WO2022197548A1

WO2022197548A1 - Methods of increasing nkg2d-positive lymphocytes in a subject and uses thereof

Info

Publication number: WO2022197548A1
Application number: PCT/US2022/019950
Authority: WO
Inventors: Christina Marie Coughlin; Laurence Allan TURKA; Anna Carol Salzberg WALDNER
Original assignee: Rubius Therapeutics, Inc.
Priority date: 2021-03-14
Filing date: 2022-03-11
Publication date: 2022-09-22
Also published as: TW202304482A

Abstract

Provided herein are methods of increasing the number of NKG2D-positive lymphocytes in a subject in need thereof, and uses of the same.

Description

METHODS OF INCREASING NKG2D-POSITIVE LYMPHOCYTES IN A SUBJECT AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Patent Application No. 63/160,858, filed on March 14, 2021. The disclosure of this prior application is considered part of the disclosure of this application, and is incorporated in its entirety into this application.

TECHNICAL FIELD

The present invention relates generally to methods of administering enucleated erythroid cells to a subject, methods of increasing NKG2D-positive lymphocytes in a subject, and methods of treating a MICA-positive cancer, a MICB-positive cancer, or a MICA/MICB-positive cancer in a subject.

SEQUENCE LISTING

This application contains a Sequence Listing that has been submitted electronically as an ASCII text file named 47472.0083WOl_ST25.txt. The ASCII text file, created on March 1, 2022, is 106 kilobytes in size. The material in the ASCII text file is hereby incorporated by reference in its entirety.

BACKGROUND

Engineered enucleated erythroid cells are in development as therapeutic agents which carry or present exogenous polypeptide(s) for patients in need thereof.

SUMMARY

The present disclosure relates to the use of MHC Class I Polypeptide-Related Sequence A (MICA) and/or MHC Class I Polypeptide-Related Sequence B (MICB) as a biomarker for identifying subjects for treatment with enucleated erythroid cells comprising 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 disclosure is based, at least in part, on the discovery that administering enucleated erythroid cells comprising 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, on its extracellular surface, and a second exogenous polypeptide comprising 4-1BBL or a functional fragment thereof, on its extracellular surface, to a subject results in an increase in the number of NKG2D-positive NK cells in the subject.

Thus, determination of MICA positivity and/or MICB positivity of a cancer in a subject is particularly useful to identify patients likely to respond to therapy with these cells. In view of this discovery, provided herein are methods of increasing the number of NKG2D-positive lymphocytes (e.g., NKG2D-positive NK cells) in a subject previously identified or diagnosed as having a MICA-positive cancer, a MICB-positive cancer, or a MICA/MICB-positive cancer; methods of treating a subject previously identified or diagnosed as having a MICA-positive cancer, a MICB-positive cancer, or a MICA/MICB-positive cancer; methods of decreasing the number and/or proliferation of MICA-positive cancer cells in a subject previously identified or diagnosed as having a MICA-positive cancer; methods of decreasing the number and/or proliferation of MICB-positive cancer cells in a subject previously identified or diagnosed as having a MICB-positive cancer; methods of decreasing the number and/or proliferation of MICA/MICB-positive cancer cells in a subject previously identified or diagnosed as having a MICA/MICB-positive cancer; methods of killing a MICA-positive cancer cell in a subject previously identified or diagnosed as having a MICA-positive cancer; methods of killing a MICB-positive cancer cell in a subject previously identified or diagnosed as having a MICB-positive cancer; methods of killing a MICA/MICB-positive cancer cell in a subject previously identified or diagnosed as having a MICA/MICB-positive cancer; and methods of reducing the volume of a solid tumor in a subject previously identified or diagnosed as having a MICA-positive cancer, a MICB-positive cancer, or a MICA/MICB-positiv cancer. Also provided herein are methods of selecting an enucleated erythroid cell comprising 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, on its extracellular surface, for a subject previously identified or diagnosed as having a MICA-positive cancer, a MICB-positive cancer, or a MICA/MICB-positive cancer.

Provided herein are methods of increasing the number of NKG2D-positive lymphocytes in a subject previously identified or diagnosed as having a MICA- positive cancer that include administering to the subject a therapeutically effective amount of a pharmaceutical composition including a population of enucleated erythroid cells including a first exogenous fusion polypeptide including (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof, on its extracellular surface. Some embodiments of any of the methods described herein further include identifying or diagnosing a subject as having a MICA-positive cancer. In some embodiments, the MICA-positive cancer is selected from the group consisting of: adrenocortical carcinoma, cholangiocarcinoma, pancreatic adenocarcinoma, kidney cancer, thyroid carcinoma, mesothelioma, skin cutaneous melanoma, colorectal cancer, cervical squamous cell carcinoma and endocervical adenocarcinoma. In some embodiments of any of the methods described herein, the MICA-positive cancer is a MICA-positive/HLA-E-negative cancer.

Also provided herein are methods of increasing the number of NKG2D- positive lymphocytes in a subject previously identified or diagnosed as having a MICB-positive cancer that include administering to the subject a therapeutically effective amount of a pharmaceutical composition including a population of enucleated erythroid cells including a first exogenous fusion polypeptide including (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof, on its extracellular surface. In some embodiments of any of the methods described herein, the method further includes identifying or diagnosing a subject as having a MICB-positive cancer. In some embodiments, the MICB-positive cancer is selected from the group consisting of: acute myeloid leukemia, lymphoid neoplasm diffuse large B-cell lymphoma, testicular germ cell tumors, stomach adenocarcinoma, ovarian serous cystadenocarcinoma, esophageal carcinoma and lung cancer. In some embodiments of any of the methods described herein, the MICB- positive cancer is a MICB-positive/HLA-E -negative cancer.

Also provided herein are methods of increasing the number of NKG2D- positive lymphocytes in a subject previously identified or diagnosed as having a MICA/MICB-positive cancer that include administering to the subject a therapeutically effective amount of a pharmaceutical composition including a population of enucleated erythroid cells including a first exogenous fusion polypeptide including (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof, on its extracellular surface. In some embodiments of any of the methods described herein, the method further includes identifying or diagnosing a subject as having a MICA/MICB-positive cancer. In some embodiments of any of the methods described herein, the MICA/MICB-positive cancer is selected from the group consisting of: adrenocortical carcinoma, acute myeloid leukemia, pancreatic adenomcarcinoma, cholangiocarcinoma, kidney cancer, cervical squamous cell carcinoma, endocervical carcinoma, colorectal cancer, mesothelioma, skin cutaneous melanoma and lung cancer. In some embodiments of any of the methods described herein, the MICA/MICB-positive cancer is a MICA/MICB-positive/HLA-E-negative cancer.

In some embodiments of any of the methods described herein, the administering results in at least a 5% increase in the number of NKG2D-positive lymphocytes in the subject as compared to the number of NKG2D-positive lymphocytes in the subject prior to the administering. In some embodiments of any of the methods described herein, the administering results in at least a 10% increase in the number of NKG2D-positive lymphocytes in the subject as compared to the number of NKG2D-positive lymphocytes in the subject prior to the administering.

In some embodiments of any of the methods described herein, the NKG2D- positive lymphocytes are NKG2D-positive NK cells.

In some embodiments of any of the methods described herein, the method results in an increase in the number of NKG2D-positive/NKG2A-negative lymphocytes in the subject. In some embodiments of any of the methods described herein, the administering step results in at least a 5% increase in the number of NKG2D-positive/NKG2A-negative lymphocytes in the subject. In some embodiments of any of the methods described herein, the administering step results in at least a 10% increase in the number of NKG2D-positive/NKG2A-negative lymphocytes in the subject. In some embodiments of any of the methods described herein, the administering step results in at least a 15% increase in the number of NKG2D-positive/NKG2A-negative lymphocytes in the subject.

In some embodiments of any of the methods described herein, the NKG2D- positive/NKG2A-negative lymphocytes are NKG2D-positive/NKG2A-negative NK cells.

In some embodiments of any of the methods described herein, the method further includes administering to the subject an NKG2A inhibitor. In some embodiments of any of the methods described herein, the NKG2A inhibitor is an antagonistic antibody that binds specifically to NKG2A. Also provided herein are methods of treating a subject previously identified or diagnosed as having a MICA-positive cancer that include administering to a subject previously identified or diagnosed as having a MICA-positive cancer a therapeutically effective amount of a pharmaceutical composition including a population of enucleated erythroid cells including a first exogenous polypeptide including (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof, on its extracellular surface. In some embodiments of any of the methods described herein, the method further includes identifying or diagnosing a subject as having a MICA-positive cancer. In some embodiments of any of the methods described herein, the MICA-positive cancer is selected from the group consisting of: adrenocortical carcinoma, cholangiocarcinoma, pancreatic adenocarcinoma, kidney cancer, thyroid carcinoma, mesothelioma, skin cutaneous melanoma, colorectal cancer, cervical squamous cell carcinoma and endocervical adenocarcinoma. In some embodiments of any of the methods described herein, the MICA-positive cancer is a MICA-positive/HLA-E-negative cancer.

Also provided herein are methods of treating a subject previously identified or diagnosed as having a MICB-positive cancer that include administering to a subject previously identified or diagnosed as having a MICB-positive cancer a therapeutically effective amount of a pharmaceutical composition including a population of enucleated erythroid cells including a first exogenous polypeptide including (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof, on its extracellular surface. Some embodiments of any of the methods described herein further include identifying or diagnosing a subject as having a MICB-positive cancer. In some embodiments of any of the methods described herein, the MICB-positive cancer is selected from the group consisting of: acute myeloid leukemia, lymphoid neoplasm diffuse large B-cell lymphoma, testicular germ cell tumors, stomach adenocarcinoma, ovarian serous cystadenocarcinoma, esophageal carcinoma and lung cancer. In some embodiments of any of the methods described herein, the MICB-positive cancer is a MICB-positive/HLA-E-negative cancer.

Also provided herein are methods of treating a subject previously identified or diagnosed as having a MICA/MICB-positive cancer that include administering to a subject previously identified or diagnosed as having a MICA/MICB-positive cancer a therapeutically effective amount of a pharmaceutical composition including a population of enucleated erythroid cells including a first exogenous polypeptide including (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof, on its extracellular surface. Some embodiments of any of the methods described herein further include identifying or diagnosing a subject as having a MICA/MICB-positive cancer. In some embodiments of any of the methods described herein, the MICA/MICB-positive cancer is selected from the group consisting of: adrenocortical carcinoma, acute myeloid leukemia, pancreatic adenomcarcinoma, cholangiocarcinoma, kidney cancer, cervical squamous cell carcinoma, endocervical carcinoma, colorectal cancer, mesothelioma, skin cutaneous melanoma and lung cancer. In some embodiments of any of the methods described herein, the MICA/MICB-positive cancer is a MICA/MICB-positive/HLA-E-negative cancer.

In some embodiments of any of the methods described herein, the administering results in an increase in the number of NKG2D-positive lymphocytes in the subject and/or an increase in the number of NKG2D-positive/NKG2A-negative lymphocytes in the subject. In some embodiments of any of the methods described herein, the NKG2D-positive lymphocytes are NKG2D-positive NK cells. In some embodiments of any of the methods described herein, the NKG2D-positive/NKG2A- negative lymphocytes are NKG2D-positive/NKG2A-negative NK cells.

Some embodiments of any of the methods described herein further include administering to the subject an NKG2A inhibitor. In some embodiments of any of the methods described herein, the NKG2A inhibitor is an antagonistic antibody that binds specifically to NKG2A.

Also provided herein are methods of decreasing the number and/or proliferation of MICA-positive cancer cells in a subject previously identified or diagnosed as having a MICA-positive cancer that include administering a therapeutically effective amount of a pharmaceutical composition including a population of enucleated erythroid cells including a first exogenous polypeptide including (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof, on its extracellular surface.

In some embodiments of any of the methods described herein, the administering results in a decrease in the number of MICA-positive cancer cells in the subject. In some embodiments of any of the methods described herein, the administering results in at least a 5% decrease in the number of MICA-positive cancer cells in the subject as compared to the number of MICA-positive cancer cells in the subject prior to the administering. In some embodiments of any of the methods described herein, the administering results in at least a 10% decrease in the number of MICA-positive cancer cells in the subject as compared to the number of MICA- positive cancer cells in the subject prior to the administering.

In some embodiments of any of the methods described herein, the administering results in a decrease in the proliferation of MICA-positive cancer cells in the subject. In some embodiments of any of the methods described herein, the administering results in at least a 5% decrease in the proliferation of MICA-positive cancer cells in the subject as compared to the proliferation of MICA-positive cancer cells in the subject prior to the administering. In some embodiments of any of the methods described herein, the administering results in at least a 10% decrease in the proliferation of MICA-positive cancer cells in the subject as compared to the proliferation of MICA-positive cancer cells in the subject prior to the administering.

Some embodiments of any of the methods described herein further include identifying or diagnosing a subject as having a MICA-positive cancer. In some embodiments of any of the methods described herein, the MICA-positive cancer is selected from the group consisting of: adrenocortical carcinoma, cholangiocarcinoma, pancreatic adenocarcinoma, kidney cancer, thyroid carcinoma, mesothelioma, skin cutaneous melanoma, colorectal cancer, cervical squamous cell carcinoma and endocervical adenocarcinoma. In some embodiments of any of the methods described herein, the MICA-positive cancer is a MICA-positive/HLA-E-negative cancer.

In some embodiments of any of the methods described herein, the administering results in a decrease in the number of MICA-positive/HLA-E-negative cancer cells in the subject. In some embodiments of any of the methods described herein, the administering results in at least a 5% decrease in the number of MICA- positive/HLA-E-negative cancer cells in the subject as compared to the number of MICA-positive/HLA-E-negative cancer cells in the subject prior to the administering. In some embodiments of any of the methods described herein, the administering results in at least a 10% decrease in the number of MICA-positive/HLA-E-negative cancer cells in the subject as compared to the number of MICA-positive/HLA-E- negative cancer cells in the subject prior to the administering. In some embodiments of any of the methods described herein, the administering results in a decrease in the proliferation of MIC A-positive/HLA-E- negative cancer cells in the subject. In some embodiments of any of the methods described herein, the administering results in at least a 5% decrease in the proliferation of MICA-positive/HLA-E-negative cancer cells in the subject as compared to the proliferation of MICA-positive/HLA-E-negative cancer cells in the subject prior to the administering. In some embodiments of any of the methods described herein, the administering results in at least a 10% decrease in the proliferation of MICA-positive/HLA-E-negative cancer cells in the subject as compared to the proliferation of MICA-positive/HLA-E-negative cancer cells in the subject prior to the administering.

Also provided herein are methods of decreasing the number and/or proliferation of MICB-positive cancer cells in a subject previously identified or diagnosed as having a MICB-positive cancer that include administering a therapeutically effective amount of a pharmaceutical composition including a population of enucleated erythroid cells including a first exogenous polypeptide including (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof, on its extracellular surface.

In some embodiments of any of the methods described herein, the administering results in a decrease in the number of MICB-positive cancer cells in the subject. In some embodiments of any of the methods described herein, the administering results in at least a 5% decrease in the number of MICB-positive cancer cells in the subject as compared to the number of MICB-positive cancer cells in the subject prior to the administering. In some embodiments of any of the methods described herein, the administering results in at least a 10% decrease in the number of MICB-positive cancer cells in the subject as compared to the number of MICB- positive cancer cells in the subject prior to the administering.

In some embodiments of any of the methods described herein, the administering results in a decrease in the proliferation of MICB-positive cancer cells in the subject. In some embodiments of any of the methods described herein, the administering results in at least a 5% decrease in the proliferation of MICB-positive cancer cells in the subject as compared to the proliferation of MICB-positive cancer cells in the subject prior to the administering. In some embodiments of any of the methods described herein, the administering results in at least a 10% decrease in the proliferation of MICB-positive cancer cells in the subject as compared to the proliferation of MICB-positive cancer cells in the subject prior to the administering.

Some embodiments of any of the methods described herein further include identifying or diagnosing a subject as having a MICB-positive cancer. In some embodiments of any of the methods described herein, the MICB-positive cancer is selected from the group consisting of: acute myeloid leukemia, lymphoid neoplasm diffuse large B-cell lymphoma, testicular germ cell tumors, stomach adenocarcinoma, ovarian serous cystadenocarcinoma, esophageal carcinoma and lung cancer. In some embodiments of any of the methods described herein, the MICB-positive cancer is a MICB-positive/HLA-E-negative cancer.

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

In some embodiments of any of the methods described herein, the administering results in a decrease in the proliferation of MICB-positive/HLA-E- negative cancer cells in the subject. In some embodiments of any of the methods described herein, the administering results in at least a 5% decrease in the proliferation of MICB-positive/HLA-E-negative cancer cells in the subject as compared to the proliferation of MICB-positive/HLA-E-negative cancer cells in the subject prior to the administering. In some embodiments of any of the methods described herein, the administering results in at least a 10% decrease in the proliferation of MICB-positive/HLA-E-negative cancer cells in the subject as compared to the proliferation of MICB-positive/HLA-E-negative cancer cells in the subject prior to the administering. Also provided herein are methods of decreasing the number and/or proliferation of MICA/MICB-positive cancer cells in a subject previously identified or diagnosed as having a MICA/MICB-positive cancer that include administering a therapeutically effective amount of a pharmaceutical composition including a population of enucleated erythroid cells including a first exogenous polypeptide including (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof, on its extracellular surface.

In some embodiments of any of the methods described herein, the administering results in a decrease in the number of MICA/MICB-positive cancer cells in the subject. In some embodiments of any of the methods described herein, the administering results in at least a 5% decrease in the number of MICA/MICB-positive cancer cells in the subject as compared to the number of MICA/MICB-positive cancer cells in the subject prior to the administering. In some embodiments of any of the methods described herein, the administering results in at least a 10% decrease in the number of MICA/MICB-positive cancer cells in the subject as compared to the number of MICA/MICB-positive cancer cells in the subject prior to the administering.

In some embodiments of any of the methods described herein, the administering results in a decrease in the proliferation of MICA/MICB-positive cancer cells in the subject. In some embodiments of any of the methods described herein, the administering results in at least a 5% decrease in the proliferation of MICA/MICB-positive cancer cells in the subject as compared to the proliferation of MICA/MICB-positive cancer cells in the subject prior to the administering. In some embodiments of any of the methods described herein, the administering results in at least a 10% decrease in the proliferation of MICA/MICB-positive cancer cells in the subject as compared to the proliferation of MICA/MICB-positive cancer cells in the subject prior to the administering.

Some embodiments of any of the methods described herein further include identifying or diagnosing a subject as having a MICA/MICB-positive cancer. In some embodiments of any of the methods described herein, the MICA/MICB-positive cancer is selected from the group consisting of: adrenocortical carcinoma, acute myeloid leukemia, pancreatic adenomcarcinoma, cholangiocarcinoma, kidney cancer, cervical squamous cell carcinoma, endocervical carcinoma, colorectal cancer, mesothelioma, skin cutaneous melanoma and lung cancer. In some embodiments of any of the methods described herein, the MICA/MICB-positive cancer is a MICA/MICB-positive/HLA-E-negative cancer.

In some embodiments of any of the methods described herein, the administering results in a decrease in the number of MICA/MICB-positive/HLA-E- negative cancer cells in the subject. In some embodiments of any of the methods described herein, the administering results in at least a 5% decrease in the number of MICA/MICB-positive/HLA-E-negative cancer cells in the subject as compared to the number of MICA/MICB-positive/HLA-E-negative cancer cells in the subject prior to the administering. In some embodiments of any of the methods described herein, the administering results in at least a 10% decrease in the number of MICA/MICB- positive/HLA-E-negative cancer cells in the subject as compared to the number of MICA/MICB-positive/HLA-E-negative cancer cells in the subject prior to the administering.

In some embodiments of any of the methods described herein, the administering results in a decrease in the proliferation of MICA/MICB-positive/HLA- E-negative cancer cells in the subject. In some embodiments of any of the methods described herein, the administering results in at least a 5% decrease in the proliferation of MICA/MICB-positive/HLA-E-negative cancer cells in the subject as compared to the proliferation of MICA/MICB-positive/HLA-E-negative cancer cells in the subject prior to the administering. In some embodiments of any of the methods described herein, the administering results in at least a 10% decrease in the proliferation of MICA/MICB-positive/HLA-E-negative cancer cells in the subject as compared to the proliferation of MICA/MICB-positive/HLA-E-negative cancer cells in the subject prior to the administering. In some embodiments of any of the methods described herein, the administering results in at least a 15% decrease in the proliferation of MICA/MICB-positive/HLA-E-negative cancer cells in the subject as compared to the proliferation of MICA/MICB-positive/HLA-E-negative cancer cells in the subject prior to the administering.

Some embodiments of any of the methods described herein further include administering to the subject an NKG2A inhibitor. In some embodiments of any of the methods described herein, the NKG2A inhibitor is an antagonistic antibody that binds specifically to NKG2A. Also provided herein are methods of inducing killing a MICA-positive cancer cell in a subject previously identified or diagnosed as having a MICA-positive cancer that include administering a therapeutically effective amount of a pharmaceutical composition including a population of enucleated erythroid cells including a first exogenous fusion polypeptide including (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof, on its extracellular surface.

In some embodiments of any of the methods described herein, the MICA- positive cancer cell is a cancer cell selected from the group consisting of: an adrenocortical carcinoma cancer cell, a cholangiocarcinoma cancer cell, a pancreatic adenocarcinoma cancer cell, a kidney cancer cancer cell, a thyroid carcinoma cancer cell, a mesothelioma cancer cell, a skin cutaneous melanoma cancer cell, a colorectal cancer cancer cell, a cervical squamous cell carcinoma cancer cell and an endocervical adenocarcinoma cancer cell.

Some embodiments of any of the methods described herein further include identifying or diagnosing the subject as having a MICA-positive cancer. In some embodiments of any of the methods described herein, the MICA-positive cancer is selected from the group consisting of: adrenocortical carcinoma, cholangiocarcinoma, pancreatic adenocarcinoma, kidney cancer, thyroid carcinoma, mesothelioma, skin cutaneous melanoma, colorectal cancer, cervical squamous cell carcinoma and endocervical adenocarcinoma.

In some embodiments of any of the methods described herein, the MICA- positive cancer is a MICA-positive/HLA-E-negative cancer. In some embodiments of any of the methods described herein, the MICA-positive cancer cell is a MICA- positive and HLA-E-negative cancer cell.

Also provided herein are methods of inducing killing a MICB-positive cancer cell in a subject previously identified or diagnosed as having a MICB-positive cancer that include administering a therapeutically effective amount of a pharmaceutical composition including a population of enucleated erythroid cells including a first exogenous fusion polypeptide including (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof, on its extracellular surface. Some embodiments of any of the methods described herein further include identifying or diagnosing the subject as having a MICB-positive cancer. In some embodiments of any of the methods described herein, the MICB-positive cancer cell is a cancer cell selected from the group consisting of: an acute myeloid leukemia cancer cell, a lymphoid neoplasm diffuse large B-cell lymphoma cancer cell, a testicular germ cell tumor cancer cell, a stomach adenocarcinoma cancer cell, a ovarian serous cystadenocarcinoma cancer cell, an esophageal carcinoma cancer cell and a lung cancer cancer cell.

In some embodiments of any of the methods described herein, the MICB- positive cancer is selected from the group consisting of: acute myeloid leukemia, lymphoid neoplasm diffuse large B-cell lymphoma, testicular germ cell tumors, stomach adenocarcinoma, ovarian serous cystadenocarcinoma, esophageal carcinoma and lung cancer.

In some embodiments of any of the methods described herein, the MICB- positive cancer is a MICB-positive/HLA-E -negative cancer. In some embodiments of any of the methods described herein, the MICB-positive cancer cell is a MICB- positive and HLA-E-negative cancer cell.

Also provided herein are methods of inducing killing a MICA/MICB-positive cancer cell in a subject previously identified or diagnosed as having a MICA/MICB- positive cancer that include administering a therapeutically effective amount of a pharmaceutical composition including a population of enucleated erythroid cells including a first exogenous fusion polypeptide including (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof, on its extracellular surface.

In some embodiments of any of the methods described herein, the MICA/MICB-positive cancer cell is a cancer cell selected from the group consisting of: an adrenocortical carcinoma cancer cell, an acute myeloid leukemia cancer cell, a pancreatic adenomcarcinoma cancer cell, a cholangiocarcinoma cancer cell, a kidney cancer cancer cell, a cervical squamous cell carcinoma cancer cell, an endocervical carcinoma cancer cell, a colorectal cancer cell, a mesothelioma cancer cell, a skin cutaneous melanoma cancer cell and a lung cancer cancer cell.

