microns, about 4.0 to about 9.5 microns, about 4.0 to about 9.0 microns, about 4.0 to about 8.5 microns, About 4.0 to about 8.0 microns, about 4.0 to about 7.5 microns, about 4.0 to about 7.0 microns, about 4.0 to about 6.5 microns, about 4.0 to about 6.0 microns, about 4.0 to about 5.5 microns, about 4.0 to about 5.0 microns, about 4.0 to about 4.5 microns, about 5.0 to about 12.0 microns, about 5.0 to about 11.5 microns, about 5.0 to about 11.0 microns, about 5.0 to about 10.5 microns, about 5.0 to about 10.0 microns, about 5.0 to about 9.5 microns, about 5.0 to about 9.0 microns, about 5.0 to about 8.5 microns, about 5.0 to about 8.0 microns, about 5.0 to about 7.5 microns, about 5.0 to about 7.0 microns, about 5.0 to about 6.5 microns, about 5.0 to about 6.0 microns, about 5.0 to about 5.5 microns, about 6.0 to about 12.0 microns, about 6.0 to about 11.5 microns, about 6.0 to about 11.0 microns, about 6.0 to about 10.5 microns, about 6.0 to about 10.0 microns, about 6.0 to about 9.5 microns, about 6.0 to about 9.0 microns, about 6.0 to about 8.5 microns, about 6.0 to about 8.0 microns, about 6.0 to about 7.5 microns, about 6.0 to about 7.0 microns, about 6.0 to about 6.5 microns, about 7.0 to about 12.0 microns, about 7.0 to about 11.5 microns microns, about 7.0 to about 11.0 microns, about 7.0 to about 10.5 microns, about 7.0 to about 10.0 microns, about 7.0 to about 9.5 microns, about 7.0 to about 9.0 microns, about 7.0 to about 8.5 microns, about 7.0 to about 8.0 microns , about 7.0 to about 7.5 microns, about 8.0 to about 12.0 microns, about 8.0 to about 11.5 microns, about 8.0 to about 11.0 microns, about 8.0 to about 10.5 microns, about 8.0 to about 10.0 microns, about 8.0 to about 9.5 microns, About 8.0 to about 9.0 microns, about 8.0 to about 8.5 microns, about 9.0 to about 12.0 microns, about 9.0 to about 11.5 microns, about 9.0 to about 11.0 microns, about 9.0 to about 10.5 microns, about 9.0 to about 10.0 microns, about 9.0 to about 9.5 microns, about 10.0 to about 12.0 microns, about 10.0 to about 11.5 microns, about 10.0 to about 11.0 microns, about 10.0 to about 10.5 microns, about 11.0 to about 12.0 microns, about 11.0 to about 11.5 microns, or about 11.5 to about 12.0 microns, and the standard deviation of the cell population is optionally less than 1, 2 or 3 microns. Enucleated erythroid diameter can be measured, for example, using an Advia 120 hematology system or a Moxi Z cytometer (Orflo).

In some embodiments, the mean hematocrit volume of the enucleated erythroid cells is from about 10 fL to about 175 fL, from about 10 fL to about 160 fL, from about 10 fL to about 140 fL, from about 10 fL to about 120 fL, about 10 fL to about 100 fL, about 10 fL to about 90 fL, about 10 fL to about 80 fL, about 10 fL to about 70 fL, about 10 fL to about 60 fL, about 10 fL to about 50 fL, about 10 fL to about About 40 fL, about 10 fL to about 30 fL, about 10 fL to about 20 fL, about 20 fL to about 175 fL, about 20 fL to about 160 fL, about 20 fL to about 140 fL, about 20 fL to about 120 fL, about 20 fL to about 100 fL, about 20 fL to about 90 fL, about 20 fL to about 80 fL, about 20 fL to about 70 fL, about 20 fL to about 60 fL, about 20 fL to about 50 fL, About 20 fL to about 40 fL, about 20 fL to about 30 FL, about 30 fL to about 175 fL, about 30 fL to about 160 fL, about 30 fL to about 140 fL, about 30 fL to about 120 fL, about 30 fL to about 100 fL, about 30 fL to about 90 fL, about 30 fL to about 80 fL, about 30 fL to about 70 fL, about 30 fL to about 60 fL, about 30 fL to about 50 fL, about 30 fL to about About 40 fL, About 40 fL to about 175 fL, About 40 fL to about 160 fL, About 40 fL to about 140 fL, About 40 fL to about 120 fL, About 40 fL to about 100 fL, About 40 fL to about 90 fL fL, about 40 fL to about 80 fL, about 40 fL to about 70 fL, about 40 fL to about 60 fL, about 40 fL to about 50 fL, about 50 fL to about 175 fL, about 50 fL to about 160 fL, About 50 fL to about 140 fL, about 50 fL to about 120 fL, about 50 fL to about 100 fL, about 50 fL to about 90 fL, about 50 fL to about 80 fL, about 50 fL to about 70 fL, about 50 fL to about 60 fL, about 60 fL to about 175 fL, about 60 fL to about 160 fL, about 60 fL to about 140 fL, about 60 fL to about 120 fL, about 60 fL to about 100 fL, about 60 fL to about About 90 fL, about 60 fL to about 80 fL, about 60 fL to about 70 fL, about 70 fL to about 175 fL, about 70 fL to about 160 fL, about 70 fL to about 140 fL, about 70 fL to about 120 fL fL, about 70 fL to about 100 fL, about 70 fL to about 90 fL, about 70 fL to about 80 fL, about 80 fL to about 175 fL, about 80 fL to about 160 fL, about 80 fL to about 140 fL, about 80 fL to about About 120 fL, about 80 fL to about 100 fL, about 80 fL to about 90 fL, about 100 fL about 175 fL, about 100 fL to about 160 fL, about 100 fL to about 140 fL, about 100 fL to about 120 fL , about 120 fL to about 175 fL, about 120 fL to about 160 fL, about 120 fL to about 140 fL, about 140 fL to about 175 fL, about 140 fL to about 160 fL, or about 160 fL to about 175 fL, and The standard deviation of the population is less than 50, 40, 30, 20, 10, 5 or 2 fL as the case may be. Mean hematocrit can be determined, for example, using a hematology analyzer (eg, a Coulter counter, a Moxi Z cell counter (Orflo) or a Sysmex hematology analyzer).

In some embodiments, the enucleated erythroid cells described herein have one or more (e.g., 2, 3, 4, or more) of the physical properties described herein, e.g., osmotic fragility, cell size, hemoglobin concentration or phosphatidylserine content. While not wishing to be bound by theory, in some embodiments, enucleated erythroid cells expressing exogenous proteins have physical characteristics similar to wild-type, unprocessed enucleated erythroid cells. Conversely, hypotonically loaded enucleated erythroid cells sometimes display abnormal physical features such as increased osmotic fragility, altered cell size, reduced hemoglobin concentration, or increased phosphatidylserine content on the outer leaflet of the cell membrane.

In some embodiments, the enucleated erythroid cells comprise exogenous proteins encoded by exogenous nucleic acids that are not retained by the cells, are not purified, or are not present entirely outside the enucleated erythroid cells. In some embodiments, the enucleated erythroid cells are in the composition lacking a stabilizer.

In some embodiments, the enucleated erythroid cells have a heme content similar to wild-type, unprocessed enucleated erythroid cells. In some embodiments, the enucleated erythroid cells comprise greater than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or greater than 10% fetal heme. In some embodiments, the enucleated erythroid cells comprise at least about 20, 22, 24, 26, 28, or 30 pg, and optionally up to about 30 pg, of total hemoglobin. In some embodiments, the hemoglobin content is determined using the Drabkin's reagent method of Example 33 of WO2015/073587.

In some embodiments, the enucleated erythroid cells have about the same phosphatidylserine content on the outer leaflet of their cell membranes as wild-type, untreated enucleated erythroid cells. Phosphatidylserine is predominantly found on the inner lobe of the cell membrane of wild-type, unprocessed, enucleated erythroid cells, whereas hypotonic loading results in distribution of phosphatidylserine to the outer lobe, where it elicits an immune response. In some embodiments, the population of enucleated erythroid cells comprises less than about 30, 25, 20, 15, 10, 9, 8, 6, 5, 4, 3, 2, or 1% cells that stain for annexin V positive cells. In some embodiments, phosphatidylserine exposure is assessed by staining for Annexin-V-FITC that preferentially binds PS and measuring FITC fluorescence by flow cytometry, e.g., using Example 54 of WO2015/073587 Methods.

In some embodiments, the population of enucleated erythroid cells comprises at least about 50%, 60%, 70%, 80%, 90% or 95% (and optionally up to 90% or 100%) GPA positive cells. In some embodiments, the presence of GPA is detected using FACS.

In some embodiments, the enucleated erythroid cells have a half-life in the individual of at least 30, 45, or 90 days.

In some embodiments, the population of cells comprising enucleated erythroid cells comprises less than about 10, 5, 4, 3, 2, or 1% echinocytes.

In some embodiments, a composition comprising a plurality of enucleated erythroid cells can be administered to an individual (eg, any individual described herein). In such embodiments, greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90% of the cells in the composition may be enucleated. In some embodiments, the cells (eg, enucleated erythroid cells) contain non-functional nuclei, eg, have been inactivated.

In some embodiments of any of the compositions described herein, the enucleated erythroid cells are human (eg, derived from human donor erythroid cell precursors) enucleated erythroid cells.

In some embodiments of any of the compositions described herein, the enucleated erythroid cells are engineered human enucleated erythroid cells. In some examples, the engineered enucleated erythroid cells comprise a single exogenous protein. In other examples, the engineered enucleated erythroid cells comprise two or more exogenous proteins (eg, any of the exemplary exogenous proteins described herein).

In some embodiments, the exogenous protein present on the engineered enucleated erythrocyte membrane can be the product of a click chemistry reaction (e.g., the exogenous protein can be attached to the cell membrane present on the cell membrane using any of the methods described herein). protein (eg, a second exogenous protein or an endogenous protein)). In some embodiments, the exogenous protein present on the membrane of the engineered enucleated erythroid cell can be the product of a ligation reaction using a sortase (e.g., the exogenous protein can be ligated to A protein present on a cell membrane (eg, a second exogenous protein or an endogenous protein)). Non-limiting examples of ligation reactions using sortases can be found in US Patent No. 10,260,038 and US Patent Publication No. 2016/0082046 Al. In some embodiments, the exogenous protein present on the membrane of the engineered enucleated erythroid cells can be a lipid-anchored protein, such as a GPI-anchored protein, N-myristoylated protein, or S-palmitoylated protein . In some embodiments, the exogenous protein present on the membrane of the engineered enucleated erythroid can be a transmembrane protein (eg, a single-pass or multi-pass transmembrane protein) or a peripheral membrane protein. In some embodiments, the exogenous protein present on the membrane of the engineered enucleated erythroid cells can be a fusion protein comprising a transmembrane domain (e.g., comprising small integral membrane protein 1 (SMIM1) or glycophorin A (GPA) fusion protein of the transmembrane domain). In some embodiments, the exogenous protein present on the membrane of the engineered enucleated erythroid cells does not have any amino acids that protrude into the extracellular space. In some embodiments, the exogenous protein present on the membrane of the engineered enucleated erythroid cells does not have any amino acids that protrude into the cytoplasm of the engineered enucleated erythroid cells. In some embodiments, the exogenous protein present on the membrane of the engineered enucleated erythroid cells has amino acids that protrude into the extracellular space and amino acids that protrude into the cytoplasm of the engineered enucleated erythroid cells . Exemplary methods of producing enucleated erythroid cells using sortagging are described in WO2014/183071 or WO2014/183066, each of which is incorporated by reference in its entirety.

The engineered enucleated erythroid cells can be expressed by one or more nucleic acids (e.g., DNA) encoding one or more exogenous proteins (e.g., any exogenous protein described herein or known in the art). vector or mRNA) into an erythroid cell precursor (eg, any erythroid cell precursor described herein or known in the art). Exemplary methods for introducing DNA expression vectors into erythroid cell precursors include, but are not limited to, liposome-mediated transfer, transformation, biolistics, transfection, and transduction, such as viral-mediated gene transfer (e.g., using Including adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, herpesvirus vectors, and retrovirus-based vectors). Other exemplary methods for introducing DNA expression vectors into erythroid cell precursors include using, for example, naked DNA, _CaPO4 precipitation, DEAE polydextrose, electroporation, protoplast fusion, lipofection, and microinjection of cells.

Optionally, eg, before and/or after introducing one or more nucleic acids encoding one or more exogenous proteins, the erythroid cell precursors are cultured under appropriate conditions to allow differentiation into engineered enucleated erythroid cells. In some embodiments, the resulting engineered enucleated erythroid cells comprise proteins associated with mature erythrocytes, such as heme (e.g., adult heme and/or fetal heme), glycophorin A, and exogenous proteins, And can be verified and quantified by standard methods (eg Western blot or FACS analysis).

In some embodiments, two or more exogenous polypeptides are encoded in a single nucleic acid (eg, a single vector). In a specific example, a single vector has different promoters for each gene, with two proteins initially transcribed into a single polypeptide with a protease cleavage site in the middle, so that subsequent proteolytic processing yields two exogenous proteins , or any other suitable configuration. In some embodiments, two or more polypeptides are encoded in two or more nucleic acids, eg, each vector encodes an exogenous polypeptide.

A nucleic acid may be, for example, DNA or RNA. Many viruses are used as gene transfer vectors, including retroviruses, Moloney murine leukemia virus (MMLV), adenoviruses, adeno-associated virus (AAV), herpes simplex virus (HSV), slow Viruses, such as human immunodeficiency virus 1 (HIV1), and spumaviruses, such as foamy virus.

In some embodiments, the enucleated erythroid cells are expanded at least 1000, 2000, 5000, 10,000, 20,000, 50,000, or 100,000-fold (and optionally at most 200,000 or 500,000-fold). In some embodiments, cell number is measured using an automated cell counter.

In some examples, enucleated erythroid cells or erythroid cell precursors can be transfected with mRNA encoding an exogenous protein to generate engineered enucleated erythroid cells. Messenger RNA can be derived from in vitro transcription of a cDNA plastid construct containing the sequence encoding the exogenous protein. For example, a cDNA sequence encoding an exogenous protein can be inserted into a cloning vector containing a promoter sequence compatible with a particular RNA polymerase. For example, the cloning vector ZAP Express® pBK-CMV (Stratagene, La Jolla, Calif., USA) contains T3 and T7 promoter sequences compatible with T3 and T7 RNA polymerases, respectively. For in vitro transcription of sense mRNA, plastids are linearized at a restriction site downstream of a stop codon corresponding to the end of the sequence encoding the exogenous protein. mRNA is transcribed from linear DNA templates using commercial kits such as, for example, the RNAMaxx® High Yield Transcription Kit (from Stratagene, La Jolla, Calif., USA). In some cases, it may be desirable to generate 5'-m7GpppG-capped mRNA. Thus, transcription of linearized cDNA templates can be performed using, for example, the mMESSAGE mMACHINE High Yield Capped RNA Transcription Kit from Ambion (Austin, Tex., USA). Transcription can be performed at 37°C for 30 minutes to 4 h in a reaction volume of 20-100 μl. Transcribed mRNA was purified from the reaction mixture by a brief treatment with DNaseI to eliminate the linearized DNA template, followed by precipitation in 70% ethanol in the presence of lithium chloride, sodium acetate or ammonium acetate. Transcribed mRNA integrity can be assessed by electrophoresis using agarose-formaldehyde gels or commercially available Novex TBE gels (Novex, Invitrogen, Carlsbad, Calif., USA).

Messenger RNA encoding exogenous proteins can be introduced into enucleated erythroid cells or erythroid cell precursors using various methods including, for example, lipofection and electroporation (van Tandeloo et al., Blood 98:49-56, 2001). For lipofection, for example, 5 μg of in vitro transcribed mRNA was incubated with cationic lipid DMRIE-C (Invitrogen) at a ratio of 1:4 in Opti-MEM (Invitrogen, Carlsbad, Calif., USA) for 5-15 minutes.

Alternatively, various other cationic lipids or cationic polymers can be used to transfect erythroid cell precursors with mRNA including, for example, DOTAP, various forms of polyethyleneimine, and poly-L-lysine (Sigma-Aldrich, Saint Louis, Mo., USA) and Superfect (Qiagen, Inc., Valencia, Calif., USA; see, for example, Bettinger et al., Nucleic Acids Res. 29:3882-3891, 2001). The resulting mRNA/lipid complexes were incubated with cells (1 - ² x 106 cells/mL) at 37°C for 2 hours, washed, and returned to culture. For electroporation, for example, about 5 to 20 x ¹⁰ cells in 500 μL of Opti-MEM (Invitrogen, Carlsbad, Calif., USA) are mixed with about 20 μg of in vitro transcribed mRNA and placed in a 0.4-cm colorimetric tube. Electroporation is performed in a medium, for example, using the Easyject Plus device (EquiBio, Kent, United Kingdom). In some cases, it may be necessary to test various voltages, capacitances, and electroporation volumes to determine the conditions used to transfect a particular mRNA into the erythroid cell precursor. In general, electroporation parameters required for efficient transfection of cells with mRNA are less deleterious than those required for DNA electroporation (van Tandeloo et al., Blood 98:49-56, 2001).

Alternatively, mRNA can be transfected into enucleated erythroid cells or erythroid cell precursors using a peptide-mediated RNA delivery strategy (see, eg, Bettinger et al., Nucleic Acids Res. 29:3882-3891, 2001). For example, the cationic lipid polyethyleneimine 2 kDA (Sigma-Aldrich, Saint Louis, Mo., USA) can be combined with melittin (Alta Biosciences, Birmingham, UK) to increase the efficiency of mRNA transfection, especially in mitotic in subsequent primary cells. Melittin can be conjugated to PEI using a disulfide cross-linker such as, for example, the heterobifunctional cross-linker succinimidyl 3-(2-pyridyldithio)propionate. In vitro transcribed mRNA was pre-incubated with melittin-PEI for 5 to 15 minutes to form RNA/peptide/lipid complexes. This complex was then added to the cells at 37°C in a 5% _CO2 humidified serum-free medium for 2 to 4 hours before being removed and the transfected cells further cultured.

In some embodiments, nucleic acids (eg, any of the exemplary nucleic acids described herein) encoding one or more exogenous proteins (eg, any of the exogenous proteins described herein or any combination of exogenous proteins) ) introduces erythroid cell precursors to generate engineered enucleated erythroid cells. In some embodiments, the exogenous protein is encoded by DNA introduced into the erythroid cell precursor. In some embodiments, the exogenous protein is encoded by RNA introduced into the erythroid cell precursor.

Nucleic acids encoding one or more exogenous proteins can be introduced into erythroid cell precursors prior to terminal differentiation into enucleated erythroid cells using various DNA techniques including, for example, transient or stable transfection and gene therapy approaches.

Viral gene transfer can be used to transfect cells with nucleic acid encoding one or more exogenous proteins. Many viruses can be used as gene transfer vectors, including Moloney murine leukemia virus (MMLV), adenovirus, adeno-associated virus (AAV), herpes simplex virus (HSV), lentiviruses such as human immunodeficiency virus 1 ( HIV1)), and foamy retroviruses such as foamy virus (see, eg, Osten et al., HEP 178:177-202, 2007). For example, retroviruses efficiently transduce mammalian cells, including human cells, and incorporate into chromosomes, providing stable gene transfer.

Nucleic acids encoding one or more exogenous proteins can be transfected into erythroid cell precursors. A suitable vector is the Moloney murine leukemia virus (MMLV) vector (Malik et al., Blood 91:2664-2671, 1998). MMLV-based vectors, an oncogenic retrovirus, are currently used in gene therapy clinical trials (Hassle et al., News Physiol. Sci. 17:87-92, 2002). For example, a DNA construct containing a cDNA encoding an exogenous protein can be generated in a MMLV vector backbone using standard molecular biology techniques. The construct is transfected into a packaging cell line such as eg PA317 cells and the viral supernatant is used to transfect producer cells such as eg PG13 cells. PG13 viral supernatants were incubated with erythroid cell precursors. The expression of exogenous protein can be monitored using FACS analysis (fluorescence-activated cell sorting), for example, using a fluorescently labeled antibody against the exogenous protein (if it is present in engineered human enucleated erythroid cells). on the film). A similar approach can be used for exogenous proteins present in the cytoplasm of engineered human enucleated erythroid cells.

Optionally, nucleic acid encoding a fluorescent tracking molecule such as, for example, green fluorescent protein (GFP) can be transfected into erythroid cell precursors using virus-based methods (Tao et al., Stem Cells 25:670- 678, 2007). Package cells containing DNA encoding enhanced green fluorescent protein (EGFP) or red fluorescent protein (e.g., DsRed-Express) are packaged using packaging cells such as, for example, the Phoenix-Eco cell line (distributed by Orbigen, San Diego, Calif.). Ectopic retroviral vectors. Packaging cell lines stably express viral proteins required for proper viral packaging, including e.g. gag, pol, and env. Phoenix-Eco cell supernatants from shed virus particles can be used to transduce erythroid cells Precursors. In some cases, transduction can be performed on specially coated surfaces (such as, for example, fragments of recombinant fibrin) to enhance gene transfer efficiency with retroviral vectors (e.g., RetroNectin, Takara Bio USA, Madison , Wis.). Cells were grown in RetroNectin-coated dishes with retroviral Phoenix-Eco supernatant and appropriate cofactors. Transduction can be repeated the next day. In this case, evaluation by FACS Percentage of erythroid cell precursors expressing EGFP or DsRed-Express. Other reporter genes that can be used to assess transduction efficiency include, for example, β-galactosidase, chloramphenicol acetyltransferase, and luciferase, as well as low-affinity Nerve growth factor receptor (LNGFR), and human cell surface CD24 antigen (Bierhuizen et al., Leukemia 13:605-613, 1999).

Non-viral vectors can be used to introduce nucleic acid encoding one or more exogenous proteins into erythroid cell precursors to generate engineered enucleated erythroid cells. A variety of delivery methods can be used to introduce non-viral vectors into erythroid cell precursors, including chemical and physical methods.

Nonviral vectors encoding exogenous proteins can be introduced into erythroid cell precursors using synthetic macromolecules such as cationic lipids and polymers (Papapetrou et al., Gene Therapy 12:S118-S130, 2005). Cationic liposomes form complexes with DNA, for example, through charge interactions. Positively charged DNA/lipid complexes bind to negatively charged cell surfaces and are taken up by cells through endocytosis. This method can be used, for example, to transfect hematopoietic cells (see, eg, Keller et al., Gene Therapy 6:931-938, 1999). For erythroid cell precursors, plastid DNA (in serum-free medium such as, for example, OptiMEM (Invitrogen, Carlsbad, CA)) was mixed with cationic liposomes (in serum-free medium) such as the commercial transfection reagent Lipofectamine™ ( Invitrogen, Carlsbad, Calif.)) and incubated for at least 20 minutes to form complexes. DNA/liposome complexes were added to erythroid cell precursors and allowed to incubate for 5-24 hours prior to analysis of transgene expression of exogenous proteins. Alternatively, other commercial liposome transfection reagents can be used (eg, In vivo GeneSHUTTLE™, Qbiogene, Carlsbad, Calif.).

Optionally, cationic polymers such as, for example, polyethyleneimine (PEI) can be used to efficiently transfect erythroid cell precursors, such as hematopoietic and cord blood-derived CD34 ⁺ cells (see, for example, Shin et al., Biochim. Biophys. Acta 1725:377-384, 2005). Human CD34 ⁺ cells were isolated from human umbilical cord blood and cultured in Iscove's modified Dulbecco's medium supplemented with 200 ng/ml stem cell factor and 20% heat-inactivated serum. Plastid DNA encoding exogenous proteins was incubated with branched or linear PEI of various sizes (from 0.8 K to 750 K) (Sigma Aldrich, Saint Louis, Mo., USA; Fermetas, Hanover, Md., USA). A PEI stock solution was prepared in 4.2 mg/mL distilled water and slightly acidified to pH 5.0 using HCl. Based on the calculation that 1 μg DNA contains 3 nmol phosphate and 1 μL PEI stock solution contains 10 nmol amine nitrogen, DNA can be mixed with PEI at various nitrogen/phosphate ratios for 30 minutes at room temperature. Isolated CD34 ⁺ cells were seeded with DNA/cation complexes, centrifuged at 280 xg for 5 minutes and incubated in culture medium for 4 hours or more until assessment of exogenous protein expression.

Plastid vectors can be introduced into suitable erythroid cell precursors using physical methods such as particle-mediated transfection, "gene guns", biolistics or particle bombardment techniques (Papapetrou, et al., Gene Therapy 12:S118-S130, 2005). In this case, DNA encoding exogenous proteins is adsorbed onto gold particles and delivered to cells by a particle gun. For example, this method can be used to transfect erythroid cell precursors, such as hematopoietic stem cells derived from umbilical cord blood (see, eg, Verma et al., Gene Therapy 5:692-699, 1998). In doing so, cord blood is isolated and diluted three-fold in phosphate-buffered saline. CD34 ⁺ cells were purified using an anti-CD34 monoclonal antibody in combination with secondary antibody-coated magnetic beads and a magnetic separation system (eg, Miltenyi MiniMac System, Auburn, Calif., USA). CD34 ⁺ enriched cells can be cultured as described herein. For transfection, plastid DNA encoding exogenous proteins is precipitated onto particles (eg, gold beads) by treatment with calcium chloride and spermtriamine. After washing the DNA-coated beads with ethanol, the beads can be delivered into cultured cells using, for example, the Biolistic PDS-1000/He system (Bio-Rad, Hercules, Calif., USA). Reporter genes such as, for example, β-galactosidase, chloramphenicol acetyltransferase, luciferase or green fluorescent protein can be used to assess transfection efficiency.

Optionally, the plastid vector can be introduced into the erythroid cell precursor using electroporation. Electroporation creates transient pores in cell membranes, allowing the introduction of various molecules, including, for example, DNA and RNA, into cells. Thus, CD34 ⁺ cells were isolated and cultured as described herein. Just before electroporation, cells are detached by centrifugation at 250xg for 10 minutes at room temperature and resuspended at ^0.2-10x106 viable cells/ml in electroporation buffer such as, For example X-VIVO 10, supplemented with 1.0% Human Serum Albumin (HSA). Add plastid DNA (1-50 µg) to an appropriate electroporation cuvette along with 500 µL of cell suspension.

Electroporation can be performed using, for example, an ECM 600 electroporator (Genetronics, San Diego, Calif., USA) at voltages ranging from 200 V to 280 V and pulse lengths ranging from 25 to 70 milliseconds. A number of alternative electroporation instruments are commercially available and can be used for this purpose (eg, Gene Pulser Xcell™, BioRad, Hercules, Calif.; Cellject Duo, Thermo Science, Milford, Mass.). Alternatively, isolated CD34 ⁺ cells can be efficiently electroporated using the following parameters: 4 mm cuvette, 1600 μE, 550 V/cm and 10 μg DNA per ⁵⁰⁰ μL of 1x105 cells/mL (Oldak et al ., Acta Biochim . Polonica 49:625-632, 2002).

Nucleofection (a form of electroporation) can also be used to transfect erythroid cell precursors. In this case, transfection is performed in a cell-type-specific solution using electrical parameters that allow direct transport of DNA (or other reagents) to the nucleus, reducing the risk of possible degradation in the cytoplasm. For example, the Human CD34 Cell Nucleofector™ Kit (from Amaxa Inc.) can be used to transfect erythroid cell precursors. In this case, ^1-5x106 cells in Human CD34 Cell Nucleofector™ Solution were mixed with 1-5 μg DNA and transfected in the Nucleofector™ instrument using the preprogrammed settings determined by the manufacturer.

Erythroid cell precursors can be transfected non-virally with conventional expression vectors which are unable to self-replicate in mammalian cells unless incorporated into the gene body. Alternatively, erythroid cell precursors can be transfected with episomal vectors that act as self-replicating genetic units in the host nucleus without incorporation into chromosomes (Papapetrou et al., Gene Therapy 12:S118-S130, 2005) . These vectors utilize genetic elements derived from viruses that normally replicate extrachromosomally following latent infection, such as, for example, EBV, human polyomavirus BK, bovine papillomavirus-1 (BPV-1), herpes simplex Virus-1 (HSV), and Simian Virus 40 (SV40). Mammalian artificial chromosomes can also be used for non-viral gene transfer (Vanderbyl et al., Exp. Hematol. 33:1470-1476, 2005).

Exogenous nucleic acids encoding one or more exogenous proteins can be assembled into expression vectors by standard molecular biology methods known in the art, such as restriction digestion, overlap extension PCR, and Gibson assembly.

The exogenous nucleic acid may comprise a gene encoding an exogenous protein not normally present on the surface of a cell (such as that of an enucleated erythrocyte) fused to a gene encoding an endogenous or native membrane protein, thereby creating Exogenous protein is expressed on the surface. For example, an exogenous gene encoding an exogenous protein can be colonized at the N-terminus (after the leader sequence of a type 1 membrane protein), at the C-terminus of a type 2 membrane protein, or at the GPI appendage of a GPI-linked membrane protein. upstream of the junction point.

A flexible amino acid linker can be introduced between the two fusion genes using standard breeding methods. For example, flexible linkers are polyglycine-polyserine linkers, such as [Gly ₄ Ser] ₃ (SEQ ID NO: 29), commonly used to generate single-chain antibody fragments from full-length antibodies (Antibody Engineering: Methods & Protocols, B. Lo, ed., Humana Press, 2004, 576 pp.), or Ala-Gly-Ser-Thr (SEQ ID NO: 68) polypeptides, such as those used to generate single-chain Arc suppressors ( Robinson & Sauer, Proc. Nat'l. Acad. Sci. USA 95: 5929-34, 1998). In some embodiments, flexible linkers provide exogenous proteins with greater flexibility and spatial freedom than equivalent constructs without flexible linkers. This adds flexibility and can be used in applications requiring binding to a target (eg, an antibody or protein, or an enzymatic reaction of a protein), where the active site must be accessible to the substrate (eg, target).

In some embodiments, provided methods comprise introducing the nucleic acid into the cell by electroporation by contacting an erythroid cell precursor with the nucleic acid and under conditions effective to deliver the nucleic acid to the cell, such as those described herein. , while delivering large nucleic acid molecules (particularly RNA, such as mRNA) to erythroid cell precursors. Suitable electroporators include, but are not limited to, Bio-Rad GENE PULSER and GENE PULSER II; Life Technologies NEON; BTX GEMINI system; and MAXCYTE electroporators. These methods do not require viral delivery or the use of viral vectors. Suitable nucleic acids include RNA, such as mRNA. Suitable nucleic acids also include DNA (including transposable elements, stable episomes, plastid DNA or linear DNA).

Conditions for electroporation of cell lines have been described by, for example, Van Tendeloo et al., Blood 98(1):49-56, 200. For the Life Technologies Neon Transfection System, suitable electroporation conditions for the methods described herein include: pulse voltages ranging from about 500 to about 2000 V, about 800 to about 1800 V, or about 850 to about 1700 V; pulse widths ranging from about 5 to about 50 msec, or about 10 to about 40 msec; and the number of pulses ranges from 1 to 2 pulses, 1 to 3 pulses, 1 to 4 pulses, or 1 to 5 pulses.

Regarding electroporation of erythroid cell precursors, particularly suitable conditions include, for example, for 4 days: a) pulse voltage 1300-1400, pulse width: 10-20 msec, pulse number: 1-3; b) pulse voltage 1400, pulse Width: 10 msec, number of pulses: 3; c) pulse voltage 1400, pulse width: 20 msec, number of pulses: 1; and d) pulse voltage 1300, pulse width: 10 msec, number of pulses: 3.

Regarding electroporation of erythroid cell precursors, particularly suitable conditions include, for example, for 8-9 days: a) pulse voltage: 1400-1600, pulse width: 20, pulse number: 1; b) pulse voltage: 1100-1300, Pulse width: 30, pulse number: 1; c) pulse voltage: 1000-1200, pulse width: 40, pulse number: 1; d) pulse voltage: 1100-1400, pulse width: 20, pulse number: 2; e) Pulse voltage: 950-1150, pulse width: 30, pulse number: 2; f) pulse voltage: 1300-1600, pulse width: 10, pulse number: 3. These conditions generally result in a transfection efficiency of at least about 60% or more (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, or at least about 97%, or more ), and the cell viability is at least about 70% or more (eg, at least about 75%, 80%, 85%, 90%, 95%, or at least about 97%, or more).

Under differentiation conditions, particularly suitable conditions for electroporation of erythroid cell precursors in culture include, for example, for 12-13 days: a) pulse voltage: 1500-1700, pulse width: 20, pulse number: 1; and b) Pulse voltage: 1500-1600, pulse width: 10, pulse number: 3. These conditions generally result in a transfection efficiency of at least about 50% or more (e.g., at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or at least about 97%). %, or more), and the cell viability is at least about 70% or more (e.g., at least about 75%, 80%, 85%, 90%, 95%, or at least about 97%, or more).

The conditions disclosed herein can be easily adjusted by those skilled in the art with reference to the Life Technologies Neon system, so that different electroporators and/or different electroporation devices can be used only by routine experimentation, and as far as the methods disclosed are concerned, this paper The specific electroporator described is not limited.

In some embodiments, using the electroporation conditions described herein, cultured erythroid cell precursors are electroporated a first time, incubated for a desired period of time (optionally under differentiation conditions), and then subjected to a second electroporation. Re-electroporation. In some embodiments, cultured erythroid cell precursors are subjected to a first electroporation, then incubated for a desired period of time (optionally under differentiating conditions), and then subjected to a second, third, fourth, fifth or second electroporation. The sixth re-electroporation. Depending on the situation, the culture period between the first and second, second and third electroporation, etc. may be changed. For example, the period between electroporations can be adjusted as desired, for example, the period can be 30 minutes, 1 hour, 6 hours, 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 48 hours, 3 days, 4 hours. days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days or 21 days. For example, 1 and 2, 1 and 3, 1 and 4, 1 and 5, 1 and 6, 1 and 7, 1 and 8, 1 and 9, 1 and 10, 1 and 11, 1 and 12, 1 Erythroid cell precursors were electroporated on days 13, 1 and 14, 1 and 15, or 1 and 16. In another example, at 2 and 3, 2 and 4, 2 and 5, 2 and 6, 2 and 7, 2 and 8, 2 and 9, 2 and 10, 2 and 11, 2 and 12, 2 Cells were electroporated on and 13, 2 and 14, 2 and 15 or 2 and 16 days. In yet another example, at 3 and 4, 3 and 5, 3 and 6, 3 and 7, 3 and 8, 3 and 9, 3 and 10, 3 and 11, 3 and 12, 3 and 13, Erythroid cell precursors were electroporated on days 3 and 14, 3 and 15 or 3 and 16. In yet another example, at 4 and 5, 4 and 6, 4 and 7, 4 and 8, 4 and 9, 4 and 10, 4 and 11, 4 and 12, 4 and 13, 4 and 14, Cells were electroporated on days 4 and 15 or 4 and 16. In yet another example, at 5 and 6, 5 and 7, 5 and 8, 5 and 9, 5 and 10, 5 and 11, 5 and 12, 5 and 13, 5 and 14, 5 and 15 or Cells were electroporated on days 5 and 16. In yet another example, on days 6 and 7, 6 and 8, 6 and 9, 6 and 10, 6 and 11, 6 and 12, 6 and 13, 6 and 14, 6 and 15, or 6 and 16 days Electroporation of erythroid cell precursors. In yet another example, erythroid cells can be treated on days 7 and 8, 7 and 9, 7 and 10, 7 and 11, 7 and 12, 7 and 13, 7 and 14, 7 and 15, or 7 and 16 Precursors for electroporation. In yet another example, the erythroid cell precursor can be electroporated on days 8 and 9, 8 and 10, 8 and 11, 8 and 12, 8 and 13, 8 and 14, 8 and 15, or 8 and 16. perforation. In yet another example, the erythroid cell precursor can be electroporated on days 9, 10, 9 and 11, 9 and 12, 9 and 13, 9 and 14, 9 and 15, or 9 and 16. In yet another example, the erythroid cell precursor can be electroporated on days 10 and 11, 10 and 12, 10 and 13, 10 and 14, 10 and 15, or 10 and 16 days. In yet another example, the erythroid cell precursor can be electroporated on days 11 and 12, 11 and 13, 11 and 14, 11 and 15, or 11 and 16. In yet another example, the erythroid cell precursor can be electroporated on days 12 and 13, 12 and 14, 12 and 15, or 12 and 16. In yet another example, the erythroid cell precursor can be electroporated on days 13 and 14, 13 and 15, or 13 and 16. In yet another example, the erythroid cell precursor can be electroporated on days 14 and 15 or 14 and 16. Optionally, the erythroid cell precursor may be electroporated more than two times, such as three, four, five or six times, and the intervals may be selected as desired at any point in the cell differentiation process.

In some embodiments, using the electroporation conditions described herein, on day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 days of differentiation Electroporation of cultured erythroid cell precursors.

In some embodiments, the engineered enucleated erythroid cells can be click-engaged engineered enucleated erythroid cells. A catalytically bond-forming polypeptide domain present in the cytoplasm or present on the membrane may be expressed, for example, on or within the erythroid cell precursor. There are a number of polypeptides that form catalytic bonds, including transpeptidases, sortases and isopeptidases, including those derived from Spy0128, a protein isolated from Streptococcus pyogenes . It has been demonstrated that cleavage of the autocatalytic isopeptide bond-forming subunit (CnaB2 domain) of Spy0128 results in two distinct polypeptides that retain specific catalytic activity for each other. The peptides in this system are called SpyTag and SpyCatcher. After mixing, the SpyTag and SpyCatcher undergo isopeptide bond formation between Aspl 17 on the SpyTag and Lys31 on the SpyCatcher (Zakeri and Howarth, JACS 132:4526, 2010). This reaction is compatible with the cellular environment and highly specific for protein/peptide conjugation (Zakeri et al., Proc. Natl. Acad. Sci. USA 109:E690-E697, 2012). SpyTag and SpyCatcher have been shown to direct post-translational topological modifications of elastin-like proteins. For example, the placement of SpyTag at the N-terminus and SpyCatcher at the C-terminus guides the formation of circular elastin-like proteins (Zhang et al, J. Am. Chem. Soc. 2013).

The components SpyTag and SpyCatcher are interchangeable such that a system in which molecule A is fused to SpyTag and molecule B is fused to SpyCatcher is functionally equivalent to a system in which molecule A is fused to SpyCatcher and molecule B is fused to SpyTag. For purposes of the present invention, when using SpyTag and SpyCatcher, it is understood that complementary molecules may be substituted in their place.

Polypeptides that form catalytic bonds (such as the SpyTag/SpyCatcher system) can be used to attach exogenous proteins to surfaces (eg, erythroid cell precursors or enucleated erythroid cells). SpyTag polypeptide sequences can be expressed on the extracellular surface of erythroid cell precursors or enucleated erythroid cells. For example, a SpyTag polypeptide can be fused to the N-terminus of a type 1 or type 3 transmembrane protein (e.g., glycophorin A), to the C-terminus of a type 2 transmembrane protein (e.g., Kell), in frame Inserted intracellularly at the extracellular terminus or extracellular loop of a multipass transmembrane protein (eg, band 3), fused to a GPI-receptor polypeptide (eg, CD55 or CD59), fused to a lipid chain-anchored polypeptide, or fused to to peripheral membrane proteins. Exogenous proteins can be fused to SpyCatcher. Nucleic acids encoding SpyCatcher fusions can be expressed and secreted by the same erythroid cell precursors or enucleated erythroid cells that express SpyTag fusions. Alternatively, nucleic acid sequences encoding SpyCatcher fusions can be produced exogenously, eg, in bacteria, fungi, insect, mammalian or cell-free production systems. Following the reaction of the SpyTag and SpyCatcher polypeptides, a covalent bond will be formed that attaches the exogenous protein to the surface of the erythroid cell precursor or the enucleated erythroid cell.

In one embodiment, the SpyTag polypeptide can be expressed as fused to the N-terminus of glycophorin A under the control of the Gatal promoter in erythroid cells. The exogenous protein fused to the SpyCatcher polypeptide sequence can be expressed in the same erythrocyte under the control of the Gatal promoter. After expression of both fusion polypeptides, an isopeptide bond will be formed between the SpyTag and SpyCatcher polypeptides, thereby forming a covalent bond between the erythroid surface and the exogenous protein.

In another embodiment, the SpyTag polypeptide can be expressed as fused to the N-terminus of glycophorin A in erythroid cell precursors or enucleated erythroid cells under the control of the Gatal promoter. The exogenous protein fused to the SpyCatcher polypeptide sequence can be expressed in a suitable mammalian cell expression system (eg HEK293 cells). Following expression of the SpyTag fusion polypeptide on erythroid cell precursors or enucleated erythroid cells, the SpyCatcher fusion polypeptide can be contacted with the cells. Under suitable reaction conditions, an isopeptide bond will be formed between the SpyTag and SpyCatcher polypeptides, thereby forming a covalent bond between the surface of the erythroid cell precursor or the surface of the enucleated erythroid cell and the exogenous protein.

Polypeptides that form catalytic bonds (such as the SpyTag/SpyCatcher system) can be used to anchor exogenous proteins in the intracellular space of erythroid cell precursors or enucleated erythroid cells. The SpyTag polypeptide sequence can be expressed in the intracellular space of erythroid cell precursors or enucleated erythroid cells by a variety of methods, including direct expression of a transgene, fusion to an endogenous intracellular protein (such as, for example, heme), fusion to an endogenous Intracellular domains of sex cell surface proteins such as, for example, band 3, glycophorin A, Kell, or structural components fused to the cytoskeleton. SpyTag sequences are not limited to polypeptide ends, and can be incorporated into internal sequences of endogenous polypeptides, allowing uninterrupted polypeptide translation and localization. Exogenous proteins can be fused to SpyCatcher. Nucleic acid sequences encoding SpyCatcher fusions can be expressed in the same erythroid cell precursors or enucleated erythroid cells that express SpyTag fusions. After the SpyTag and SpyCatcher peptides react, a covalent bond will be formed that anchors the exogenous protein in the intracellular space of the erythroid cell precursor or the enucleated erythroid cell.

In one embodiment, erythroid cell precursors or enucleated erythroid cells can intracellularly express SpyTag fused to heme beta. Erythroid cell precursors or enucleated erythroid cells can be genetically modified with a gene sequence including a heme promoter, a β-globin gene, and a SpyTag sequence such that after translation, the intracellular β-globin is fused to the SpyTag at its C-terminus . In addition, erythroid cell precursors or enucleated erythroid cells express a gene directed by the Gatal promoter that encodes SpyCatcher kinesin expression (eg, phenylalanine hydroxylase (PAH) expression), thereby rendering the cell Intein (eg, PAH) is fused to SpyCatcher at its N-terminus. After both fusion proteins are expressed, the SpyTag-bound β-globin is linked to a SpyCatcher-binding protein (eg, PAH) in the intracellular space via an isopeptide bond, thereby anchoring the protein (eg, PAH) to the β-globin and are preserved during maturity.