Some embodiments of any of the methods described herein further include identifying or diagnosing the subject as having a MICA/MICB-positive cancer. In some embodiments of any of the methods described herein, the MICA/MICB-positive cancer is selected from the group consisting of: adrenocortical carcinoma, acute myeloid leukemia, pancreatic adenomcarcinoma, cholangiocarcinoma, kidney cancer, cervical squamous cell carcinoma, endocervical carcinoma, colorectal cancer, mesothelioma, skin cutaneous melanoma, and lung cancer.

In some embodiments of any of the methods described herein, the MICA/MICB-positive cancer is a MICA/MICB-positive/HLA-E -negative cancer. In some embodiments of any of the methods described herein, the MICA/MICB-positive cancer cell is a MICA/MICB-positive and HLA-E -negative cancer cell.

In some embodiments of any of the methods described herein, the killing includes necrosis. In some embodiments of any of the methods described herein, the killing includes apoptosis. In some embodiments of any of the methods described herein, the killing is mediated via NK-cell mediated cytolysis. In some embodiments of any of the methods described herein, the administering also results in the killing of non-MICA-positive and non-MICB-positive cancer cells within the subject.

Also provided herein are methods of reducing the volume of a solid tumor in a subject previously identified or diagnosed as having a MICA-positive cancer that include administering a therapeutically effective amount of a population of enucleated erythroid cells including a first exogenous fusion polypeptide including (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof, on its extracellular surface.

Some embodiments of the methods described herein further include identifying or diagnosing a subject as having a MICA-positive cancer. In some embodiments of any of the methods described herein, the MICA-positive cancer is selected from the group consisting of: adrenocortical carcinoma, cholangiocarcinoma, pancreatic adenocarcinoma, kidney cancer, thyroid carcinoma, mesothelioma, skin cutaneous melanoma, colorectal cancer, cervical squamous cell carcinoma and endocervical adenocarcinoma. In some embodiments of any of the methods described herein, the MICA-positive cancer is a MICA-posihve/HLA-E-negative cancer. Also provided herein are methods of reducing the volume of a solid tumor in a subject previously identified or diagnosed as having a MICB-positive cancer that include administering a therapeutically effective amount of a population of enucleated erythroid cells including a first exogenous fusion polypeptide including (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof, on its extracellular surface.

Also provided herein are methods of reducing the volume of a solid tumor in a subject previously identified or diagnosed as having a MICA/MICB-positive cancer that include administering a therapeutically effective amount of a population of enucleated erythroid cells including a first exogenous fusion polypeptide including (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof, on its extracellular surface.

In some embodiments of any of the methods described herein, the administering results in at least a 5% decrease in the volume of the solid tumor in the subject as compared to the volume of the solid tumor prior to the administering. In some embodiments of any of the methods described herein, the administering results in at least a 10% decrease in the volume of the solid tumor in the subject as compared to the volume of the solid tumor prior to the administering. In some embodiments of any of the methods described herein, the administering results in at least a 15% decrease in the volume of the solid tumor in the subject as compared to the volume of the solid tumor prior to the administering.

In some embodiments of any of the methods described herein, the enucleated erythroid cell includes at least 1,000 copies of the first exogenous fusion polypeptide.

In some embodiments of any of the methods described herein, the enucleated erythroid cell is made by a process including: introducing into a nucleated erythroid cell precursor a nucleic acid encoding the first exogenous fusion polypeptide; and culturing the nucleated erythroid cell precursor under conditions sufficient for expression of the first exogenous fusion polypeptide and enucleation of the nucleated erythroid cell precursor.

In some embodiments of any of the methods described herein, the enucleated erythroid cell further includes a second exogenous polypeptide including 4-1BBL or a functional fragment thereof, where the second exogenous polypeptide is present on the extracellular surface of the enucleated erythroid cell. In some embodiments, the enucleated erythroid cell includes at least 1,000 copies of the second exogenous polypeptide.

In some embodiments of any of the methods described herein, the enucleated erythroid cell is made by a process including: introducing into a nucleated erythroid cell precursor a nucleic acid encoding the first exogenous fusion polypeptide or a functional fragment thereof, and a nucleic acid encoding the second exogenous polypeptide; and culturing the nucleated erythroid cell precursor under conditions sufficient for expression of the first exogenous fusion polypeptide and the second exogenous polypeptide, and enucleation of the nucleated erythroid cell precursor.

In some embodiments of any of the methods described herein, the enucleated erythroid cell is not a hypotonically-dialyzed cell. In some embodiments of any of the methods described herein, the enucleated erythroid cell does not include a sortase- transfer signature.

In some embodiments of any of the methods described herein, the subject is a human and the enucleated erythroid cell is a human cell.

Also provided herein are kits that include: a pharmaceutical composition including an enucleated erythroid cell including a first exogenous fusion polypeptide including (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof, on its extracellular surface; and instructions for performing any of the methods described herein.

In some embodiments of any of the kits described herein, the enucleated erythroid cell includes at least 1,000 copies of the first exogenous fusion polypeptide.

In some embodiments of any of the kits described herein, the enucleated erythroid cell is made by a process including: introducing into a nucleated erythroid cell precursor a nucleic acid encoding the first exogenous fusion polypeptide; and culturing the nucleated erythroid cell precursor under conditions sufficient for expression of the first exogenous fusion polypeptide and enucleation of the nucleated erythroid cell precursor.

In some embodiments of any of the kits described herein, the enucleated erythroid cell further includes a second exogenous polypeptide including 4-1BBL or a functional fragment thereof, where the second exogenous polypeptide is present on the extracellular surface of the enucleated erythroid cell. In some embodiments of any of the kits described herein, the enucleated erythroid cell includes at least 1,000 copies of the second exogenous polypeptide.

In some embodiments of any of the kits described herein, the enucleated erythroid cell is made by a process including: introducing into a nucleated erythroid cell precursor a nucleic acid encoding the first exogenous fusion polypeptide and a nucleic acid encoding the second exogenous polypeptide; and culturing the nucleated erythroid cell precursor under conditions sufficient for expression of the first exogenous fusion polypeptide and the second exogenous polypeptide, and enucleation of the nucleated erythroid cell precursor.

In some embodiments of any of the kits described herein, the enucleated erythroid cell is not a hypotonically-dialyzed cell. In some embodiments of any of the kits described herein, the enucleated erythroid cell does not include a sortase-transfer signature. In some embodiments of any of the kits described herein, the enucleated erythroid cell is a human cell.

Also provided herein are methods of selecting a pharmaceutical composition including a population of enucleated erythroid cells including a first exogenous fusion polypeptide including (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof, on its extracellular surface, for a subject previously identified or diagnosed as having a MICA-positive cancer.

In some embodiments of any of the methods described herein further include identifying or diagnosing the subject as having a MICA-positive cancer. In some embodiments of any of the methods described herein, the MICA-positive cancer is selected from the group consisting of: adrenocortical carcinoma, cholangiocarcinoma, pancreatic adenocarcinoma, kidney cancer, thyroid carcinoma, mesothelioma, skin cutaneous melanoma, colorectal cancer, cervical squamous cell carcinoma and endocervical adenocarcinoma. In some embodiments of the methods described herein, the MICA-positive cancer is a MICA-positive/HLA-E-negative cancer.

Also provided herein are methods of selecting a pharmaceutical composition including a population of enucleated erythroid cells including a first exogenous fusion polypeptide including (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof, on its extracellular surface, for a subject previously identified or diagnosed as having a MICB-positive cancer.

Some embodiments of any of the methods described herein further include identifying or diagnosing the subject as having a MICB-positive cancer. In some embodiments of any of the methods described herein, the MICB-positive cancer is selected from the group consisting of: acute myeloid leukemia, lymphoid neoplasm diffuse large B-cell lymphoma, testicular germ cell tumors, stomach adenocarcinoma, ovarian serous cystadenocarcinoma, esophageal carcinoma and lung cancer. In some embodiments of any of the methods described herein, the MICB-positive cancer is a MICB-positive/HLA-E-negative cancer.

Also provided herein are methods of selecting a pharmaceutical composition including a population of enucleated erythroid cells including a first exogenous fusion polypeptide including (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof, on its extracellular surface, for a subject previously identified or diagnosed as having a MICA/MICB-positive cancer. Some embodiments of any of the methods described herein further include identifying or diagnosing the subject as having a MICA/MICB-positive cancer. In some embodiments of any of the methods described herein, the MICA/MICB-positive cancer is selected from the group consisting of: adrenocortical carcinoma, acute myeloid leukemia, pancreatic adenomcarcinoma, cholangiocarcinoma, kidney cancer, cervical squamous cell carcinoma, endocervical carcinoma, colorectal cancer, mesothelioma, skin cutaneous melanoma and lung cancer. In some embodiments of any of the methods described herein, the MICA/MICB-positive cancer is a MICA/MICB-positive/HLA-E-negative cancer.

The term “engineered enucleated erythroid cell” means an enucleated erythroid cell (e.g., a human enucleated erythroid cell) that comprises one or more (e.g., two, three, four, five, or six) exogenous polypeptide(s) (e.g., any combination of the exemplary exogenous polypeptides described herein or known in the art). For example, an engineered enucleated erythroid cell can have one or more exogenous polypeptide(s) present in its cytosol. In some examples, an engineered enucleated erythroid cell can have one or more exogenous polypeptide(s) present on its extracellular surface. In some examples, an engineered enucleated erythroid cell can have (i) one or more exogenous polypeptide(s) present in its cytosol and (ii) one or more exogenous polypeptides present on its extracellular surface. Non-limiting examples of engineered enucleated erythroid cells include click-conjugated enucleated erythroid cells, enucleated erythroid cells that have been hypotonically loaded, and enucleated erythroid cells that have been loaded by physical manipulation (e.g., any of the exemplary types of physical manipulation described herein or known in the art). Additional non-limiting aspects of engineered enucleated erythroid cells are described herein.

The term “conjugated enucleated erythroid cell” means an engineered enucleated erythroid cell that has at least one exogenous polypeptide conjugated to another polypeptide (e.g., an endogenous polypeptide of an enucleated red blood cell or different exogenous polypeptide) present on the extracellular surface of an engineered enucleated erythroid cells through the catalytic activity of an enzyme(s) and/or peptide sequence(s), and/or a chemical reaction.

The term “hypotonically-loaded enucleated erythroid cell” means an engineered enucleated erythroid cell that was generated, at least in part, by exposing an enucleated erythroid cell or an erythroid cell precursor to a low ionic strength buffer (e.g., any of the exemplary low ionic strength buffers described herein) comprising one or more exogenous polypeptide(s). Non-limiting examples of methods that can be used to generate a hypotonically-loaded enucleated erythroid cell are described herein. Additional methods for generating a hypotonically-loaded enucleated erythroid cell are known in the art.

The term “enucleated erythroid cell loaded by physical manipulation” means an enucleated erythroid cell that was generated, at least in part, by physically manipulating an erythroid cell precursor in a manner that results in the introduction of a nucleic acid encoding one or more exogenous polypeptide(s) (e.g., any of the exemplary exogenous polypeptides described herein or known in the art) and/or exogenous polypeptides into the erythroid cell precursor. Non-limiting examples of physical manipulation that can be used to introduce a nucleic acid encoding one or more exogenous polypeptide(s) into an erythroid cell precursor include electroporation and particle-mediated transfection. Additional examples of physical manipulation that can be used to introduce a nucleic acid encoding one or more exogenous polypeptide(s) into an erythroid cell precursor are known in the art.

The term “exogenous polypeptide” refers to a polypeptide that is introduced into or onto a cell, or is caused to be expressed by the cell by introducing an exogenous nucleic acid encoding the polypeptide into the cell or into a precursor of the cell. In some embodiments, an exogenous polypeptide is a polypeptide encoded by an exogenous nucleic acid that was introduced into the cell or a precursor of the cell, which nucleic acid is optionally not retained by the cell. In some embodiments, an exogenous polypeptide is a polypeptide conjugated to the surface of the cell by chemical or enzymatic means. Non-limiting classes of exogenous polypeptides include enzymes, interleukins, cytokine receptors, Fc-binding molecules, T-cell activating ligands, T-cell receptors, immune inhibitory molecules, MHC molecules, APC-binding molecules, autoantigens, allergens, toxins, targeting agents, receptor ligands (e.g., receptor agonists or receptor antagonists), and antibodies or antibody fragments.

The term “on the extracellular surface” when used in the context of an exogenous polypeptide means a (1) an exogenous polypeptide that is physically attached to or at least partially embedded in the membrane of an enucleated erythroid cell (e.g., a transmembrane polypeptide, a peripheral membrane polypeptide, a lipid- anchored polypeptide (e.g., a GPI-anchor, an N-myristoylated polypeptide, or a S- palmitoylated polypeptide)) or (2) an exogenous polypeptide that is stably bound to its cognate receptor, where the cognate receptor is physically attached to the membrane of an enucleated erythroid cell (e.g., a ligand bound to its cognate receptor, where the cognate receptor is physically attached to the membrane of the enucleated erythroid cell). Non-limiting methods for determining the presence of an exogenous polypeptide on the extracellular surface of an enucleated erythroid cell include fluorescence-activated cell sorting (FACS), immunohistochemistry, cell-fractionation assays, and Western blotting.

The term “erythroid cell precursor” means a mammalian cell that is capable of eventually differentiating/devel oping into an enucleated erythroid cell. In some embodiments, the erythroid cell precursor is a cord blood stem cell, a CD34⁺ cell, a hematopoietic stem/progenitor cell (HSC, HSPC), a spleen colony forming (CFU-S) cell, a common myeloid progenitor (CMP) cell, a blastocyte colony-forming cell, a burst forming unit-erythroid/erythrocyte (BFU-E), a megakaryocyte-erythroid progenitor (MEP) cell, an erythroid colony-forming unit, or colony-forming unit erythrocyte (CFU-E), an induced pluripotent stem cell (iPSC), a mesenchymal stem cell (MSC), or a combination thereof.

The term “subject” refers to any mammal. In some embodiments, the subject or “subject in need of treatment” may be a primate (e.g., a human, a simian (e.g., a monkey (e.g., marmoset or baboon), or an ape (e.g., a gorilla, chimpanzee, orangutan, or gibbon)), a rodent (e.g., a mouse, a guinea pig, a hamster, or a rat), a rabbit, a dog, a cat, a horse, a sheep, a cow, a pig, or a goat. In some embodiments, the subject or “subject suitable for treatment” may be a non-human mammal, especially mammals that are conventionally used as models for demonstrating therapeutic efficacy in humans (e.g., a mouse, a pig, a rat, or a non-human primate) may be employed. In some examples, a subject can be previously diagnosed or identified as being in need of treatment by a medical professional (e.g., a physician, a laboratory technician, a physician’s assistant, a nurse, or a clinical laboratory technician) (e.g., previously diagnosed or identified as having a MICA-positive cancer or previously diagnosed or identified as having a MICA-positive and HLA-E -negative cancer; previously diagnosed or identified as having a MICB-positive cancer or previously diagnosed or identified as having a MICB-positive and HLA-E-negative cancer; or previously diagnosed or identified as having a MICA/MICB-positive cancer or previously diagnosed or identified as having a MICA/MICB-positive and HLA-E-negative cancer;).

As used herein, “treating” means a reduction in 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 “MICA” means a MICA polypeptide or a MICA mRNA including wild type human MICA polypeptides and wild type human MICA mRNAs, and variants thereof (e.g., truncated or mutated forms). The term “MICA” excludes soluble MICA polypeptides. Non-limiting examples of methods of detecting a level of MICA are described herein.

As used herein, the term “MICA-positive cancer” means a cancer comprising a MICA-positive cancer cell. Non-limiting examples of MIC A-positive cancers are described herein.

As used herein, the term “MICA-positive cancer cell” means a cancer cell having a level of MICA (MICA polypeptide or MICA mRNA) that is greater than a reference level of MICA. Non-limiting examples of reference levels of MICA are described herein. Non-limiting examples of MICA-positive cancer cells are also described herein.

As used herein, the term “MICB” means a MICB polypeptide or a MICB mRNA including wild type human MICB polypeptides and wild type human MICB mRNAs, and variants thereof (e.g., truncated or mutated forms). The term “MICB” excludes soluble MICB polypeptides. Non-limiting examples of methods of detecting a level of MICB are described herein.

As used herein, the term “MICB-positive cancer” means a cancer comprising a MICB-positive cancer cell. Non-limiting examples of MICB-positive cancers are described herein.

As used herein, the term “MICB-positive cancer cell” means a cancer cell having a level of MICB (MICB polypeptide or MICB mRNA) that is greater than a reference level of MICB. Non-limiting examples of reference levels of MICB are described herein. Non-limiting examples of MICB-positive cancer cells are also described herein. As used herein, the term “MICA/MICB-positive cancer” means a cancer comprising a MICA/MICB-positive cancer cell or a cancer comprising a MICA/MICB-positive cancer cell. Non-limiting examples of MIC A/MICB-positive cancers are described herein.

As used herein, the term “MICA/MICB-positive cancer cell” means a cancer cell having a level of MICA (MICA polypeptide or MICA mRNA) that is greater than a reference level of MICA and a level of MICB (MICB polypeptide or MICB mRNA) that is greater than a reference level of MICB. Non-limiting examples of MICA/MICB-positive cancer cells are also described herein.

As used herein, the term “HLA-E” means a human leukocyte antigen-E (HLA- E) polypeptide or a HLA-E mRNA including wild type human HLA-E polypeptides and mRNAs encoding wild type human HLA-E, and variants thereof (e.g., truncated or mutated forms). Non-limiting examples of methods of detecting a level of HLA-E are described herein.

As used herein, the term “HLA-E-negative cancer” means a cancer having an HLA-E-negative cancer cell.

As used herein, the term “HLA-E-negative cancer cell” means a cancer cell having a level of HLA-E (HLA-E polypeptide or HLA-E mRNA) that is less than a reference level of HLA-E. Non-limiting examples of reference levels of HLA-E are described herein.

As used herein, the term “NKG2D” means a NKG2D polypeptide or a NKG2D mRNA including wild type human NKG2D polypeptides and mRNAs encoding wild type human NKG2D, and variants thereof (e.g., truncated or mutated forms). The term “NKG2D” also includes all known isoforms of NKG2D polypeptide and NKG2D mRNA. Non-limiting examples of methods of detecting a level of NKG2D are described herein.

As used herein, the term “NKG2D-positive lymphocyte” means a lymphocyte having a level of NKG2D (NKG2D polypeptide or NKG2D mRNA) that is greater than a reference level of NKG2D. Non-limiting examples of reference levels of NKG2D are described herein.

As used herein, the term “NKG2A” means a NK group 2 member A (NKG2A) polypeptide or a NKG2A mRNA including wild type human NKG2A polypeptides and mRNAs encoding wild type human NKG2A, and variants thereof (e.g., truncated or mutated forms). Non-limiting examples of methods of detecting a level of NKG2A are also described herein.

The nkg2a gene encodes two isoforms, NKG2A and NKG2B, with the latter lacking the stem region. The term “NKG2A” as used herein does not include NKG2B polypeptides or mRNA transcripts encoding NKG2B polypeptides.

As used herein, the term “NKG2A-negative lymphocyte” means a lymphocyte having a level of NKG2A (NKG2A polypeptide or NKG2A mRNA) that is less than a reference level of NKG2A. Non-limiting examples of reference levels of NKG2A are described herein.

As used herein, the term “NKG2A-positive lymphocyte” means a lymphocyte having a level of NKG2A (NKG2A polypeptide or NKG2A mRNA) that is greater than a reference level of NKG2A. Non-limiting examples of reference levels of NKG2A are described herein.

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

As used herein, the term “NK cell-mediated cytolysis” means an NK cell having the ability to release lytic granules, where the lytic granules are used to induce killing of other cells. In some embodiments, the lytic granules include, at least, perforin and granzymes.

As used herein, the term “trafficking” refers to NK cells in the blood of a subject. For example, an “increase in the trafficking of NKG2D-positive NK cells in a subject” refers to an increase in NKG2D-positive NK cells in the blood of a subject. In another example, an “increase in the trafficking of NKG2D-positive/NKG2A- negative NK cells in a subject” refers to an increase in NKG2D-positive/NKG2A- negative NK cells in the blood of a subject.

As used herein, the term “sortase transfer signature” means an exogenous polypeptide or polypeptide(s) that includes a sequence that can be created by a sortase reaction. Non-limiting examples of polypeptides and polypeptides that lack a sortase transfer signature are as described in WO 2017/123646, which is incorporated by reference in its entirety.

Unless otherwise defined, 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 disclosure belongs. Methods and materials are described herein for use in the present disclosure; other, suitable 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 disclosure will be apparent from the following detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a graph showing the maximum fold change from baseline in the ratio of NKG2D-positive lymphocytes to NKG2A-positive lymphocytes in whole blood samples obtained from human subjects before (baseline) and after (post-treatment) administration of human enucleated erythroid cells genetically engineered to express

IL-15/IL-15RA and 4-1BBL.

FIG. 2A is a graph showing the maximum fold change from baseline in the percentage of CD45⁺ CD56⁺ lymphocytes expressing NKG2D in whole blood samples obtained from human subjects before (baseline) and after (post-treatment) administration of greater than or equal to 1 x 10¹⁰ human enucleated erythroid cells genetically engineered to express IL-15/IL-15RA and 4-1BBL. Post-treatment measurements were conducted across multiple dosing cycles.

FIG. 2B is a graph showing the maximum fold change from baseline in the percentage of CD45⁺ CD16⁺ CD56^dim lymphocytes expressing NKG2D in whole blood samples obtained from human subjects before (baseline) and after (post treatment) administration of greater than or equal to 1 x 10¹⁰ human enucleated erythroid cells genetically engineered to express IL-15/IL-15RA and 4-1BBL. Post treatment measurements were conducted across multiple dosing cycles.

FIG. 3A is a graph showing the maximum fold change from baseline in the absolute number of circulating NK cells (CD3 CD16⁺ CD56⁺) in whole blood samples obtained from human subjects before (baseline) and after (post-treatment) administration of greater than or equal to 1 x 10¹⁰ human enucleated erythroid cells genetically engineered to express IL-15/IL-15RA and 4-1BBL. Post-treatment measurements were conducted across multiple dosing cycles.

FIG. 3B is a graph showing the maximum fold change from baseline in the percentage of CD8⁺ memory T cells expressing granzyme B (% GrB⁺ of CD8⁺CD45RA) in whole blood samples obtained from human subjects before (baseline) and after (post-treatment) administration of greater than or equal to 1 x 10¹⁰ human enucleated erythroid cells genetically engineered to express IL-15/IL-15RA and 4-1BBL. Post-treatment measurements were conducted across multiple dosing cycles.

FIG. 4A is a graph showing the maximum fold change from baseline in the percent of CD4⁺ T cells of total T cells (CD3⁺) in whole blood samples obtained from human subjects before (baseline) and after (post-treatment) administration of greater than or equal to 1 x 10¹⁰ human enucleated erythroid cells genetically engineered to express IL-15/IL-15RA and 4-1BBL. Post-treatment measurements were conducted across multiple dosing cycles.

FIG. 4B is a graph showing the maximum fold change from baseline in the percent of CD8⁺ T cells of total T cells (CD3⁺) in whole blood samples obtained from human subjects before (baseline) and after (post-treatment) administration of greater than or equal to 1 x 10¹⁰ human enucleated erythroid cells genetically engineered to express IL-15/IL-15RA and 4-1BBL. Post-treatment measurements were conducted across multiple dosing cycles.

DETAILED DESCRIPTION

Provided herein are methods of increasing the number of NKG2D-positive lymphocytes (e.g., NKG2D-positive NK cells) in a subject previously identified or diagnosed as having a MICA-positive cancer that include administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a population of enucleated erythroid cells including a first exogenous fusion polypeptide including (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof, on its extracellular surface. Also provided herein are methods of increasing the number of NKG2D-positive lymphocytes (e.g., NKG2D-positive NK cells) in a subject previously identified or diagnosed as having a MICB-positive cancer that include administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a population of enucleated erythroid cells including a first exogenous fusion polypeptide including (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof, on its extracellular surface. Also provided herein are methods of increasing the number of NKG2D-positive lymphocytes (e.g., NKG2D-positive NK cells) in a subject previously identified or diagnosed as having a MICA/MICB-positive cancer that include adminsitering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a population of enucleated erythroid cells including a first exogenous fusion polypeptide including (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof, on its extracellular surface.