In another embodiment, a SpyTag polypeptide can be expressed as an exogenous protein fused to an erythroid cell precursor or an enucleated erythroid cell. SpyCatcher polypeptides can be expressed as fused to the C-terminus of glycophorin A (intracellularly) within the same erythroid cell precursor or enucleated erythroid cells. After expression of both fusion polypeptides, an isopeptide bond will be formed between the SpyTag and SpyCatcher polypeptides, thereby forming a covalent bond between the membrane-anchored endogenous erythroid polypeptide and the exogenous protein.

Other molecular fusions can be formed between polypeptides and include direct or indirect junctions. Polypeptides can be joined to each other directly or indirectly via a linker. Linkers can be peptides, polymers, aptamers or nucleic acids. Polymers can be, for example, natural, synthetic, linear or branched. The exogenous protein may comprise a heterologous fusion protein comprising a first polypeptide and a second polypeptide comprising a protein directly linked to each other or having an intervening linker sequence and/or other sequences at one or both ends. peptide. Attachment to the linker can be via covalent or ionic bonding.

In some embodiments, the engineered enucleated erythroid cells are hypotonically loaded human enucleated erythroid cells. For hypotonic loading/lysis, erythroid cell precursors or enucleated erythroid cells are exposed to a low ionic strength buffer to rupture. The exogenous protein is distributed in the cell. Enucleated erythrocytes or erythroid cell precursors can be lysed in a hypotonic manner by adding a 30-50 fold excess volume of 5 mM phosphate buffer (pH 8) to the isolated enucleated erythroid pellet. Lysed cell membranes were obtained by centrifugation. The solubilised cell membrane pellet is resuspended and incubated in low ionic strength buffer in the presence of exogenous protein, for example, for 30 minutes. Alternatively, lysed cell membranes can be incubated with exogenous proteins for as little as a minute, or as many as several days, depending on the optimal conditions determined for efficient loading of enucleated erythroid cells or erythroid precursors. For hypotonic loading of nucleic acids encoding one or more exogenous proteins (e.g., any exogenous protein described herein or known in the art), the nucleic acid can be suspended in a hypotonic Tris-HCl solution (pH 7.0) and Injected into erythroid cell precursors. The Tris-HCl concentration may be from about 20 mmol/l to about 150 mmol/l, depending on optimal conditions determined for efficient loading of enucleated erythroid cells.

Alternatively, erythroid cell precursors or enucleated erythroid cells can be loaded with exogenous proteins using controlled dialysis against a hypotonic solution to swell the cells and form pores in the cell membrane (see, e.g., U.S. Patent No. 4,327,710; 5,753,221; 6,495,351 and 10,046,009). For example, cell pellets were resuspended in 10 mM HEPES, 140 mM NaCl, 5 mM glucose, pH 7.4, and resuspended in 10 mM NaH ₂ PO ₄ , 10 mM NaHCO ₃ , 20 mM glucose, and 4 mM MgCl ₂ , pH 7.4 low ionic strength buffer for dialysis. After 30-60 minutes, the cells were dialyzed against a solution of ₁₆ mM _NaH2PO4 , pH 7.4 containing exogenous protein for an additional 30-60 minutes. All these procedures can advantageously be performed at a temperature of 4 °C. In some cases, it may be beneficial to load large quantities of erythroid cell precursors or enucleated erythroid cells by means of dialysis, and specific equipment designed for this purpose may be used (see, e.g., U.S. Pat. Nos. 4,327,710, 6,139,836 and No. 6,495,351).

Exemplary methods of making enucleated erythroid cells (e.g., reticulocytes or erythrocytes) comprising exogenous polypeptides are described, for example, in WO2015/073587, WO2015/153102, WO2020/243006, and WO2020/219909, each of which is incorporated by reference in its entirety way incorporated.

In some embodiments, the population of enucleated erythroid cells comprises less than 1% viable enucleated cells, eg, comprises no detectable viable enucleated cells. In some embodiments, enucleation is measured by FACS using nuclear staining. In some embodiments, at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80% (and optionally up to about 70, 80, 90, or 100%) of the population of enucleated erythroid cells ) comprising one or more (eg, 2, 3, 4 or more) exogenous polypeptides. In some embodiments, the expression of exogenous polypeptides can be measured by FACS using labeled antibodies directed against the polypeptides. In some embodiments, at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80% (and optionally up to about 70, 80, 90, or 100%) of a population of cells are enucleated , and comprising one or more (eg, 2, 3, 4 or more) exogenous polypeptides. In some embodiments, the population of enucleated erythroid cells comprises about 1x10 ⁹ - 2x10 ⁹ , 2x10 ⁹ - 5x10 ⁹ , 5x10 ⁹ - 1x10 ¹⁰ , 1x10 ¹⁰ - 2x10 ¹⁰ , 2x10 ¹⁰ - 5x10 ¹⁰ , 5x10 ¹⁰ - 1x10 ¹¹ , 1x10 ¹¹ - 2x10 ¹¹ , 2x10 ¹¹ - 5x10 ¹¹ , 5x10 ¹¹ - 1x10 ¹² , 1x10 ¹² - 2x10 ¹² , 2x10 ¹² - 5x10 ¹² or 5x10 ¹² - 1x10 ¹³ cells.

Any of the enucleated erythroid cells described herein may be formulated as described, eg, in WO 2020/219909 (incorporated herein by reference). Method of treating an individual by increasing the number of NKp30 positive lymphocytes

Also provided herein is a method of increasing the number of NKp30-positive lymphocytes (e.g., NKp30-positive NK cells) in an individual previously identified or diagnosed as having a B7-H6-positive cancer comprising administering to the individual a therapeutically effective amount of enucleated erythroid cells ( For example, any of the exemplary enucleated erythroid cells described herein) comprising on their extracellular surface an exogenous fusion polypeptide comprising (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof.

Also provided herein is a method of increasing the number of NKp30-positive lymphocytes (e.g., NKp30-positive NK cells) in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of enucleated erythroid cells (e.g., any of the exemplary enucleated cells described herein) erythroid cells) comprising on their extracellular surface an exogenous fusion polypeptide comprising (i) IL-15 or a functional fragment thereof, and (ii) an IL-15 receptor α or a functional fragment thereof; and an exogenous polypeptide comprising a 4-1BBL polypeptide or a functional fragment thereof.

Some embodiments of these methods also include administering to the individual an NKG2A inhibitor (eg, any of the exemplary NKG2A inhibitors described herein).

In some embodiments of these methods, administering comprises intravenously administering to the individual.

Some embodiments of any of the methods described herein also include administering to the individual one or more additional therapeutic agents. In some embodiments, the one or more additional therapeutic agents are cancer therapeutics. In some embodiments, the cancer therapeutic is selected from the group consisting of inhibitors of immune checkpoint molecules (e.g., any of the exemplary inhibitors of immune checkpoint molecules described herein), chemotherapeutic agents, therapeutic antibodies, chimeric antigen receptor T cells, kinase inhibitors, or soluble cytokines. In some embodiments, one or more additional therapeutic agents can be administered to a subject substantially simultaneously with any of the compositions provided herein.

In some embodiments, one or more additional therapeutic agents can be administered to the individual before or after administration of any of the compositions described herein to the individual.

Some embodiments of these methods can further comprise identifying or diagnosing the individual as having a B7-H6 positive cancer. Some embodiments of these methods can further comprise identifying or diagnosing the individual as having a B7-H6 positive and HLA-E negative cancer.

For example, in some embodiments, one or more additional therapeutic agents include blocking, reducing and/or inhibiting PD-1 and PD-L1 or PD-L2, and/or blocking, reducing and/or inhibiting PD-1 and PD-L1 or PD-L2 binding agents (as non-limiting examples, one or more nivolumab (ONO-4538/BMS-936558, MDX1106, OPDIVO, BRISTOL MYERS SQUIBB), pembrolizumab ( pembrolizumab (KEYTRUDA, MERCK), pidilizumab (CT-011, CURE TECH), MK-3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), and MPDL328OA (ROCHE)). In some embodiments, the one or more additional therapeutic agents include blocking, reducing and/or inhibiting the activity of CTLA-4 and/or the interaction of CTLA-4 with one or more of its receptors (e.g., CD80, CD86, AP2M1, SHP- 2 and PPP2R5A) binding agents. For example, in some embodiments, the agent that inhibits CTLA-4 activity is an antibody, such as ipilimumab (MDX-010, MDX-101, Yervoy, BRISTOL MYERS SQUIBB) and/or tremelimumab ( tremelimumab) (PFIZER). Furthermore, in some embodiments, the one or more additional therapeutic agents include blocking antibodies that target immune checkpoint molecules, such as, for example, BTLA, HVEM, TIM3, GALS, LAG3, VISTA, KIR, 2B4, CD160 (also known as BY55 ), CGEN-15049, CHK 1 and CHK2 kinases, A2aR, CEACAM (eg, CEACAM-1, CEACAM-3 and/or CEACAM-5), GITR, GITRL, Galectin-9, CD244, CD160, TIGIT, SIRPα, ICOS, CD172a, TMIGD2 and various B-7 family ligands (including but not limited to B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7- H5, B7-H6 and B7-H7).

In some embodiments of these methods, the methods result in an increase (e.g., an increase of at least 5%, an increase of at least 10%, an increase of at least 15%, an increase of at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% increase, at least 80% increase, at least 85% increase, at least 90% increase, at least 95% increase, at least 100% increase, at least 120% increase, at least 140% increase, at least 160% increase, at least 160% increase, at least 180%, at least 200% increase, at least 220% increase, at least 240% increase, at least 260% increase, at least 280% increase, or at least 300% increase (e.g., compared to NKp30-positive cells (e.g., NKp30-positive cells) in the subject prior to administration NK cells) compared to the number of).

In some embodiments of these methods, the methods result in an increase (e.g., an increase of at least 5%, an increase of at least 10%, an increase in the number of NKp30 positive/NKG2A negative lymphocytes (e.g., NKp30 positive/NKG2A negative NK cells) in the individual At least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least At least 65% increase, at least 70% increase, at least 75% increase, at least 80% increase, at least 85% increase, at least 90% increase, at least 95% increase, at least 100% increase, at least 120% increase, at least 140% increase, increase At least 160%, at least 180% increase, at least 200% increase, at least 220% increase, at least 240% increase, at least 260% increase, at least 280% increase, or at least 300% increase (e.g., compared to NKp30-positive in an individual prior to administration /NKG2A-negative cells (eg, NKp30-positive/NKG2A-negative NK cells) compared to the number).

In some embodiments of these methods, the methods result in an increased (e.g., at least 5% increase, at least 10% increase, at least 15% increase, at least 15% increase, At least 20% increase, at least 25% increase, at least 30% increase, at least 35% increase, at least 40% increase, at least 45% increase, at least 50% increase, at least 55% increase, at least 60% increase, at least 65% increase, increase At least 70% increase, at least 75% increase, at least 80% increase, at least 85% increase, at least 90% increase, at least 95% increase, at least 100% increase, at least 120% increase, at least 140% increase, at least 160% increase, increase At least 180%, at least 200% increase, at least 220% increase, at least 240% increase, at least 260% increase, at least 280% increase, or at least 300% increase (e.g., compared to NKp30-positive cells (e.g., NKp30 positive NK cells) compared to the number of transported).

In some embodiments of these methods, the methods result in increased (e.g., at least 5% increase, at least 10% increase, at least 10% increase, at least 5% increase, at least 10% increase, At least 15% increase, at least 20% increase, at least 25% increase, at least 30% increase, at least 35% increase, at least 40% increase, at least 45% increase, at least 50% increase, at least 55% increase, at least 60% increase, At least 65% increase, at least 70% increase, at least 75% increase, at least 80% increase, at least 85% increase, at least 90% increase, at least 95% increase, at least 100% increase, at least 120% increase, at least 140% increase, An increase of at least 160%, an increase of at least 180%, an increase of at least 200%, an increase of at least 220%, an increase of at least 240%, an increase of at least 260%, an increase of at least 280%, or an increase of at least 300% (e.g., compared to NKp30 in a subject prior to administration Trafficked numbers of positive/NKG2A-negative cells (eg, NKp30-positive/NKG2A-negative NK cells) compared).

In some embodiments, the individual has been previously diagnosed or identified as having a B7-H6 positive cancer (eg, any of the exemplary B7-H6 positive cancers described herein). In some embodiments, the individual has been previously diagnosed or identified as having a B7-H6 positive and HLA-E negative cancer. Some embodiments of these methods can further comprise identifying or diagnosing the individual as having a B7-H6 positive cancer. Some embodiments of these methods can further comprise identifying or diagnosing the individual as having a B7-H6 positive and HLA-E-negative cancer.

Non-limiting examples of B7-H6 positive cancers include: adrenocortical carcinoma, cholangiocarcinoma, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, acute myeloid leukemia, lymphoid Tumors, diffuse large b-cell lymphoma, renal carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, renal chromophobe, liver carcinoma, lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, mesothelioma, ovary cancer, pancreatic cancer, rectal cancer, skin cancer, gastric cancer, testicular cancer, thymoma, thyroid cancer, uterine cancer, uterine endometrial cancer, uterine carcinosarcoma, and uveal melanoma.

In some embodiments, the individual is an adult human individual. In some embodiments, the individual is a pediatric human individual.

Enucleated erythroid cells can be administered to an individual as described herein using any dosage and with any frequency. Methods of treating individuals with B7-H6 positive cancer

Also provided herein are methods of treating an individual previously identified or diagnosed as having a B7-H6-positive cancer, such as any of the exemplary B7-H6-positive cancers described herein, comprising administering a drug to a patient previously identified or diagnosed as having a B7-H6 Individuals positive for cancer are administered a therapeutically effective amount of enucleated erythroid cells (such as any of the exemplary enucleated erythroid cells described herein) comprising an exogenous fusion polypeptide on their extracellular surface, the exogenous The derived fusion polypeptide comprises (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof.

Also provided herein are methods of treating an individual with a B7-H6-positive cancer, such as any of the exemplary B7-H6-positive cancers described herein, comprising administering to an individual previously identified or diagnosed as having a B7-H6-positive cancer A therapeutically effective amount of enucleated erythroid cells (such as any of the exemplary enucleated erythroid cells described herein) comprising on their extracellular surface an exogenous fusion polypeptide comprising ( i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof; and an exogenous polypeptide comprising a 4-1BBL polypeptide or a functional fragment thereof.

In some embodiments of these methods, administering comprises intravenously administering to the individual.

Some embodiments of these methods also include administering to the individual an NKG2A inhibitor (eg, any of the exemplary NKG2A inhibitors described herein).

Some embodiments of any of the methods described herein also include administering to the individual one or more additional therapeutic agents. In some embodiments, the one or more additional therapeutic agents are cancer therapeutics. In some embodiments, the cancer therapeutic is selected from the group consisting of inhibitors of immune checkpoint molecules (e.g., any of the exemplary inhibitors of immune checkpoint molecules described herein), chemotherapeutic agents, therapeutic antibodies, chimeric antigen receptor T cells, kinase inhibitors, or soluble cytokines. In some embodiments, one or more additional therapeutic agents can be administered to a subject substantially simultaneously with any of the compositions provided herein. In some embodiments, one or more additional therapeutic agents can be administered to the individual before or after administration of any of the compositions described herein to the individual.

For example, in some embodiments, one or more additional therapeutic agents include blocking, reducing and/or inhibiting PD-1 and PD-L1 or PD-L2, and/or blocking, reducing and/or inhibiting PD-1 and PD-L1 or PD-L2 binding agents (as non-limiting examples, one or more of nivolumab (ONO-4538/BMS-936558, MDX1106, OPDIVO, BRISTOL MYERS SQUIBB), pembrolizumab (KEYTRUDA, MERCK ), Pidizumab (CT-011, CURE TECH), MK-3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), and MPDL328OA (ROCHE)). In some embodiments, the one or more additional therapeutic agents include blocking, reducing and/or inhibiting the activity of CTLA-4 and/or the interaction of CTLA-4 with one or more of its receptors (e.g., CD80, CD86, AP2M1, SHP- 2 and PPP2R5A) binding agents. For example, in some embodiments, the agent that inhibits CTLA-4 activity is an antibody, such as ipilimumab (MDX-010, MDX-101, Yervoy, BRISTOL MYERS SQUIBB) and/or tremelimumab (PFIZER). Furthermore, in some embodiments, the one or more additional therapeutic agents include blocking antibodies that target immune checkpoint molecules, such as, for example, BTLA, HVEM, TIM3, GALS, LAG3, VISTA, KIR, 2B4, CD160 (also known as BY55 ), CGEN-15049, CHK 1 and CHK2 kinases, A2aR, CEACAM (eg, CEACAM-1, CEACAM-3 and/or CEACAM-5), GITR, GITRL, Galectin-9, CD244, CD160, TIGIT, SIRPα, ICOS, CD172a, TMIGD2 and various B-7 family ligands (including but not limited to B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7- H5, B7-H6 and B7-H7).

Some embodiments of these methods can further comprise identifying or diagnosing the individual as having a B7-H6 positive cancer. Some embodiments of these methods may further comprise identifying or diagnosing the individual as having a B7-H6 positive and HLA-E negative cancer.

Non-limiting examples of B7-H6 positive cancers include: adrenocortical carcinoma, cholangiocarcinoma, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, acute myeloid leukemia, lymphoid Tumors, diffuse large b-cell lymphoma, renal carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, renal chromophobe, liver carcinoma, lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, mesothelioma, ovary cancer, pancreatic cancer, rectal cancer, skin cancer, gastric cancer, testicular cancer, thymoma, thyroid cancer, uterine cancer, uterine endometrial cancer, uterine carcinosarcoma, and uveal melanoma.

In some embodiments, the individual is an adult human individual. In some embodiments, the individual is a pediatric human individual.

Enucleated erythroid cells can be administered to an individual as described herein using any dosage and with any frequency. Method of treating an individual by reducing the number and/or proliferation of B7-H6 positive cancer cells

Also provided herein is the reduction of B7-H6 positive cancer cells (such as described herein) in an individual previously identified or diagnosed as having a B7-H6 positive cancer (such as any B7-H6 positive cancer described herein or known in the art). or any B7-H6 positive cancer cells known in the art) to quantify and/or proliferate, the method comprising administering a therapeutically effective amount of exogenous fusion polypeptide enucleated erythroid cells (for example, any of the exemplified erythrocytes described herein enucleated erythroid cells), the exogenous fusion polypeptide comprises (i) IL-15 or its functional fragments, and (ii) IL-15 receptor α or its functional fragments on its extracellular surface.

Also provided herein is reducing the number of B7-H6 positive cancer cells (such as any B7-H6 positive cancer cells described herein or known in the art) in an individual previously identified or diagnosed as having a B7-H6 positive cancer and/or or a method of proliferation comprising administering a therapeutically effective amount of enucleated erythroid cells (e.g., any of the exemplary enucleated erythroid cells described herein) comprising exogenously fused erythroid cells on their extracellular surfaces A polypeptide, the exogenous fusion polypeptide comprising (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor α or a functional fragment thereof; and an exogenous polypeptide comprising a 4-1BBL polypeptide or a functional fragment thereof .

In some embodiments of these methods, administering comprises intravenously administering to the individual.

Some embodiments of these methods also include administering to the individual an NKG2A inhibitor (eg, any of the exemplary NKG2A inhibitors described herein).

In some embodiments, the B7-H6 positive cancer cells are B7-H6 positive and HLA-E negative cancer cells.

Some embodiments of any of the methods described herein also include administering to the individual one or more additional therapeutic agents. In some embodiments, the one or more additional therapeutic agents are cancer therapeutics. In some embodiments, the cancer therapeutic is selected from the group consisting of inhibitors of immune checkpoint molecules (e.g., any of the exemplary inhibitors of immune checkpoint molecules described herein), chemotherapeutic agents, therapeutic antibodies, chimeric antigen receptor T cells, kinase inhibitors, or soluble cytokines. In some embodiments, one or more additional therapeutic agents can be administered to a subject substantially simultaneously with any of the compositions provided herein. In some embodiments, one or more additional therapeutic agents can be administered to the individual before or after administration of any of the compositions described herein to the individual.

For example, in some embodiments, one or more additional therapeutic agents include blocking, reducing and/or inhibiting PD-1 and PD-L1 or PD-L2, and/or blocking, reducing and/or inhibiting PD-1 and PD-L1 or PD-L2 binding agents (as non-limiting examples, one or more of nivolumab (ONO-4538/BMS-936558, MDX1106, OPDIVO, BRISTOL MYERS SQUIBB), pembrolizumab (KEYTRUDA, MERCK ), Pidizumab (CT-011, CURE TECH), MK-3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), and MPDL328OA (ROCHE)). In some embodiments, the one or more additional therapeutic agents include blocking, reducing and/or inhibiting the activity of CTLA-4 and/or the interaction of CTLA-4 with one or more of its receptors (e.g., CD80, CD86, AP2M1, SHP- 2 and PPP2R5A) binding agents. For example, in some embodiments, the agent that inhibits CTLA-4 activity is an antibody, such as ipilimumab (MDX-010, MDX-101, Yervoy, BRISTOL MYERS SQUIBB) and/or tremelimumab (PFIZER). Furthermore, in some embodiments, the one or more additional therapeutic agents include blocking antibodies that target immune checkpoint molecules, such as, for example, BTLA, HVEM, TIM3, GALS, LAG3, VISTA, KIR, 2B4, CD160 (also known as BY55 ), CGEN-15049, CHK 1 and CHK2 kinases, A2aR, CEACAM (eg, CEACAM-1, CEACAM-3 and/or CEACAM-5), GITR, GITRL, Galectin-9, CD244, CD160, TIGIT, SIRPα, ICOS, CD172a, TMIGD2 and various B-7 family ligands (including but not limited to B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7- H5, B7-H6 and B7-H7).

Some embodiments of these methods can further comprise identifying or diagnosing the individual as having a B7-H6 positive cancer. Some embodiments of these methods can further comprise identifying or diagnosing the individual as having a B7-H6 positive and HLA-E negative cancer.

In some embodiments of these methods, the method results in a reduction (e.g., at least 5% reduction, at least 10% reduction, at least 15% reduction, at least 20% reduction, at least 25% reduction) in the number of B7-H6 positive cancer cells in the individual , at least 30% reduction, at least 35% reduction, at least 40% reduction, at least 45% reduction, at least 50% reduction, at least 55% reduction, at least 60% reduction, at least 65% reduction, at least 70% reduction, at least 75% reduction , a reduction of at least 80%, a reduction of at least 85%, a reduction of at least 90%, or a reduction of at least 95%, or a reduction of about 5% to a reduction of about 99% (or any subrange of this range described herein) (e.g., with the administration compared with the number of B7-H6 positive cancer cells in the former individual).

In some embodiments of these methods, the method results in a reduction in the number of B7-H6 positive and HLA-E negative cancer cells in the individual (e.g., at least a 5% reduction, at least a 10% reduction, at least a 15% reduction, at least a 20% reduction , at least 25% reduction, at least 30% reduction, at least 35% reduction, at least 40% reduction, at least 45% reduction, at least 50% reduction, at least 55% reduction, at least 60% reduction, at least 65% reduction, at least 70% reduction , a reduction of at least 75%, a reduction of at least 80%, a reduction of at least 85%, a reduction of at least 90%, or a reduction of at least 95%, or a reduction of about 5% to a reduction of about 99% (or any subrange of this range described herein) ( For example, compared to the number of B7-H6 positive and HLA-E negative cancer cells in the individual prior to administration).

In some embodiments of these methods, the method results in a reduction (e.g., at least a 5% reduction, at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction in the proliferation of B7-H6 positive cancer cells in the individual , at least 30% reduction, at least 35% reduction, at least 40% reduction, at least 45% reduction, at least 50% reduction, at least 55% reduction, at least 60% reduction, at least 65% reduction, at least 70% reduction, at least 75% reduction , a reduction of at least 80%, a reduction of at least 85%, a reduction of at least 90%, or a reduction of at least 95%, or a reduction of about 5% to a reduction of about 99% (or any subrange of this range described herein) (e.g., with the administration hyperplasia of B7-H6 positive cancer cells in former individuals).

In some embodiments of these methods, the method results in a reduction (e.g., at least a 5% reduction, at least a 10% reduction, at least a 15% reduction, at least a 20% reduction) in the proliferation of B7-H6 positive and HLA-E negative cancer cells in the individual , at least 25% reduction, at least 30% reduction, at least 35% reduction, at least 40% reduction, at least 45% reduction, at least 50% reduction, at least 55% reduction, at least 60% reduction, at least 65% reduction, at least 70% reduction , a reduction of at least 75%, a reduction of at least 80%, a reduction of at least 85%, a reduction of at least 90%, or a reduction of at least 95%, or a reduction of about 5% to a reduction of about 99% (or any subrange of this range described herein) ( For example, compared to proliferation of B7-H6 positive and HLA-E negative cancer cells in an individual prior to administration).

Some embodiments of these methods can further comprise identifying or diagnosing the individual as having a B7-H6 positive cancer. Some embodiments of these methods can further comprise identifying or diagnosing the individual as having a B7-H6 positive and HLA-E-negative cancer.

Non-limiting examples of B7-H6 positive cancers include: adrenocortical carcinoma, cholangiocarcinoma, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, acute myeloid leukemia, lymphoid Tumors, diffuse large b-cell lymphoma, renal carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, renal chromophobe, liver carcinoma, lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, mesothelioma, ovary cancer, pancreatic cancer, rectal cancer, skin cancer, gastric cancer, testicular cancer, thymoma, thyroid cancer, uterine cancer, uterine endometrial cancer, uterine carcinosarcoma, and uveal melanoma.

In some embodiments, the individual is an adult human individual. In some embodiments, the individual is a pediatric human individual.

Enucleated erythroid cells can be administered to an individual as described herein using any dosage and with any frequency. Method of treating an individual by inducing killing of B7-H6 positive cancer cells

Also provided herein are methods of inducing the killing of B7-H6 positive cancer cells (such as any of the exemplary B7-H6 positive cancer cells described herein) in an individual previously identified or diagnosed as having a B7-H6 positive cancer comprising administering a treatment An effective number of enucleated erythroid cells (e.g., any of the exemplary enucleated erythroid cells described herein) comprising on their extracellular surface an exogenous fusion polypeptide comprising (i ) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof.

Also provided herein are methods of killing B7-H6-positive cancer cells (such as any of the exemplary B7-H6-positive cancer cells described herein) in an individual previously identified or diagnosed as having a B7-H6-positive cancer comprising administering a therapeutically effective A quantity of enucleated erythroid cells (such as any of the exemplary enucleated erythroid cells described herein) comprising on their extracellular surface an exogenous fusion polypeptide comprising (i) IL -15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof; and an exogenous polypeptide comprising a 4-1BBL polypeptide or a functional fragment thereof.

In some embodiments, the killing of B7-H6 positive cancer cells is necrosis. In some embodiments, the killing of B7-H6 positive cancer cells is apoptosis. In some embodiments, the killing is NK cell mediated cytolysis or NK cell mediated cytotoxicity.

Some embodiments of these methods also include administering to the individual an NKG2A inhibitor (eg, any of the exemplary NKG2A inhibitors described herein).

In some embodiments of these methods, administering comprises intravenously administering to the individual.

Some embodiments of any of the methods described herein also include administering to the individual one or more additional therapeutic agents. In some embodiments, the one or more additional therapeutic agents are cancer therapeutics. In some embodiments, the cancer therapeutic is selected from the group consisting of inhibitors of immune checkpoint molecules (e.g., any of the exemplary inhibitors of immune checkpoint molecules described herein), chemotherapeutic agents, therapeutic antibodies, chimeric antigen receptor T cells, kinase inhibitors, or soluble cytokines. In some embodiments, one or more additional therapeutic agents can be administered to a subject substantially simultaneously with any of the compositions provided herein. In some embodiments, one or more additional therapeutic agents can be administered to the individual before or after administration of any of the compositions described herein to the individual.

For example, in some embodiments, one or more additional therapeutic agents include blocking, reducing and/or inhibiting PD-1 and PD-L1 or PD-L2, and/or blocking, reducing and/or inhibiting PD-1 and PD-L1 or PD-L2 binding agents (as non-limiting examples, one or more of nivolumab (ONO-4538/BMS-936558, MDX1106, OPDIVO, BRISTOL MYERS SQUIBB), pembrolizumab (KEYTRUDA, MERCK ), Pidizumab (CT-011, CURE TECH), MK-3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), and MPDL328OA (ROCHE)). In some embodiments, the one or more additional therapeutic agents include blocking, reducing and/or inhibiting the activity of CTLA-4 and/or the interaction of CTLA-4 with one or more of its receptors (e.g., CD80, CD86, AP2M1, SHP- 2 and PPP2R5A) binding agents. For example, in some embodiments, the agent that inhibits CTLA-4 activity is an antibody, such as ipilimumab (MDX-010, MDX-101, Yervoy, BRISTOL MYERS SQUIBB) and/or tremelimumab (PFIZER). Furthermore, in some embodiments, the one or more additional therapeutic agents include blocking antibodies that target immune checkpoint molecules, such as, for example, BTLA, HVEM, TIM3, GALS, LAG3, VISTA, KIR, 2B4, CD160 (also known as BY55 ), CGEN-15049, CHK 1 and CHK2 kinases, A2aR, CEACAM (eg, CEACAM-1, CEACAM-3 and/or CEACAM-5), GITR, GITRL, Galectin-9, CD244, CD160, TIGIT, SIRPα, ICOS, CD172a, TMIGD2 and various B-7 family ligands (including but not limited to B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7- H5, B7-H6 and B7-H7).

Some embodiments of these methods can further comprise identifying or diagnosing the individual as having a B7-H6 positive cancer. Some embodiments of these methods can further comprise identifying or diagnosing the individual as having a B7-H6 positive and HLA-E negative cancer.

Non-limiting examples of B7-H6 positive cancers include: adrenocortical carcinoma, cholangiocarcinoma, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, acute myeloid leukemia, lymphoid Tumors, diffuse large b-cell lymphoma, renal carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, renal chromophobe, liver carcinoma, lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, mesothelioma, ovary cancer, pancreatic cancer, rectal cancer, skin cancer, gastric cancer, testicular cancer, thymoma, thyroid cancer, uterine cancer, uterine endometrial cancer, uterine carcinosarcoma, and uveal melanoma.

In some embodiments, the individual is an adult human individual. In some embodiments, the individual is a pediatric human individual.

Enucleated erythroid cells can be administered to an individual as described herein using any dosage and with any frequency. Method of treating an individual by reducing the volume of a solid tumor in the individual

Also provided herein are methods of reducing the volume of a solid tumor in an individual previously identified or diagnosed as having a B7-H6 positive cancer, such as any exemplary B7-H6 cancer described herein or known in the art, comprising administering a treatment An effective number of enucleated erythroid cells (e.g., any of the exemplary enucleated erythroid cells described herein) comprising on their extracellular surface an exogenous fusion polypeptide comprising (i ) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof.

Also provided herein is a method of reducing the volume of a solid tumor in an individual previously identified or diagnosed as having a B7-H6-positive cancer, such as any exemplary B7-H6-positive cancer described herein or known in the art, comprising administering A therapeutically effective amount of enucleated erythroid cells (such as any of the exemplary enucleated erythroid cells described herein) comprising on their extracellular surface an exogenous fusion polypeptide comprising (i ) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof; and an exogenous polypeptide comprising a 4-1BBL polypeptide or a functional fragment thereof.

Some embodiments of these methods also include administering to the individual an NKG2A inhibitor (eg, any of the exemplary NKG2A inhibitors described herein).

In some embodiments of these methods, administering comprises intravenously administering to the individual.

Some embodiments of any of the methods described herein also include administering to the individual one or more additional therapeutic agents. In some embodiments, the one or more additional therapeutic agents are cancer therapeutics. In some embodiments, the cancer therapeutic is selected from the group consisting of inhibitors of immune checkpoint molecules (e.g., any of the exemplary inhibitors of immune checkpoint molecules described herein), chemotherapeutic agents, therapeutic antibodies, chimeric antigen receptor T cells, kinase inhibitors, or soluble cytokines. In some embodiments, one or more additional therapeutic agents can be administered to a subject substantially simultaneously with any of the compositions provided herein. In some embodiments, one or more additional therapeutic agents can be administered to the individual before or after administration of any of the compositions described herein to the individual.

For example, in some embodiments, one or more additional therapeutic agents include blocking, reducing and/or inhibiting PD-1 and PD-L1 or PD-L2, and/or blocking, reducing and/or inhibiting PD-1 and PD-L1 or PD-L2 binding agents (as non-limiting examples, one or more of nivolumab (ONO-4538/BMS-936558, MDX1106, OPDIVO, BRISTOL MYERS SQUIBB), pembrolizumab (KEYTRUDA, MERCK ), Pidizumab (CT-011, CURE TECH), MK-3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), and MPDL328OA (ROCHE)). In some embodiments, the one or more additional therapeutic agents include blocking, reducing and/or inhibiting the activity of CTLA-4 and/or the interaction of CTLA-4 with one or more of its receptors (e.g., CD80, CD86, AP2M1, SHP- 2 and PPP2R5A) binding agents. For example, in some embodiments, the agent that inhibits CTLA-4 activity is an antibody, such as ipilimumab (MDX-010, MDX-101, Yervoy, BRISTOL MYERS SQUIBB) and/or tremelimumab (PFIZER). Furthermore, in some embodiments, the one or more additional therapeutic agents include blocking antibodies that target immune checkpoint molecules, such as, for example, BTLA, HVEM, TIM3, GALS, LAG3, VISTA, KIR, 2B4, CD160 (also known as BY55 ), CGEN-15049, CHK 1 and CHK2 kinases, A2aR, CEACAM (eg, CEACAM-1, CEACAM-3 and/or CEACAM-5), GITR, GITRL, Galectin-9, CD244, CD160, TIGIT, SIRPα, ICOS, CD172a, TMIGD2 and various B-7 family ligands (including but not limited to B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7- H5, B7-H6 and B7-H7).

Some embodiments of these methods can further comprise identifying or diagnosing the individual as having a B7-H6 positive cancer. Some embodiments of these methods can further comprise identifying or diagnosing the individual as having a B7-H6 positive and HLA-E negative cancer.

Non-limiting examples of B7-H6 positive cancers include: adrenocortical carcinoma, cholangiocarcinoma, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, acute myeloid leukemia, lymphoid Tumors, diffuse large b-cell lymphoma, renal carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, renal chromophobe, liver carcinoma, lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, mesothelioma, ovary cancer, pancreatic cancer, rectal cancer, skin cancer, gastric cancer, testicular cancer, thymoma, thyroid cancer, uterine cancer, uterine endometrial cancer, uterine carcinosarcoma, and uveal melanoma.

In some embodiments of these methods, the method results in a reduction in the volume of the solid tumor in the individual (e.g., at least 5% reduction, at least 10% reduction, at least 15% reduction, at least 20% reduction, at least 25% reduction, at least 30% reduction %, at least 35% reduction, at least 40% reduction, at least 45% reduction, at least 50% reduction, at least 55% reduction, at least 60% reduction, at least 65% reduction, at least 70% reduction, at least 75% reduction, at least 80% reduction %, a reduction of at least 85%, a reduction of at least 90%, or a reduction of at least 95%, or a reduction of about 5% to a reduction of about 99% (or any sub-range of this range described herein) (e.g., compared to that of a solid tumor prior to administration) compared to volume).

In some embodiments, the individual is an adult human individual. In some embodiments, the individual is a pediatric human individual.

Enucleated erythroid cells can be administered to an individual as described herein using any dosage and with any frequency. Dosage and Administration

Methods of administering enucleated erythroid cells (e.g., reticulocytes) comprising (e.g., expressing) an exogenous agent (e.g., a polypeptide) are described in, e.g., WO2015/073587, WO2015/153102, and WO 2019/173798, each as Incorporated by reference in its entirety.

In particular examples, the enucleated erythroid cells described herein can be administered to an individual, such as a mammal (eg, a human). Exemplary mammals that may be treated include, but are not limited to, humans, domesticated animals (such as dogs, cats, and the like), farm animals (such as cattle, sheep, sheep, horses, and the like), and laboratory animals (such as monkeys , rats, mice, rabbits, horses and the like). The methods described herein are applicable to human therapy as well as veterinary applications.

In some embodiments, the enucleated erythroid cells are administered to the patient every 1, 2, 3, 4, 5, or 6 months.

In some embodiments, a dose of enucleated erythroid cells comprises about 1x10 ⁸ - 1x10 ¹³ , about 1x10 ⁸ - 1x10 ⁹ , about 1x10 ⁹ - 2x10 ⁹ , 2x10 ⁹ - 5x10 ⁹ , 5x10 ⁹ - 1x10 ¹⁰ , 1x10 ¹⁰ - 2x10 ¹⁰ , 2x10 ¹⁰ - 5x10 ¹⁰ , 5x10 ¹⁰ - 1x10 ¹¹ , 1x10 ¹¹ - 2x10 ¹¹ , 2x10 ¹¹ - 5x10 ¹¹ , 5x10 ^{11 -} 1x10 ¹² , 1x10 ¹² - 2x10 ¹² , 2x10 ¹¹ - 2x10, 2x10 ¹² - 2x10, 2x10 - 5 ¹³ cells.

Engineered erythroid cells can be administered to an individual in a formulation suitable for parenteral, intralesional, buccal, ophthalmic, intravenous, intraorgan, or other routes of administration.

The engineered erythroid cells can be administered to the individual as frequently as several times a day, or can be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently , such as every few months or even a year or less. The frequency of dosage will be apparent to the skilled person and will depend on many factors such as, but not limited to, the type and severity of the disease being treated, the type and age of the individual, and the like.

In some embodiments, the B7-H6 positive cancer is selected from the group consisting of adrenocortical carcinoma, cholangiocarcinoma, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, Acute myelogenous leukemia, Lymphoid neoplasms, Diffuse large B-cell lymphoma, Renal carcinoma, Renal clear cell carcinoma, Renal papillary cell carcinoma, Kidney chromophobe, Liver carcinoma, Lung cancer, Lung adenocarcinoma, Lung squamous cell carcinoma , mesothelioma, ovarian cancer, pancreatic cancer, rectal cancer, skin cancer, gastric cancer, testicular cancer, thymoma, thyroid cancer, uterine cancer, uterine endometrial cancer, uterine carcinosarcoma, and uveal melanoma. set

Also provided herein are kits comprising any of the compositions provided herein. For example, provided herein is a kit comprising a pharmaceutical composition comprising enucleated erythroid cells (e.g., any of the exemplary enucleated erythroid cells described herein) comprising exogenous A fusion polypeptide comprising (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof; and instructions for performing any of the methods described herein. In some embodiments, the enucleated erythroid cells comprise at least 1,000 copies, at least 10,000 copies, or at least 15,000 copies of the exogenous fusion polypeptide. In some embodiments, the enucleated erythroid cells comprise at least 1,000 copies, at least 10,000 copies, at least 15,000 copies, at least 20,000 copies, at least 25,000 copies, at least 30,000 copies, at least 40,000 copies, at least 50,000 copies , at least 60,000 copies, at least 80,000 copies, at least 100,000 copies, at least 200,000 copies, at least 300,000 copies, at least 400,000 copies, at least 500,000 copies, or at least 600,000 copies of the exogenous polypeptide.

In another example, provided herein is a kit comprising a pharmaceutical composition comprising an enucleated erythroid cell (e.g., any of the exemplary enucleated erythroid cells described herein) having on its extracellular surface An exogenous fusion polypeptide comprising (i) IL-15 or a functional fragment thereof, and (ii) an IL-15 receptor alpha or a functional fragment thereof; and a 4-1BBL polypeptide or a functional fragment thereof an exogenous polypeptide, wherein the exogenous polypeptide is present on the surface of an enucleated erythroid cell; and instructions for performing any of the methods described herein.

In some embodiments, the composition is formulated as described in, eg, WO 2020/219909 (incorporated herein by reference). Any of the enucleated erythroid cells described herein can be formulated for administration to an individual.

Further provided herein are kits comprising one or more sterile containers (e.g., sterile conical tubes, sterile Petri dishes, sterile vials (e.g., borosilicate glass vials), and sterile plastic bags (two) containing any of the compositions described herein. - 2-Ethylhexylphthalate (DEHP)-plasticized polyvinyl chloride (PVC) bags, or n-butyryl tris(n-hexyl)citrate (BTHC)-plasticized PVC bags). In some In embodiments, any of the kits provided herein can further include instructions for administering any of the compositions to an individual in need thereof.