Also provided herein are methods of increasing the number of NKG2D- positive lymphocytes (e.g., NKG2D-positive NK cells) in a subject previously identified or diagnosed as having a MICA-positive, a MICB-positive, or a MICA/MICB-positive cancer that include administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a population of enucleated erythroid cells including a first exogenous fusion polypeptide including (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof, on its extracellular surface; and a second exogenous polypeptide including 4-1BBL or a functional fragment thereof, on its extracellular surface. Some embodiments of these methods result in at least a 1% increase, at least a 2% increase, at least a 3% increase, at least a 4% increase, at least a 5% increase, at least a 6% increase, at least a 7% increase, at least a 8% increase, at least a 9% increase, at least a 10% increase, at least a 15% increase, at least a 20% increase, at least a 25% increase, at least a 30% increase, at least a 35% increase, at least a 40% increase, at least a 45% increase, at least a 50% increase, at least a 55% increase, at least a 60% increase, at least a 65% increase, at least a 70% increase, at least a 75% increase, at least a 80% increase, at least a 85% increase, at least a 90% increase, at least a 95% increase, at least a 100% increase, at least a 150% increase, at least a 200% increase, at least a 250% increase, at least a 300% increase, at least a 350% increase, at least a 400% increase, at least a 450% increase, at least a 500% increase, at least a 550% increase, at least a 600% increase, at least a 650% increase, at least a 700% increase, at least a 750% increase, at least a 800% incrase, at least a 850% increase, at least a 900% increase, at least a 950% increase, or at least a 1000% increase in the number of NKG2D-positive lymphocytes (e.g., NKG2D-positive NK cells) in the subject (e.g., as compared to the number of NKG2D-positive lymphocytes (e.g., NKG2D-positive NK cells) in the subject prior to the administering). Some embodiments of these methods result in about a 1% increase to about a 1,000% increase, about a 1% increase to about a 900% increase, about a 1% increase to about a 800% increase, about a 1% increase to about a 700% increase, about a 1% increase to about a 600% increase, about a 1% increase to about a 500% increase, about a 1% increase to about a 400% increase, about a 1% increase to about a 300% increase, about a 1% increase to about a 200% increase, about a 1% increase to about a 150% increase, about a 1% increase to about a 100% increase, about a 1% increase to about a 90% increase, about a 1% increase to about a 80% increase, about a 1% increase to about a 70% increase, about a 1% increase to about a 60% increase, about a 1% increase to about a 50% increase, about a 1% increase to about a 40% increase, about a 1% increase to about a 30% increase, about a 1% increase to about a 20% increase, about a 1% increase to about a 10% increase, about a 1% increase to about a 5% increase, about a 5% increase to about a 1,000% increase, about a 5% increase to about a 900% increase, about a 5% increase to about a 800% increase, about a 5% increase to about a 700% increase, about a 5% increase to about a 600% increase, about a 5% increase to about a 500% increase, about a 5% increase to about a 400% increase, about a 5% increase to about a 300% increase, about a 5% increase to about a 200% increase, about a 5% increase to about a 150% increase, about a 5% increase to about a 100% increase, about a 5% increase to about a 90% increase, about a 5% increase to about a 80% increase, about a 5% increase to about a 70% increase, about a 5% increase to about a 60% increase, about a 5% increase to about a 50% increase, about a 5% increase to about a 40% increase, about a 5% increase to about a 30% increase, about a 5% increase to about a 20% increase, about a 5% increase to about a 10% increase, about a 10% increase to about a 1,000% increase, about a 10% increase to about a 900% increase, about a 10% increase to about a 800% increase, about a 10% increase to about a 700% increase, about a 10% increase to about a 600% increase, about a 10% increase to about a 500% increase, about a 10% increase to about a 400% increase, about a 10% increase to about a 300% increase, about a 10% increase to about a 200% increase, about a 10% increase to about a 150% increase, about a 10% increase to about a 100% increase, about a 10% increase to about a 90% increase, about a 10% increase to about a 80% increase, about a 10% increase to about a 70% increase, about a 10% increase to about a 60% increase, about a 10% increase to about a 50% increase, about a 10% increase to about a 40% increase, about a 10% increase to about a 30% increase, about a 10% increase to about a 20% increase, about a 20% increase to about a 1,000% increase, about a 20% increase to about a 900% increase, about a 20% increase to about a 800% increase, about a 20% increase to about a 700% increase, about a 20% increase to about a 600% increase, about a 20% increase to about a 500% increase, about a 20% increase to about a 400% increase, about a 20% increase to about a 300% increase, about a 20% increase to about a 200% increase, about a 20% increase to about a 150% increase, about a 20% increase to about a 100% increase, about a 20% increase to about a 90% increase, about a 20% increase to about a 80% increase, about a 20% increase to about a 70% increase, about a 20% increase to about a 60% increase, about a 20% increase to about a 50% increase, about a 20% increase to about a 40% increase, about a 20% increase to about a 30% increase, about a 30% increase to about a 1,000% increase, about a 30% increase to about a 900% increase, about a 30% increase to about a 800% increase, about a 30% increase to about a 700% increase, about a 30% increase to about a 600% increase, about a 30% increase to about a 500% increase, about a 30% increase to about a 400% increase, about a 30% increase to about a 300% increase, about a 30% increase to about a 200% increase, about a 30% increase to about a 150% increase, about a 30% increase to about a 100% increase, about a 30% increase to about a 90% increase, about a 30% increase to about a 80% increase, about a 30% increase to about a 70% increase, about a 30% increase to about a 60% increase, about a 30% increase to about a 50% increase, about a 30% increase to about a 40% increase, about a 40% increase to about a 1,000% increase, about a 40% increase to about a 900% increase, about a 40% increase to about a 800% increase, about a 40% increase to about a 700% increase, about a 40% increase to about a 600% increase, about a 40% increase to about a 500% increase, about a 40% increase to about a 400% increase, about a 40% increase to about a 300% increase, about a 40% increase to about a 200% increase, about a 40% increase to about a 150% increase, about a 40% increase to about a 100% increase, about a 40% increase to about a 90% increase, about a 40% increase to about a 80% increase, about a 40% increase to about a 70% increase, about a 40% increase to about a 60% increase, about a 40% increase to about a 50% increase, about a 50% increase to about a 1,000% increase, about a 50% increase to about a 900% increase, about a 50% increase to about a 800% increase, about a 50% increase to about a 700% increase, about a 50% increase to about a 600% increase, about a 50% increase to about a 500% increase, about a 50% increase to about a 400% increase, about a 50% increase to about a 300% increase, about a 50% increase to about a 200% increase, about a 50% increase to about a 150% increase, about a 50% increase to about a 100% increase, about a 50% increase to about a 90% increase, about a 50% increase to about a 80% increase, about a 50% increase to about a 70% increase, about a 50% increase to about a 60% increase, about a 60% increase to about a 1,000% increase, about a 60% increase to about a 900% increase, about a 60% increase to about a 800% increase, about a 60% increase to about a 700% increase, about a 60% increase to about a 600% increase, about a 60% increase to about a 500% increase, about a 60% increase to about a 400% increase, about a 60% increase to about a 300% increase, about a 60% increase to about a 200% increase, about a 60% increase to about a 150% increase, about a 60% increase to about a 100% increase, about a 60% increase to about a 90% increase, about a 60% increase to about a 80% increase, about a 60% increase to about a 70% increase, about a 70% increase to about a 1,000% increase, about a 70% increase to about a 900% increase, about a 70% increase to about a 800% increase, about a 70% increase to about a 700% increase, about a 70% increase to about a 600% increase, about a 70% increase to about a 500% increase, about a 70% increase to about a 400% increase, about a 70% increase to about a 300% increase, about a 70% increase to about a 200% increase, about a 70% increase to about a 150% increase, about a 70% increase to about a 100% increase, about a 70% increase to about a 90% increase, about a 70% increase to about a 80% increase, about a 80% increase to about a 1,000% increase, about a 80% increase to about a 900% increase, about a 80% increase to about a 800% increase, about a 80% increase to about a 700% increase, about a 80% increase to about a 600% increase, about a 80% increase to about a 500% increase, about a 80% increase to about a 400% increase, about a 80% increase to about a 300% increase, about a 80% increase to about a 200% increase, about a 80% increase to about a 150% increase, about a 80% increase to about a 100% increase, about a 80% increase to about a 90% increase, about a 90% increase to about a 1,000% increase, about a 90% increase to about a 900% increase, about a 90% increase to about a 800% increase, about a 90% increase to about a 700% increase, about a 90% increase to about a 600% increase, about a 90% increase to about a 500% increase, about a 90% increase to about a 400% increase, about a 90% increase to about a 300% increase, about a 90% increase to about a 200% increase, about a 90% increase to about a 150% increase, about a 90% increase to about a 100% increase, about a 100% increase to about a 1,000% increase, about a 100% increase to about a 900% increase, about a 100% increase to about a 800% increase, about a 100% increase to about a 700% increase, about a 100% increase to about a 600% increase, about a 100% increase to about a 500% increase, about a 100% increase to about a 400% increase, about a 100% increase to about a 300% increase, about a 100% increase to about a 200% increase, about a 100% increase to about a 150% increase, about a 150% increase to about a 1,000% increase, about a 150% increase to about a 900% increase, about a 150% increase to about a 800% increase, about a 150% increase to about a 700% increase, about a 150% increase to about a 600% increase, about a 150% increase to about a 500% increase, about a 150% increase to about a 400% increase, about a 150% increase to about a 300% increase, about a 150% increase to about a 200% increase, about a 200% increase to about a 1,000% increase, about a 200% increase to about a 900% increase, about a 200% increase to about a 800% increase, about a 200% increase to about a 700% increase, about a 200% increase to about a 600% increase, about a 200% increase to about a 500% increase, about a 200% increase to about a 400% increase, about a 200% increase to about a 300% increase, about a 300% increase to about a 1,000% increase, about a 300% increase to about a 900% increase, about a 300% increase to about a 800% increase, about a 300% increase to about a 700% increase, about a 300% increase to about a 600% increase, about a 300% increase to about a 500% increase, about a 300% increase to about a 400% increase, about a 400% increase to about a 1,000% increase, about a 400% increase to about a 900% increase, about a 400% increase to about a 800% increase, about a 400% increase to about a 700% increase, about a 400% increase to about a 600% increase, about a 400% increase to about a 500% increase, about a 500% increase to about a 1,000% increase, about a 500% increase to about a 900% increase, about a 500% increase to about a 800% increase, about a 500% increase to about a 700% increase, about a 500% increase to about a 600% increase, about a 600% increase to about a 1,000% increase, about a 600% increase to about a 900% increase, about a 600% increase to about a 800% increase, about a 600% increase to about a 700% increase, about a 700% increase to about a 1,000% increase, about a 700% increase to about a 900% increase, about a 700% increase to about a 800% increase, about a 800% increase to about a 1,000% increase, about a 800% increase to about a 900% increase, or about a 900% increase to about a 1,000% increase, in the number of NKG2D-positive lymphocytes (e.g., NKG2D-positive NK cells) in the subject (e.g., as compared to the number of NKG2D-positive lymphocytes (e.g., NKG2D- positive NK cells) in the subject prior to the administering).

Some embodiments of these methods result in at least a 1% increase, at least a 2% increase, at least a 3% increase, at least a 4% increase, at least a 5% increase, at least a 6% increase, at least a 7% increase, at least a 8% increase, at least a 9% increase, at least a 10% increase, at least a 15% increase, at least a 20% increase, at least a 25% increase, at least a 30% increase, at least a 35% increase, at least a 40% increase, at least a 45% increase, at least a 50% increase, at least a 55% increase, at least a 60% increase, at least a 65% increase, at least a 70% increase, at least a 75% increase, at least a 80% increase, at least a 85% increase, at least a 90% increase, at least a 95% increase, at least a 100% increase, at least a 200% increase, at least a 250% increase, at least a 300% increase, at least a 350% increase, at least a 400% increase, at least a 450% increase, at least a 500% increase, at least a 550% increase, at least a 600% increase, at least a 650% increase, at least a 700% increase, at least a 750% increase, at least a 800% increase, at least a 850% increase, at least a 900% increase, at least a 950% increase, or at least a 1,000% increase, in the number of NKG2D-positive/NKG2A-negative lymphocytes (e.g., NKG2D-positive/NKG2A- negative NK cells) in the subject (e.g., as compared to the number of NKG2D- positive/NKG2A-negative lymphocytes (e.g., NKG2D-positive/NKG2A-negative NK cells) in the subject prior to the administering). Some embodiments of these methods result in about a 1% increase to about a 1,000% increase (or any of the exemplary subranges of this range described herein), in the number of NKG2D- positive/NKG2A-negative lymphocytes (e.g., NKG2D-positive/NKG2A-negative NK cells) in the subject (e.g., as compared to the number of NKG2D-positive/NKG2A- negative lymphocytes (e.g., NKG2D-positive/NKG2A-negative NK cells) in the subject prior to the administering). Some embodiments of the methods described herein, result in at least a 1% increase, at least a 2% increase, at least a 3% increase, at least a 4% increase, at least a 5% increase, at least a 6% increase, at least a 7% increase, at least a 8% increase, at least a 9% increase, at least a 10% increase, at least a 15% increase, at least a 20% increase, at least a 25% increase, at least a 30% increase, at least a 35% increase, at least a 40% increase, at least a 45% increase, at least a 50% increase, at least a 55% increase, at least a 60% increase, at least a 65% increase, at least a 70% increase, at least a 75% increase, at least a 80% increase, at least a 85% increase, at least a 90% increase, at least a 95% increase, at least a 100% increase, at least a 200% increase, at least a 250% increase, at least a 300% increase, at least a 350% increase, at least a 400% increase, at least a 450% increase, at least a 500% increase, at least a 550% increase, at least a 600% increase, at least a 650% increase, at least a 700% increase, at least a 750% increase, at least a 800% increase, at least a 850% increase, at least a 900% increase, at least a 950% increase, or at least a 1000% increase in the trafficking of NKG2D-positive lymphocytes (e.g., NKG2D-positive NK cells) in a subject (e.g., as compared to the trafficking of NKG2D-positive lymphocytes (e.g., NKG2D-positive NK cells) in the subject prior to the administering). Some embodiments of any of the methods described herein, result in about a 1% increase to about a 1,000% increase (or any of the exemplary subranges of this range described herein) in the trafficking of NKG2D-positive lymphocytes (e.g., NKG2D-positive NK cells) in a subject (e.g., as compared to the trafficking of NKG2D-positive lymphocytes (e.g., NKG2D-positive NK cells) in the subject prior to the administering).

Some embodiments of the methods described herein, result in at least a 1% increase, at least a 2% increase, at least a 3% increase, at least a 4% increase, at least a 5% increase, at least a 6% increase, at least a 7% increase, at least a 8% increase, at least a 9% increase, at least a 10% increase, at least a 15% increase, at least a 20% increase, at least a 25% increase, at least a 30% increase, at least a 35% increase, at least a 40% increase, at least a 45% increase, at least a 50% increase, at least a 55% increase, at least a 60% increase, at least a 65% increase, at least a 70% increase, at least a 75% increase, at least a 80% increase, at least a 85% increase, at least a 90% increase, at least a 95% increase, at least a 100% increase, at least a 200% increase, at least a 250% increase, at least a 300% increase, at least a 350% increase, at least a 400% increase, at least a 450% increase, at least a 500% increase, at least a 550% increase, at least a 600% increase, at least a 650% increase, at least a 700% increase, at least a 750% increase, at least a 800% increase, at least a 850% increase, at least a 900% increase, at least a 950% increase, or at least a 1000% increase, in the trafficking of NKG2D-positive/NKG2A-negative lymphocytes (e.g., NKG2D- positive/NKG2A-negative NK cells) in a subject (e.g., as compared to the trafficking of NKG2D-positive/NKG2A-negative lymphocytes (e.g., NKG2D-positive/NKG2A- negative NK cells) in the subject prior to the administering). Some embodiments of any of the methods described herein, result in about a 1% increase to about a 1,000% increase (or any of the exemplary subranges of this range described herein) in the trafficking of NKG2D-positive/NKG2A-negative lymphocytes (e.g., NKG2D- positive/NKG2A-negative NK cells) in a subject (e.g., as compared to the trafficking ofNKG2D-positive/NKG2A-negative lymphocytes (e.g., NKG2D-positive/NKG2A- negative NK cells) in the subject prior to the administering).

Some embodiments of the methods described herein result in the at least a 0.1- fold increase, at least a 0.2-fold increase, at least a 0.3-fold increase, at least a 0.4-fold increase, at least a 0.5-fold increase, at least a 0.6-fold increase, at least a 0.7-fold increase, at least a 0.8-fold increase, at least a 0.9-fold increase, at least a 1.0-fold increase, at least a 1.5-fold increase, at least a 2.0-fold increase, at least a 2.5-fold increase, at least a 3.0-fold increase, at least a 3.5-fold increase, at least a 4.0-fold increase, at least a 4.5-fold increase, at least a 5.0-fold increase, at least a 5.5-fold increase, at least a 6.0-fold increase, at least a 6.5-fold increase, at least a 7.0-fold increase, at least a 7.5-fold increase, at least a 8.0-fold incrase, at least a 8.5-fold increase, at least a 9.0-fold increase, at least a 9.5-fold increase, or at least a 10-fold increase in the maximum fold change in the ratio of NKG2D-positive lymphocytes (e.g., NKG2A-positive NK cells) to NKG2A-positive lymphocytes (e.g., NKGA2A- positive NK cells) in the blood of a subject (e.g., as compared to the ratio of NKG2D- positive lymphocytes (e.g., NKG2D-positive NK cells) to NKG2A-positive lymphocytes (e.g., NKG2A-positive NK cells) in the blood of the subject prior to the administering). Some embodiments of the methods described herein result about a 0.01-fold increase to about a 10-fold increase, about 0.01-fold increase to about a 9- fold increase, about a 0.01-fold increase to about a 8-fold increase, about a 0.01-fold increase to about a 7-fold increase, about a 0.01-fold increase to about a 6-fold increase, about a 0.01-fold increase to about a 5-fold increase, about a 0.01-fold increase to about a 4-fold increase, about a 0.01 -fold increase to about a 3-fold increase, about a 0.01-fold increase to about a 2-fold increase, about a 0.01-fold increase to about a 1-fold increase, about a 0.01 -fold increase to about a 0.5-fold increase, about a 0.01 -fold increase to a 0.1-fold increase, about a 0.1 -fold increase to about a 10-fold increase, about 0.1-fold increase to about a 9-fold increase, about a 0.1-fold increase to about a 8-fold increase, about a 0.1-fold increase to about a 7-fold increase, about a 0.1 -fold increase to about a 6-fold increase, about a 0.1 -fold increase to about a 5-fold increase, about a 0.1 -fold increase to about a 4-fold increase, about a 0.1-fold increase to about a 3 -fold increase, about a 0.1-fold increase to about a 2-fold increase, about a 0.1 -fold increase to about a 1-fold increase, about a 0.1 -fold increase to about a 0.5-fold increase, about a 0.5-fold increase to about a 10-fold increase, about a 0.5-fold increase to about a 9-fold increase, about a 0.5-fold increase to about a 8-fold increase, about a 0.5-fold increase to about a 7-fold increase, about a 0.5-fold increase to about a 6-fold increase, about a 0.5-fold increase to about a 5-fold increase, about a 0.5-fold increase to about a 4-fold increase, about a 0.5-fold increase to about a 3-fold increase, about a 0.5-fold increase to about a 2-fold increase, about a 0.5-fold increase to about a 1-fold increase, about a 1-fold increase to about a 10-fold increase, about a 1-fold increase to about a 9-fold increase, about a 1-fold increase to about a 8-fold increase, about a 1-fold increase to about a 7-fold increase, about a 1- fold increase to about a 6-fold increase, about a 1-fold increase to about a 5-fold increase, about a 1-fold increase to about a 4-fold increase, about a 1-fold increase to about a 3-fold increase, about a 1-fold increase to about a 2-fold increase, about a 2- fold increase to about a 10-fold increase, about a 2-fold increase to about a 9-fold increase, about a 2-fold increase to about a 8-fold increase, about a 2-fold increase to about a 7-fold increase, about a 2-fold increase to about a 6-fold increase, about a 2- fold increase to about a 5-fold increase, about a 2-fold increase to about a 4-fold increase, about a 2-fold increase to about a 3-fold increase, about a 3-fold increase to about a 10-fold increase, about a 3-fold increase to about a 9-fold increase, about a 3- fold increase to about a 8-fold increase, about a 3-fold increase to about a 7-fold increase, about a 3-fold increase to about a 6-fold increase, about a 3-fold increase to about a 5-fold increase, about a 3-fold increase to about a 4-fold increase, about a 4- fold increase to about a 10-fold increase, about a 4-fold increase to about a 9-fold increase, about a 4-fold increase to about a 8-fold increase, about a 4-fold increase to about a 7-fold increase, about a 4-fold increase to about a 6-fold increase, about a 4- fold increase to about a 5-fold increase, about a 5-fold increase to about a 10-fold increase, about a 5-fold increase to about a 9-fold increase, about a 5-fold increase to about a 8-fold increase, about a 5-fold increase to about a 7-fold increase, about a 5- fold increase to about a 6-fold increase, about a 6-fold increase to about a 10-fold increase, about a 6-fold increase to about a 9-fold increase, about a 6-fold increase to about a 8-fold increase, about a 6-fold increase to about a 7-fold increase, about a 7- fold increase to about a 10-fold increase, about a 7-fold increase to about a 9-fold increase, about a 7-fold increase to about a 8-fold increase, about a 8-fold increase to about a 10-fold increase, about a 8-fold increase to about a 9-fold increase, or about a 9-fold increase to about a 10-fold increase in the maximum fold change in the ratio of NKG2D-positive lymphocytes (e.g., NKG2D-positive NK cells) to NKG2A-positive lymphocytes (e.g., NKGA2A-positive NK cells) in the blood of a subject (e.g., as compared to the ratio of NKG2D-positive lymphocytes (e.g., NKG2D-positive NK cells) to NKG2A-positive lymphocytes (e.g., NKG2A-positive NK cells) in the blood of the subject prior to the administering).

Some embodiments of the methods described herein result in at least a 1% increase, at least a 2% increase, at least a 3% increase, at least a 4% increase, at least a 5% increase, at least a 6% increase, at least a 7% increase, at least a 8% increase, at least a 9% increase, at least a 10% increase, at least a 15% increase, at least a 20% increase, at least a 25% increase, at least a 30% increase, at least a 35% increase, at least a 40% increase, at least a 45% increase, at least a 50% increase, at least a 55% increase, at least a 60% increase, at least a 65% increase, at least a 70% increase, at least a 75% increase, at least a 80% increase, at least a 85% increase, at least a 90% increase, at least a 95% increase, at least a 100% increase, at least a 150% increase, at least a 200% increase, at least a 250% increase, at least a 300% increase, at least a 350% increase, at least a 400% increase, at least a 450% increase, at least a 500% increase, at least a 550% increase, at least a 600% increase, at least a 650% increase, at least a 700% increase, at least a 750% increase, at least a 800% increase, at least a 850% increase, at least a 900% increase, at least a 950% increase, or at least a 1000% increase in the absolute number of NK cells in the blood of the subject (e.g., as compared to the absolute number of NK cells in the blood of the subject prior to the administering). Some embodiments of any of the methods described herein, result in about a 1% increase to about a 1,000% increase (or any of the exemplary subranges of this range described herein)in the absolute number of NK cells in the blood of the subject (e.g., as compared to the absolute number of NK cells in the blood of the subject prior to the administering).

Some embodiments of the methods described herein result in the at least a 0.1- fold increase, at least a 0.2-fold increase, at least a 0.3-fold increase, at least a 0.4-fold increase, at least a 0.5-fold increase, at least a 0.6-fold increase, at least a 0.7-fold increase, at least a 0.8-fold increase, at least a 0.9-fold increase, at least a 1.0-fold increase, at least a 1.5-fold increase, at least a 2.0-fold increase, at least a 2.5-fold increase, at least a 3.0-fold increase, at least a 3.5-fold increase, at least a 4.0-fold increase, at least a 4.5-fold increase, or at least a 5.0-fold increase in the maximum fold increase in the absolute number of NK cells in the blood of the subject (e.g., as compared to the maximum fold increase in the absolute number of NK cells in the blood of the subject prior to the administering). Some embodiments of the methods described herein result about a 0.01 -fold increase to about a 5-fold increase, about a 0.01-fold increase to about a 4.5-fold increase, about a 0.01-fold increase to about a 4- fold increase, about a 0.01-fold increase to about a 3.5-fold increase, about a 0.01-fold increase to about a 3-fold increase, about a 0.01 -fold increase to about a 2.5-fold increase, about a 0.01-fold increase to about a 2-fold increase, about a 0.01-fold increase to about a 1.5-fold increase, about a 0.01 -fold increase to about a 1-fold increase, about a 0.01-fold increase to about a 0.8-fold increase, about a 0.01-fold increase to about a 0.6-fold increase, about a 0.01-fold increase to about a 0.4-fold increase, about a 0.01-fold increase to about a 0.2-fold increase, about a 0.01-fold increase to about a 0.1-fold increase, about a 0.01-fold increase to about a 0.05-fold increase, about a 0.05-fold increase to about a 5-fold increase, about a 0.05-fold increase to about a 4.5-fold increase, about a 0.05-fold increase to about a 4-fold increase, about a 0.05-fold increase to about a 3.5-fold increase, about a 0.05-fold increase to about a 3-fold increase, about a 0.05-fold increase to about a 2.5-fold increase, about a 0.05-fold increase to about a 2-fold increase, about a 0.05-fold increase to about a 1.5-fold increase, about a 0.05-fold increase to about a 1-fold increase, about a 0.05-fold increase to about a 0.8-fold increase, about a 0.05-fold increase to about a 0.6-fold increase, about a 0.05-fold increase to about a 0.4-fold increase, about a 0.05-fold increase to about a 0.2-fold increase, about a 0.05-fold increase to about a 0.1 -fold increase, about a 0.1 -fold increase to about a 5-fold increase, about a 0.1-fold increase to about a 4.5-fold increase, about a 0.1-fold increase to about a 4-fold increase, about a 0.1-fold increase to about a 3.5-fold increase, about a 0.1 -fold increase to about a 3-fold increase, about a 0.1 -fold increase to about a 2.5-fold increase, about a 0.1 -fold increase to about a 2-fold increase, about a 0.1-fold increase to about a 1.5-fold increase, about a 0.1-fold increase to about a 1- fold increase, about a 0.1-fold increase to about a 0.8-fold increase, about a 0.1 -fold increase to about a 0.6-fold increase, about a 0.1-fold increase to about a 0.4-fold increase, about a 0.1 -fold increase to about a 0.2-fold increase, about a 0.2-fold increase to about a 5-fold increase, about a 0.2-fold increase to about a 4.5-fold increase, about a 0.2-fold increase to about a 4-fold increase, about a 0.2-fold increase to about a 3.5-fold increase, about a 0.2-fold increase to about a 3-fold increase, about a 0.2-fold increase to about a 2.5-fold increase, about a 0.2-fold increase to about a 2- fold increase, about a 0.2-fold increase to about a 1.5-fold increase, about a 0.2-fold increase to about a 1-fold increase, about a 0.2-fold increase to about a 0.8-fold increase, about a 0.2-fold increase to about a 0.6-fold increase, about a 0.2-fold increase to about a 0.4-fold increase, about a 0.4-fold increase to about a 5-fold increase, about a 0.4-fold increase to about a 4.5-fold increase, about a 0.4-fold increase to about a 4-fold increase, about a 0.4-fold increase to about a 3.5-fold increase, about a 0.4-fold increase to about a 3-fold increase, about a 0.4-fold increase to about a 2.5-fold increase, about a 0.4-fold increase to about a 2-fold increase, about a 0.4-fold increase to about a 1.5-fold increase, about a 0.4-fold increase to about a 1- fold increase, about a 0.4-fold increase to about a 0.8-fold increase, about a 0.4-fold increase to about a 0.6-fold increase, about a 0.6-fold increase to about a 5-fold increase, about a 0.6-fold increase to about a 4.5-fold increase, about a 0.6-fold increase to about a 4-fold increase, about a 0.6-fold increase to about a 3.5-fold increase, about a 0.6-fold increase to about a 3-fold increase, about a 0.6-fold increase to about a 2.5-fold increase, about a 0.6-fold increase to about a 2-fold increase, about a 0.6-fold increase to about a 1.5-fold increase, about a 0.6-fold increase to about a 1- fold increase, about a 0.6-fold increase to about a 0.8-fold increase, about a 0.8-fold increase to about a 5-fold increase, about a 0.8-fold increase to about a 4.5-fold increase, about a 0.8-fold increase to about a 4-fold increase, about a 0.8-fold increase to about a 3.5-fold increase, about a 0.8-fold increase to about a 3-fold increase, about a 0.8-fold increase to about a 2.5-fold increase, about a 0.8-fold increase to about a 2- fold increase, about a 0.8-fold increase to about a 1.5-fold increase, about a 0.8-fold increase to about a 1-fold increase, about a 1-fold increase to about a 5-fold increase, about a 1-fold increase to about a 4.5-fold increase, about a 1-fold increase to about a 4-fold increase, about a 1-fold increase to about a 3.5-fold increase, about a 1-fold increase to about a 3-fold increase, about a 1-fold increase to about a 2.5-fold increase, about a 1-fold increase to about a 2-fold increase, about a 1-fold increase to about a 1.5-fold increase, about a 1.5-fold increase to about a 5-fold increase, about a

1.5-fold increase to about a 4.5-fold increase, about a 1.5-fold increase to about a 4- fold increase, about a 1.5-fold increase to about a 3.5-fold increase, about a 1.5-fold increase to about a 3-fold increase, about a 1.5-fold increase to about a 2.5-fold increase, about a 1.5-fold increase to about a 2-fold increase, about a 2-fold increase to about a 5-fold increase, about a 2-fold increase to about a 4.5-fold increase, about a 2-fold increase to about a 4-fold increase, about a 2-fold increase to about a 3.5-fold increase, about a 2-fold increase to about a 3-fold increase, about a 2-fold increase to about a 2.5-fold increase, about a 2.5-fold increase to about a 5-fold increase, about a

2.5-fold increase to about a 4.5-fold increase, about a 2.5-fold increase to about a 4- fold increase, about a 2.5-fold increase to about a 3.5-fold increase, about a 2.5-fold increase to about a 3-fold increase, about a 3-fold increase to about a 5-fold increase, about a 3-fold increase to about a 4.5-fold increase, about a 3-fold increase to about a 4-fold increase, about a 3-fold increase to about a 3.5-fold increase, about a 3.5-fold increase to about a 5-fold increase, about a 3.5-fold increase to about a 4.5-fold increase, about a 3.5-fold increase to about a 4-fold increase, about a 4-fold increase to about a 5-fold increase, about a 4-fold increase to about a 4.5-fold increase, or about a 4.5-fold increase to about a 5-fold increase, in the maximum fold increase in the absolute number of NK cells in the blood of the subject (e.g., as compared to the maximum fold increase in the absolute number of NK cells in the blood of the subject prior to the administering).

Some embodiments of the methods described herein result in the at least a 0.1- fold increase, at least a 0.2-fold increase, at least a 0.3-fold increase, at least a 0.4-fold increase, at least a 0.5-fold increase, at least a 0.6-fold increase, at least a 0.7-fold increase, at least a 0.8-fold increase, at least a 0.9-fold increase, at least a 1.0-fold increase, at least a 1.5-fold increase, at least a 2.0-fold increase, at least a 2.5-fold increase, at least a 3.0-fold increase, at least a 3.5-fold increase, at least a 4.0-fold increase, at least a 4.5-fold increase, or at least a 5.0-fold increase in the maximum fold increase in the absolute number of CD56^bnght lymphocytes (e.g., CD56^bnght NK cells) in the blood of the subject (e.g., as compared to the maximum fold increase in the absolute number of CD56^bnght lymphocytes (e.g., CD56^bnght NK cells) in the blood of the subject prior to the administering). Some embodiments of the methods described herein result about a 0.01 -fold increase to about a 5-fold increase (or any of the exemplary subranges of this range described herein) in the maximum fold increase in the absolute number of CD56^bnght lymphocytes (e.g., CD56^bnghtNK cells) in the blood of the subject (e.g., as compared to the maximum fold increase in the absolute number of CD56^bnght lymphocytes (e.g., CD56^bnght NK cells) in the blood of the subject prior to the administering).

Some embodiments of the methods described herein result in the at least a 0.1- fold increase, at least a 0.2-fold increase, at least a 0.3-fold increase, at least a 0.4-fold increase, at least a 0.5-fold increase, at least a 0.6-fold increase, at least a 0.7-fold increase, at least a 0.8-fold increase, at least a 0.9-fold increase, at least a 1.0-fold increase, at least a 1.5-fold increase, at least a 2.0-fold increase, at least a 2.5-fold increase, at least a 3.0-fold increase, at least a 3.5-fold increase, at least a 4.0-fold increase, at least a 4.5-fold increase, or at least a 5.0-fold increase in the maximum fold increase in the absolute number of CD16⁺ CD56^dim lymphocytes (e.g., CD16⁺ CD56^dim NK cells) in the blood of the subject (e.g., as compared to the maximum fold increase in the absolute number of CD16⁺ CD56^dim lymphocytes (e.g., CD16⁺

CD56^dim NK cells) in the blood of the subject prior to the administering). Some embodiments of the methods described herein result about a 0.01 -fold increase to about a 5-fold increase (or any of the exemplary subranges of this range described herein), in the maximum fold increase in the absolute number of CD16⁺ CD56^dim lymphocytes (e.g., CD16⁺ CD56^dimNK cells) in the blood of the subject (e.g., as compared to the maximum fold increase in the absolute number of CD16⁺ CD56^dim lymphocytes (e.g., CD16⁺ CD56^dim NK cells) in the blood of the subject prior to the administering).

Some embodiments of the methods described herein result in the at least a 0.1- fold increase, at least a 0.2-fold increase, at least a 0.3-fold increase, at least a 0.4-fold increase, at least a 0.5-fold increase, at least a 0.6-fold increase, at least a 0.7-fold increase, at least a 0.8-fold increase, at least a 0.9-fold increase, at least a 1.0-fold increase, at least a 1.5-fold increase, at least a 2.0-fold increase, at least a 2.5-fold increase, at least a 3.0-fold increase, at least a 3.5-fold increase, at least a 4.0-fold increase, at least a 4.5-fold increase, or at least a 5.0-fold increase in the maximum fold increase in the absolute number of HLA-DR lymphocytes (e.g., HLA-DR NK cells) in the blood of the subject (e.g., as compared to the maximum fold increase in the absolute number of HLA-DR lymphocytes (e.g., HLA-DR NK cells) in the blood of the subject prior to the administering). Some embodiments of the methods described herein result about a 0.01 -fold increase to about a 5-fold increase (or any of the exemplary subranges of this range described herein) in the maximum fold increase in the absolute number of HLA-DR lymphocytes (e.g., HLA-DR NK cells) in the blood of the subject (e.g., as compared to the maximum fold increase in the absolute number of HLA-DR lymphocytes (e.g., HLA-DR NK cells) in the blood of the subject prior to the administering).

Provided herein are methods of treating a subject previously identified or diagnosed as having a MICA-positive cancer that include administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a population of enucleated erythroid cells including a first exogenous fusion polypeptide that includes (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof, on its extracellular surface. Also provided herein are methods of treating a subject previously identified or diagnosed as having a MICA-positive cancer that include administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising a population of enucleated erythroid cells including a first exogenous fusion polypeptide including (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof, on its extracellular surface; and a second exogenous polypeptide including 4-1BBL or a functional fragment thereof, on its extracellular surface.

Provided herein are methods of decreasing the number and/or proliferation of MICA-positive cancer cells in a subject previously identified or diagnosed as having a MICA-positive cancer that include administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a population of enucleated erythroid cells including a first exogenous fusion polypeptide that includes (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof, on its extracellular surface. Also provided herein are methods of decreasing the number and/or proliferation of MICA-positive cancer cells in a subject previously identified or diagnosed as having a MICA-positive cancer that include administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a population of enucleated erythroid cells including a first exogenous fusion polypeptide including (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof, on its extracellular surface; and a second exogenous polypeptide including 4-1BBL or a functional fragment thereof, on its extracellular surface.

Some embodiments 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 a 8% reduction, at least a 9% reduction, at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 35% reduction, at least a 40% reduction, at least a 45% reduction, at least a 50% reduction, at least a 55% reduction, at least a 60% reduction, at least a 65% reduction, at least a 70% reduction, at least a 75% reduction, at least a 80% reduction, at least a 85% reduction, at least a 90% reduction, at least a 95% reduction, or at least a 99% reduction, in the number of MIC A-positive cancer cells in the subject (e.g., as compared to the number of MICA-positive cancer cells in the subject prior to the administering). Some embodiments of these methods result in about a 1% reduction to about a 99% reduction, about a 1% reduction to about a 90% reduction, about a 1% reduction to about a 80% reduction, about a 1% reduction to about a 70% reduction, about a 1% reduction to about a 60% reduction, about a 1% reduction to about a 50% reduction, about a 1% reduction to about a 40% reduction, about a 1% reduction to about a 30% reduction, about a 1% reduction to about a 20% reduction, about a 1% reduction to about a 10% reduction, about a 5% reduction to about a 99% reduction, about a 5% reduction to about a 90% reduction, about a 5% reduction to about a 80% reduction, about a 5% reduction to about a 70% reduction, about a 5% reduction to about a 60% reduction, about a 5% reduction to about a 50% reduction, about a 5% reduction to about a 40% reduction, about a 5% reduction to about a 30% reduction, about a 5% reduction to about a 20% reduction, about a 5% reduction to about a 10% reduction, about a 10% reduction to about a 99% reduction, about a 10% reduction to about a 90% reduction, about a 10% reduction to about a 80% reduction, about a 10% reduction to about a 70% reduction, about a 10% reduction to about a 60% reduction, about a 10% reduction to about a 50% reduction, about a 10% reduction to about a 40% reduction, about a 10% reduction to about a 30% reduction, about a 10% reduction to about a 20% reduction, about a 20% reduction to about a 99% reduction, about a 20% reduction to about a 90% reduction, about a 20% reduction to about a 80% reduction, about a 20% reduction to about a 70% reduction, about a 20% reduction to about a 60% reduction, about a 20% reduction to about a 50% reduction, about a 20% reduction to about a 40% reduction, about a 20% reduction to about a 30% reduction, about a 30% reduction to about a 99% reduction, about a 30% reduction to about a 90% reduction, about a 30% reduction to about a 80% reduction, about a 30% reduction to about a 70% reduction, about a 30% reduction to about a 60% reduction, about a 30% reduction to about a 50% reduction, about a 30% reduction to about a 40% reduction, about a 40% reduction to about a 99% reduction, about a 40% reduction to about a 90% reduction, about a 40% reduction to about a 80% reduction, about a 40% reduction to about a 70% reduction, about a 40% reduction to about a 60% reduction, about a 40% reduction to about a 50% reduction, about a 50% reduction to about a 99% reduction, about a 50% reduction to about a 90% reduction, about a 50% reduction to about a 80% reduction, about a 50% reduction to about a 70% reduction, about a 50% reduction to about a 60% reduction, about a 60% reduction to about a 99% reduction, about a 60% reduction to about a 90% reduction, about a 60% reduction to about a 80% reduction, about a 60% reduction to about a 70% reduction, about a 70% reduction to about a 99% reduction, about a 70% reduction to about a 90% reduction, about a 70% reduction to about a 80% reduction, about a 80% reduction to about a 99% reduction, about a 80% reduction to about a 90% reduction, or about a 90% reduction to about a 99% reduction, in the number of MICA-positive cancer cells in the subject (e.g., as compared to the number of MICA-positive cancer cells in the subject prior to the administering).

Some embodiments 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 a 8% reduction, at least a 9% reduction, at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 35% reduction, at least a 40% reduction, at least a 45% reduction, at least a 50% reduction, at least a 55% reduction, at least a 60% reduction, at least a 65% reduction, at least a 70% reduction, at least a 75% reduction, at least a 80% reduction, at least a 85% reduction, at least a 90% reduction, at least a 95% reduction, or at least a 99% reduction, in the number of MIC A-positive/HLA-E-negative cancer cells in the subject (e.g., as compared to the number of MICA-positive/HLA-E negative cancer cells in the subject prior to the administering). Some embodiments of these methods result in about a 1% reduction to about a 99% reduction (or any of the exemplary subranges of this range described herein) in the number of MIC A-positive/HLA-E-negative cancer cells in the subject (e.g., as compared to the number of MIC A-positive/HLA-E- negative cancer cells in the subject prior to the administering).

Some embodiments of these methods results 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 a 8% reduction, at least a 9% reduction, at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 35% reduction, at least a 40% reduction, at least a 45% reduction, at least a 50% reduction, at least a 55% reduction, at least a 60% reduction, at least a 65% reduction, at least a 70% reduction, at least a 75% reduction, at least a 80% reduction, at least a 85% reduction, at least a 90% reduction, at least a 95% reduction, or at least a 99% reduction, in the proliferation of MICA-positive cancer cells in the subject (e.g., as compared to the proliferation of MICA-positive cancer cells in the subject prior to the administering). Some embodiments of these methods results in about a 1% reduction to about a 99% reduction (or any of the exemplary subranges of this range described herein) in the proliferation of MICA-positive cancer cells in the subject (e.g., as compared to the proliferation of MICA-positive cancer cells in the subject prior to the administering).

Some embodiments of these methods results 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 a 8% reduction, at least a 9% reduction, at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 35% reduction, at least a 40% reduction, at least a 45% reduction, at least a 50% reduction, at least a 55% reduction, at least a 60% reduction, at least a 65% reduction, at least a 70% reduction, at least a 75% reduction, at least a 80% reduction, at least a 85% reduction, at least a 90% reduction, at least a 95% reduction, or at least a 99% reduction, in the proliferation of MICA-positive/HLA-E-negative cancer cells in the subject (e.g., as compared to the proliferation of MIC A-positive/HLA-E negative cancer cells in the subject prior to the administering). Some embodiments of these methods results in about a 1% reduction to about a 99% reduction (or any of the exemplary subranges of this range described herein) in the proliferation of MICA-positive and HLA-E negative cancer cells in the subject (e.g., as compared to the proliferation of MICA- positive and HLA-E negative cancer cells in the subject prior to the administering).

Provided herein are methods of inducing killing a MICA-positive cancer cell in a subject previously identified or diagnosed as having a MICA-positive cancer that include administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a population of enucleated erythroid cells including an first exogenous fusion polypeptide that includes (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof, on its extracellular surface. Also provided herein are methods of killing a MICA-positive cancer cell in a subject previously identified or diagnosed as having a MICA-positive cancer that include administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a population of enucleated erythroid cells including a first exogenous fusion polypeptide including (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof, on its extracellular surface; and a second exogenous polypeptide including 4-1BBL or a functional fragment thereof, on its extracellular surface.

Provided herein are methods of treating a subject previously identified or diagnosed as having a MICB-positive cancer that include administering to the subject previously identified or diagnosed as having a MICB-positive cancer a therapeutically effective amount of a pharmaceutical composition comprising a population of enucleated erythroid cells including a first exogenous fusion polypeptide that includes (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof, on its extracellular surface. Also provided herein are methods of treating a subject previously identified or diagnosed as having a MICB-positive cancer that include administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a population of enucleated erythroid cells including a first exogenous fusion polypeptide including (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof, on its extracellular surface; and a second exogenous polypeptide including 4-1BBL or a functional fragment thereof, on its extracellular surface.

Provided herein are methods of decreasing the number and/or proliferation of MICB-positive cancer cells in a subject previously identified or diagnosed as having a MICB-positive cancer that include administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a population of enucleated erythroid cells including a first exogenous fusion polypeptide that includes (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof, on its extracellular surface. Also provided herein are methods of decreasing the number and/or proliferation of MICB-positive cancer cells in a subject previously identified or diagnosed as having a MICB-positive cancer that include administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a population of enucleated erythroid cells including a first exogenous fusion polypeptide including (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof, on its extracellular surface; and a second exogenous polypeptide including 4-1BBL or a functional fragment thereof, on its extracellular surface.

Some embodiments 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 a 8% reduction, at least a 9% reduction, at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 35% reduction, at least a 40% reduction, at least a 45% reduction, at least a 50% reduction, at least a 55% reduction, at least a 60% reduction, at least a 65% reduction, at least a 70% reduction, at least a 75% reduction, at least a 80% reduction, at least a 85% reduction, at least a 90% reduction, at least a 95% reduction, or at least a 99% reduction, in the number of MICB-positive cancer cells in the subject (e.g., as compared to the number of MICB-positive cancer cells in the subject prior to the administering). Some embodiments of these methods result in about a 1% reduction to about a 99% reduction (or any of the exemplary subranges of this range described herein) in the number of MICB-positive cancer cells in the subject (e.g., as compared to the number of MICB-positive cancer cells in the subject prior to the administering).

Some embodiments 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 a 8% reduction, at least a 9% reduction, at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 35% reduction, at least a 40% reduction, at least a 45% reduction, at least a 50% reduction, at least a 55% reduction, at least a 60% reduction, at least a 65% reduction, at least a 70% reduction, at least a 75% reduction, at least a 80% reduction, at least a 85% reduction, at least a 90% reduction, at least a 95% reduction, or at least a 99% reduction, in the number of MICB-positive/HLA-E-negative cancer cells in the subject (e.g., as compared to the number of MICB-positive/HLA-E negative cancer cells in the subject prior to the administering). Some embodiments of these methods result in about a 1% reduction to about a 99% reduction (or any of the exemplary subranges of this range described herein) in the number of MICB-positive/HLA-E-negative cancer cells in the subject (e.g., as compared to the number of MICB-positive/HLA-E-negative cancer cells in the subject prior to the administering).

Some embodiments of these methods results 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 a 8% reduction, at least a 9% reduction, at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 35% reduction, at least a 40% reduction, at least a 45% reduction, at least a 50% reduction, at least a 55% reduction, at least a 60% reduction, at least a 65% reduction, at least a 70% reduction, at least a 75% reduction, at least a 80% reduction, at least a 85% reduction, at least a 90% reduction, at least a 95% reduction, or at least a 99% reduction, in the proliferation of MICB-positive cancer cells in the subject (e.g., as compared to the proliferation of MICB-positive cancer cells in the subject prior to the administering). Some embodiments of these methods results in about a 1% reduction to about a 99% reduction (or any of the exemplary subranges of this range described herein) in the proliferation of MICB-positive cancer cells in the subject (e.g., as compared to the proliferation of MICB-positive cancer cells in the subject prior to the administering).

Some embodiments of these methods results 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 a 8% reduction, at least a 9% reduction, at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 35% reduction, at least a 40% reduction, at least a 45% reduction, at least a 50% reduction, at least a 55% reduction, at least a 60% reduction, at least a 65% reduction, at least a 70% reduction, at least a 75% reduction, at least a 80% reduction, at least a 85% reduction, at least a 90% reduction, at least a 95% reduction, or at least a 99% reduction, in the proliferation of MICB-positive/HLA-E-negative cancer cells in the subject (e.g., as compared to the proliferation of MICB-positive/HLA-E negative cancer cells in the subject prior to the administering). Some embodiments of these methods results in about a 1% reduction to about a 99% reduction (or any of the exemplary subranges of this range described herein) in the proliferation of MICB-positive/HLA-E-negative cancer cells in the subject (e.g., as compared to the proliferation of MICB- positive/HLA-E-negative cancer cells in the subject prior to the administering).

Provided herein are methods of inducing killing a MICB-positive cancer cell in a subject previously identified or diagnosed as having a MICB-positive cancer that include administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a population of enucleated erythroid cells including an first exogenous fusion polypeptide that includes (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof, on its extracellular surface. Also provided herein are methods of killing a MICB-positive cancer cell in a subject previously identified or diagnosed as having a MICB-positive cancer that include administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a population of enucleated erythroid cells including a first exogenous fusion polypeptide including (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof, on its extracellular surface; and a second exogenous polypeptide including 4-1BBL or a functional fragment thereof, on its extracellular surface. Provided herein are methods of treating a subject previously identified or diagnosed as having a MICA/MICB-positive cancer that include administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a population of enucleated erythroid cells including a first exogenous fusion polypeptide that includes (i) IL-15 or a functional fragment thereof, and (ii) IL- 15 receptor alpha or a functional fragment thereof, on its extracellular surface. Also provided herein are methods of treating a subject previously identified or diagnosed as having a MICA/MICB-positive cancer that include administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a population of enucleated erythroid cells including a first exogenous fusion polypeptide including (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof, on its extracellular surface; and a second exogenous polypeptide including 4-1BBL or a functional fragment thereof, on its extracellular surface.