Some embodiments of the kits described herein include suitable single dosage forms of any of the compositions described herein. For example, a single dosage form of any of the compositions described herein can have volumes such as: about 0.5 mL to about 2 L, about 0.5 mL to about 1800 mL, about 0.5 mL to about 1500 mL, about 0.5 mL to about 1200 mL, about 0.5 mL to about 1000 mL, about 0.5 mL to about 800 mL, about 0.5 mL to about 600 mL, about 0.5 mL to about 400 mL, about 0.5 mL to about 200 mL, about 0.5 mL to about 180 mL, about 0.5 mL to about 160 mL, about 0.5 mL to about 140 mL, about 0.5 mL to about 120 mL, about 0.5 mL to about 100 mL, about 0.5 mL to about 80 mL, about 0.5 mL to about 60 mL, about 0.5 mL to about 40 mL, about 0.5 mL to about 20 mL, about 0.5 mL to about 10 mL, about 0.5 mL to about 5 mL, about 1.0 mL to about 2 L, about 1.0 mL to about 1800 mL, about 1.0 mL to about 1500 mL , about 1.0 mL to about 1200 mL, about 1.0 mL to about 1000 mL, about 1.0 mL to about 800 mL, about 1.0 mL to about 600 mL, about 1.0 mL to about 400 mL, about 1.0 mL to about 200 mL, about 1.0 mL to about 180 mL, about 1.0 mL to about 160 mL, about 1.0 mL to about 140 mL, about 1.0 mL to about 120 mL, about 1.0 mL to about 100 mL, about 1.0 mL to about 80 mL, about 1.0 mL to about 60 mL, about 1.0 mL to about 40 mL, about 1.0 mL to about 20 mL, about 1.0 mL to about 10 mL, about 1.0 mL to about 5 mL, about 5 mL to about 2 L, about 5 mL to about 1800 mL, about 5 mL to about 1500 mL, about 5 mL to about 1200 mL, about 5 mL to about 1000 mL, about 5 mL to about 800 mL, about 5 mL to about 600 mL, about 5 mL to about 400 mL , about 5 mL to about 200 mL, about 5 mL to about 180 mL, about 5 mL to about 160 mL, about 5 mL to about 140 mL, about 5 mL to about 120 mL, about 5 mL to about 100 mL, about 5 mL to about 80 mL, about 5 mL to about 60 mL, about 5 mL to about 40 mL, about 5 mL to about 20 mL, about 5 mL to about 10 mL, about 10 mL to about 2 L, about 10 mL to about 1800 mL, about 10 mL to about 1500 mL, about 10 mL to about 1200 mL, about 10 mL to about 1000 mL, about 10 mL to About 800 mL, about 10 mL to about 600 mL, about 10 mL to about 400 mL, about 10 mL to about 200 mL, about 10 mL to about 180 mL, about 10 mL to about 160 mL, about 10 mL to about 140 mL, about 10 mL to about 120 mL, about 10 mL to about 100 mL, about 10 mL to about 80 mL, about 10 mL to about 60 mL, about 10 mL to about 40 mL, about 10 mL to about 20 mL, About 20 mL to about 2 L, about 20 mL to about 1800 mL, about 20 mL to about 1500 mL, about 20 mL to about 1200 mL, about 20 mL to about 1000 mL, about 20 mL to about 800 mL, about 20 mL to about 600 mL, about 20 mL to about 400 mL, about 20 mL to about 200 mL, about 20 mL to about 180 mL, about 20 mL to about 160 mL, about 20 mL to about 140 mL, about 20 mL to About 120 mL, about 20 mL to about 100 mL, about 20 mL to about 80 mL, about 20 mL to about 60 mL, about 20 mL to about 40 mL, about 40 mL to about 2 L, about 40 mL to about 1800 mL, about 40 mL to about 1500 mL, about 40 mL to about 1200 mL, about 40 mL to about 1000 mL, about 40 mL to about 800 mL, about 40 mL to about 600 mL, about 40 mL to about 400 mL, About 40 mL to about 200 mL, about 40 mL to about 180 mL, about 40 mL to about 160 mL, about 40 mL to about 140 mL, about 40 mL to about 120 mL, about 40 mL to about 100 mL, about 40 mL to about 80 mL, about 40 mL to about 60 mL, about 60 mL to about 2 L, about 60 mL to about 1800 mL, about 60 mL to about 1500 mL, about 60 mL to about 1200 mL, about 60 mL to About 1000 mL, about 60 mL to about 800 mL, about 60 mL to about 600 mL, about 60 mL to about 400 mL, about 60 mL to about 200 mL, about 60 mL to about 180 mL, about 60 mL to about 160 mL, about 60 mL to about 140 mL, about 60 mL to about 120 mL, about 60 mL to about 100 mL, about 60 mL to about 80 mL, about 80 mL to about 2 L, about 80 mL to about 1800 mL, About 80 mL to about 1500 mL, about 80 mL to about 1200 mL, about 80 mL to about 1000 mL, about 80 mL to about 800 mL, about 80 mL to about 600 mL, about 80 mL to about 400 mL, about 80 mL to about 200 mL, about 80 mL to about 180 mL, about 80 mL to about 160 mL, about 80 mL to about 140 mL, about 80 mL to about 120 mL , about 80 mL to about 100 mL, about 100 mL to about 2 L, about 100 mL to about 1800 mL, about 100 mL to about 1500 mL, about 100 mL to about 1200 mL, about 100 mL to about 1000 mL, about 100 mL to about 800 mL, about 100 mL to about 600 mL, about 100 mL to about 400 mL, about 100 mL to about 200 mL, about 100 mL to about 180 mL, about 100 mL to about 160 mL, about 100 mL to about 140 mL, about 100 mL to about 120 mL, about 200 mL to about 2 L, about 200 mL to about 1800 mL, about 200 mL to about 1500 mL, about 200 mL to about 1200 mL, about 200 mL to about 1000 mL, about 200 mL to about 800 mL, about 200 mL to about 600 mL, about 200 mL to about 500 mL, about 200 mL to about 450 mL, about 200 mL to about 400 mL, about 200 mL to about 350 mL , about 200 mL to about 300 mL, about 200 mL to about 250 mL, about 300 mL to about 2 L, about 300 mL to about 1800 mL, about 300 mL to about 1500 mL, about 300 mL to about 1200 mL, about 300 mL to about 1000 mL, about 300 mL to about 800 mL, about 300 mL to about 600 mL, about 300 mL to about 500 mL, about 300 mL to about 450 mL, about 300 mL to about 400 mL, about 300 mL to about 350 mL, about 400 mL to about 2 L, about 400 mL to about 1800 mL, about 400 mL to about 1500 mL, about 400 mL to about 1200 mL, about 400 mL to about 1000 mL, about 400 mL to about 800 mL, about 400 mL to about 600 mL, about 400 mL to about 500 mL, about 400 mL to about 450 mL, about 500 mL to about 2 L, about 500 mL to about 1800 mL, about 500 mL to about 1500 mL , about 500 mL to about 1200 mL, about 500 mL to about 1000 mL, about 500 mL to about 800 mL, about 500 mL to about 600 mL, about 600 mL to about 2 L, about 600 mL to about 1800 mL, about 600 mL to about 1500 mL, about 600 mL to about 1200 mL, about 600 mL to about 1000 mL, about 600 mL to about 800 mL, about 800 mL to about 2 L, about 800 mL to about 1800 mL, about 800 mL to About 1500 mL, about 800 mL to about 1200 mL, about 800 mL to about 1000 mL, about 1000 mL to about 2 L, about 1000 mL to about 1800 mL, about 1000 mL to about 1500 mL, about 1000 mL to about 1200 mL, about 1200 mL to about 2 L, about 1200 mL to about 1800 mL, about 1200 mL to about 1500 mL, about 1500 mL to about 2 L, about 1500 mL to about 1800 mL, or about 1800 mL to about 2 L . example Example 1. Generation of Erythroid Cells Genetically Engineered to Express the First Fusion Polypeptide IL-15 and IL-15/IL-15RA fusion constructs

Various DNA constructs encoding fusion polypeptides were prepared for expression in erythroid cells, as shown in Table 5 below. Table 5. IL-15 and IL-15/IL-15RA fusion constructs and polypeptides. SEQ ID NO refers to the amino acid sequence. Construct/ peptide illustrate SEQ ID NO : V3 IL-15 GPA Signal Peptide (SEQ ID NO: 27) - Mature Human IL-15 (SEQ ID NO: 16) - Linker - HA - Linker (SEQ ID NO: 57) - GPA (SEQ ID NO: 36) 38 V4 IL-15 + IL-15RA (extracellular) GPA signal peptide (SEQ ID NO: 27) - mature human IL-15 (SEQ ID NO: 16) - flexible linker (SEQ ID NO: 29) - mature human extracellular IL-15RA (SEQ ID NO: 22) - Linker-HA- Linker (SEQ ID NO: 57) - GPA (SEQ ID NO: 36) 40 V5 IL-15 + IL-15RA (sushi domain + 13 aa) GPA signal peptide (SEQ ID NO: 27) - mature human IL-15 (SEQ ID NO: 16) - flexible linker (SEQ ID NO: 29) - mature human IL-15RA (sushi domain + 13 aa) (SEQ ID NO: 25) - Linker-HA-Linker (SEQ ID NO: 57) - GPA (SEQ ID NO: 36) 42 V3.1 GPA Signal Peptide (SEQ ID NO: 27) - Mature Human IL-15 (SEQ ID NO: 16) - Linker (SEQ ID NO: 35) - GPA (SEQ ID NO: 36) 44 V4.1 GPA signal peptide (SEQ ID NO: 27) - mature human IL-15 (SEQ ID NO: 16) - flexible linker (SEQ ID NO: 29) - mature human extracellular IL-15RA (SEQ ID NO: 22) - Linker (SEQ ID NO: 35) - GPA (SEQ ID NO: 36) 46 IL-15/IL-15RA fusion polypeptide Mature human IL-15 (SEQ ID NO: 16) - flexible linker (SEQ ID NO: 29) - mature human extracellular IL-15RA (SEQ ID NO: 22) 31 IL-15/IL-15RA (sushi domain + 13 aa) fusion polypeptide Mature human IL-15 (SEQ ID NO: 16) - flexible linker (SEQ ID NO: 29) - mature human IL-15RA (sushi domain + 13 aa) (SEQ ID NO: 25) 33

The DNA constructs were cloned into multiple selection sites of lentiviral vectors under the control of the MSCV promoter sequence for expression in erythroid cells as described below. Generating lentiviral vectors

The constructs were cloned into multiple selection sites of the lentiviral vector pCDH under the control of the MSCV promoter sequence (System Biosciences). Lentivirus was produced in 293T cells by transfecting cells with pPACKH1 (System Biosciences) and the pCDH lentiviral vector containing the construct. Place cells in fresh medium. Viral supernatants were collected 48 hours after medium change by centrifugation at 1,500 rpm for 5 minutes. The supernatant was collected, filtered, and frozen at -80°C in aliquots. Expansion and differentiation of erythroid cells

Peripheral mobilized human CD34 ⁺ cells derived from normal human donors were used. The expansion/differentiation procedure consisted of 3 stages. In the first stage, thawed CD34 ⁺ erythroid cell precursors were cultured in Iscove's MDM (IMDM) medium containing recombinant human insulin, human transferrin, recombinant human stem cell factor, and recombinant human interleukin-3. In the second stage, erythroid cells were cultured in IMDM medium supplemented with recombinant human insulin, human transferrin, human recombinant stem cell factor, human recombinant erythropoietin, and L-glutamine. In the third stage, erythroid cells were cultured in IMDM medium supplemented with human transferrin, recombinant human insulin, human recombinant erythropoietin, and heparin. Cultures were maintained at 37°C in a 5% CO2 incubator. Transduction of erythroid cell precursors

Erythroid cell precursors were transduced during Phase 1 of the culture process described above. Combine erythroid cell precursors in culture medium with lentiviral supernatant and polybrene. Infection was achieved by spin-inoculation, spinning the plate at 2,000 rpm for 90 minutes at room temperature. After spin seeding, cells were incubated overnight at 37°C. antibody binding

Binding of PE-labeled anti-IL-15-RA antibodies (eg, anti-IL-15RA antibody (JM7A4) (ab91270), AbCam) was used to verify the expression of the first exogenous polypeptide in engineered erythroid cells. Antibody binding was measured by flow cytometry for APC fluorescence or PE fluorescence. Circles were set based on stained, non-transduced cells. Example 2. Generation of erythroid cells genetically engineered to express 4-1BBL 4-1BBL construct

DNA constructs were prepared for expression in erythroid cells as shown in Table 6 below: Table 6. 4-1BBL constructs. SEQ ID NO refers to the amino acid sequence construct illustrate SEQ ID NO: 4-1BBL GPA signal peptide (SEQ ID NO: 21) - human extracellular 4-1BBL (SEQ ID NO: 41) - 4-1BBL linker (SEQ ID NO: 39) - GPA (SEQ ID NO: 25) 43 Generating lentiviral vectors

The 4-1BBL gene construct was constructed as shown in Table 6. The genes were cloned into multiple selection sites in the lentiviral vector pCDH (from System Biosciences) with the MSCV promoter sequence. Lentivirus was produced in 293T cells by transfecting cells with pPACKH1 (System Biosciences) and pCDH lentiviral vector containing the 4-1BBL gene. Place cells in fresh medium. Viral supernatants were collected 48 hours after medium change by centrifugation at 1,500 rpm for 5 minutes. The supernatant was collected, filtered, and frozen at -80°C in aliquots. Expansion and differentiation of erythroid cells

Peripheral mobilized human CD34 ⁺ cells derived from normal human donors were purchased frozen from AllCells Inc. The expansion/differentiation procedure consisted of 3 stages. In the first stage, thawed CD34 ⁺ erythroid cell precursors were cultured in Iscove's MDM (IMDM) medium containing recombinant human insulin, human transferrin, recombinant human recombinant stem cell factor, and recombinant human interleukin-3. In the second stage, erythroid cells were cultured in Iscove's MDM medium supplemented with recombinant human insulin, human transferrin, human recombinant stem cell factor, human recombinant erythropoietin and L-glutamine. In the third stage, erythroid cells were cultured in Iscove's MDM medium supplemented with human transferrin, recombinant human insulin, human recombinant erythropoietin and heparin. Cultures were maintained at 37°C in a 5% CO2 incubator. Transduction of erythroid cell precursors

Transduce the erythroid cell precursors during step 1 of the above culture procedure. Combine erythroid cells in culture medium with lentiviral supernatant and polybrene. Infection was achieved by spin-inoculation, spinning the plate at 2,000 rpm for 90 minutes at room temperature. After spin seeding, cells were incubated overnight at 37°C. antibody binding

Binding of PE-labeled anti-4-1BBL antibody (eg, purified anti-human 4-1BB ligand (CD137L) antibody, BioLegend) was used to verify the expression of 4-1BBL in engineered erythroid cells. Antibody binding was measured by flow cytometry to measure PE fluorescence. Circles were set based on stained, non-transduced cells. Example 3. Generation of Erythroid Cells Genetically Engineered to Express First and Second Exogenous Polypeptides Generating lentiviral vectors

Genes encoding IL-15/IL-15-RA fusion protein and 4-1BBL were constructed. Each gene was cloned into multiple selection sites of the lentiviral vector pCDH (System Biosciences) controlled by the MSCV promoter sequence, so that one vector contained the IL-15/IL-15RA gene and the other vector contained 4-1BBL Gene. Lentiviruses were produced in 293T cells by co-transfecting cells with pPACKH1 (System Biosciences) and pCDH lentiviral vectors containing the IL-15/IL-15-RA gene and pCDH lentiviral vectors containing the 4-1BBL gene. Place cells in fresh medium. Viral supernatants were collected 48 hours after medium change by centrifugation at 1,500 rpm for 5 minutes. The supernatant was collected, filtered, and frozen at -80°C in aliquots.

Alternatively, construct IL-15/IL-15-RA fusion protein and 4-1BBL gene, and colonize into the multiple selection site of the lentiviral vector pCDH (System Biosciences) controlled by the MSCV promoter sequence, so that a single vector includes IL-15/IL-15RA gene and 4-1BBL gene. Lentivirus was produced in 293T cells by co-transfecting cells with pPACKH1 (System Biosciences) and pCDH lentiviral vector containing IL-15/IL-15-RA gene and 4-1BBL gene. Place cells in fresh medium. Viral supernatants were collected 48 hours after medium change by centrifugation at 1,500 rpm for 5 minutes. The supernatant was collected, filtered, and frozen at -80°C in aliquots. Expansion and differentiation of erythroid cells

Peripheral mobilized human CD34 ⁺ cells derived from normal human donors were purchased frozen from AllCells Inc. The expansion/differentiation procedure consisted of 3 stages. In the first stage, thawed CD34 ⁺ erythroid cell precursors were cultured in Iscove's MDM (IMDM) medium containing recombinant human insulin, human transferrin, recombinant human stem cell factor, and recombinant human interleukin-3. In the second stage, erythroid cells were cultured in Iscove's MDM (IMDM) medium supplemented with recombinant human insulin, human transferrin, human recombinant stem cell factor, human recombinant erythropoietin and L-glutamine. In the third stage, erythroid cells were cultured in Iscove's MDM (IMDM) medium supplemented with human transferrin, recombinant human insulin, human recombinant erythropoietin and heparin. Cultures were maintained at 37°C in a 5% CO2 incubator. Transduction of erythroid cell precursors

Erythroid cell precursors were transduced during step 1 of the culture procedure described above. Combine erythroid cells in culture medium with lentiviral supernatant and polybrene. Infection was achieved by spin-inoculation, spinning the plate at 2,000 rpm for 90 minutes at room temperature. After spin seeding, cells were incubated overnight at 37°C. antibody binding

Binding of PE-labeled anti-IL-15-RA antibodies (e.g. anti-IL-15RA antibody (JM7A4) (ab91270), AbCam) for validation of IL-15/IL-15-RA expression in engineered erythroid cells . Binding of PE-labeled anti-4-1BBL antibody (eg, purified anti-human 4-1BB ligand (CD137L) antibody, BioLegend) was used to verify the expression of 4-1BBL in engineered erythroid cells. Antibody binding was measured by flow cytometry to measure PE fluorescence. Circles were set based on stained, non-transduced cells. Example 4. Enucleated erythroid cells genetically engineered to express IL-15/IL-15RA and 4-1BBL resulted in increased lymphocytes, NKp30-positive lymphocytes, and CD8 ⁺ T cells in vitro

Erythroid cells comprising IL-15/IL-15RA and 4-1BBL were prepared substantially as described in Example 3.

Frozen peripheral blood mononuclear cells (PBMC) were thawed, resuspended in RPMI medium containing 10% fetal bovine serum, and plated at 100,000 cells per well in 96-well round bottom dishes. PBMCs are co-cultured with any of the following: (a) enucleated erythroid blood cells comprising the first exogenous protein on their extracellular surface, the first exogenous protein comprising (i) IL-15 or its function fragment, and (ii) IL-15 receptor alpha or a functional fragment thereof; and a second exogenous polypeptide comprising 4-1BBL (200,000 cells per well); (b) recombinant human IL-15 ("rhIL15"; 0.1 ng/mL) and anti-4-1BB stimulatory antibody (1 nM), which was cross-linked with the secondary antibody (2.5 nM), or (c) media only (control). Cultures were incubated for 8 days and then analyzed by flow cytometry using the following antibody staining: anti-CD56 antibody (clonal 5.1H11, Biolegend), anti-NKp30 (clonal p30-15, Biolegend) and LIVE/DEAD™ Fixable Aqua Dead Cell Stain (Invitrogen) labeled dead cells (to allow confinement of live cells) and were subsequently analyzed by flow cytometry (Novocyte).

After 8 days, co-culture of PBMCs with enucleated erythroid cells resulted in an increase in the total number of NK cells compared to media alone control and treatment with rhIL-15 and anti-4-1BB stimulatory antibody (Fig. 1) (not shown) , and the proportion of NKp30-positive lymphocytes increases, the enucleated erythrocytes contain an exogenous fusion protein and an exogenous fusion polypeptide comprising 4-1BBL on its extracellular surface, and the exogenous fusion protein comprises (i) IL- 15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof. Example 5. Cell surface markers in fresh whole blood from patients before and at various time points during treatment with enucleated erythroid cells genetically engineered to express IL-15/IL-15RA and 4-1BBL

Cell surface markers in fresh whole blood by flow cytometry before and during treatment with erythroid cells containing IL-15/IL-15RA and 4-1BBL (prepared substantially as described in Example 3) Multiple time points were evaluated in patients. Whole blood was collected from patients at pre-treatment baseline and 4 hours, 1 day, 2 days, 7 days, and 14 days post-treatment for the first dosing cycle and 2 days, 7 days, and 14 days post-treatment following subsequent dosing cycles. Analysis by Flow Cytometry Whole blood was stained with fluorophore-conjugated antibodies using standard techniques. Multiple antibody panels were used, with separate tubes for each panel and flow cytometry analysis. All panels include markers for identification of lymphocyte, NK cell and T cell populations as well as markers for activation status. The data were obtained using the following panels: Group 1 - CD3, CD8, CD16/CD56, CD45, CD45RA, CCR7, HLA-DR and live/dead staining; Group 2 - CD3, CD4, Foxp3, CD25, CD127, Ki67, Live/Dead staining; Group 3 - CD45, CD16, CD56, NKG2D, NKp30, Live/Dead staining). The data were then analyzed as population frequency for the percentage of established maternal fences (eg, %NKp30+ of CD45+CD56+ viable lymphocytes). In the case of Group 1, quantitative cell population number data per volume of blood were also analyzed against the included quantitative count controls. Data are reported as the maximum fold change observed from baseline for each parameter of interest (number of cells per μL of whole blood or % of parent population).

For example, cell surface markers were assessed by flow cytometry in viable lymphocytes (CD45+) in fresh whole blood at baseline (before treatment) and at various time points after treatment. Patients administered ≥1× ¹⁰ erythroid cells (comprising IL-15/IL-15RA and 4-1BBL) prepared substantially as described in Example 3 had an increased percentage of CD45 ⁺ CD56+ lymphocytes positive for NKp30 ( Figure 2A), the percentage of NKp30-positive CD45 ⁺ CD56dimCD16 ⁺ lymphocytes increased (Figure 2B), and the percentage of NKp30-positive CD45 ⁺ CD56 ⁺ CD16 ⁺ lymphocytes increased (Figure 2C). The number of circulating CD3 ⁻ CD16/56 ⁺ cells was increased in patients administered > 1× ^{10 10} erythroid cells (comprising IL-15/IL-15RA and 4-1BBL) prepared substantially as described in Example 3 ( FIG. 3A ) and CD8 ⁺ memory T cells expressing granzyme B (GrB ⁺ CD8 ⁺ CD45RA ⁻ ) were increased ( FIG. 3B ). Patients administered ≥1 x 1010 erythroid cells (comprising IL- ¹⁵ /IL-15RA and 4-1BBL) prepared substantially as described in Example 3 showed CD3 ⁺ CD4 ⁺ lymphocytes (CD4 ⁺ T cells) (Fig. 4A) or the number of CD3 ⁺ CD8 ⁺ lymphocytes (CD8 ⁺ T cells) (Fig. 4B) changed little. Sequence Appendix SEQ ID NO: identifier sequence 1 NKp30 (NP 001138938.1) polypeptide MAWMLLLILIMVHPGSCALWVSQPPEIRTLEGSSAFLPCSFNASQGRLAIGSVTWFRDEVVPGKEVRNGTPEFRGRLAPLASSRFLHDHQAELHIRDVRGHDASIYVCRVEVLGLGVGTGNGTRLVVEKEHPQLGAGTVLLLRAGFYAVSFLSVAVGSTVYYQGKYAKSTLSGFPQL 2 Nucleic acid sequence encoding SEQ ID NO: 1 ATGGCCTGGATGCTGTTGCTCATCTTGATCATGGTCCATCCAGGATCCTGTGCTCTCTGGGTGTCCCAGCCCCCTGAGATTCGTACCCTGGAAGGATCCTCTGCCTTCCTGCCCTGCTCCTTCAATGCCAGCCAAGGGAGACTGGCCATTGGCTCCGTCACGTGGTTCCGAGATGAGGTGGTTCCAGGGAAGGAGGTGAGGAATGGAACCCCAGAGTTCAGGGGCCGCCTGGCCCCACTTGCTTCTTCCCGTTTCCTCCATGACCACCAGGCTGAGCTGCACATCCGGGACGTGCGAGGCCATGACGCCAGCATCTACGTGTGCAGAGTGGAGGTGCTGGGCCTTGGTGTCGGGACAGGGAATGGGACTCGGCTGGTGGTGGAGAAAGAACATCCTCAGCTAGGGGCTGGTACAGTCCTCCTCCTTCGGGCTGGATTCTATGCTGTCAGCTTTCTCTCTGTGGCCGTGGGCAGCACCGTCTATTACCAGGGCAAATATGCCAAATCTACTCTCTCCGGATTCCCCCAACTCTGA 3 NKp30 (NP 001138939.1) polypeptide MAWMLLLILIMVHPGSCALWVSQPPEIRTLEGSSAFLPCSFNASQGRLAIGSVTWFRDEVVPGKEVRNGTPEFRGRLAPLASSRFLHDHQAELHIRDVRGHDASIYVCRVEVLGLGVGTGNGTRLVVEKEHPQLGAGTVLLLRAGFYAVSFLSVAVGSTVYYQGKCHCHMGTHCHSSDGPRGVIPEPRCP 4 Nucleic acid sequence encoding SEQ ID NO: 3 ATGGCCTGGATGCTGTTGCTCATCTTGATCATGGTCCATCCAGGATCCTGTGCTCTCTGGGTGTCCCAGCCCCCTGAGATTCGTACCCTGGAAGGATCCTCTGCCTTCCTGCCCTGCTCCTTCAATGCCAGCCAAGGGAGACTGGCCATTGGCTCCGTCACGTGGTTCCGAGATGAGGTGGTTCCAGGGAAGGAGGTGAGGAATGGAACCCCAGAGTTCAGGGGCCGCCTGGCCCCACTTGCTTCTTCCCGTTTCCTCCATGACCACCAGGCTGAGCTGCACATCCGGGACGTGCGAGGCCATGACGCCAGCATCTACGTGTGCAGAGTGGAGGTGCTGGGCCTTGGTGTCGGGACAGGGAATGGGACTCGGCTGGTGGTGGAGAAAGAACATCCTCAGCTAGGGGCTGGTACAGTCCTCCTCCTTCGGGCTGGATTCTATGCTGTCAGCTTTCTCTCTGTGGCCGTGGGCAGCACCGTCTATTACCAGGGCAAATGCCACTGTCACATGGGAACACACTGCCACTCCTCAGATGGGCCCCGAGGAGTGATTCCAGAGCCCAGATGTCCCTAG 5 NKp30 (NP 667341.1) polypeptide MAWMLLLILIMVHPGSCALWVSQPPEIRTLEGSSAFLPCSFNASQGRLAIGSVTWFRDEVVPGKEVRNGTPEFRGRLAPLASSRFLHDHQAELHIRDVRGHDASIYVCRVEVLGLGVGTGNGTRLVVEKEHPQLGAGTVLLLRAGFYAVSFLSVAVGSTVYYQGKCLTWKGPRRQLPAVVPAPPLPPPCGSSAHLLPPVPGG 6 Nucleic acid sequence encoding SEQ ID NO: 5 CATGGCCTGGATGCTGTTGCTCATCTTGATCATGGTCCATCCAGGATCCTGTGCTCTCTGGGTGTCCCAGCCCCCTGAGATTCGTACCCTGGAAGGATCCTCTGCCTTCCTGCCCTGCTCCTTCAATGCCAGCCAAGGGAGACTGGCCATTGGCTCCGTCACGTGGTTCCGAGATGAGGTGGTTCCAGGGAAGGAGGTGAGGAATGGAACCCCAGAGTTCAGGGGCCGCCTGGCCCCACTTGCTTCTTCCCGTTTCCTCCATGACCACCAGGCTGAGCTGCACATCCGGGACGTGCGAGGCCATGACGCCAGCATCTACGTGTGCAGAGTGGAGGTGCTGGGCCTTGGTGTCGGGACAGGGAATGGGACTCGGCTGGTGGTGGAGAAAGAACATCCTCAGCTAGGGGCTGGTACAGTCCTCCTCCTTCGGGCTGGATTCTATGCTGTCAGCTTTCTCTCTGTGGCCGTGGGCAGCACCGTCTATTACCAGGGCAAATGTCTGACCTGGAAAGGTCCAAGAAGGCAGCTGCCGGCTGTGGTCCCAGCGCCCCTCCCACCACCATGTGGGAGCTCAGCACATCTGCTTCCCCCAGTCCCAGGAGGCTGA 7 B7-H6 (NP 001189368.1) polypeptide MTWRAAASTCAALLILLWALTTEGDLKVEMMAGGTQITPLNDNVTIFCNIFYSQPLNITSMGITWFWKSLTFDKEVKVFEFFGDHQEAFRPGAIVSPWRLKSGDASLRLPGIQLEEAGEYRCEVVVTPLKAQGTVQLEVVASPASRLLLDQVGMKENEDKYMCESSGFYPEAINITWEKQTQKFPHPIEISEDVITGPTIKNMDGTFNVTSCLKLNSSQEDPGTVYQCVVRHASLHTPLRSNFTLTAARHSLSETEKTDNFSIHWWPISFIGVGLVLLIVLIPWKKICNKSSSAYTPLKCILKHWNSFDTQTLKKEHLIFFCTRAWPSYQLQDGEAWPPEGSVNINTIQQLDVFCRQEGKWSEVPYVQAFFALRDNPDLCQCCRIDPALLTVTSGKSIDDNSTKSEKQTPREHSDAVPDAPILPVSPIWEPPPATTSTTPVLSSQPPTLLLPLQ 8 Nucleic acid sequence encoding SEQ ID NO: 7 ATGACGTGGAGGGCTGCCGCCTCCACGTGCGCGGCGCTCCTGATTCTGCTGTGGGCGCTGACGACCGAAGGTGATCTGAAAGTAGAGATGATGGCAGGGGGGACTCAGATCACACCCCTGAATGACAATGTCACCATATTCTGCAATATCTTTTATTCCCAACCCCTCAACATCACGTCTATGGGTATCACCTGGTTTTGGAAGAGTCTGACGTTTGACAAAGAAGTCAAAGTCTTTGAATTTTTTGGAGATCACCAAGAGGCATTCCGACCTGGAGCCATTGTGTCTCCATGGAGGCTGAAGAGTGGGGACGCCTCACTGCGGCTGCCTGGAATCCAGCTGGAGGAAGCAGGAGAGTACCGATGTGAGGTGGTGGTCACCCCTCTGAAGGCACAGGGAACAGTCCAGCTTGAAGTTGTGGCTTCCCCAGCCAGCAGATTGTTGCTGGATCAAGTGGGCATGAAAGAGAATGAAGACAAATATATGTGTGAGTCAAGTGGGTTCTACCCAGAGGCTATTAATATAACATGGGAGAAGCAGACCCAGAAGTTTCCCCATCCCATAGAGATTTCTGAGGATGTCATCACTGGTCCCACCATCAAGAATATGGATGGCACATTTAATGTCACTAGCTGCTTGAAGCTGAACTCCTCTCAGGAAGACCCTGGGACTGTCTACCAGTGTGTGGTACGGCATGCGTCCTTGCATACCCCCTTGAGGAGCAACTTTACCCTGACTGCTGCTCGGCACAGTCTTTCTGAAACTGAGAAGACAGATAATTTTTCCATTCATTGGTGGCCTATTTCATTCATTGGTGTTGGACTGGTTTTATTAATTGTTTTGATTCCTTGGAAAAAGATATGTAACAAATCATCTTCAGCCTATACTCCTCTCAAGTGCATTCTGAAACACTGGAACTCCTTTGACACTCAGACTCTGAAGAAAGAGCACCTCATATTCTTTTGCACTCGGGCATGGCCGTCTTACCAGCTGCAGGATG GGGAGGCTTGGCCTCCTGAGGGAAGTGTTAATATTAATACTATTCAACAACTAGATGTTTTCTGCAGACAGGAGGGCAAATGGTCCGAGGTTCCTTATGTGCAAGCCTTCTTTGCCTTGCGAGACAACCCAGATCTTTGTCAGTGTTGTAGAATTGACCCTGCTCTCCTAACAGTTACATCAGGCAAGTCCATAGATGATAATTCCACAAAGTCTGAGAAACAAACCCCTAGGGAACACTCGGATGCAGTTCCGGATGCCCCAATCCTTCCTGTCTCCCCTATCTGGGAACCTCCTCCAGCCACAACATCAACAACTCCAGTTCTATCCTCCCAACCCCCAACTTTACTGTTACCCCTACAGTAA 9 NKG2A (NP 002250.2) polypeptide MDNQGVIYSDLNLPPNPKRQQRKPKGNKNSILATEQEITYAELNLQKASQDFQGNDKTYHCKDLPSAPEKLIVGILGIICLILMASVVTIVVIPSTLIQRHNNSSLNTRTQKARHCGHCPEEWITYSNSCYYIGKERRTWEESLLACTSKNSSLLSIDNEEEMKFLSIISPSSWIGVFRNSSHHPWVTMNGLAFKHEIKDSDNAELNCAVLQVNRLKSAQCGSSIIYHCKHKL 10 Nucleic acid sequence encoding SEQ ID NO: 9 ATGGATAACCAAGGAGTAATCTACTCAGACCTGAATCTGCCCCCAAACCCAAAGAGGCAGCAACGAAAACCTAAAGGCAATAAAAACTCCATTTTAGCAACTGAACAGGAAATAACCTATGCGGAATTAAACCTTCAAAAAGCTTCTCAGGATTTTCAAGGGAATGACAAAACCTATCACTGCAAAGATTTACCATCAGCTCCAGAGAAGCTCATTGTTGGGATCCTGGGAATTATCTGTCTTATCTTAATGGCCTCTGTGGTAACGATAGTTGTTATTCCCTCTACATTAATACAGAGGCACAACAATTCTTCCCTGAATACAAGAACTCAGAAAGCACGTCATTGTGGCCATTGTCCTGAGGAGTGGATTACATATTCCAACAGTTGTTACTACATTGGTAAGGAAAGAAGAACTTGGGAAGAGAGTTTGCTGGCCTGTACTTCGAAGAACTCCAGTCTGCTTTCTATAGATAATGAAGAAGAAATGAAATTTCTGTCCATCATTTCACCATCCTCATGGATTGGTGTGTTTCGTAACAGCAGTCATCATCCATGGGTGACAATGAATGGTTTGGCTTTCAAACATGAGATAAAAGACTCAGATAATGCTGAACTTAACTGTGCAGTGCTACAAGTAAATCGACTTAAATCAGCCCAGTGTGGATCTTCAATAATATATCATTGTAAGCATAAGCTTTAG 11 NKG2A (NP_998823.1) polypeptide MDNQGVIYSDLNLPPNPKRQQRKPKGNKSSILATEQEITYAELNLQKASQDFQGNDKTYHCKDLPSAPEKLIVGILGIICLILMASVVTIVVIPSTLIQRHNNSSLNTRTQKARHCGHCPEEWITYSNSCYYIGKERRTWEESLLACTSKNSSLLSIDNEEEMKFLSIISPSSWIGVFRNSSHHPWVTMNGLAFKHEIKDSDNAELNCAVLQVNRLKSAQCGSSIIYHCKHKL 12 Nucleic acid sequence encoding SEQ ID NO: 11 ATGGATAACCAAGGAGTAATCTACTCAGACCTGAATCTGCCCCCAAACCCAAAGAGGCAGCAACGAAAACCTAAAGGCAATAAAAACTCCATTTTAGCAACTGAACAGGAAATAACCTATGCGGAATTAAACCTTCAAAAAGCTTCTCAGGATTTTCAAGGGAATGACAAAACCTATCACTGCAAAGATTTACCATCAGCTCCAGAGAAGCTCATTGTTGGGATCCTGGGAATTATCTGTCTTATCTTAATGGCCTCTGTGGTAACGATAGTTGTTATTCCCTCTACATTAATACAGAGGCACAACAATTCTTCCCTGAATACAAGAACTCAGAAAGCACGTCATTGTGGCCATTGTCCTGAGGAGTGGATTACATATTCCAACAGTTGTTACTACATTGGTAAGGAAAGAAGAACTTGGGAAGAGAGTTTGCTGGCCTGTACTTCGAAGAACTCCAGTCTGCTTTCTATAGATAATGAAGAAGAAATGAAATTTCTGTCCATCATTTCACCATCCTCATGGATTGGTGTGTTTCGTAACAGCAGTCATCATCCATGGGTGACAATGAATGGTTTGGCTTTCAAACATGAGATAAAAGACTCAGATAATGCTGAACTTAACTGTGCAGTGCTACAAGTAAATCGACTTAAATCAGCCCAGTGTGGATCTTCAATAATATATCATTGTAAGCATAAGCTTTAG 13 HLA-E (NP 005507.3) polypeptide MVDGTLLLLLSEALALTQTWAGSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRFDNDAASPRMVPRAPWMEQEGSEYWDRETRSARDTAQIFRVNLRTLRGYYNQSEAGSHTLQWMHGCELGPDGRFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDASEAEHQRAYLEDTCVEWLHKYLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQQDGEGHTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPVTLRWKPASQPTIPIVGIIAGLVLLGSVVSGAVVAAVIWRKKSSGGKGGSYSKAEWSDSAQGSESHSL 14 Nucleic acid sequence encoding SEQ ID NO: 12 ATGGTAGATGGAACCCTCCTTTTACTCCTCTCGGAGGCCCTGGCCCTTACCCAGACCTGGGCGGGCTCCCACTCCTTGAAGTATTTCCACACTTCCGTGTCCCGGCCCGGCCGCGGGGAGCCCCGCTTCATCTCTGTGGGCTACGTGGACGACACCCAGTTCGTGCGCTTCGACAACGACGCCGCGAGTCCGAGGATGGTGCCGCGGGCGCCGTGGATGGAGCAGGAGGGGTCAGAGTATTGGGACCGGGAGACACGGAGCGCCAGGGACACCGCACAGATTTTCCGAGTGAATCTGCGGACGCTGCGCGGCTACTACAATCAGAGCGAGGCCGGGTCTCACACCCTGCAGTGGATGCATGGCTGCGAGCTGGGGCCCGACGGGCGCTTCCTCCGCGGGTATGAACAGTTCGCCTACGACGGCAAGGATTATCTCACCCTGAATGAGGACCTGCGCTCCTGGACCGCGGTGGACACGGCGGCTCAGATCTCCGAGCAAAAGTCAAATGATGCCTCTGAGGCGGAGCACCAGAGAGCCTACCTGGAAGACACATGCGTGGAGTGGCTCCACAAATACCTGGAGAAGGGGAAGGAGACGCTGCTTCACCTGGAGCCCCCAAAGACACACGTGACTCACCACCCCATCTCTGACCATGAGGCCACCCTGAGGTGCTGGGCCCTGGGCTTCTACCCTGCGGAGATCACACTGACCTGGCAGCAGGATGGGGAGGGCCATACCCAGGACACGGAGCTCGTGGAGACCAGGCCTGCAGGGGATGGAACCTTCCAGAAGTGGGCAGCTGTGGTGGTGCCTTCTGGAGAGGAGCAGAGATACACGTGCCATGTGCAGCATGAGGGGCTACCCGAGCCCGTCACCCTGAGATGGAAGCCGGCTTCCCAGCCCACCATCCCCATCGTGGGCATCATTGCTGGCCTGGTTCTCCTTGGATCTGTGGTCTCTGGAGCTGTGGTTGCTGCTGTGATATGGAGGAAGAAGAGCT CAGGTGGAAAAGGAGGGAGCTACTCTAAGGCTGAGTGGAGCGACAGTGCCCAGGGGTCTGAGTCTCACAGCTTGTAA 15 immature human IL-15 MRISKPHLRSISIQCYLCLLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 16 mature human IL-15 NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 17 Nucleic acid encoding mature human IL-15 (SEQ ID NO: 16) AACTGGGTGAACGTTATTAGTGACCTTAAAAAGATCGAAGATTTGATACAGTCAATGCACATAGACGCGACGCTTTATACAGAATCTGATGTACATCCTTCATGCAAGGTTACTGCTATGAAGTGTTTTCTTCTCGAACTCCAAGTAATAAGTCTTGAGAGCGGAGATGCGAGCATTCATGACACCGTTGAGAATCTTATTATATTGGCTAACAACTCTCTGTCCAGCAATGGTAATGTGACAGAAAGCGGGTGTAAGGAGTGCGAGGAACTCGAGGAGAAGAACATCAAAGAGTTCTTGCAGTCTTTCGTCCATATTGTCCAGATGTTCATAAATACTAGC 18 Nucleic acid encoding mature human IL-15 (SEQ ID NO: 16) AACTGGGTAAACGTCATAAGCGACCTCAAAAAGATCGAGGACCTTATACAGTCTATGCACATAGATGCGACACTTTACACGGAATCAGACGTGCACCCGTCCTGCAAAGTCACAGCCATGAAGTGCTTTCTTCTCGAACTGCAGGTAATTTCTCTCGAATCAGGTGACGCATCTATCCACGACACAGTTGAAAATCTTATTATCCTGGCTAATAACTCCCTTAGCTCAAACGGCAATGTCACCGAGAGTGGATGTAAAGAATGTGAGGAACTTGAAGAGAAAAACATAAAGGAATTCTTGCAGAGTTTCGTTCATATTGTGCAAATGTTCATCAATACTAGT 19 Immature full-length human IL-15 receptor alpha MAPRRARGCRTLGLPALLLLLLLRPPATRGITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVE MEAMEALPVTWGTSSRDEDLENCSHHL 20 Mature full-length human IL-15 receptor alpha ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSLLECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTTVAISTSTVLLCGLSAVSLLALACYLKSRQTPPLASTLEDSH twenty one immature extracellular human IL-15 receptor alpha MAPRRARGCRTLGLPALLLLLLRPPATRGITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTT twenty two Mature extracellular human IL-15 receptor alpha ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTT twenty three Nucleic acid sequence encoding SEQ ID NO: 22 (mature extracellular human IL-15 receptor alpha) ATCACATGCCCACCGCCCATGTCTGTTGAACACGCAGACATTTGGGTTAAAAGTTACTCACTTTACTCACGCGAGAGATATATATGCAACAGCGGCTTCAAGCGCAAAGCAGGCACTAGTAGTCTTACAGAGTGCGTGCTCAATAAAGCTACAAATGTAGCTCATTGGACTACTCCTAGTCTCAAATGCATTCGGGACCCCGCGCTTGTGCACCAGAGACCTGCGCCGCCGTCCACAGTGACGACAGCTGGTGTAACCCCCCAACCTGAATCCCTTAGTCCGTCTGGTAAAGAACCGGCGGCGTCTTCACCTTCCAGCAATAATACTGCGGCGACAACAGCCGCGATAGTTCCTGGATCCCAACTCATGCCGTCAAAGTCTCCTTCAACGGGAACGACAGAGATCTCTTCACATGAAAGTTCTCATGGAACACCGAGCCAAACTACGGCAAAGAACTGGGAACTGACTGCCTCAGCAAGCCACCAGCCGCCAGGGGTGTACCCGCAAGGGCACTCAGATACTACT twenty four Human IL-15 receptor sushi domain ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR 25 Human IL-15 receptor sushi domain + 13 aa of IL-15 receptor alpha ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLETCVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPS 26 Nucleic acid sequence encoding SEQ ID NO: 25 ATCACCTGCCCGCCTCCCATGAGCGTGGAACACGCGGACATTTGGGTTAAGAGCTACAGTCTTTACAGCCGGGAGCGCTATATCTGCAACTCAGGGTTTAAGCGGAAAGCAGGGACATCAAGTTTGACAGAATGTGTGTTGAACAAGGCTACAAATGTTGCTCACTGGACCACGCCATCTTTGAAGTGTATCCGAGATCCCGCGCTTGTCCATCAGCGCCCAGCGCCT 27 GPA signal peptide MYGKIIFVLLLSEIVSISA 28 Nucleic acid encoding SEQ ID NO: 33 ATGTATGGAAAAATAATCTTTGTATTACTATTGTCAGAAATTGTGAGCATATCAGCA 29 Linker GGGGSGGGGSGGGGS 30 (GGGGS) _n (GGGGS) _n , wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 31 Mature human IL-15 + (G4S) ₃ linker + mature extracellular soluble human IL-15 receptor alpha NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGGSGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTT 32 Nucleic acid sequence encoding SEQ ID NO: 31 AACTGGGTGAACGTTATTAGTGACCTTAAAAAGATCGAAGATTTGATACAGTCAATGCACATAGACGCGACGCTTTATACAGAATCTGATGTACATCCTTCATGCAAGGTTACTGCTATGAAGTGTTTTCTTCTCGAACTCCAAGTAATAAGTCTTGAGAGCGGAGATGCGAGCATTCATGACACCGTTGAGAATCTTATTATATTGGCTAACAACTCTCTGTCCAGCAATGGTAATGTGACAGAAAGCGGGTGTAAGGAGTGCGAGGAACTCGAGGAGAAGAACATCAAAGAGTTCTTGCAGTCTTTCGTCCATATTGTCCAGATGTTCATAAATACTAGCGGGGGAGGTGGCTCTGGTGGAGGCGGGAGTGGCGGGGGCGGCTCAATCACATGCCCACCGCCCATGTCTGTTGAACACGCAGACATTTGGGTTAAAAGTTACTCACTTTACTCACGCGAGAGATATATATGCAACAGCGGCTTCAAGCGCAAAGCAGGCACTAGTAGTCTTACAGAGTGCGTGCTCAATAAAGCTACAAATGTAGCTCATTGGACTACTCCTAGTCTCAAATGCATTCGGGACCCCGCGCTTGTGCACCAGAGACCTGCGCCGCCGTCCACAGTGACGACAGCTGGTGTAACCCCCCAACCTGAATCCCTTAGTCCGTCTGGTAAAGAACCGGCGGCGTCTTCACCTTCCAGCAATAATACTGCGGCGACAACAGCCGCGATAGTTCCTGGATCCCAACTCATGCCGTCAAAGTCTCCTTCAACGGGAACGACAGAGATCTCTTCACATGAAAGTTCTCATGGAACACCGAGCCAAACTACGGCAAAGAACTGGGAACTGACTGCCTCAGCAAGCCACCAGCCGCCAGGGGTGTACCCGCAAGGGCACTCAGATACTACT 33 Mature human IL-15 + (G4S) ₃ linker + IL-15 receptor alpha sushi domain + 13 aa of IL-15 receptor alpha NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGGSGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAPPSLTECVLNKATNVAHWTTPSLK 34 Nucleic acid sequence encoding SEQ ID NO: 33 AACTGGGTAAACGTCATAAGCGACCTCAAAAAGATCGAGGACCTTATACAGTCTATGCACATAGATGCGACACTTTACACGGAATCAGACGTGCACCCGTCCTGCAAAGTCACAGCCATGAAGTGCTTTCTTCTCGAACTGCAGGTAATTTCTCTCGAATCAGGTGACGCATCTATCCACGACACAGTTGAAAATCTTATTATCCTGGCTAATAACTCCCTTAGCTCAAACGGCAATGTCACCGAGAGTGGATGTAAAGAATGTGAGGAACTTGAAGAGAAAAACATAAAGGAATTCTTGCAGAGTTTCGTTCATATTGTGCAAATGTTCATCAATACTAGTGGCGGGGGGGGAAGCGGTGGTGGAGGGAGCGGGGGTGGTGGATCCATCACCTGCCCGCCTCCCATGAGCGTGGAACACGCGGACATTTGGGTTAAGAGCTACAGTCTTTACAGCCGGGAGCGCTATATCTGCAACTCAGGGTTTAAGCGGAAAGCAGGGACATCAAGTTTGACAGAATGTGTGTTGAACAAGGCTACAAATGTTGCTCACTGGACCACGCCATCTTTGAAGTGTATCCGAGATCCCGCGCTTGTCCATCAGCGCCCAGCGCCTCCCTCC 35 Linker GGSGGSGGGGGSGGGSGGGSGGGS 36 GPA peptide LSTTEVAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRAHEVSEISVRTVYPPEEETGERVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDDTDVPLSSVEIEENPETSDQ 37 Nucleic acid sequence encoding SEQ ID NO: 36 TTAAGTACCACTGAGGTGGCAATGCACACTTCAACCTCTTCTTCAGTCACAAAGAGTTACATCTCATCACAGACAAATGATACGCACAAACGGGACACATATGCAGCCACTCCTAGAGCTCATGAAGTTTCAGAAATTTCTGTTAGAACTGTTTACCCTCCAGAAGAGGAAACCGGAGAAAGGGTACAACTTGCCCATCATTTCTCTGAACCAGAGATAACACTCATTATTTTTGGGGTGATGGCTGGTGTTATTGGAACGATCCTCTTAATTTCTTACGGTATTCGCCGACTGATAAAGAAAAGCCCATCTGATGTAAAACCTCTCCCCTCACCTGACACAGACGTGCCTTTAAGTTCTGTTGAAATAGAAAATCCAGAGACAAGTGATCAA 38 IL-15 V3 construct MYGKIIFVLLLSEIVSISANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGSGGSGGYPYDVPDYAGGGSGGGSLSTTEVAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRAHEVSEISVRTVYPPEEETGERVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDTDVPLSSVEIENPETSDQ 39 Nucleic acid sequence encoding SEQ ID NO: 38 (IL-15 V3) ATGTATGGAAAAATAATCTTTGTATTACTATTGTCAGAAATTGTGAGCATATCAGCAAACTGGGTAAATGTGATTTCAGACCTGAAAAAAATCGAAGACCTTATTCAATCCATGCACATCGACGCGACACTTTATACTGAATCAGACGTACACCCGTCCTGTAAGGTTACTGCGATGAAGTGCTTTCTGTTGGAATTGCAAGTGATCTCCCTCGAATCAGGGGATGCATCCATTCATGATACCGTCGAGAATTTGATCATTCTGGCAAATAACTCCCTCAGTAGTAACGGGAATGTGACCGAGTCTGGGTGTAAGGAGTGCGAAGAGTTGGAGGAAAAGAATATCAAAGAATTCCTTCAGTCCTTTGTTCACATCGTGCAAATGTTTATTAATACATCTGGAGGATCTGGCGGGTCTGGAGGCTACCCCTATGACGTGCCCGACTATGCCGGCGGAGGGTCTGGAGGCGGTTCCTTAAGTACCACTGAGGTGGCAATGCACACTTCAACCTCTTCTTCAGTCACAAAGAGTTACATCTCATCACAGACAAATGATACGCACAAACGGGACACATATGCAGCCACTCCTAGAGCTCATGAAGTTTCAGAAATTTCTGTTAGAACTGTTTACCCTCCAGAAGAGGAAACCGGAGAAAGGGTACAACTTGCCCATCATTTCTCTGAACCAGAGATAACACTCATTATTTTTGGGGTGATGGCTGGTGTTATTGGAACGATCCTCTTAATTTCTTACGGTATTCGCCGACTGATAAAGAAAAGCCCATCTGATGTAAAACCTCTCCCCTCACCTGACACAGACGTGCCTTTAAGTTCTGTTGAAATAGAAAATCCAGAGACAAGTGATCAA 40 IL-15/IL-15Ra V4 construct MYGKIIFVLLLSEIVSISANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGGSGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTGGSGGSGGYPYDVPDYAGGGSGGGSLSTTEVAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRAHEVSEISVRTVYPPEEETGERVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDTDVPLSSVEIENPETSDQ 41 Nucleic acid sequence encoding SEQ ID NO: 40 (IL-15-V4) ATGTATGGAAAAATAATCTTTGTATTACTATTGTCAGAAATTGTGAGCATATCAGCAAACTGGGTGAACGTTATTAGTGACCTTAAAAAGATCGAAGATTTGATACAGTCAATGCACATAGACGCGACGCTTTATACAGAATCTGATGTACATCCTTCATGCAAGGTTACTGCTATGAAGTGTTTTCTTCTCGAACTCCAAGTAATAAGTCTTGAGAGCGGAGATGCGAGCATTCATGACACCGTTGAGAATCTTATTATATTGGCTAACAACTCTCTGTCCAGCAATGGTAATGTGACAGAAAGCGGGTGTAAGGAGTGCGAGGAACTCGAGGAGAAGAACATCAAAGAGTTCTTGCAGTCTTTCGTCCATATTGTCCAGATGTTCATAAATACTAGCGGGGGAGGTGGCTCTGGTGGAGGCGGGAGTGGCGGGGGCGGCTCAATCACATGCCCACCGCCCATGTCTGTTGAACACGCAGACATTTGGGTTAAAAGTTACTCACTTTACTCACGCGAGAGATATATATGCAACAGCGGCTTCAAGCGCAAAGCAGGCACTAGTAGTCTTACAGAGTGCGTGCTCAATAAAGCTACAAATGTAGCTCATTGGACTACTCCTAGTCTCAAATGCATTCGGGACCCCGCGCTTGTGCACCAGAGACCTGCGCCGCCGTCCACAGTGACGACAGCTGGTGTAACCCCCCAACCTGAATCCCTTAGTCCGTCTGGTAAAGAACCGGCGGCGTCTTCACCTTCCAGCAATAATACTGCGGCGACAACAGCCGCGATAGTTCCTGGATCCCAACTCATGCCGTCAAAGTCTCCTTCAACGGGAACGACAGAGATCTCTTCACATGAAAGTTCTCATGGAACACCGAGCCAAACTACGGCAAAGAACTGGGAACTGACTGCCTCAGCAAGCCACCAGCCGCCAGGGGTGTACCCGCAAGGGCACTCAGATACTACTGGAGGATCTGGCGGGTCTGGAGGCTACCCCT ATGACGTGCCCGACTATGCCGGCGGAGGGTCTGGAGGCGGTTCCTTAAGTACCACTGAGGTGGCAATGCACACTTCAACCTCTTCTTCAGTCACAAAGAGTTACATCTCATCACAGACAAATGATACGCACAAACGGGACACATATGCAGCCACTCCTAGAGCTCATGAAGTTTCAGAAATTTCTGTTAGAACTGTTTACCCTCCAGAAGAGGAAACCGGAGAAAGGGTACAACTTGCCCATCATTTCTCTGAACCAGAGATAACACTCATTATTTTTGGGGTGATGGCTGGTGTTATTGGAACGATCCTCTTAATTTCTTACGGTATTCGCCGACTGATAAAGAAAAGCCCATCTGATGTAAAACCTCTCCCCTCACCTGACACAGACGTGCCTTTAAGTTCTGTTGAAATAGAAAATCCAGAGACAAGTGATCAA 42 IL-15/IL-15Ra V5 MYGKIIFVLLLSEIVSISANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGGSGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSGGSGGSGGYPYDVPDYAGGGSGGGSLSTTEVAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRAHEVSEISVRTVYPPEEETGERVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDTDVPLSSVEIENPETSDQ 43 Nucleic acid sequence encoding SEQ ID NO: 42 (IL-15 V5) ATGTATGGAAAAATAATCTTTGTATTACTATTGTCAGAAATTGTGAGCATATCAGCAAACTGGGTAAACGTCATAAGCGACCTCAAAAAGATCGAGGACCTTATACAGTCTATGCACATAGATGCGACACTTTACACGGAATCAGACGTGCACCCGTCCTGCAAAGTCACAGCCATGAAGTGCTTTCTTCTCGAACTGCAGGTAATTTCTCTCGAATCAGGTGACGCATCTATCCACGACACAGTTGAAAATCTTATTATCCTGGCTAATAACTCCCTTAGCTCAAACGGCAATGTCACCGAGAGTGGATGTAAAGAATGTGAGGAACTTGAAGAGAAAAACATAAAGGAATTCTTGCAGAGTTTCGTTCATATTGTGCAAATGTTCATCAATACTAGTGGCGGGGGGGGAAGCGGTGGTGGAGGGAGCGGGGGTGGTGGATCCATCACCTGCCCGCCTCCCATGAGCGTGGAACACGCGGACATTTGGGTTAAGAGCTACAGTCTTTACAGCCGGGAGCGCTATATCTGCAACTCAGGGTTTAAGCGGAAAGCAGGGACATCAAGTTTGACAGAATGTGTGTTGAACAAGGCTACAAATGTTGCTCACTGGACCACGCCATCTTTGAAGTGTATCCGAGATCCCGCGCTTGTCCATCAGCGCCCAGCGCCTCCCTCCGGAGGATCTGGCGGGTCTGGAGGCTACCCCTATGACGTGCCCGACTATGCCGGCGGAGGGTCTGGAGGCGGTTCCTTAAGTACCACTGAGGTGGCAATGCACACTTCAACCTCTTCTTCAGTCACAAAGAGTTACATCTCATCACAGACAAATGATACGCACAAACGGGACACATATGCAGCCACTCCTAGAGCTCATGAAGTTTCAGAAATTTCTGTTAGAACTGTTTACCCTCCAGAAGAGGAAACCGGAGAAAGGGTACAACTTGCCCATCATTTCTCTGAACCAGAGATAACACTCATTATTTTTGGGGTGATGGCTG GTGTTATTGGAACGATCCTCTTAATTTCTTACGGTATTCGCCGACTGATAAAGAAAAGCCCATCTGATGTAAAACCTCTCCCCTCACCTGACACAGACGTGCCTTTAAGTTCTGTTGAAATAGAAAATCCAGGACAAGTGATCAA 44 IL-15 V3.1 construct MYGKIIFVLLLSEIVSISANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGSGGSGGGGGSGGGSGGGSGGGSLSTTEVAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRAHEVSEISVRTVYPPEEETGERVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDTDVPLSSVEIENPETSDQ 45 Nucleic acid sequence encoding SEQ ID NO: 44 (IL-15 V3.1) ATGTATGGAAAAATAATCTTTGTATTACTATTGTCAGAAATTGTGAGCATATCAGCAAACTGGGTAAATGTGATTTCAGACCTGAAAAAAATCGAAGACCTTATTCAATCCATGCACATCGACGCGACACTTTATACTGAATCAGACGTACACCCGTCCTGTAAGGTTACTGCGATGAAGTGCTTTCTGTTGGAATTGCAAGTGATCTCCCTCGAATCAGGGGATGCATCCATTCATGATACCGTCGAGAATTTGATCATTCTGGCAAATAACTCCCTCAGTAGTAACGGGAATGTGACCGAGTCTGGGTGTAAGGAGTGCGAAGAGTTGGAGGAAAAGAATATCAAAGAATTCCTTCAGTCCTTTGTTCACATCGTGCAAATGTTTATTAATACATCTGGAGGATCTGGCGGGTCTGGAGGCGGCGGCGGCAGCGGCGGCGGCAGCGGCGGAGGGTCTGGAGGCGGTTCCTTAAGTACCACTGAGGTGGCAATGCACACTTCAACCTCTTCTTCAGTCACAAAGAGTTACATCTCATCACAGACAAATGATACGCACAAACGGGACACATATGCAGCCACTCCTAGAGCTCATGAAGTTTCAGAAATTTCTGTTAGAACTGTTTACCCTCCAGAAGAGGAAACCGGAGAAAGGGTACAACTTGCCCATCATTTCTCTGAACCAGAGATAACACTCATTATTTTTGGGGTGATGGCTGGTGTTATTGGAACGATCCTCTTAATTTCTTACGGTATTCGCCGACTGATAAAGAAAAGCCCATCTGATGTAAAACCTCTCCCCTCACCTGACACAGACGTGCCTTTAAGTTCTGTTGAAATAGAAAATCCAGAGACAAGTGATCAA 46 IL-15/IL-15Ra V4.1 construct MYGKIIFVLLLSEIVSISANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGGSGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTGGSGGSGGGGGSGGGSGGGSGGGSLSTTEVAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRAHEVSEISVRTVYPPEEETGERVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDTDVPLSSVEIENPETSDQ 47 Nucleic acid sequence encoding SEQ ID NO: 46 (IL-15-V4.1) ATGTATGGAAAAATAATCTTTGTATTACTATTGTCAGAAATTGTGAGCATATCAGCAAACTGGGTGAACGTTATTAGTGACCTTAAAAAGATCGAAGATTTGATACAGTCAATGCACATAGACGCGACGCTTTATACAGAATCTGATGTACATCCTTCATGCAAGGTTACTGCTATGAAGTGTTTTCTTCTCGAACTCCAAGTAATAAGTCTTGAGAGCGGAGATGCGAGCATTCATGACACCGTTGAGAATCTTATTATATTGGCTAACAACTCTCTGTCCAGCAATGGTAATGTGACAGAAAGCGGGTGTAAGGAGTGCGAGGAACTCGAGGAGAAGAACATCAAAGAGTTCTTGCAGTCTTTCGTCCATATTGTCCAGATGTTCATAAATACTAGCGGGGGAGGTGGCTCTGGTGGAGGCGGGAGTGGCGGGGGCGGCTCAATCACATGCCCACCGCCCATGTCTGTTGAACACGCAGACATTTGGGTTAAAAGTTACTCACTTTACTCACGCGAGAGATATATATGCAACAGCGGCTTCAAGCGCAAAGCAGGCACTAGTAGTCTTACAGAGTGCGTGCTCAATAAAGCTACAAATGTAGCTCATTGGACTACTCCTAGTCTCAAATGCATTCGGGACCCCGCGCTTGTGCACCAGAGACCTGCGCCGCCGTCCACAGTGACGACAGCTGGTGTAACCCCCCAACCTGAATCCCTTAGTCCGTCTGGTAAAGAACCGGCGGCGTCTTCACCTTCCAGCAATAATACTGCGGCGACAACAGCCGCGATAGTTCCTGGATCCCAACTCATGCCGTCAAAGTCTCCTTCAACGGGAACGACAGAGATCTCTTCACATGAAAGTTCTCATGGAACACCGAGCCAAACTACGGCAAAGAACTGGGAACTGACTGCCTCAGCAAGCCACCAGCCGCCAGGGGTGTACCCGCAAGGGCACTCAGATACTACTGGAGGATCTGGCGGGTCTGGAGGCGGCGGCG GCAGCGGCGGCGGCAGCGGCGGAGGGTCTGGAGGCGGTTCCTTAAGTACCACTGAGGTGGCAATGCACACTTCAACCTCTTCTTCAGTCACAAAGAGTTACATCTCATCACAGACAAATGATACGCACAAACGGGACACATATGCAGCCACTCCTAGAGCTCATGAAGTTTCAGAAATTTCTGTTAGAACTGTTTACCCTCCAGAAGAGGAAACCGGAGAAAGGGTACAACTTGCCCATCATTTCTCTGAACCAGAGATAACACTCATTATTTTTGGGGTGATGGCTGGTGTTATTGGAACGATCCTCTTAATTTCTTACGGTATTCGCCGACTGATAAAGAAAAGCCCATCTGATGTAAAACCTCTCCCCTCACCTGACACAGACGTGCCTTTAAGTTCTGTTGAAATAGAAAATCCAGAGACAAGTGATCAA 48 extracellular 4-1BBL polypeptide ACPWAVSGARASPPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPPEIPAGL 49 Nucleic acid sequence encoding SEQ ID NO: 48 (extracellular 4-1BBL) GCCTGCCCCTGGGCCGTGTCCGGGGCTCGCGCCTCGCCCGGCTCCGCGGCCAGCCCGAGACTCCGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA 50 4-1BBL construct MYGKIIFVLLLSEIVSISAACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSEGGSGGSGGGPEDEPGSGSGGGSGGGSLSTTEVAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRAHEVSEISVRTVYPPEEETGERVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDTDVPLSSVEIENPETSDQ 51 Nucleic acid sequence encoding SEQ ID NO: 50 (4-1BBL construct) ATGTATGGAAAAATAATCTTTGTATTACTATTGTCAGAAATTGTGAGCATATCAGCAGCCTGCCCCTGGGCCGTGTCCGGGGCTCGCGCCTCGCCCGGCTCCGCGGCCAGCCCGAGACTCCGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAAGGAGGATCTGGCGGGTCTGGAGGCGGCCCCGAGGACGAGCCCGGCAGCGGCAGCGGCGGAGGGTCTGGAGGCGGTTCCTTAAGTACCACTGAGGTGGCAATGCACACTTCAACCTCTTCTTCAGTCACAAAGAGTTACATCTCATCACAGACAAATGATACGCACAAACGGGACACATATGCAGCCACTCCTAGAGCTCATGAAGTTTCAGAAATTTCTGTTAGAACTGTTTACCCTCCAGAAGAGGAAACCGGAGAAAGGGTACAACTTGCCCATCATTTCTCTGAACCAGAGATAACACTCATTATTTTTGGGGTGATGGCTGGTG TTATTGGAACGATCCTCTTAATTTCTTACGGTATTCGCCGACTGATAAAGAAAAGCCCATCTGATGTAAAACCTCTCCCCTCACCTGACACAGACGTGCCTTTAAGTTCTGTTGAAATAGAAAATCCAGAGACAAGTGATCAA 52 4-1BBL linker GGSGGSGGGPEDEPGSGSGGGSGGGS 53 Nucleic acid sequence encoding SEQ ID NO: 52 GGAGGATCTGGCGGGTCTGGAGGCGGCCCCGAGGACGAGCCCGGCAGCGGCAGCGGCGGAGGGTCTGGAGGCGGTTCC 54 Full-length GPA Amino Acids MYGKIIFVLLLSAIVSISALSTTEVAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRAHEVSEISVRTVYPPEEETGERVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDDVPLSSVEIEENPETSDQ 55 SMIM1 polypeptide MQPQESHVHYSRWEDGSRDGVSLGAVSSTEEASRCRRISQRLCTGKLGIAMKVLGGVALFWIIFILGYLTGYYVHKCK 56 G4S linker GGGGS 57 Linker-HA-Linker GGSGGSGGYPYDVPDYAGGGSGGGS 58 Linker GSGSGSGSGSEDEDEDEDEDGSGSGSGSGS 59 Linker GGGGSGGGGSGGGGSGGGGS 60 Linker GSGSGSGSEDGSGSGSGS 61 Linker GSGSGSGSGSGSGSGSGSGS 62 Linker GCGGSGGGGSGGGGS 63 Linker SGRGGGGSGGGGSGGGGSGGGGSSPA 64 Linker GGGGSGGGGSGGGGSGGGGSGGGG 65 Snorkel linker SGRGASSGSSGSGSQKKPRYEIRWKVVVISAILALVVLTVISLIILIMLWGSGMQSPA 66 Ig heavy chain V region 3 message peptide MGWSCIILFLVATATGVHS 67 light chain leader MRVPAQLLGLLLLWLPGARC 68 amino acid linker AGST