Provided herein are methods of decreasing the number and/or proliferation of MICA/MICB-positive cancer cells in a subject previously identified or diagnosed as having a MICA/MICB-positive cancer that include administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a population of enucleated erythroid cells including a first exogenous fusion polypeptide that includes (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof, on its extracellular surface. Also provided herein are methods of decreasing the number and/or proliferation of MICA/MICB-positive cancer cells in a subject previously identified or diagnosed as having a MICA/MICB-positive cancer that include administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a population of enucleated erythroid cells including a first exogenous fusion polypeptide including (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof, on its extracellular surface; and a second exogenous polypeptide including 4-1BBL or a functional fragment thereof, on its extracellular surface.

Some embodiments 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 a 8% reduction, at least a 9% reduction, at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 35% reduction, at least a 40% reduction, at least a 45% reduction, at least a 50% reduction, at least a 55% reduction, at least a 60% reduction, at least a 65% reduction, at least a 70% reduction, at least a 75% reduction, at least a 80% reduction, at least a 85% reduction, at least a 90% reduction, at least a 95% reduction, or at least a 99% reduction, in the number of MICA/MICB-positive cancer cells in the subject (e.g., as compared to the number of MICA/MICB-positive cancer cells in the subject prior to the administering). Some embodiments of these methods result in about a 1% reduction to about a 99% reduction (or any of the exemplary subranges of this range described herein) in the number of MICA/MICB-positive cancer cells in the subject (e.g., as compared to the number of MICA/MICB-positive cancer cells in the subject prior to the administering).

Some embodiments 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 a 8% reduction, at least a 9% reduction, at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 35% reduction, at least a 40% reduction, at least a 45% reduction, at least a 50% reduction, at least a 55% reduction, at least a 60% reduction, at least a 65% reduction, at least a 70% reduction, at least a 75% reduction, at least a 80% reduction, at least a 85% reduction, at least a 90% reduction, at least a 95% reduction, or at least a 99% reduction, in the number of MICA/MICB-positive/HLA-E-negative cancer cells in the subject (e.g., as compared to the number of MICA/MICB-positive/HLA-E negative cancer cells in the subject prior to the administering). Some embodiments of these methods result in about a 1% reduction to about a 99% reduction (or any of the exemplary subranges of this range described herein) in the number of MICA/MICB-positive/HLA-E-negative cancer cells in the subject (e.g., as compared to the number of MICA/MICB- positive/HLA-E-negative cancer cells in the subject prior to the administering).

Some embodiments of these methods results 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 a 8% reduction, at least a 9% reduction, at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 35% reduction, at least a 40% reduction, at least a 45% reduction, at least a 50% reduction, at least a 55% reduction, at least a 60% reduction, at least a 65% reduction, at least a 70% reduction, at least a 75% reduction, at least a 80% reduction, at least a 85% reduction, at least a 90% reduction, at least a 95% reduction, or at least a 99% reduction, in the proliferation of MICA/MICB-positive cancer cells in the subject (e.g., as compared to the proliferation of MICA/MICB-positive cancer cells in the subject prior to the administering). Some embodiments of these methods results in about a 1% reduction to about a 99% reduction (or any of the exemplary subranges of this range described hereinjin the proliferation of MICA/MICB-positive cancer cells in the subject (e.g., as compared to the proliferation of MICA/MICB-positive cancer cells in the subject prior to the administering).

Some embodiments of these methods results 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 a 8% reduction, at least a 9% reduction, at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 35% reduction, at least a 40% reduction, at least a 45% reduction, at least a 50% reduction, at least a 55% reduction, at least a 60% reduction, at least a 65% reduction, at least a 70% reduction, at least a 75% reduction, at least a 80% reduction, at least a 85% reduction, at least a 90% reduction, at least a 95% reduction, or at least a 99% reduction, in the proliferation of MICA/MICB-positive/HLA-E-negative cancer cells in the subject (e.g., as compared to the proliferation of MICA/MICB-positive/HLA-E negative cancer cells in the subject prior to the administering). Some embodiments of these methods results in about a 1% reduction to about a 99% reduction (or any of the exemplary subranges of this range described herein) in the proliferation of MICA/MICB-positive/HLA-E-negative cancer cells in the subject (e.g., as compared to the proliferation of MICA/MICB-positive/HLA-E-negative cancer cells in the subject prior to the administering).

Provided herein are methods of inducing killing a MICA/MICB-positive cancer cell in a subject previously identified or diagnosed as having a MICA/MICB- positive cancer that include administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a population of enucleated erythroid cells including an first exogenous fusion polypeptide that includes (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof, on its extracellular surface. Also provided herein are methods of killing a MICA/MICB-positive cancer cell in a subject previously identified or diagnosed as having a MICA/MICB-positive cancerthat include administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a population of enucleated erythroid cells including a first exogenous fusion polypeptide including (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof, on its extracellular surface; and a second exogenous polypeptide including 4-1BBL or a functional fragment thereof, on its extracellular surface.

Provided herein are methods of reducing the volume of a solid tumor in a subject previously identified or diagnosed as having a MICA-positive cancer, a MICB-positive cancer, or a MICA/MICB-positive cancer, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a population of enucleated erythroid cells including an first exogenous fusion polypeptide that includes (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof, on its extracellular surface. Also provided herein are methods of reducing the volume of a solid tumor in a subject previously identified or diagnosed as having a MICA-positive cancer, a MICB-positive cancer, or a MICA/MICB-positive cancer that include administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a population of enucleated erythroid cells including a first exogenous fusion polypeptide including (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof, on its extracellular surface; and a second exogenous polypeptide including 4-1BBL or a functional fragment thereof, on its extracellular surface.

Some embodiments 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 a 8% reduction, at least a 9% reduction, at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 35% reduction, at least a 40% reduction, at least a 45% reduction, at least a 50% reduction, at least a 55% reduction, at least a 60% reduction, at least a 65% reduction, at least a 70% reduction, at least a 75% reduction, at least a 80% reduction, at least a 85% reduction, at least a 90% reduction, at least a 95% reduction, or at least a 99% reduction, in the volume of the solid tumor in the subject (e.g., as compared to the volume of the solid tumor prior to the administering).

Some embodiments of these methods result in about a 1% reduction to about a 99% reduction (or any of the exemplary subranges of this range described herein) in the volume of the solid tumor in the subject (e.g., as compared to the volume of the solid tumor prior to the administering).

Also provided herein are methods of increasing the ratio of the concentration ofNKG2D to the concentration ofNKG2A on the cell surface of lymphocytes (e.g., NK cells) in a subject previously identified or diagnosed as having a MICA-positive, a MICB-positive, or a MICA/MICB-positive cancer that include adminsitering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a population of enucleated erythroid cells including an first exogenous fusion polypeptide that includes (i) IL-15 or a functional fragment thereof, and (ii) IL- 15 receptor alpha or a functional fragment thereof, on its extracellular surface. Also provided herein are methods of increasing the ratio of the concentration of NKG2D- positive lymphocytes (e.g., NKG2D-positive NK cells) to the concentration of NKG2A-positive lymphocytes (e.g., NKG2A-positive NK cells) in a subject previously identified or diagnosed as having a MICA-positive, a MICB-positive, or a MICA/MICB-positive cancer that include adminsitering to the subject a pharmaceutical composition comprising a population of enucleated erythroid cells including a first exogenous fusion polypeptide including (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof, on its extracellular surface; and a second exogenous polypeptide including 4-1BBL or a functional fragment thereof, on its extracellular surface.

Some embodiments of these methods result in at least a 0.1 -fold increase, at least a 0.2-fold increase, at least a 0.3-fold increase, at least a 0.4-fold increase, at least a 0.5-fold increase, at least a 0.6-fold increase, at least a 0.7-fold increase, at least a 0.8-fold increase, at least a 0.9-fold increase, at least a 1.0-fold increase, at least a 1.5-fold increase, at least a 2.0-fold increase, at least a 2.5-fold increase, at least a 3.0-fold increase, at least a 3.5-fold increase, at least a 4.0-fold increase, at least a 4.5-fold increase, at least a 5.0-fold increase, at least a 5.5-fold increase, at least a 6.0-fold increase, at least a 6.5-fold increase, at least a 7.0-fold increase, at least a 7.5-fold increase, at least a 8.0-fold incrase, at least a 8.5-fold increase, at least a 9.0-fold increase, at least a 9.5-fold increase, or at least a 10-fold increase, in the ratio of the concentration of NKG2D to the concentration of NKG2A on the cell surface of lymphocytes (e.g., NK cells) in the subject (e.g., as compared to the ratio of the concentration of NKG2D to the concentration of NKG2A on the cell surface of lymphocytes (e.g., NK cells) in the subject prior to the administering). Some embodiments of these methods result in about a 0.01 -fold increase to about a 10-fold increase (or any of the exemplary subranges of this range described herein) in the ratio of the concentration of NKG2D to the concentration of NKG2A on the cell surface of lymphocytes (e.g., NK cells) in the subject (e.g., as compared to the ratio of the concentration of NKG2D to the concentration of NKG2A on the cell surface of lymphocytes (e.g., NK cells) in the subject prior to the administering).

Some embodiments of the methods described herein result in the at least a 0.1- fold increase, at least a 0.2-fold increase, at least a 0.3-fold increase, at least a 0.4-fold increase, at least a 0.5-fold increase, at least a 0.6-fold increase, at least a 0.7-fold increase, at least a 0.8-fold increase, at least a 0.9-fold increase, at least a 1.0-fold increase, at least a 1.5-fold increase, at least a 2.0-fold increase, at least a 2.5-fold increase, at least a 3.0-fold increase, at least a 3.5-fold increase, at least a 4.0-fold increase, at least a 4.5-fold increase, at least a 5.0-fold increase, at least a 5.5-fold increase, at least a 6.0-fold increase, at least a 6.5-fold increase, at least a 7.0-fold increase, at least a 7.5-fold increase, at least a 8.0-fold incrase, at least a 8.5-fold increase, at least a 9.0-fold increase, at least a 9.5-fold increase, or at least a 10-fold increase in the maximum fold change in the ratio of NKG2D-positive lymphocytes (e.g., NKG2D-positive NK cells) to NKG2A-positive lymphocytes (e.g., NKG2A- positive NK cells) in the blood of a subject (e.g., as compared to the ratio of NKG2D- positive lymphocytes (e.g., NKG2D-positive NK cells) to NKG2A-positive lymphocytes (e.g., NKG2A-positive NK cells) in the blood of the subject prior to the administering). Some embodiments of the methods described herein result about a 0.01-fold increase to about a 10-fold increase (or any of the exemplary subranges of this range described herein) in the maximum fold change in the ratio of NKG2D- positive lymphocytes (e.g., NKG2D-positive NK cells) to NKG2A-positive lymphocytes (e.g., NKG2A-positive NK cells) in the blood of a subject (e.g., as compared to the ratio of NKG2D-positive lymphocytes (e.g., NKG2D-positive NK cells) to NKG2A-positive lymphocytes (e.g., NKG2A-positive NK cells) in the blood of the subject prior to the administering).

Some embodiments of the methods described herein result in at least a 1% increase, at least a 2% increase, at least a 3% increase, at least a 4% increase, at least a 5% increase, at least a 6% increase, at least a 7% increase, at least a 8% increase, at least a 9% increase, at least a 10% increase, at least a 15% increase, at least a 20% increase, at least a 25% increase, at least a 30% increase, at least a 35% increase, at least a 40% increase, at least a 45% increase, at least a 50% increase, at least a 55% increase, at least a 60% increase, at least a 65% increase, at least a 70% increase, at least a 75% increase, at least a 80% increase, at least a 85% increase, at least a 90% increase, at least a 95% increase, at least a 100% increase, at least a 150% increase, at least a 200% increase, at least a 250% increase, at least a 300% increase, at least a 350% increase, at least a 400% increase, at least a 450% increase, at least a 500% increase, at least a 550% increase, at least a 600% increase, at least a 650% increase, at least a 700% increase, at least a 750% increase, at least a 800% increase, at least a 850% increase, at least a 900% increase, at least a 950% increase, or at least a 1000% increase in the absolute number of NK cells in the blood of the subject (e.g., as compared to the absolute number of NK cells in the blood of the subject prior to the administering). Some embodiments of any of the methods described herein, result in about a 1% increase to about a 1,000% increase (or any of the exemplary subranges of this range described herein) in the absolute number of NK cells in the blood of the subject (e.g., as compared to the absolute number of NK cells in the blood of the subject prior to the administering).

Some embodiments of the methods described herein result in the at least a 0.1- fold increase, at least a 0.2-fold increase, at least a 0.3-fold increase, at least a 0.4-fold increase, at least a 0.5-fold increase, at least a 0.6-fold increase, at least a 0.7-fold increase, at least a 0.8-fold increase, at least a 0.9-fold increase, at least a 1.0-fold increase, at least a 1.5-fold increase, at least a 2.0-fold increase, at least a 2.5-fold increase, at least a 3.0-fold increase, at least a 3.5-fold increase, at least a 4.0-fold increase, at least a 4.5-fold increase, or at least a 5.0-fold increase in the maximum fold increase in the absolute number of NK cells in the blood of the subject (e.g., as compared to the maximum fold increase in the absolute number of NK cells in the blood of the subject prior to the administering). Some embodiments of the methods described herein result about a 0.01 -fold increase to about a 5-fold increase (or any of the exemplary subranges of this range described herein) 0 in the maximum fold increase in the absolute number of NK cells in the blood of the subject (e.g., as compared to the maximum fold increase in the absolute number of NK cells in the blood of the subject prior to the administering).

Some embodiments of the methods described herein result in the at least a 0.1- fold increase, at least a 0.2-fold increase, at least a 0.3-fold increase, at least a 0.4-fold increase, at least a 0.5-fold increase, at least a 0.6-fold increase, at least a 0.7-fold increase, at least a 0.8-fold increase, at least a 0.9-fold increase, at least a 1.0-fold increase, at least a 1.5-fold increase, at least a 2.0-fold increase, at least a 2.5-fold increase, at least a 3.0-fold increase, at least a 3.5-fold increase, at least a 4.0-fold increase, at least a 4.5-fold increase, or at least a 5.0-fold increase in the maximum fold increase in the absolute number of CD56^bnght lymphocytes (e.g., CD56^bnght NK cells) in the blood of the subject (e.g., as compared to the maximum fold increase in the absolute number of CD56^bnght lymphocytes (e.g., CD56^bnght NK cells) in the blood of the subject prior to the administering). Some embodiments of the methods described herein result about a 0.01 -fold increase to about a 5-fold increase (or any of the exemplary subranges of this range described herein) in the maximum fold increase in the absolute number of CD56^bnght lymphocytes (e.g., CD56^bnght NK cells) in the blood of the subject (e.g., as compared to the maximum fold increase in the absolute number of CD56^bnght lymphocytes (e.g., CD56^bnght NK cells) in the blood of the subject prior to the administering).

CD56^dim NK cells) in the blood of the subject prior to the administering). Some embodiments of the methods described herein result about a 0.01 -fold increase to about a 5-fold increase (or any of the exemplary subranges of this range described herein) in the maximum fold increase in the absolute number of CD16⁺ CD56^dim lymphocytes (e.g., CD16⁺ CD56^dimNK cells) in the blood of the subject (e.g., as compared to the maximum fold increase in the absolute number of CD16⁺ CD56^dim lymphocytes (e.g., CD16⁺ CD56^dim NK cells) in the blood of the subject prior to the administering).

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

NKG2D

As used herein, a NKG2D polypeptide refers to the amino acid sequence encoded by the killer cell lectin like receptor K1 (KLRKl) gene. KLRKl gene is also known as CD314, NKG2D, and NKG2-D. An exemplary NKG2D polypeptide includes without limitation amino acid sequences corresponding to NCBI reference sequence: NP_031386.2.

In some embodiments, a NKG2D polypeptide includes an amino acid 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

MGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCRENA SPFFFCCFIAVAMGIRFIIMVTIWSAVFLNSLFNQEVQIPLTESYCGPCPKNWIC YKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWM GLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTY ICMQRTV (SEQ ID NO: 1) (with or without the signal sequence).

In some embodiments, a NKG2D polypeptide is encoded by a nucleic acid 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

ATGGGGTGGATTCGTGGTCGGAGGTCTCGACACAGCTGGGAGATGAGTGA

ATTTCATAATTATAACTTGGATCTGAAGAAGAGTGATTTTTCAACACGATG

GCAAAAGCAAAGATGTCCAGTAGTCAAAAGCAAATGTAGAGAAAATGCA

TCTCCATTTTTTTTCTGCTGCTTCATCGCTGTAGCCATGGGAATCCGTTTCA

TTATTATGGTAACAATATGGAGTGCTGTATTCCTAAACTCATTATTCAACC

AAGAAGTTCAAATTCCCTTGACCGAAAGTTACTGTGGCCCATGTCCTAAA

AACTGGATATGTTACAAAAATAACTGCTACCAATTTTTTGATGAGAGTAA

AAACTGGTATGAGAGCCAGGCTTCTTGTATGTCTCAAAATGCCAGCCTTCT

GAAAGTATACAGCAAAGAGGACCAGGATTTACTTAAACTGGTGAAGTCAT

ATCATTGGATGGGACTAGTACACATTCCAACAAATGGATCTTGGCAGTGG

GAAGATGGCTCCATTCTCTCACCCAACCTACTAACAATAATTGAAATGCA

GAAGGGAGACTGTGCACTCTATGCCTCGAGCTTTAAAGGCTATATAGAAA

ACTGTTCAACTCCAAATACGTACATCTGCATGCAAAGGACTGTGTAA (SEQ

ID NO: 2).

In some embodiments, the methods include detecting the number of NKG2D- positive lymphocytes (e.g., NKG2D-positive NK cells) in the subject. Any appropriate sample containing NKG2D-positive lymphocytes (e.g., NKG2D-positive NK cells) can be obtained from a subject. In some embodiments, a whole blood sample, a plasma sample, a serum sample, a sample containing a cell fraction (PBMC) of a whole blood sample, a tissue sample, a tumor sample, a biopsy sample, and a laser capture dissected biological sample (e.g., tumor or tissue sample) can be obtained and assessed to identify the number of NKG2D-positive lymphocytes (e.g., NKG2D-positive NK cells). In some embodiments, detecting the number of NKG2D- positive lymphocytes (e.g., NKG2D-positive NK cells) is done prior to, simultaneously with, or after, administering a therapeutically effective amount of a pharmaceutical composition comprising a population of enucleated erythroid cells (e.g., any of the exemplary enucleated erythroid cells described herein). In some embodiments, the NKG2D-positive lymphocytes (e.g., NKG2D-positive NK cells) can be determined using any an antibody that binds specifically to NKG2D or an antigen-binding fragment thereof (e.g., anti-NKG2D antibody clone 149810 (R&D Systems) and anti-NKG2D mAh clone 1D11 (BD Biosciences)). In some embodiments, NKG2D polypeptide expression can be assessed using fluorescence- assisted cell sorting (FACS) (e.g., using compensation beads (e.g., Bangs beads)) or assessed by other immunological based methods including, without limitation, Western blot analysis, immunohistochemical staining, ELISA, tissue array analysis, in situ hybridization, and immunofluoresence, using, e.g., an antibody that binds specifically to NKG2D or an antigen-binding fragment thereof. In some embodiments, NKG2D-positive lymphocytes (e.g., NKG2D-positive NK cells) can be determined by measuring NKG2D 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.

As noted herein, an NKG2D-positive lymphocyte (e.g., a NKG2D-positive NK cell) is determined by comparing the level of NKG2D in the lymphocyte (e.g.,

NK cell) as compared to a reference level (e.g., a level of NKG2D in a control non- activated or resting lymphocyte (e.g., NK cell), or a level in a cell that is not a lymphocyte (e.g., not an NK cell). In some embodiments, the reference level of NKG2D can be one transcript per one million transcripts. In some embodiments, a NKG2D-positive lymphocyte (e.g., a NKG2D-positive NK cell) is identified based a flow cytometry gating strategy using fluorescence minus one control samples, fluorescence compensation, and qualification experience.

MICA As used herein, a MICA polypeptide refers to the amino acid sequence encoded by the MHC class I polypeptide-related sequence A (MICA) gene. An exemplary MICA polypeptide includes without limitation amino acid sequence corresponding to NCBI reference sequences: NP_000238.1, NP_001170990.1, NP_001276081.1, andNP_001276083.1. MICA, a member of the major histocompatability complex class I, is as a cell-surface ligand for NKG2D.

In some embodiments, a MICA polypeptide can include 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 MGLGPVFLLLAGIFPFAPPGAAAEPHSLRYNLTVLSWDGSVOSGFLTEVHLD GQPFLRCDRQKCRAKPQGQWAEDVLGNKTWDRETRDLTGNGKDLRMTLAH IKDQKEGLHSLQEIRVCEIHEDNSTRSSQHFYYDGELFLSQNLETKEWTMPQS SRAQTLAMNVRNFLKEDAMKTKTHYHAMHADCLQELRRYLKSGVVLRRTV PPMVNVTRSEASEGNITVTCRASGFYPWNITLSWRQDGVSLSHDTQQWGDV LPDGNGTYQTWVATRICQGEEQRFTCYMEHSGNHSTHPVPSGKVLVLQSHW QTFHV S AV AAAAIFVIIIFYVRCCKKKTS AAEGPELVSLQVLDQHPV GTSDHR DATQLGFQPLMSDLGSTGSTEGA (SEQ ID NO: 3) (with or without its signal peptide). The underlined portion of SEQ ID NO: 3 above indicates the signal peptide.

In some embodiments, a MICA polypeptide is encoded by a nucleic acid 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

ATGGGGCTGGGCCCGGTCTTCCTGCTTCTGGCTGGCATCTTCCCTTTTGCA CCTCCGGGAGCTGCTGCTGAGCCCCACAGTCTTCGTTATAACCTCACGGTG CTGTCCTGGGATGGATCTGTGCAGTCAGGGTTTCTCACTGAGGTACATCTG GATGGTCAGCCCTTCCTGCGCTGTGACAGGCAGAAATGCAGGGCAAAGCC CCAGGGACAGTGGGCAGAAGATGTCCTGGGAAATAAGACATGGGACAGA GAGAC C AGAGACTT GAC AGGGAAC GGA A AGGAC CTC AGGAT GAC C CT GG CTCATATCAAGGACCAGAAAGAAGGCTTGCATTCCCTCCAGGAGATTAGG

GTCTGTGAGATCCATGAAGACAACAGCACCAGGAGCTCCCAGCATTTCTA

CTACGATGGGGAGCTCTTCCTCTCCCAAAACCTGGAGACTAAGGAATGGA

CAATGCCCCAGTCCTCCAGAGCTCAGACCTTGGCCATGAACGTCAGGAAT

TTCTTGAAGGAAGATGCCATGAAGACCAAGACACACTATCACGCTATGCA

TGCAGACTGCCTGCAGGAACTACGGCGATATCTAAAATCCGGCGTAGTCC

TGAGGAGAACAGTGCCCCCCATGGTGAATGTCACCCGCAGCGAGGCCTCA

GAGGGCAACATTACCGTGACATGCAGGGCTTCTGGCTTCTATCCCTGGAA

TATCACACTGAGCTGGCGTCAGGATGGGGTATCTTTGAGCCACGACACCC

AGCAGTGGGGGGATGTCCTGCCTGATGGGAATGGAACCTACCAGACCTGG

GTGGCCACCAGGATTTGCCAAGGAGAGGAGCAGAGGTTCACCTGCTACAT

GGAACACAGCGGGAATCACAGCACTCACCCTGTGCCCTCTGGGAAAGTGC

TGGTGCTTCAGAGTCATTGGCAGACATTCCATGTTTCTGCTGTTGCTGCTG

CTGCTATTTTTGTTATTATTATTTTCTATGTCCGTTGTTGTAAGAAGAAAAC

AT C AGCT GC AGAGGGTCC AGAGCT C GT GAGC CT GC AGGTC CT GGATC AAC

ACCCAGTTGGGACGAGTGACCACAGGGATGCCACACAGCTCGGATTTCAG

CCTCTGATGTCAGATCTTGGGTCCACTGGCTCCACTGAGGGCGCCTAG

(SEQ ID NO: 4).

In some embodiments, a MICA polypeptide can include 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

MGLGPVFLLLAGIFPFAPPGAAAEPHSLRYNLTVLSWDGSVOSGFLAEVHLD GQPFLRYDRQKCRAKPQGQWAEDVLGNKTWDRETRDLTGNGKDLRMTLA HIKDQKEGLHSLQEIRVCEIHEDNSTRSSQHFYYDGELFLSQNLETEEWTVPQ SSRAQTLAMNVRNFLKEDAMKTKTHYHAMHADCLQELRRYLESGVVLRRT VPPMVNVTRSEASEGNITVTCRASSFYPRNIILTWRQDGVSLSHDTQQWGDV LPDGNGTYQTWVATRICRGEEQRFTCYMEHSGNHSTHPVPSGKVLVLQSHW QTFHVSAVAAGCCYFCYYYFLCPLL (SEQ ID NO: 5) (with or without its signal peptide). The underlined portion of SEQ ID NO: 5 above indicates the signal peptide.

ATGGGGCTGGGCCCGGTCTTTCTGCTTCTGGCTGGCATCTTCCCTTTTGCA

CCTCCGGGAGCTGCTGCTGAGCCCCACAGTCTTCGTTATAACCTCACGGTG

CTGTCCTGGGATGGATCTGTGCAGTCAGGGTTTCTTGCTGAGGTACATCTG

GATGGTCAGCCCTTCCTGCGCTATGACAGGCAGAAATGCAGGGCAAAGCC

CCAGGGACAGTGGGCAGAAGATGTCCTGGGAAATAAGACATGGGACAGA

GAGAC C AGGGACTT GAC AGGGAAC GGA A AGGAC CTC AGGAT GAC C CT GG

CTCATATCAAGGACCAGAAAGAAGGCTTGCATTCCCTCCAGGAGATTAGG

GTCTGTGAGATCCATGAAGACAACAGCACCAGGAGCTCCCAGCATTTCTA

CTACGATGGGGAGCTCTTCCTCTCCCAAAACCTGGAGACTGAGGAATGGA

CAGTGCCCCAGTCCTCCAGAGCTCAGACCTTGGCCATGAACGTCAGGAAT

TTCTTGAAGGAAGATGCCATGAAGACCAAGACACACTATCACGCTATGCA

TGCAGACTGCCTGCAGGAACTACGGCGATATCTAGAATCCGGCGTAGTCC

TGAGGAGAACAGTGCCCCCCATGGTGAATGTCACCCGCAGCGAGGCCTCA

GAGGGCAACATCACCGTGACATGCAGGGCTTCCAGCTTCTATCCCCGGAA

TATCATACTGACCTGGCGTCAGGATGGGGTATCTTTGAGCCACGACACCC

AGCAGTGGGGGGATGTCCTGCCTGATGGGAATGGAACCTACCAGACCTGG

GTGGCCACCAGGATTTGCCGAGGAGAGGAGCAGAGGTTCACCTGCTACAT

GGAACACAGCGGGAATCACAGCACTCACCCTGTGCCCTCTGGGAAAGTGC

TGGTGCTTCAGAGTCATTGGCAGACATTCCATGTTTCTGCTGTTGCTGCTG

GCTGCTGCTATTTTTGTTATTATTATTTTCTATGTCCGTTGTTGTAA (SEQ ID

NO: 6).

MTLAHIKDQKEGLHSLQEIRVCEIHEDNSTRSSQHFYYDGELFLSQNLETEEW TVPQSSRAQTLAMNVRNFLKEDAMKTKTHYHAMHADCLQELRRYLESGVV LRRTVPPMVNVTRSEASEGNITVTCRASSFYPRNIILTWRQDGVSLSHDTQQW GDVLPDGNGTYQTWVATRICRGEEQRFTCYMEHSGNHSTHPVPSGKVLVLQ SHW QTFHV S AV AAGCCYF CYYYFLCPLL (SEQ ID NO: 7) (with or without its signal peptide). In some embodiments, a MICA polypeptide is encoded by a nucleic acid 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

ATGACCCTGGCTCATATCAAGGACCAGAAAGAAGGCTTGCATTCCCTCCA

GGAGATTAGGGTCTGTGAGATCCATGAAGACAACAGCACCAGGAGCTCCC

AGCATTTCTACTACGATGGGGAGCTCTTCCTCTCCCAAAACCTGGAGACTG

AGGAATGGACAGTGCCCCAGTCCTCCAGAGCTCAGACCTTGGCCATGAAC

GTCAGGAATTTCTTGAAGGAAGATGCCATGAAGACCAAGACACACTATCA

CGCTATGCATGCAGACTGCCTGCAGGAACTACGGCGATATCTAGAATCCG

GCGTAGTCCTGAGGAGAACAGTGCCCCCCATGGTGAATGTCACCCGCAGC

GAGGCCTCAGAGGGCAACATCACCGTGACATGCAGGGCTTCCAGCTTCTA

TCCCCGGAATATCATACTGACCTGGCGTCAGGATGGGGTATCTTTGAGCC

AC GAC ACC C AGC AGT GGGGGGAT GTC CT GC CT GAT GGGAAT GGAAC CT AC

C AGAC CT GGGT GGC C AC C AGGATTTGC CGAGGAGAGGAGC AGAGGTT C A

CCTGCTACATGGAACACAGCGGGAATCACAGCACTCACCCTGTGCCCTCT

GGGAAAGTGCTGGTGCTTCAGAGTCATTGGCAGACATTCCATGTTTCTGCT

GTTGCTGCTGGCTGCTGCTATTTTTGTTATTATTATTTTCTATGTCCGTTGTT

GTAA (SEQ ID NO: 8).

MGQRDQGLDRERKGPQDDPGSYQGPERRNFLKEDAMKTKTHYHAMHADCL QELRRYLESGVVLRRTVPPMVNVTRSEASEGNITVTCRASSFYPRNIILTWRQ DGVSLSHDTQQWGDVLPDGNGTYQTWVATRICRGEEQRFTCYMEHSGNHST HPVPSGKVLVLQSHWQTFHVSAVAAGCCYFCYYYFLCPLL (SEQ ID NO: 9) (with or without its signal peptide).

ATGGGACAGAGAGACCAGGGACTTGACAGGGAACGGAAAGGACCTCAGG ATGACCCTGGCTCATATCAAGGACCAGAAAGAAGGAATTTCTTGAAGGAA

GATGCCATGAAGACCAAGACACACTATCACGCTATGCATGCAGACTGCCT

GCAGGAACTACGGCGATATCTAGAATCCGGCGTAGTCCTGAGGAGAACA

GTGCCCCCCATGGTGAATGTCACCCGCAGCGAGGCCTCAGAGGGCAACAT

CACCGTGACATGCAGGGCTTCCAGCTTCTATCCCCGGAATATCATACTGAC

CTGGCGTCAGGATGGGGTATCTTTGAGCCACGACACCCAGCAGTGGGGGG

ATGTCCTGCCTGATGGGAATGGAACCTACCAGACCTGGGTGGCCACCAGG

ATTT GCC GAGGAGAGGAGC AGAGGTT C AC CT GCT AC AT GGAAC AC AGCG

GGAATCACAGCACTCACCCTGTGCCCTCTGGGAAAGTGCTGGTGCTTCAG

AGTCATTGGCAGACATTCCATGTTTCTGCTGTTGCTGCTGGCTGCTGCTAT

TTTTGTTATTATTATTTTCTATGTCCGTTGTTGTAA (SEQ ID NO: 10).

In some embodiments, the methods include detecting the presence of MIC A- positive cancer cells in the subject. Any appropriate sample containing MICA- positive cancer cells can be obtained from a subject. In some embodiments, a whole blood sample, a plasma sample, a serum sample, a sample containing a cell fraction (PBMC) of a whole blood sample, a tissue sample, a tumor sample, a biopsy sample, and a laser capture dissected biological sample (e.g., tumor or tissue sample) can be obtained and assessed to identify the the number of MICA-positive cancer cells. In some embodiments, detecting the MICA-positive cancer cells is done prior to, simultaneously with, or after, administering the enucleated erythroid cells. In some embodiments, the MICA-positive cancer cells can be determined using an antibody that binds specifically to MICA (e.g., on the cell surface of cancer cells) or an antigen-binding fragment thereof (e.g., anti-MICA antibody ab62540 (Abeam), LS- B9176 (LSBio), and 1E2C8 (ThermoFisher Scientific)). MICA polypeptide expression can be assessed using fluorescence-associated cell sorting (FACS) and can also be assessed by other immunological based methods including, without limitation, Western blotting, immunohistochemical staining, ELISA, and immunofluoresence. In some embodiments, MICA-positive cells can be determined by measuring MICA 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 reference levels of MICA useful for identifying a MICA-positive cancer cell or a MICA-positive cancer can be a level of MICA in a non-cancerous cell, a median level of MICA in multiple tumor biopsy samples, one standard of deviation higher than a median level of MICA in multiple tumor biopsy samples of the same type of cancer or multiple different cancers, quarter of a standard of deviation higher than a median level of MICA in multiple tumor biopsy samples of the same type of cancer or multiple different cancers, half a standard of deviation higher than a median level of MICA in multiple tumor biopsy samples of the same type of cancer or multiple different cancers, three quarters of a standard deviation higher than a median level of MICA in multiple tumor biopsy samples of the same type of cancer or multiple different cancers, one and half standard deviation higher than a median level of MICA in multiple tumor biopsy samples of the same type of cancer or multiple different cancers, or two standard deviations higher than a median level of MICA in multiple tumor biopsy samples of the same type of cancer or multiple different cancers. In some embodiments, the reference level of MICA can be one transcript per one million transcripts.

In some embodiments, a reference level of MICA is a level of MICA in normal (non-cancerous) tissue of the same type. In some embodiments, a reference level of MICA is a level of MICA in a healthy tissue that is proximal to a solid tumor. In some embodiments, the reference level is a level that is 50% greater than the level of MICA in the neighboring tissue or a corresponding healthy tissue.

MICB

As used herein, a MICB polypeptide refers to the amino acid sequence encoded by the MHC class I polypeptide-related sequence B (MICB) gene. An exemplary MICB polypeptide includes without limitation amino acid sequence corresponding to NCBI reference sequences: NP_001276089.1, NP_001276090.1, and NP_005922.2. MICB, a member of the major histocompatability complex class I, is as a cell-surface ligand for NKG2D.

In some embodiments, a MICB polypeptide can include 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

MVLSQDGSVQSGFLAEGHLDGQPFLRYDRQKRRAKPQGQWAENVLGAKTW

DTETEDLTENGQDLRRTLTHIKDQKGGLHSLQEIRVCEIHEDSSTRGSRHFYY DGELFLSQNLETQESTVPQSSRAQTLAMNVTNFWKEDAMKTKTHYRAMQA DCLQKLQRYLKSGVAIRRTVPPMVNVTCSEVSEGNITVTCRASSFYPRNITLT WRQDGVSLSHNTQQWGDVLPDGNGTYQTWVATRIRQGEEQRFTCYMEHSG NHGTHPVPSGKALVLQSQRTDFPYVSAAMPCFVIIIILCVPCCKKKTSAAEGPE LVSLQVLDQHPVGTGDHRDAAQLGFQPLMSATGSTGSTEGT (SEQ ID NO:

11) (with or without its signal peptide).

In some embodiments, a MICB polypeptide is encoded by a nucleic acid 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

ATGGTGCTGTCCCAGGATGGATCTGTGCAGTCAGGGTTTCTCGCTGAGGG

ACATCTGGATGGTCAGCCCTTCCTGCGCTATGACAGGCAGAAACGCAGGG

CAAAGCCCCAGGGACAGTGGGCAGAAAATGTCCTGGGAGCTAAGACCTG

GGACACAGAGACCGAGGACTTGACAGAGAATGGGCAAGACCTCAGGAGG

ACCCTGACTCATATCAAGGACCAGAAAGGAGGCTTGCATTCCCTCCAGGA

GATTAGGGTCTGTGAGATCCATGAAGACAGCAGCACCAGGGGCTCCCGGC

ATTTCTACTACGATGGGGAGCTCTTCCTCTCCCAAAACCTGGAGACTCAAG

AATCGACAGTGCCCCAGTCCTCCAGAGCTCAGACCTTGGCTATGAACGTC

ACAAATTTCTGGAAGGAAGATGCCATGAAGACCAAGACACACTATCGCGC

TATGCAGGCAGACTGCCTGCAGAAACTACAGCGATATCTGAAATCCGGGG

TGGCCATCAGGAGAACAGTGCCCCCCATGGTGAATGTCACCTGCAGCGAG

GTCTCAGAGGGCAACATCACCGTGACATGCAGGGCTTCCAGCTTCTATCC

CCGGAATATCACACTGACCTGGCGTCAGGATGGGGTATCTTTGAGCCACA

ACACCCAGCAGTGGGGGGATGTCCTGCCTGATGGGAATGGAACCTACCAG

AC CT GGGT GGCC AC C AGGATT C GC C AAGGAGAGGAGC AGAGGTT C ACCT

GCTACATGGAACACAGCGGGAATCACGGCACTCACCCTGTGCCCTCTGGG

AAGGCGCTGGTGCTTCAGAGTCAACGGACAGACTTTCCATATGTTTCTGCT

GCTATGCCATGTTTTGTTATTATTATTATTCTCTGTGTCCCTTGTTGCAAGA

AGAAAACATCAGCGGCAGAGGGTCCAGAGCTTGTGAGCCTGCAGGTCCTG

GATCAACACCCAGTTGGGACAGGAGACCACAGGGATGCAGCACAGCTGG

GATTTCAGCCTCTGATGTCAGCTACTGGGTCCACTGGTTCCACTGAGGGCA

CCTAG (SEQ ID NO: 12). In some embodiments, a MICB polypeptide can include 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

MGLGRVLLFLAVAFPFAPPAAAAEPHSLRYNLMVLSODGSVOSGFLAEGHLD GQPFLRYDRQKRRAKPQGQWAENVLGAKTWDTETEDLTENGQDLRRTLTHI KDQKGVPQSSRAQTLAMNVTNFWKEDAMKTKTHYRAMQADCLQKLQRYL KSGVAIRRTVPPMVNVTCSEVSEGNITVTCRASSFYPRNITLTWRQDGVSLSH NTQQWGDVLPDGNGTYQTWVATRIRQGEEQRFTCYMEHSGNHGTHPVPSG KALVLQSQRTDFPYVSAAMPCFVIIIILCVPCCKKKTSAAEGPELVSLQVLDQH PV GTGDHRDAAQLGF QPLMS ATGSTGSTEGT (SEQ ID NO: 13) (with or without its signal peptide). The underlined portion of SEQ ID NO: 13 above indicates the signal peptide.

ATGGGGCTGGGCCGGGTCCTGCTGTTTCTGGCCGTCGCCTTCCCTTTTGCA

CCCCCGGCAGCCGCCGCTGAGCCCCACAGTCTTCGTTACAACCTCATGGT

GCTGTCCCAGGATGGATCTGTGCAGTCAGGGTTTCTCGCTGAGGGACATC

TGGATGGTCAGCCCTTCCTGCGCTATGACAGGCAGAAACGCAGGGCAAAG

CCCCAGGGACAGTGGGCAGAAAATGTCCTGGGAGCTAAGACCTGGGACA

CAGAGACCGAGGACTTGACAGAGAATGGGCAAGACCTCAGGAGGACCCT

GACTCATATCAAGGACCAGAAAGGAGTGCCCCAGTCCTCCAGAGCTCAGA

CCTTGGCTATGAACGTCACAAATTTCTGGAAGGAAGATGCCATGAAGACC

AAGACACACTATCGCGCTATGCAGGCAGACTGCCTGCAGAAACTACAGCG

ATATCTGAAATCCGGGGTGGCCATCAGGAGAACAGTGCCCCCCATGGTGA

ATGTCACCTGCAGCGAGGTCTCAGAGGGCAACATCACCGTGACATGCAGG

GCTTCCAGCTTCTATCCCCGGAATATCACACTGACCTGGCGTCAGGATGG

GGTATCTTTGAGCCACAACACCCAGCAGTGGGGGGATGTCCTGCCTGATG

GGAATGGAACCTACCAGACCTGGGTGGCCACCAGGATTCGCCAAGGAGA

GGAGCAGAGGTTCACCTGCTACATGGAACACAGCGGGAATCACGGCACTC

ACCCTGTGCCCTCTGGGAAGGCGCTGGTGCTTCAGAGTCAACGGACAGAC TTTCCATATGTTTCTGCTGCTATGCCATGTTTTGTTATTATTATTATTCTCTG TGTCCCTTGTTGCAAGAAGAAAACATCAGCGGCAGAGGGTCCAGAGCTTG TGAGCCTGCAGGTCCTGGATCAACACCCAGTTGGGACAGGAGACCACAGG GATGCAGCACAGCTGGGATTTCAGCCTCTGATGTCAGCTACTGGGTCCAC TGGTTCCACTGAGGGCACCTAG (SEQ ID NO: 14).

MGLGRVLLFLAVAFPFAPPAAAAEPHSLRYNLMVLSODGSVOSGFLAEGHLD

GQPFLRYDRQKRRAKPQGQWAENVLGAKTWDTETEDLTENGQDLRRTLTHI

KDQKGGLHSLQEIRVCEIHEDSSTRGSRHFYYDGELFLSQNLETQESTVPQSSR

AQTLAMNVTNFWKEDAMKTKTHYRAMQADCLQKLQRYLKSGVAIRRTVPP

MVNVTCSEVSEGNITVTCRASSFYPRNITLTWRQDGVSLSHNTQQWGDVLPD

GN GTY QTWV ATRIRQ GEEQRFT C YMEH S GNHGTHP VP S GKALVLQ S QRTDF

PYVSAAMPCFVIIIILCVPCCKKKTSAAEGPELVSLQVLDQHPVGTGDHRDAA

QLGFQPLMSATGSTGSTEGT (SEQ ID NO: 15) (with or without its signal peptide). The underlined portion of SEQ ID NO: 15 above indicates the signal peptide.

ATGGGGCTGGGCCGGGTCCTGCTGTTTCTGGCCGTCGCCTTCCCTTTTGCA

CCCCCGGCAGCCGCCGCTGAGCCCCACAGTCTTCGTTACAACCTCATGGT

GCTGTCCCAGGATGGATCTGTGCAGTCAGGGTTTCTCGCTGAGGGACATC

TGGATGGTCAGCCCTTCCTGCGCTATGACAGGCAGAAACGCAGGGCAAAG

CCCCAGGGACAGTGGGCAGAAAATGTCCTGGGAGCTAAGACCTGGGACA

CAGAGACCGAGGACTTGACAGAGAATGGGCAAGACCTCAGGAGGACCCT

GACTCATATCAAGGACCAGAAAGGAGGCTTGCATTCCCTCCAGGAGATTA

GGGTCTGTGAGATCCATGAAGACAGCAGCACCAGGGGCTCCCGGCATTTC

TACTACGATGGGGAGCTCTTCCTCTCCCAAAACCTGGAGACTCAAGAATC

GACAGTGCCCCAGTCCTCCAGAGCTCAGACCTTGGCTATGAACGTCACAA ATTTCTGGAAGGAAGATGCCATGAAGACCAAGACACACTATCGCGCTATG

CAGGCAGACTGCCTGCAGAAACTACAGCGATATCTGAAATCCGGGGTGGC

CATCAGGAGAACAGTGCCCCCCATGGTGAATGTCACCTGCAGCGAGGTCT

CAGAGGGCAACATCACCGTGACATGCAGGGCTTCCAGCTTCTATCCCCGG

AATATCACACTGACCTGGCGTCAGGATGGGGTATCTTTGAGCCACAACAC

CCAGCAGTGGGGGGATGTCCTGCCTGATGGGAATGGAACCTACCAGACCT

GGGTGGC C ACC AGGATT C GC C AAGGAGAGGAGC AGAGGTT C AC CT GCT A

CATGGAACACAGCGGGAATCACGGCACTCACCCTGTGCCCTCTGGGAAGG

CGCTGGTGCTTCAGAGTCAACGGACAGACTTTCCATATGTTTCTGCTGCTA

TGCCATGTTTTGTTATTATTATTATTCTCTGTGTCCCTTGTTGCAAGAAGAA

AACATCAGCGGCAGAGGGTCCAGAGCTTGTGAGCCTGCAGGTCCTGGATC

AAC AC CC AGTT GGGAC AGGAGAC C AC AGGGAT GC AGC AC AGCTGGGATT

TCAGCCTCTGATGTCAGCTACTGGGTCCACTGGTTCCACTGAGGGCACCTA

G (SEQ ID NO: 16).

In some embodiments, the methods include detecting the presence of MICB- positive cancer cells in the subject. Any appropriate sample containing MICB- positive cancer cells can be obtained from a subject. In some embodiments, a whole blood sample, a plasma sample, a serum sample, a sample containing a cell fraction (PBMC) of a whole blood sample, a tissue sample, a tumor sample, a biopsy sample, and a laser capture dissected biological sample (e.g., tumor or tissue sample) can be obtained and assessed to identify the the number of MICB-positive cancer cells. In some embodiments, detecting the MICB-positive cancer cells is done prior to, simultaneously with, or after, administering the enucleated erythroid cells. In some embodiments, the MICB-positive cancer cells can be determined using an antibody that binds specifically to MICB (e.g., on the cell surface of cancer cells) or an antigen binding fragment thereof (e.g., anti-MICB antibody MM0473-3C37 (Abeam), anti- MICB antibody 3C37 (Abeam), and anti-MICB antibody ARG56879 (Arigo Biolaboratories)). MICB polypeptide expression can be assessed using fluorescence- associated cell sorting (FACS) and can also be assessed by other immunological based methods including, without limitation, Western blotting, immunohistochemical staining, ELISA, and immunofluoresence. In some embodiments, MICB-positive cells can be determined by measuring MICB 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 reference levels of MICB useful for identifying a MICB-positive cancer cell or a MICB-positive cancer can be a level of MICB in a non-cancerous cell, a median level of MICB in multiple tumor biopsy samples, one standard of deviation higher than a median level of MICB in multiple tumor biopsy samples of the same type of cancer or multiple different cancers, quarter of a standard of deviation higher than a median level of MICB in multiple tumor biopsy samples of the same type of cancer or multiple different cancers, half a standard of deviation higher than a median level of MICB in multiple tumor biopsy samples of the same type of cancer or multiple different cancers, three quarters of a standard deviation higher than a median level of MICB in multiple tumor biopsy samples of the same type of cancer or multiple different cancers, one and half standard deviation higher than a median level of MICB in multiple tumor biopsy samples of the same type of cancer or multiple different cancers, or two standard deviations higher than a median level of MICB in multiple tumor biopsy samples of the same type of cancer or multiple different cancers. In some embodiments, the reference level of MICB can be one transcript per one million transcripts.

In some embodiments, a reference level of MICB is a level of MICB in normal (non-cancerous) tissue of the same type. In some embodiments, a reference level of MICB is a level of MICB in a healthy tissue that is proximal to a solid tumor. In some embodiments, the reference level is a level that is 50% greater than the level of MICB in the neighboring tissue or a corresponding healthy tissue.

NKG2A

As used herein, a NKG2A polypeptide refers to the amino acid sequence encoded by the killer cell lectin like receptor Cl (KLRC1) gene. NKG2A forms a complex with KLRD1/CD94. An exemplary KLRC1 polypeptide includes without limitation amino acid sequence corresponding to NCBI reference sequences: NP_001291377.1, NP_002250.2 and NP_998823.1.

In some embodiments, a NKG2A polypeptide can include 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

MDNQGVIYSDLNLPPNPKRQQRKPKGNKNSILATEQEITYAELNLQKASQDF QGNDKTYHCKDLPSAPEKLIVGILGIICLILMASVVTIVVIPSTLIQRHNNSSLN TRTQKARHCGHCPEEWITYSNSCYYIGKERRTWEESLLACTSKNSSLLSIDNE EEMKFLSIISPSSWIGVFRNSSHHPWVTMNGLAFKHEIKDSDNAELNCAVLQV NRLKSAQCGSSIIYHCKHKL (SEQ ID NO: 17).