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1 is a graph showing ex vivo treatment of PBMCs with enucleated erythroid cells comprising a first exogenous polypeptide on their extracellular surface and a second exogenous polypeptide comprising 4-1BBL (" RBC-IL15/IL15RA-4-1BBL"), the first exogenous polypeptide comprises (i) IL-15 or its functional fragment and (ii) IL-15 receptor α or its functional fragment, compared with the culture medium and with rhIL-15 resulted in an increased proportion of NKp30-positive lymphocytes compared with anti-4-1BB stimulatory antibody treatment.

Figures 2A-2C are graphs showing the following relative to baseline in patients administered ≥1 x ¹⁰ erythroid cells (comprising IL-15/IL-15RA and 4-1BBL) prepared substantially as described in Example 3 Plots of maximum fold change: percentage of CD56 ⁺ lymphocytes positive for NKp30 (Figure 2A), percentage of CD56dimCD16 ⁺ lymphocytes positive for NKp30 (Figure 2B), and percentage of CD56 ⁺ CD16 ⁺ lymphocytes positive for NKp30 (Figure 2C) . Cell surface markers were assessed by flow cytometry in fresh whole blood in viable lymphocytes (CD45 ⁺ ) at baseline (before treatment) and at various time points after treatment. Post-treatment measurements were performed over multiple dosing cycles.

Figures 3A-3B are graphs showing the following relative to baseline in patients administered ≥1 x ¹⁰ erythroid cells (comprising IL-15/IL-15RA and 4-1BBL) prepared substantially as described in Example 3 Graphs of maximum fold change: Absolute number of CD3 ⁻ CD16/56 ⁺ NK cells/μl (Fig. 3A) and percentage of CD8 ⁺ memory T cells expressing granzyme B (%GrB ⁺ of CD3 ⁺ CD8 ⁺ CD45RA- ⁾ (Fig. 3B). Cell surface markers were assessed by flow cytometry in fresh whole blood in viable lymphocytes (CD45 ⁺ ) at baseline (before treatment) and at various time points after treatment. Post-treatment measurements were performed over multiple dosing cycles.

Figures 4A-4B are graphs showing the following relative to baseline in patients administered ≥1 x ¹⁰ erythroid cells (comprising IL-15/IL-15RA and 4-1BBL) prepared substantially as described in Example 3 Graph of maximum fold change: percentage of CD3 ⁺ lymphocytes positive for CD4 (Figure 4A) and percentage of CD3 ⁺ lymphocytes positive for CD8 (Figure 4B). Cell surface markers were assessed by flow cytometry in fresh whole blood in viable lymphocytes (CD45 ⁺ ) at baseline (before treatment) and at various time points after treatment. Post-treatment measurements were performed over multiple dosing cycles.

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Claims (96)

A method of increasing the number of NKp30 positive lymphocytes in an individual in need thereof previously identified or diagnosed with a B7-H6 positive cancer comprising administering to the individual a therapeutically effective amount of a pharmaceutical composition comprising an enucleated A population of erythrocytes comprising on their extracellular surface a first exogenous fusion polypeptide comprising (i) IL-15 or a functional fragment thereof, and ( ii) IL-15 receptor alpha or a functional fragment thereof. The method of claim 1, wherein the administration results in an increase in the number of NKp30-positive lymphocytes in the individual by at least 5% compared to the number of NKp30-positive lymphocytes in the individual before the administration. The method of claim 1, wherein the administration results in an increase in the number of NKp30-positive lymphocytes in the individual by at least 10% compared to the number of NKp30-positive lymphocytes in the individual before the administration. The method according to any one of claims 1 to 3, wherein the NKp30-positive lymphocytes are NKp30-positive NK cells. The method of claim 1, wherein the method further results in an increase in the number of NKp30-positive/NKG2A-negative lymphocytes in the individual. The method of claim 5, wherein the administering step results in an increase in the number of NKp30-positive/NKG2A-negative lymphocytes in the individual by at least 5%. The method of claim 6, wherein the administering step results in an increase in the number of NKp30-positive/NKG2A-negative lymphocytes in the individual by at least 10%. The method according to any one of claims 5 to 7, wherein the NKp30-positive/NKG2A-negative lymphocytes are NKp30-positive/NKG2A-negative NK cells. The method of claim 1, wherein the administering step results in an increase in the number of NKp30-positive, CD45-positive, CD56-positive lymphocytes in the individual. The method according to claim 9, wherein the administering step results in an increase in the percentage of NKp30-positive CD45-positive, CD56-positive lymphocytes in the individual by at least 1.2 times. The method of claim 10, wherein the step of administering results in an increase in the percentage of NKp30-positive CD45-positive, CD56-positive lymphocytes in the individual by at least 1.5 times. The method of claim 11, wherein the step of administering results in an increase in the percentage of NKp30-positive CD45-positive, CD56-positive lymphocytes in the individual by at least 2.0 times. The method according to any one of claims 9 to 12, wherein the NKp30-positive, CD45-positive, and CD56-positive lymphocytes are NKp30-positive, CD45-positive, and CD56-positive NK cells. The method of claim 1, wherein the administering step results in an increase in the number of NKp30-positive, CD45-positive, CD16-positive, CD56-dim lymphocytes in the individual. The method of claim 14, wherein the step of administering results in an increase in the percentage of NKp30-positive CD56-dim, CD16-positive, CD45-positive lymphocytes in the individual by at least 1.2 times. The method of claim 15, wherein the step of administering results in an increase in the percentage of NKp30-positive CD56-dim, CD16-positive, CD45-positive lymphocytes in the individual by at least 1.5 times. The method of claim 16, wherein the step of administering results in an increase in the percentage of NKp30-positive CD56-dim, CD16-positive, CD45-positive lymphocytes in the individual by at least 1.7 times. The method according to any one of claims 14 to 17, wherein the NKp30-positive, CD45-positive, CD16-positive, CD56-dim lymphocytes are NKp30-positive, CD45-positive, CD16-positive, CD56-dim NK cells. The method of claim 1, wherein the administering step results in an increase in the number of NKp30-positive, CD45-positive, CD16-positive and CD56-positive lymphocytes in the individual. The method of claim 19, wherein the step of administering results in an increase in the percentage of NKp30-positive CD56-positive, CD16-positive, CD45-positive lymphocytes in the individual by at least 1.5 times. The method of claim 20, wherein the administering step results in an increase in the percentage of NKp30-positive CD56-positive, CD16-positive, CD45-positive lymphocytes in the individual by at least 2.0 times. The method of claim 21, wherein the administering step results in an increase in the percentage of NKp30-positive CD56-positive, CD16-positive, CD45-positive lymphocytes in the individual by at least 2.5 times. The method of claim 22, wherein the step of administering results in an increase in the percentage of NKp30-positive CD56-positive, CD16-positive, CD45-positive lymphocytes in the individual by at least 3.0 times. The method of claim 23, wherein the administering step results in an increase in the percentage of NKp30-positive CD56-positive, CD16-positive, CD45-positive lymphocytes in the individual by at least 3.5 times. The method according to any one of claims 19 to 24, wherein the NKp30-positive, CD56-positive, CD16-positive, and CD45-positive lymphocytes are NKp30-positive, CD56-positive, CD16-positive, and CD45-positive NK cells. The method of any one of claims 1 to 25, wherein the administering step does not result in a significant degree of myeloid cytotoxicity in the individual. The method of any one of claims 1 to 26, wherein the method further comprises identifying or diagnosing the individual as having a B7-H6 positive cancer. The method of claim 27, wherein the B7-H6 positive cancer is selected from the group consisting of adrenocortical cancer, cholangiocarcinoma, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, esophagus cancer, eye cancer, head and neck cancer Carcinoma, acute myelogenous leukemia, lymphoid neoplasms, diffuse large b-cell lymphoma, renal carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, renal chromophobe, liver carcinoma, lung cancer, lung adenocarcinoma, lung squamous Cell carcinoma, mesothelioma, ovarian cancer, pancreatic cancer, rectal cancer, skin cancer, stomach cancer, testicular cancer, thymoma, thyroid cancer, uterine cancer, uterine corpus endometrial cancer, uterine carcinosarcoma, and uveal melanoma . The method of claim 27 or 28, wherein the B7-H6 positive cancer is a B7-H6 positive/HLA-E negative cancer. The method of any one of claims 1 to 29, wherein the method further comprises administering an NKG2A inhibitor to the individual. The method of claim 30, wherein the NKG2A inhibitor is an antagonistic antibody that specifically binds to NKG2A. A method of treating an individual previously identified or diagnosed as having a B7-H6 positive cancer, the method comprising administering to the individual previously identified or diagnosed as having a B7-H6 positive cancer a therapeutically effective amount of a pharmaceutical composition, the pharmaceutical composition The substance comprises a group of enucleated erythroid cells comprising on their extracellular surface a first exogenous polypeptide comprising (i) IL-15 or a functional fragment thereof , and (ii) IL-15 receptor alpha or a functional fragment thereof. The method of claim 32, wherein the B7-H6 positive cancer is selected from the group consisting of adrenocortical cancer, cholangiocarcinoma, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer Carcinoma, acute myelogenous leukemia, lymphoid neoplasms, diffuse large b-cell lymphoma, renal carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, renal chromophobe, liver carcinoma, lung cancer, lung adenocarcinoma, lung squamous Cell carcinoma, mesothelioma, ovarian cancer, pancreatic cancer, rectal cancer, skin cancer, stomach cancer, testicular cancer, thymoma, thyroid cancer, uterine cancer, uterine corpus endometrial cancer, uterine carcinosarcoma, and uveal melanoma . The method of claim 32 or 33, wherein the B7-H6 positive cancer is a B7-H6 positive/HLA-E negative cancer. The method of any one of claims 32 to 34, wherein the step of administering does not result in a significant degree of myeloid cytotoxicity in the individual. The method of any one of claims 32 to 35, wherein the method further comprises diagnosing or identifying the individual as having a B7-H6 positive cancer. The method of any one of claims 32 to 36, wherein the method further comprises administering an NKG2A inhibitor to the individual. The method of claim 37, wherein the NKG2A inhibitor is an antagonistic antibody that specifically binds to NKG2A. A method of reducing the number and/or proliferation of B7-H6 positive cancer cells in an individual previously identified or diagnosed with a B7-H6 positive cancer, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising A population of enucleated erythroid cells comprising on their extracellular surface a first exogenous polypeptide comprising (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof. The method of claim 39, wherein the administration results in a reduction in the number of B7-H6 positive cancer cells in the individual. The method of claim 40, wherein the administering results in at least a 5% reduction in the number of B7-H6 positive cancer cells in the individual compared to the number of B7-H6 positive cancer cells in the individual before the administration. The method of claim 40, wherein the administering results in at least a 10% reduction in the number of B7-H6 positive cancer cells in the individual compared to the number of B7-H6 positive cancer cells in the individual before the administration. The method of claim 39, wherein the administration results in a reduction in the number of B7-H6 positive/HLA-E negative cancer cells in the individual. The method of claim 43, wherein the administration results in the number of B7-H6 positive/HLA-E negative cancer cells in the individual compared to the number of B7-H6 positive/HLA-E negative cancer cells in the individual before the administration Reduced by at least 5%. The method of claim 43, wherein the administration results in the number of B7-H6 positive/HLA-E negative cancer cells in the individual compared to the number of B7-H6 positive/HLA-E negative cancer cells in the individual before the administration Reduce by at least 10%. The method of claim 39, wherein the administration results in decreased proliferation of B7-H6 positive cancer cells in the individual. The method of claim 46, wherein the administering results in at least a 5% reduction in the proliferation of B7-H6 positive cancer cells in the individual compared to the proliferation of B7-H6 positive cancer cells in the individual before the administration. The method of claim 46, wherein the administering results in at least a 10% reduction in proliferation of B7-H6 positive cancer cells in the individual compared to proliferation of B7-H6 positive cancer cells in the individual prior to the administration. The method of claim 39, wherein the administration results in decreased proliferation of B7-H6 positive/HLA-E negative cancer cells in the individual. The method of claim 49, wherein the administration results in proliferation of B7-H6 positive/HLA-E negative cancer cells in the individual compared to proliferation of B7-H6 positive/HLA-E negative cancer cells in the individual prior to the administration Reduced by at least 5%. The method of claim 49, wherein the administration results in proliferation of B7-H6 positive/HLA-E negative cancer cells in the individual compared to proliferation of B7-H6 positive/HLA-E negative cancer cells in the individual prior to the administration Reduce by at least 10%. The method of any one of claims 39 to 51, wherein the step of administering does not result in a significant degree of myeloid cytotoxicity in the individual. The method of any one of claims 39 to 52, wherein the method further comprises identifying or diagnosing the individual as having a B7-H6 positive cancer. The method of claim 53, wherein the B7-H6 positive cancer is selected from the group consisting of adrenocortical carcinoma, cholangiocarcinoma, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer Carcinoma, acute myelogenous leukemia, lymphoid neoplasms, diffuse large b-cell lymphoma, renal carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, renal chromophobe, liver carcinoma, lung cancer, lung adenocarcinoma, lung squamous Cell carcinoma, mesothelioma, ovarian cancer, pancreatic cancer, rectal cancer, skin cancer, stomach cancer, testicular cancer, thymoma, thyroid cancer, uterine cancer, uterine corpus endometrial cancer, uterine carcinosarcoma, and uveal melanoma . The method of claim 53 or 54, wherein the B7-H6 positive cancer is a B7-H6 positive/HLA-E negative cancer. The method of any one of claims 39 to 55, wherein the method further comprises administering an NKG2A inhibitor to the individual. The method of claim 56, wherein the NKG2A inhibitor is an antagonistic antibody that specifically binds to NKG2A. A method of inducing the killing of B7-H6 positive cancer cells in an individual previously identified or diagnosed as having a B7-H6 positive cancer, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an enucleated A population of erythrocytes comprising on their extracellular surface an exogenous fusion polypeptide comprising (i) IL-15 or a functional fragment thereof, and (ii) IL-15 15 receptor alpha or functional fragments thereof. The method of claim 58, wherein killing comprises necrosis. The method of claim 58, wherein killing comprises apoptosis. The method of claim 58, wherein the killing is mediated by NK cell mediated cytolysis. The method according to any one of claims 58 to 61, wherein the B7-H6 positive cancer cells are cancer cells selected from the group consisting of adrenocortical carcinoma, cholangiocarcinoma, bladder cancer, breast cancer, cervical cancer, colon cancer, Colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, acute myeloid leukemia, lymphoid neoplasms, diffuse large b-cell lymphoma, kidney cancer, renal clear cell carcinoma, renal papillary cell carcinoma, renal chromophobe, Liver cancer, lung cancer, lung adenocarcinoma, squamous cell carcinoma of the lung, mesothelioma, ovarian cancer, pancreatic cancer, rectal cancer, skin cancer, stomach cancer, testicular cancer, thymoma, thyroid cancer, uterine cancer, uterine endometrium carcinoma, uterine carcinosarcoma, and uveal melanoma. The method according to any one of claims 58 to 62, wherein the B7-H6 positive cancer cells are B7-H6 positive and HLA-E negative cancer cells. The method of any one of claims 58 to 63, wherein the step of administering does not result in a significant degree of myeloid cytotoxicity in the individual. The method of any one of claims 58 to 64, wherein the individual has previously been identified or diagnosed as having a B7-H6 positive cancer. The method of claim 65, wherein the B7-H6 positive cancer is selected from the group consisting of adrenocortical cancer, cholangiocarcinoma, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, esophagus cancer, eye cancer, head and neck cancer Carcinoma, acute myelogenous leukemia, lymphoid neoplasms, diffuse large b-cell lymphoma, renal carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, renal chromophobe, liver carcinoma, lung cancer, lung adenocarcinoma, lung squamous Cell carcinoma, mesothelioma, ovarian cancer, pancreatic cancer, rectal cancer, skin cancer, stomach cancer, testicular cancer, thymoma, thyroid cancer, uterine cancer, uterine corpus endometrial cancer, uterine carcinosarcoma, and uveal melanoma . The method of claim 65 or 66, wherein the B7-H6 positive cancer is a B7-H6 positive/HLA-E negative cancer. The method of any one of claims 58 to 67, wherein the method further comprises administering an NKG2A inhibitor to the individual. The method of claim 68, wherein the NKG2A inhibitor is an antagonistic antibody that specifically binds to NKG2A. A method of reducing the volume of a solid tumor in an individual previously identified or diagnosed as having a B7-H6 positive cancer, the method comprising administering a therapeutically effective amount of a population of enucleated erythroid cells in which A first exogenous fusion polypeptide comprising (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof is included on the extracellular surface of the cell. The method of claim 70, wherein the administering results in at least a 5% reduction in the volume of the solid tumor in the individual compared to the volume of the solid tumor before the administering. The method of claim 70, wherein the administering results in a reduction in the volume of the solid tumor in the individual by at least 10% compared to the volume of the solid tumor before the administering. The method of any one of claims 70 to 72, wherein the step of administering does not result in a significant degree of myeloid cytotoxicity in the individual. The method according to any one of claims 70 to 73, wherein the B7-H6 positive cancer is selected from the group consisting of adrenocortical carcinoma, cholangiocarcinoma, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, esophagus Carcinoma, eye cancer, head and neck cancer, acute myeloid leukemia, lymphoid neoplasms, diffuse large b-cell lymphoma, kidney cancer, renal clear cell carcinoma, renal papillary cell carcinoma, renal chromophobe, liver cancer, lung cancer, lung Adenocarcinoma, squamous cell carcinoma of the lung, mesothelioma, ovarian cancer, pancreatic cancer, rectal cancer, skin cancer, gastric cancer, testicular cancer, thymoma, thyroid cancer, uterine cancer, endometrial cancer of the uterine body, carcinosarcoma of the uterus , and uveal melanoma. The method of any one of claims 70 to 74, wherein the B7-H6 positive cancer is a B7-H6 positive/HLA-E negative cancer. The method of any one of claims 70 to 75, wherein the step of administering does not result in a significant degree of myeloid cytotoxicity in the individual. The method of any one of claims 70 to 76, wherein the method further comprises diagnosing or identifying the individual as having a B7-H6 positive cancer. The method of any one of claims 70 to 77, wherein the method further comprises administering an NKG2A inhibitor to the individual. The method of claim 78, wherein the NKG2A inhibitor is an antagonistic antibody that specifically binds to NKG2A. The method according to any one of claims 1 to 79, wherein the enucleated erythroid blood cells comprise at least 1,000 copies of the exogenous fusion polypeptide. The method according to any one of claims 1 to 80, wherein the enucleated erythroid blood cells are produced by the following method, the method comprising: introducing a nucleic acid encoding a first exogenous polypeptide into the nucleated erythroid cell precursor; and The nucleated erythroid cell precursor is cultured under conditions sufficient to express the exogenous polypeptide and enucleate the nucleated erythroid cell precursor. The method according to any one of claims 1 to 81, wherein the enucleated erythrocytes further comprise a second exogenous polypeptide comprising 4-1BBL or a functional fragment thereof, wherein the second exogenous The polypeptide is present on the extracellular surface of the enucleated erythroid cells. The method of claim 82, wherein the enucleated erythroid blood cells comprise at least 1,000 copies of the second exogenous polypeptide. The method of claim 82 or 83, wherein the enucleated erythroid blood cells are produced by the following method, the method comprising: introducing a nucleic acid encoding a first exogenous polypeptide comprising 4-1BBL or a functional fragment thereof and a nucleic acid encoding a second exogenous polypeptide into the nucleated erythroid cell precursor; and The nucleated erythroid cell precursor is cultured under conditions sufficient to express the first exogenous polypeptide and the second exogenous polypeptide and to enucleate the nucleated erythroid cell precursor. The method according to any one of claims 1 to 84, wherein the enucleated erythroid cells are not hypodialyzed cells. The method of any one of claims 1 to 85, wherein the enucleated erythroid blood cells do not comprise a sortase-transferred feature. The method of claim 86, wherein the subject is a human and the enucleated erythroid cells are human cells. A kit comprising: A pharmaceutical composition comprising an enucleated erythroid cell comprising a first exogenous polypeptide on its extracellular surface, the first exogenous polypeptide comprising (i) IL-15 or its function fragments, and (ii) IL-15 receptor alpha or functional fragments thereof; and Instructions for carrying out the method of any one of claims 1 to 66. The kit according to claim 88, wherein the enucleated erythroid blood cells comprise at least 1,000 copies of the first exogenous polypeptide. The set of claim 88 or 89, wherein the enucleated erythroid cells are produced by the following method, the method comprising: introducing a nucleic acid encoding a first exogenous polypeptide into the nucleated erythroid cell precursor; and The nucleated erythroid cell precursor is cultured under conditions sufficient to express the first exogenous polypeptide and enucleate the nucleated erythroid cell precursor. The set according to any one of claims 88 to 90, wherein the enucleated erythroid cells further comprise a second exogenous polypeptide comprising 4-1BBL or a functional fragment thereof, wherein the second exogenous The polypeptide is present on the extracellular surface of the enucleated erythroid cells. The set according to claim 91, wherein the enucleated erythroid blood cells comprise at least 1,000 copies of the second exogenous polypeptide. The set of claim 91 or 92, wherein the enucleated erythroid blood cells are produced by the following method, the method comprising: introducing a nucleic acid encoding a first exogenous polypeptide and a nucleic acid encoding a second exogenous polypeptide into the nucleated erythroid cell precursor; and The nucleated erythroid cell precursor is cultured under conditions sufficient to express the first exogenous polypeptide and the second exogenous polypeptide and to enucleate the nucleated erythroid cell precursor. The set according to any one of claims 88 to 93, wherein the enucleated erythroid cells are not hypotonic cells. The kit according to any one of claims 88 to 94, wherein the enucleated erythroid blood cells do not comprise sortase-transferred features. The set according to any one of claims 88 to 95, wherein the enucleated erythroid cells are human cells.

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