In some embodiments, a NKG2A polypeptide is encoded by a nucleic acid 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

ATGGATAACCAAGGAGTAATCTACTCAGACCTGAATCTGCCCCCAAACCC

AAAGAGGC AGC AAC GAAAAC CT AAAGGC AATAAAAACT C C ATTTT AGC A

ACTGAACAGGAAATAACCTATGCGGAATTAAACCTTCAAAAAGCTTCTCA

GGATTTTCAAGGGAATGACAAAACCTATCACTGCAAAGATTTACCATCAG

CTCCAGAGAAGCTCATTGTTGGGATCCTGGGAATTATCTGTCTTATCTTAA

TGGCCTCTGTGGTAACGATAGTTGTTATTCCCTCTACATTAATACAGAGGC

ACAACAATTCTTCCCTGAATACAAGAACTCAGAAAGCACGTCATTGTGGC

CATTGTCCTGAGGAGTGGATTACATATTCCAACAGTTGTTACTACATTGGT

AAGGAAAGAAGAACTTGGGAAGAGAGTTTGCTGGCCTGTACTTCGAAGA

ACTCCAGTCTGCTTTCTATAGATAATGAAGAAGAAATGAAATTTCTGTCCA

TCATTTCACCATCCTCATGGATTGGTGTGTTTCGTAACAGCAGTCATCATC

CATGGGTGACAATGAATGGTTTGGCTTTCAAACATGAGATAAAAGACTCA

GATAATGCTGAACTTAACTGTGCAGTGCTACAAGTAAATCGACTTAAATC

AGCCCAGTGTGGATCTTCAATAATATATCATTGTAAGCATAAGCTTTAG

(SEQ ID NO: 18).

MDNQGVIYSDLNLPPNPKRQQRKPKGNKSSILATEQEITYAELNLQKASQDFQ

GNDKTYHCKDLPSAPEKLIVGILGIICLILMASVVTIVVIPSTLIQRHNNSSLNT

RTQKARHCGHCPEEWITYSNSCYYIGKERRTWEESLLACTSKNSSLLSIDNEE EMKFL SII SP S S WIGVFRN S SHHPWVTMN GL AFKHEIKD SDN AELN C AVLQ VN RLKSAQCGSSIIYHCKHKL (SEQ ID NO: 19).

ATGGATAACCAAGGAGTAATCTACTCAGACCTGAATCTGCCCCCAAACCC

AAAGAGGC AGC AAC GAAAAC CT AAAGGC AATAAAAACT C C ATTTT AGC A

ACTGAACAGGAAATAACCTATGCGGAATTAAACCTTCAAAAAGCTTCTCA

GGATTTTCAAGGGAATGACAAAACCTATCACTGCAAAGATTTACCATCAG

CTCCAGAGAAGCTCATTGTTGGGATCCTGGGAATTATCTGTCTTATCTTAA

TGGCCTCTGTGGTAACGATAGTTGTTATTCCCTCTACATTAATACAGAGGC

ACAACAATTCTTCCCTGAATACAAGAACTCAGAAAGCACGTCATTGTGGC

CATTGTCCTGAGGAGTGGATTACATATTCCAACAGTTGTTACTACATTGGT

AAGGAAAGAAGAACTTGGGAAGAGAGTTTGCTGGCCTGTACTTCGAAGA

ACTCCAGTCTGCTTTCTATAGATAATGAAGAAGAAATGAAATTTCTGTCCA

TCATTTCACCATCCTCATGGATTGGTGTGTTTCGTAACAGCAGTCATCATC

CATGGGTGACAATGAATGGTTTGGCTTTCAAACATGAGATAAAAGACTCA

GATAATGCTGAACTTAACTGTGCAGTGCTACAAGTAAATCGACTTAAATC

AGCCCAGTGTGGATCTTCAATAATATATCATTGTAAGCATAAGCTTTAG(S

EQ ID NO: 20).

In some embodiments, the methods include detecting the level of NKG2A in lymphocytes (e.g., NK cells) in the subject. Any appropriate sample containing NKG2A-negative or NKG2D-positive/NKG2A-negative lymphocytes (e.g., NKG2A- negative or NKG2D-positive/NKG2A-negative NK cells) can be obtained from a subject. In some embodiments, a whole blood sample, a plasma sample, a serum sample, a sample containing a cell fraction (PBMC) of a whole blood sample, a tissue sample, a tumor sample, a biopsy sample, and a laser capture dissected biological sample (e.g., tumor or tissue sample) can be obtained and assessed to identify the number of NKG2A-negative or NKG2D-positive/NKG2A-negative lymphocytes (e.g., NKG2A-negative or NKG2D-positive/NKG2A-negative NK cells). In some embodiments, detecting the NKG2A level in lymphocytes (e.g., NK cells) is done prior to, simultaneously with, or after, administering the enucleated erythroid cells. In some embodiments, NKG2A-negative lymphocytes (e.g., NKG2A-negative NK cells) can be determined using an antibody that binds specifically to NKG2A or an antigen binding fragment thereof (e.g., Beckman Coulter, clone Z199.1, and anti-NKG2A mAh clone 131411 (BD Biosciences). In some embodiments, the level of NKG2A in a lymphocyte (e.g., an NK cell) can be determined using fluorescence-assisted cell sorting. The level of NKG2A in a lymphocyte (e.g., an NK cell) can also be assessed by other immunological based methods including, without limitation, Western blotting, immunohistochemical staining, ELISA, and immunofluoresence. In some embodiments, NKG2A-positive lymphocytes (e.g., NKG2A-positive NK 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 reference levels ofNKG2A useful for identifying aNKG2A- negative lymphocyte (e.g., a NKG2A-negative NK cell) are known in the art. For example, a NKG2A-positive lymphocyte (e.g., a NKG2A-positive NK cell) is identified based on a flow cytometry gating strategy using fluorescence minus one control samples, fluorescence compensation, and qualification experience.

In some embodiments, the methods include modulating the activity of an NKG2A polypeptide. In some embodiments, the method further includes administering to the subject an NKG2A inhibitor (e.g., antagonistic antibodies). For example, the method can include administering monalizumab (see, Andre et ak, Cell 175(7): 1731-1743; 2018), dasatinib (Chang et ak, Front. Immunol. 9:31521; 2019), HY-0102, or S095029 (NCT05162755) to the subject, before, simultaneously with, or after administering any of the enucleated erythroid cells described herein.

HLA-E

As used herein, an HLA-E polypeptide refers to an amino acid sequence encoded by the major histocompatibility complex, class I, E (HLA-E) gene. An exemplary HLA-E polypeptide includes without limitation amino acid sequence corresponding to NCBI reference sequences: NP_005507.3.

In some embodiments, an HLA-E polypeptide can include an amino acid 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

MVDGTLLLLLSEALALTOTWAGSHSLKYFHTSVSRPGRGEPRFISVGYVDDT QFVRFDNDAASPRMVPRAPWMEQEGSEYWDRETRSARDTAQIFRVNLRTLR GYYNQSEAGSHTLQWMHGCELGPDGRFLRGYEQFAYDGKDYLTLNEDLRS WT AVDT AAQI SEQKSND ASE AEHQRAYLEDT C VEWLHKYLEKGKETLLHLE PPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQQDGEGHTQDTELVETRP AGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPVTLRWKPASQPTIPIVGII AGL VLLGS V V S GAV V AAVIWRKKS S GGKGGS Y SKAEWSD S AQGS ESH SL (SEQ ID NO: 21) (with or without the signal peptide). The signal peptide is underlined in SEQ ID NO: 21.

In some embodiments, a HLA-E polypeptide is encoded by a nucleic acid 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

ATGGTAGATGGAACCCTCCTTTTACTCCTCTCGGAGGCCCTGGCCCTTACC

CAGACCTGGGCGGGCTCCCACTCCTTGAAGTATTTCCACACTTCCGTGTCC

CGGCCCGGCCGCGGGGAGCCCCGCTTCATCTCTGTGGGCTACGTGGACGA

CACCCAGTTCGTGCGCTTCGACAACGACGCCGCGAGTCCGAGGATGGTGC

CGCGGGCGCCGTGGATGGAGCAGGAGGGGTCAGAGTATTGGGACCGGGA

GACACGGAGCGCCAGGGACACCGCACAGATTTTCCGAGTGAATCTGCGGA

CGCTGCGCGGCTACTACAATCAGAGCGAGGCCGGGTCTCACACCCTGCAG

TGGATGCATGGCTGCGAGCTGGGGCCCGACGGGCGCTTCCTCCGCGGGTA

TGAACAGTTCGCCTACGACGGCAAGGATTATCTCACCCTGAATGAGGACC

TGCGCTCCTGGACCGCGGTGGACACGGCGGCTCAGATCTCCGAGCAAAAG

TCAAATGATGCCTCTGAGGCGGAGCACCAGAGAGCCTACCTGGAAGACAC

ATGCGTGGAGTGGCTCCACAAATACCTGGAGAAGGGGAAGGAGACGCTG

CTTCACCTGGAGCCCCCAAAGACACACGTGACTCACCACCCCATCTCTGA

CCATGAGGCCACCCTGAGGTGCTGGGCCCTGGGCTTCTACCCTGCGGAGA

TCACACTGACCTGGCAGCAGGATGGGGAGGGCCATACCCAGGACACGGA

GCTCGTGGAGACCAGGCCTGCAGGGGATGGAACCTTCCAGAAGTGGGCA

GCTGTGGTGGTGCCTTCTGGAGAGGAGCAGAGATACACGTGCCATGTGCA

GCATGAGGGGCTACCCGAGCCCGTCACCCTGAGATGGAAGCCGGCTTCCC AGCCCACCATCCCCATCGTGGGCATCATTGCTGGCCTGGTTCTCCTTGGAT CTGTGGTCTCTGGAGCTGTGGTTGCTGCTGTGATATGGAGGAAGAAGAGC TCAGGTGGAAAAGGAGGGAGCTACTCTAAGGCTGAGTGGAGCGACAGTG CCCAGGGGTCTGAGTCTCACAGCTTGTAA (SEQ ID NO: 22). In some embodiments, the methods include detecting the level of HLA-E in cancer cells or in a cancer in the subject. Any appropriate sample containing HLA-E cancer cells can be obtained from a subject. In some embodiments, whole blood, blood plasma, cell fraction (PBMC) of whole blood, a tissue sample, a biopsy sample, and laser capture microdissection of a tissue sample or biopsy sample can be obtained and assessed to identify the level of HLA-E in cancer cells in a subject. In some embodiments, detecting the HLA-E level in cancer cells is done prior to, simultaneously with, or after, administering the enucleated erythroid cells. In some embodiments, HLA-E- negative cancer cells can be determined using an antibody that binds specifically to HLA-E or an antigen-binding fragment thereof (e.g., Beckman Coulter, clone Z199.1). In some embodiments, the level of HLA-E in a cancer cell can be determined using fluorescence-assisted cell sorting. The level of HLA-E in a cancer cell can also be assessed by other immunological based methods including, without limitation, Western blotting, immunohistochemical staining, ELISA, and immunofluoresence. In some embodiments, HLA-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 examples, a HLA-E-negative cancer cell is a cancer cell that has loss of heterozygosity at the HLA-E gene locus.

Exemplary reference levels of HLA-E useful for identifying a HLA-E- negative cancer cell can be a level of HLA-E present in a non-cancerous cell or a cancer cell not having a loss of heterozygosity at the HLA-E gene locus. Exemplary levels of HLA-E can be determined using the methods described in Seliger et al., Oncotarget 7(41):67360-67372, 2016). In some embodiments, a reference level of HLA-E is a level of HLA-E in a healthy tissue that is proximal to a solid tumor. In some embodiments, the reference level is a level that is 50% less than the level of HLA-E in the neighboring tissue or a corresponding healthy tissue. First Exogenous Polypeptides

In some embodiments, this disclosure features an enucleated erythroid cell that includes 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, on its extracellular surface.

In some embodiments, the IL-15 includes the immature form of wild-type human IL-15. In some embodiments, the IL-15 includes an amino acid 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

MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVIS DLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHD TVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 23), or a functional fragment thereof. In some embodiments, the IL-15 includes a sequence of SEQ ID NO: 23.

In some embodiments, the IL-15 includes the mature form of wild type human IL-15, or a functional fragment thereof. In some embodiments, the IL-15 includes an amino acid 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

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLE SGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ MFINTS (SEQ ID NO: 16), or a functional fragment thereof. In some embodiments, the IL-15 includes a sequence of SEQ ID NO: 24.

In some embodiments, the IL-15 is encoded by a nucleic acid 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

AACTGGGTGAACGTTATTAGTGACCTTAAAAAGATCGAAGATTTGATACA

GTCAATGCACATAGACGCGACGCTTTATACAGAATCTGATGTACATCCTTC

ATGCAAGGTTACTGCTATGAAGTGTTTTCTTCTCGAACTCCAAGTAATAAG

TCTTGAGAGCGGAGATGCGAGCATTCATGACACCGTTGAGAATCTTATTA

TATTGGCTAACAACTCTCTGTCCAGCAATGGTAATGTGACAGAAAGCGGG TGTAAGGAGTGCGAGGAACTCGAGGAGAAGAACATCAAAGAGTTCTTGC AGTCTTTCGTCCATATTGTCCAGATGTTCATAAATACTAGC (SEQ ID NO:

25).

AACT GGGT A A AC GT CAT A AGC GAC CTC AA A A AGAT C GAGGAC C TT AT AC A

GTCTATGCACATAGATGCGACACTTTACACGGAATCAGACGTGCACCCGT

CCTGCAAAGTCACAGCCATGAAGTGCTTTCTTCTCGAACTGCAGGTAATTT

CTCTCGAATCAGGTGACGCATCTATCCACGACACAGTTGAAAATCTTATTA

TCCTGGCTAATAACTCCCTTAGCTCAAACGGCAATGTCACCGAGAGTGGA

TGTAAAGAATGTGAGGAACTTGAAGAGAAAAACATAAAGGAATTCTTGC

AGAGTTTCGTTCATATTGTGCAAATGTTCATCAATACTAGT (SEQ ID NO:

26).

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

In some embodiments, the interleukin- 15 receptor alpha (IL-15RA) or a functional fragment thereof can include the immature form of wild-type human IL- 15RA. In some embodiments, the IL-15RA can include 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

MAPRRARGCRTLGLPALLLLLLLRPPATRGITCPPPMSVEHADIWVKSYSLYS

RERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAP

PSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPST

GTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVL

LCGLSAVSLLACYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHH

L (SEQ ID NO: 27, or a functional fragment thereof. In some embodiments, the IL-

15RA polypeptide includes a sequence of SEQ ID NO: 27.

In some embodiments, the IL-15RA includes the mature form of wild-type human IL-15RA. In some embodiments, the IL-15RA includes an amino acid 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

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNV AHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSN NTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASH QPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVEMEAM EALP VTW GTS SRDEDLEN C SHHL (SEQ ID NO: 28), or a functional fragment thereof. In some embodiments, the IL-15RA includes a sequence of SEQ ID NO: 28.

In some embodiments, the IL-15RA includes an extracellular portion of an IL- 15RA polypeptide. For example, the IL-15RA polypeptide may lack the transmembrane domain of wild type IL-15RA, and optionally, the intracellular domain of wild type IL-15RA. In some embodiments, the IL-15RA includes the immature form of an extracellular wild type human IL-15RA. In some embodiments, the IL-15RA polypeptide can include 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 MAPRRARGCRTLGLPALLLLLLLRPPATRGITCPPPMSVEHADIWVKSYSLYS RERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAP PSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPST GTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTT (SEQ ID NO: 29), or a functional fragment thereof. In some embodiments, an IL-15RA polypeptide includes a sequence of SEQ ID NO: 29.

In some embodiments, the IL-15RA includes the mature form of an extracellular wild-type human IL-15RA. In some embodiments, the IL-15RA can include 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

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNV AHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSN NTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASH QPPGVYPQGHSDTT (SEQ ID NO: 30), or a functional fragment thereof. In some embodiments, an IL-15RA polypeptide includes a sequence of SEQ ID NO: 30.

In some embodiments, the IL-15RA is encoded by a nucleic acid 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

ATCACATGCCCACCGCCCATGTCTGTTGAACACGCAGACATTTGGGTTAA

AAGTTACTCACTTTACTCACGCGAGAGATATATATGCAACAGCGGCTTCA

AGCGCAAAGCAGGCACTAGTAGTCTTACAGAGTGCGTGCTCAATAAAGCT

ACAAATGTAGCTCATTGGACTACTCCTAGTCTCAAATGCATTCGGGACCCC

GCGCTTGTGCACCAGAGACCTGCGCCGCCGTCCACAGTGACGACAGCTGG

TGTAACCCCCCAACCTGAATCCCTTAGTCCGTCTGGTAAAGAACCGGCGG

CGTCTTCACCTTCCAGCAATAATACTGCGGCGACAACAGCCGCGATAGTT

CCTGGATCCCAACTCATGCCGTCAAAGTCTCCTTCAACGGGAACGACAGA

GATCTCTTCACATGAAAGTTCTCATGGAACACCGAGCCAAACTACGGCAA

AGAACTGGGAACTGACTGCCTCAGCAAGCCACCAGCCGCCAGGGGTGTAC

CCGCAAGGGCACTCAGATACTACT (SEQ ID NO: 31).

In some embodiments, the IL-15RA or functional fragment thereof includes the “sushi domain” in exon 2 of the extracellular domain of the receptor (Wei et al., J Immunol. 2001; 167:277-282). In some embodiments, the IL-15RA or functional fragment thereof that includes the sushi domain of wild type human IL-15RA includes an amino acid sequence that it 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

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNV AHWTTPSLKCIR (SEQ ID NO: 32), or a functional fragment thereof. In some embodiments, the IL-15RA or functional fragment thereof includes the sushi domain of wild-type human IL-15RA that includes a sequence of SEQ ID NO: 32.

In some embodiments, the IL-15RA includes the sushi domain of wild type human IL15RA or a functional fragment thereof and 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 additional amino acids of wild type human IL-15RA. In some embodiments, the IL-15RA or a functional fragment thereof includes the sushi domain of wild type human IL-15RA and 13 additional amino acids of wild type human IL-15RA. In some embodiments, the IL-15RA includes an amino acid 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

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNV AHWTTPSLKCIRDPALVHQRPAPPS (SEQ ID NO: 33), or a functional fragment thereof. In some embodiments, an IL-15RA polypeptide includes a sequence of SEQ ID NO: 33.

ATCACCTGCCCGCCTCCCATGAGCGTGGAACACGCGGACATTTGGGTTAA GAGCTACAGTCTTTACAGCCGGGAGCGCTATATCTGCAACTCAGGGTTTA AGCGGAAAGCAGGGACATCAAGTTTGACAGAATGTGTGTTGAACAAGGC TACAAATGTTGCTCACTGGACCACGCCATCTTTGAAGTGTATCCGAGATCC CGCGCTTGTCCATCAGCGCCCAGCGCCTCCCTCC (SEQ ID NO: 34).

In some embodiments, the functional fragment of the 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 the 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 comprises the IL-15RA sushi domain. In some embodiments, the IL-15RA fragments or variants retain at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of the ability of wild type human IL-15RA to bind an IL-15, as measured by assays well known in the art, e.g., ELISA, surface plasmon resonance (SPR) binding analysis, and co- immunoprecipitation. In some embodiments, IL-15RA variants or fragments retain at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the ability of wild type human IL-15RA polypeptide to induce IL-15 -mediated signal transduction, as measured by assays well-known in the art, e.g., electromobility shift assays, ELISAs and other immunoassays. In some embodiments, a functional fragment of the IL-15RA polypeptide includes one or both of the sushi domain and the transmembane domain of IL-15RA polypeptide. In some embodiments, a functional fragment of the IL-15-RA polypeptide includes the sushi domain. In some embodiments, a functional fragment of the IL-15-RA polypeptide includes the 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 that includes an amino acid sequence set forth 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 an amino acid sequence of SEQ ID NO: 35. In some embodiments, the first exogenous polypeptide includes a leader (signal) sequence that includes the amino acid sequence of SEQ ID NO: 35 an IL-15 polypeptide, and an IL-15RA polypeptide. In some embodiments, the first exogenous polypeptide includes a leader (signal) sequence that includes the amino acid sequence of SEQ ID NO: 35, a mature human IL-15 polypeptide that includes the amino acid sequence of SEQ ID NO: 24, and an IL-15RA polypeptide that includes the amino acid sequence of SEQ ID NO: 30. In some embodiments, the mature human IL-15 polypeptide and the IL-15RA polypeptide are connected by a flexible linker having an amino acid sequence of SEQ ID NO: 37.

In some embodiments, one or more linkers are disposed between the IL-15 or the functional fragment thereof, and IL-15RA or the functional fragment thereof. Any of the linkers provided herein may be used. In some embodiments, the linker is a peptide that 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 long. In some embodiments, the linker is long enough to preserve the ability of IL-15 to bind to the IL-15RA. In other embodiments, the linker is long enough to preserve the ability of the IL-15/IL-15RA complex to bind to the bg IL-15 receptor complex and to act as an agonist to mediate IL-15 signal transduction. In some embodiments, the linker includes an amino acid sequence listed in Table 2. In some embodiments, the linker comprises the amino acid sequence (GGGGS)_n (SEQ ID NO: 38), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, the linker consists of the (GGGGS)_n linker (SEQ ID NO: 30), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, the linker includes the amino acid sequence GGGGS GGGGS GGGGS (SEQ ID NO: 37).

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

In some embodiments, the first exogenous polypeptide includes an IL-15 polypeptide or a functional fragment thereof and an extracellular region of the IL- 15Ra polypeptide. In some embodiments, the first exogenous polypeptide includes the amino acid sequence of SEQ ID NO: 39. In some embodiments, the first exogenous polypeptide includes as 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 NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLE SGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ MFINTSGGGGSGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSG FKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAG VTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHE SSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTT (SEQ ID NO: 39).

In some embodiments, an first exogenous polypeptide is encoded by a nucleic acid 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 AACTGGGTGAACGTTATTAGTGACCTTAAAAAGATCGAAGATTTGATACA

GTCAATGCACATAGACGCGACGCTTTATACAGAATCTGATGTACATCCTTC

ATGCAAGGTTACTGCTATGAAGTGTTTTCTTCTCGAACTCCAAGTAATAAG

TCTTGAGAGCGGAGATGCGAGCATTCATGACACCGTTGAGAATCTTATTA

T ATT GGCT AAC AACTCTCT GTC C AGC AAT GGT AATGT GAC AGAAAGC GGG

TGTAAGGAGTGCGAGGAACTCGAGGAGAAGAACATCAAAGAGTTCTTGC

AGTCTTTCGTCCATATTGTCCAGATGTTCATAAATACTAGCGGGGGAGGTG

GCTCTGGTGGAGGCGGGAGTGGCGGGGGCGGCTCAATCACATGCCCACCG

CCCATGTCTGTTGAACACGCAGACATTTGGGTTAAAAGTTACTCACTTTAC

TCACGCGAGAGATATATATGCAACAGCGGCTTCAAGCGCAAAGCAGGCA

CTAGTAGTCTTACAGAGTGCGTGCTCAATAAAGCTACAAATGTAGCTCAT

TGGACTACTCCTAGTCTCAAATGCATTCGGGACCCCGCGCTTGTGCACCAG

AGACCTGCGCCGCCGTCCACAGTGACGACAGCTGGTGTAACCCCCCAACC

TGAATCCCTTAGTCCGTCTGGTAAAGAACCGGCGGCGTCTTCACCTTCCAG

CAATAATACTGCGGCGACAACAGCCGCGATAGTTCCTGGATCCCAACTCA

TGCCGTCAAAGTCTCCTTCAACGGGAACGACAGAGATCTCTTCACATGAA

AGTTCTCATGGAACACCGAGCCAAACTACGGCAAAGAACTGGGAACTGA

CTGCCTCAGCAAGCCACCAGCCGCCAGGGGTGTACCCGCAAGGGCACTCA

GATACTACT (SEQ ID NO: 40).

In some embodiments, the first exogenous polypeptide comprises an IL-15 polypeptide and the sushi domain of the IL-15RA polypeptide. In some embodiments, the first exogenous polypeptide includes the amino acid sequence of SEQ ID NO: 41. In some embodiments, the first exogenous polypeptide includes as 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

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLE SGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ MFINTSGGGGSGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSG FKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPS (SEQ ID NO: 41).

In some embodiments, an first exogenous polypeptide is encoded by a nucleic acid 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 ACT GGGT A A AC GT CAT A AGC GAC CTC AA A A AGAT C GAGGAC C TT AT AC A

GTCTATGCACATAGATGCGACACTTTACACGGAATCAGACGTGCACCCGT

CCTGCAAAGTCACAGCCATGAAGTGCTTTCTTCTCGAACTGCAGGTAATTT

CTCTCGAATCAGGTGACGCATCTATCCACGACACAGTTGAAAATCTTATTA

TCCTGGCTAATAACTCCCTTAGCTCAAACGGCAATGTCACCGAGAGTGGA

TGTAAAGAATGTGAGGAACTTGAAGAGAAAAACATAAAGGAATTCTTGC

AGAGTTTCGTTCATATTGTGCAAATGTTCATCAATACTAGTGGCGGGGGG

GGA AGC GGT GGT GGAGGGAGC GGGGGT GGT GGAT C CAT CACCTGCCC GC

CTCCCATGAGCGTGGAACACGCGGACATTTGGGTTAAGAGCTACAGTCTT

TACAGCCGGGAGCGCTATATCTGCAACTCAGGGTTTAAGCGGAAAGCAGG

GACATCAAGTTTGACAGAATGTGTGTTGAACAAGGCTACAAATGTTGCTC

ACTGGACCACGCCATCTTTGAAGTGTATCCGAGATCCCGCGCTTGTCCATC

AGCGCCCAGCGCCTCCCTCC (SEQ ID NO: 42).

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 (e.g., an IL-15 binding fragment). Any of the IL-15 polypeptides described herein may be combined with any of the IL-15RA polypeptides described herein to form the first exogenous polypeptide. In some embodiments, the IL-15 polypeptide or a functional fragment thereof and the extracellular portion of an IL-15RA polypeptide or a functional fragment thereof are present as a complex. In some embodiments, the IL-15 polypeptide and the extracellular portion of an IL-15RA polypeptide are present as a fusion polypeptide (e.g., a first exogenous fusion polypeptide). In some embodiments, the IL-15 polypeptide is linked to the extracellular portion of the IL-15RA polypeptide by a linker. In some embodiments, the IL-15 polypeptide and the IL-15RA polypeptide are present as a complex. The components of an IL-15/IL-15RA complex may be directly fused, using either non-covalent bonds or covalent bonds (e.g., by combining amino acid sequences via peptide bonds).

In some embodiments, the first exogenous polypeptide, the IL-15 polypeptide, IL-15RA polypeptide, or IL-15/IL-15RA complex are not released from the erythroid cell (e.g., the enucleated erythroid cell). In some embodiments the IL-15 polypeptide, IL-15RA polypeptide, or IL-15/IL-15RA complex or fusion polypeptide are attached to the erythroid cell membrane (e.g., the membrane of an enucleated erythroid cell).

In some embodiments, the first exogenous polypeptide further includes a polypeptide sequence (e.g. 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 cell membrane is heterologous to another polypeptide in the first exogenous polypeptide. In some embodiments, the polypeptide sequence that anchors the polypeptide to the erythroid cell membrane is heterologous to the IL-15 polypeptide and/or the IL-15RA polypeptide. In some embodiments, the polypeptide sequence that anchors the polypeptide to the erythroid cell membrane is a GPA sequence.

In some embodiments, other polypeptides useful for anchoring the first exogenous polypeptides to the erythroid cell membrane (e.g., the membrane of an enucleated erythroid cell) are known to the skilled person and are contemplated for inclusion in the exogenous polypeptides comprising IL-15, IL-15RA, or 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 anchor or transmembrane domain can include a type 1 membrane polypeptide or a transmembrane portion thereof. For example, in some embodiments of the first exogenous polypeptide, the anchor or transmembrane domain comprises a type 1 membrane polypeptide or a transmembrane portion thereof 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; Erythroblast Membrane Associated Protein (ERMAP); junctional adhesion molecule A (JAM- A); neuroplastin (NPTN); AMIG02; and DS Cell Adhesion Molecule Like 1 (DSCAML1). In some embodiments, the anchor or transmembrane domain comprises or consists of a type 2 membrane polypeptide 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 polypeptide 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 polypeptide. In some embodiments of the first exogenous polypeptide, the GPI-linked membrane polypeptide anchor is selected from the group consisting of CD59; CD55; and Semaphorin 7A (SEMA7A).

In some embodiments, the anchor or transmembrane domain can include small integral membrane protein 1 (SMIM1) or a transmembrane portion thereof. In some embodiments, the anchor or transmembrane domain includes glycophorin A (GPA), or a fragment thereof (e.g., a transmembrane portion thereof). In some embodiments, the anchor or transmembrane domain include an amino acid sequence provided in Table 1.

In some embodiments, the first exogenous polypeptide includes an IL-15 polypeptide or a functional fragment thereof and a transmembrane region of the wild- type human IL-15RA. In some embodiments, the first exogenous polypeptide includes an IL-15 polypeptide or a functional fragment thereof and a transmembrane region (e.g., 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 an IL-15 polypeptide, an IL-15RA polypeptide, or an IL- 15/IL-15RA polypeptide. Suitable linkers include, without limitation, any linker amino acid sequence provided in Table 2. In some embodiments, the linker between the anchor or transmembrane domain, e.g., GPA, and an IL-15 polypeptide, an IL- 15RA polypeptide, or an 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: 41.

In some embodiments, the first exogenous polypeptide can further include an anchor. In some embodiments, the first exogenous polypeptide can comprise the amino acid sequence of SEQ ID NO: 44 (which is encoded by nucleic acid sequence SEQ ID NO: 45), an interleukin- 15 (IL-15) polypeptide, and an extracellular portion of an interleukin- 15 receptor alpha (IL-15RA) polypeptide. In some embodiments, the first exogenous polypeptide can comprise an anchor that includes the amino acid sequence of SEQ ID NO: 44, mature human IL-15 that includes the amino acid sequence of SEQ ID NO: 24, and mature human extracellular IL-15RA that includes the amino acid sequence of SEQ ID NO: 30 whereby the mature human IL-15 amino acid sequence and the mature human extracellular IL-15 RA amino acid sequence are connected by a flexible linker that includes the amino acid sequence of SEQ ID NO: 37.

In some embodiments, the exogenous fusion polypeptide comprises: a signal peptide (e.g., a GPA signal peptide) that includes the amino acid sequence of SEQ ID NO: 35, a mature human IL-15 that includes an amino acid sequence of SEQ ID NO: 24, a flexible linker (e.g., connecting the mature human IL-15 and the mature human extracellular IL-15RA) that includes the amino acid sequence of SEQ ID NO: 37, a mature human extracellular IL-15RA that includes the amino acid sequence of SEQ ID NO: 30, a linker that includes the amino acid sequence of SEQ ID NO: 43, and an anchor that includes an amino acid sequence of SEQ ID NO: 44. In some embodiments, the exogenous fusion polypeptide comprises (e.g., from N-terminus to C-terminus): a mature human IL-15 that includes an amino acid sequence of SEQ ID NO: 24, a flexible linker (e.g., connecting the mature human IL-15 and the mature human extracellular IL-15RA) that includes the amino acid sequence of SEQ ID NO: 37, a mature human extracellular IL-15RA that includes the amino acid sequence of SEQ ID NO: 30, a linker that includes the amino acid sequence of SEQ ID NO: 43, and an anchor that includes an amino acid sequence of SEQ ID NO: 44. In some embodiments, the exogenous fusion polypeptide includes a sequence of SEQ ID NO: 54.

In some embodiments, an first exogenous polypeptide includes amino acid 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

MYGKIIFVLLLSEIVSISANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCK VTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEE LEEKNIKEFLQSFVHIVQMFINTSGGSGGSGGYPYDVPDYAGGGSGGGSLSTT EV AMHTSTS S S VTKS YIS SQTNDTHKRDTY AATPRAHEV SEIS VRTVYPPEEET GERVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDT DVPLSSVEIENPETSDQ (SEQ ID NO: 46), or a functional fragment thereof.

ATGTATGGAAAAATAATCTTTGTATTACTATTGTCAGAAATTGTGAGCATA

T C AGC AA AC T GGGT A A AT GT GATTT C AGAC CT GA AAA A A AT C GA AGAC CT

TATTCAATCCATGCACATCGACGCGACACTTTATACTGAATCAGACGTAC

ACCCGTCCTGTAAGGTTACTGCGATGAAGTGCTTTCTGTTGGAATTGCAAG

TGATCTCCCTCGAATCAGGGGATGCATCCATTCATGATACCGTCGAGAATT

TGATCATTCTGGCAAATAACTCCCTCAGTAGTAACGGGAATGTGACCGAG

TCTGGGTGTAAGGAGTGCGAAGAGTTGGAGGAAAAGAATATCAAAGAAT

TCCTTCAGTCCTTTGTTCACATCGTGCAAATGTTTATTAATACATCTGGAG

GATCTGGCGGGTCTGGAGGCTACCCCTATGACGTGCCCGACTATGCCGGC

GGAGGGTCTGGAGGCGGTTCCTTAAGTACCACTGAGGTGGCAATGCACAC

TTCAACCTCTTCTTCAGTCACAAAGAGTTACATCTCATCACAGACAAATGA

TACGCACAAACGGGACACATATGCAGCCACTCCTAGAGCTCATGAAGTTT

CAGAAATTTCTGTTAGAACTGTTTACCCTCCAGAAGAGGAAACCGGAGAA

AGGGTACAACTTGCCCATCATTTCTCTGAACCAGAGATAACACTCATTATT

TTTGGGGTGATGGCTGGTGTTATTGGAACGATCCTCTTAATTTCTTACGGT

ATTCGCCGACTGATAAAGAAAAGCCCATCTGATGTAAAACCTCTCCCCTC

ACCTGACACAGACGTGCCTTTAAGTTCTGTTGAAATAGAAAATCCAGAGA

CAAGTGATCAA (SEQ ID NO: 47).

MYGKIIFVLLLSEIVSISANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCK VTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEE LEEKNIKEFLQSFVHIVQMFINTSGGGGSGGGGSGGGGSITCPPPMSVEHADI WVKS YSLYSRERYICNSGFKRKAGTS SLTEC VLNKATNV AHWTTPSLKCIRD PALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGS QLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSD TT GGS GGS GGYPYD VPD Y AGGGS GGGSL STTEV AMHTS TS S S VTKS YI S S QT NDTHKRDTYAATPRAHEVSEISVRTVYPPEEETGERVQLAHHFSEPEITLIIFG VMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDTDVPLSSVEIENPETSDQ (SEQ ID NO: 48), or a functional fragment thereof.

ATGTATGGAAAAATAATCTTTGTATTACTATTGTCAGAAATTGTGAGCATA

TCAGCAAACTGGGTGAACGTTATTAGTGACCTTAAAAAGATCGAAGATTT

GATACAGTCAATGCACATAGACGCGACGCTTTATACAGAATCTGATGTAC

ATCCTTCATGCAAGGTTACTGCTATGAAGTGTTTTCTTCTCGAACTCCAAG

TAATAAGTCTTGAGAGCGGAGATGCGAGCATTCATGACACCGTTGAGAAT

CTTATTATATTGGCTAACAACTCTCTGTCCAGCAATGGTAATGTGACAGAA

AGC GGGT GT A AGGAGT GC GAGGA ACT C GAGGAGA AGA AC AT C A A AGAGT

TCTTGCAGTCTTTCGTCCATATTGTCCAGATGTTCATAAATACTAGCGGGG

GAGGTGGCTCTGGTGGAGGCGGGAGTGGCGGGGGCGGCTCAATCACATG

CCCACCGCCCATGTCTGTTGAACACGCAGACATTTGGGTTAAAAGTTACTC

ACTTTACTCACGCGAGAGATATATATGCAACAGCGGCTTCAAGCGCAAAG

CAGGCACTAGTAGTCTTACAGAGTGCGTGCTCAATAAAGCTACAAATGTA

GCTCATTGGACTACTCCTAGTCTCAAATGCATTCGGGACCCCGCGCTTGTG

CACCAGAGACCTGCGCCGCCGTCCACAGTGACGACAGCTGGTGTAACCCC

CCAACCTGAATCCCTTAGTCCGTCTGGTAAAGAACCGGCGGCGTCTTCAC

CTTCCAGCAATAATACTGCGGCGACAACAGCCGCGATAGTTCCTGGATCC

CAACTCATGCCGTCAAAGTCTCCTTCAACGGGAACGACAGAGATCTCTTC

ACATGAAAGTTCTCATGGAACACCGAGCCAAACTACGGCAAAGAACTGG

GAACTGACTGCCTCAGCAAGCCACCAGCCGCCAGGGGTGTACCCGCAAGG

GCACTCAGATACTACTGGAGGATCTGGCGGGTCTGGAGGCTACCCCTATG

ACGTGCCCGACTATGCCGGCGGAGGGTCTGGAGGCGGTTCCTTAAGTACC

ACTGAGGTGGCAATGCACACTTCAACCTCTTCTTCAGTCACAAAGAGTTA

CATCTCATCACAGACAAATGATACGCACAAACGGGACACATATGCAGCCA

CTCCTAGAGCTCATGAAGTTTCAGAAATTTCTGTTAGAACTGTTTACCCTC

C AGAAGAGGAAAC C GGAGAAAGGGT AC AACTT GCC C AT C ATTT CTCTGAA

CCAGAGATAACACTCATTATTTTTGGGGTGATGGCTGGTGTTATTGGAACG

ATCCTCTTAATTTCTTACGGTATTCGCCGACTGATAAAGAAAAGCCCATCT GATGTAAAACCTCTCCCCTCACCTGACACAGACGTGCCTTTAAGTTCTGTT GAAATAGAAAATCCAGAGACAAGTGATCAA (SEQ ID NO: 49)

MYGKIIFVLLLSEIVSISANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCK VTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEE LEEKNIKEFLQSFVHIVQMFINTSGGGGSGGGGSGGGGSITCPPPMSVEHADI WVKS YSLYSRERYICNSGFKRKAGTS SLTEC VLNKATNV AHWTTPSLKCIRD PALVHQRPAPPSGGSGGSGGYPYDVPDYAGGGSGGGSLSTTEVAMHTSTSSS VTKSYIS SQTNDTHKRDTY AATPRAHEV SEIS VRTVYPPEEETGERV QLAHHF SEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDTDVPLSSVEIENP ETSDQ (SEQ ID NO: 50), or a functional fragment thereof.

ATGTATGGAAAAATAATCTTTGTATTACTATTGTCAGAAATTGTGAGCATA

TCAGCAAACTGGGTAAACGTCATAAGCGACCTCAAAAAGATCGAGGACCT

TATACAGTCTATGCACATAGATGCGACACTTTACACGGAATCAGACGTGC

ACCCGTCCTGCAAAGTCACAGCCATGAAGTGCTTTCTTCTCGAACTGCAG

GTAATTTCTCTCGAATCAGGTGACGCATCTATCCACGACACAGTTGAAAA

TCTTATTATCCTGGCTAATAACTCCCTTAGCTCAAACGGCAATGTCACCGA

GAGTGGATGTAAAGAATGTGAGGAACTTGAAGAGAAAAACATAAAGGAA

TTCTTGCAGAGTTTCGTTCATATTGTGCAAATGTTCATCAATACTAGTGGC

GGGGGGGGA AGC GGT GGT GGAGGGAGC GGGGGT GGT GGAT C C ATC AC CT

GCCCGCCTCCCATGAGCGTGGAACACGCGGACATTTGGGTTAAGAGCTAC

AGTCTTTACAGCCGGGAGCGCTATATCTGCAACTCAGGGTTTAAGCGGAA

AGCAGGGACATCAAGTTTGACAGAATGTGTGTTGAACAAGGCTACAAATG

TTGCTCACTGGACCACGCCATCTTTGAAGTGTATCCGAGATCCCGCGCTTG

TCCATCAGCGCCCAGCGCCTCCCTCCGGAGGATCTGGCGGGTCTGGAGGC

TACCCCTATGACGTGCCCGACTATGCCGGCGGAGGGTCTGGAGGCGGTTC CTTAAGTACCACTGAGGTGGCAATGCACACTTCAACCTCTTCTTCAGTCAC AAAGAGTTACATCTCATCACAGACAAATGATACGCACAAACGGGACACAT ATGCAGCCACTCCTAGAGCTCATGAAGTTTCAGAAATTTCTGTTAGAACTG TTTACCCTCCAGAAGAGGAAACCGGAGAAAGGGTACAACTTGCCCATCAT TTCTCT GAAC C AGAGAT AAC ACT C ATT ATTTTTGGGGTGAT GGCT GGT GTT ATTGGAACGATCCTCTTAATTTCTTACGGTATTCGCCGACTGATAAAGAAA AGCCCATCTGATGTAAAACCTCTCCCCTCACCTGACACAGACGTGCCTTTA AGTT C T GTT GA A AT AGA A A AT C C AGAGAC A AGT GAT C A A (SEQ ID NO: 51).

In some embodiments, the first exogenous polypeptide includes amino acid 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

MYGKIIFVLLLSEIVSISANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCK

VTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEE

LEEKNIKEFLQSFVHIVQMFINTSGGSGGSGGGGGSGGGSGGGSGGGSLSTTE

VAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRAHEVSEISVRTVYPPEEET

GERVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDT

DVPLSSVEIENPETSDQ (SEQ ID NO: 52), or a functional fragment thereof.

ATGTATGGAAAAATAATCTTTGTATTACTATTGTCAGAAATTGTGAGCATA

TCAGCAAACTGGGTAAATGTGATTTCAGACCTGAAAAAAATCGAAGACCT

TATTCAATCCATGCACATCGACGCGACACTTTATACTGAATCAGACGTAC

ACCCGTCCTGTAAGGTTACTGCGATGAAGTGCTTTCTGTTGGAATTGCAAG

TGATCTCCCTCGAATCAGGGGATGCATCCATTCATGATACCGTCGAGAATT

TGATCATTCTGGCAAATAACTCCCTCAGTAGTAACGGGAATGTGACCGAG

TCTGGGTGTAAGGAGTGCGAAGAGTTGGAGGAAAAGAATATCAAAGAAT

TCCTTCAGTCCTTTGTTCACATCGTGCAAATGTTTATTAATACATCTGGAG

GAT CT GGC GGGT C T GGAGGC GGC GGC GGC AGC GGC GGC GGC AGC GGC GG

AGGGTCTGGAGGCGGTTCCTTAAGTACCACTGAGGTGGCAATGCACACTT

CAACCTCTTCTTCAGTCACAAAGAGTTACATCTCATCACAGACAAATGAT ACGCACAAACGGGACACATATGCAGCCACTCCTAGAGCTCATGAAGTTTC

AGAAATTTCTGTTAGAACTGTTTACCCTCCAGAAGAGGAAACCGGAGAAA

GGGTACAACTTGCCCATCATTTCTCTGAACCAGAGATAACACTCATTATTT

TTGGGGTGATGGCTGGTGTTATTGGAACGATCCTCTTAATTTCTTACGGTA

TTCGCCGACTGATAAAGAAAAGCCCATCTGATGTAAAACCTCTCCCCTCA

CCTGACACAGACGTGCCTTTAAGTTCTGTTGAAATAGAAAATCCAGAGAC

AAGTGATCAA (SEQ ID NO: 53).

MYGKIIFVLLLSEIVSISANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCK VTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEE LEEKNIKEFLQSFVHIVQMFINTSGGGGSGGGGSGGGGSITCPPPMSVEHADI WVKS YSLYSRERYICNSGFKRKAGTS SLTEC VLNKATNV AHWTTPSLKCIRD PALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGS QLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSD TTGGSGGSGGGGGSGGGSGGGSGGGSLSTTEVAMHTSTSSSVTKSYISSQTN DTHKRDTY AATPRAHEV SEI S VRTV YPPEEET GERV QL AHHF SEPEITLIIF GV MAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDTDVPLSSVEIENPETSDQ (SEQ ID NO: 54), or a functional fragment thereof.

ATGTATGGAAAAATAATCTTTGTATTACTATTGTCAGAAATTGTGAGCATA

TCAGCAAACTGGGTGAACGTTATTAGTGACCTTAAAAAGATCGAAGATTT

GATACAGTCAATGCACATAGACGCGACGCTTTATACAGAATCTGATGTAC

ATCCTTCATGCAAGGTTACTGCTATGAAGTGTTTTCTTCTCGAACTCCAAG

TAATAAGTCTTGAGAGCGGAGATGCGAGCATTCATGACACCGTTGAGAAT

CTTATTATATTGGCTAACAACTCTCTGTCCAGCAATGGTAATGTGACAGAA

AGC GGGT GT A AGGAGT GC GAGGA ACT C GAGGAGA AGA AC AT C A A AGAGT

TCTTGCAGTCTTTCGTCCATATTGTCCAGATGTTCATAAATACTAGCGGGG GAGGTGGCTCTGGTGGAGGCGGGAGTGGCGGGGGCGGCTCAATCACATG

CCCACCGCCCATGTCTGTTGAACACGCAGACATTTGGGTTAAAAGTTACTC

ACTTTACTCACGCGAGAGATATATATGCAACAGCGGCTTCAAGCGCAAAG

CAGGCACTAGTAGTCTTACAGAGTGCGTGCTCAATAAAGCTACAAATGTA

GCTCATTGGACTACTCCTAGTCTCAAATGCATTCGGGACCCCGCGCTTGTG

CACCAGAGACCTGCGCCGCCGTCCACAGTGACGACAGCTGGTGTAACCCC

CCAACCTGAATCCCTTAGTCCGTCTGGTAAAGAACCGGCGGCGTCTTCAC

CTTCCAGCAATAATACTGCGGCGACAACAGCCGCGATAGTTCCTGGATCC

CAACTCATGCCGTCAAAGTCTCCTTCAACGGGAACGACAGAGATCTCTTC

ACATGAAAGTTCTCATGGAACACCGAGCCAAACTACGGCAAAGAACTGG

GAACTGACTGCCTCAGCAAGCCACCAGCCGCCAGGGGTGTACCCGCAAGG

GCACTCAGATACTACTGGAGGATCTGGCGGGTCTGGAGGCGGCGGCGGCA

GCGGCGGCGGCAGCGGCGGAGGGTCTGGAGGCGGTTCCTTAAGTACCACT

GAGGTGGCAATGCACACTTCAACCTCTTCTTCAGTCACAAAGAGTTACAT

CTCATCACAGACAAATGATACGCACAAACGGGACACATATGCAGCCACTC

CTAGAGCT CAT GAAGTTT C AGAAATTT CT GTTAGAACT GTTT ACC CT C C AG

AAGAGGAAACCGGAGAAAGGGTACAACTTGCCCATCATTTCTCTGAACCA

GAGATAACACTCATTATTTTTGGGGTGATGGCTGGTGTTATTGGAACGATC

CTCTTAATTTCTTACGGTATTCGCCGACTGATAAAGAAAAGCCCATCTGAT

GTAAAACCTCTCCCCTCACCTGACACAGACGTGCCTTTAAGTTCTGTTGAA

ATAGAAAATCCAGAGACAAGTGATCAA (SEQ ID NO: 55).

In some embodiments, the exogenous fusion polypeptide includes an amino acid sequence of SEQ ID NO: 46. In some embodiments, the exogenous fusion polypeptides includes an amino acid sequence of SEQ ID NO: 48. In some embodiments, the exogenous fusion polypeptide includes an amino acid sequence of SEQ ID NO: 50. In some embodiments, the exogenous fusion polypeptide includes an amino acid sequence of SEQ ID NO: 52. In some embodiments, the exogenous fusion polypeptide includes an amino acid sequence of SEQ ID NO: 54.

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

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

As used herein 4-1BBL polypeptide refers 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 surfaces of cells of the immune system. See Alderson et ak, 1994, Eur.

J. Immunol. 24:2219-2227.

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

In some embodiments, a 4-1BBL includes an amino acid 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

ACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNV LLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRV VAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLL HLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE (SEQ ID NO: 56), or a functional fragment thereof. In some embodiments, the 4- 1BBL includes a sequence of SEQ ID NO: 56.

In some embodiments, the 4-1BBL is encoded by a nucleic acid 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

GCCTGCCCCTGGGCCGTGTCCGGGGCTCGCGCCTCGCCCGGCTCCGCGGC CAGCCCGAGACTCCGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCG GC CTCTT GGAC CT GCGGC AGGGC AT GTTT GC GC AGCT GGTGGC CC AAAAT GTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGC AGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAG

CTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTG

CGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCA

CCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGT

GGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCA

GGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTC

ACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACA

GTCTTGGGACTCTTCCGGGTGACCCCCGAAATCCCAGCCGGACTCCCTTCA

CCGAGGTCGGAA (SEQ ID NO: 57).

In some embodiments, the second exogenous polypeptide includes an amino acid 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

MYGKIIFVLLLSEIVSISAACPWAVSGARASPGSAASPRLREGPELSPDDPAGL LDLRQ GMF AQLV AQNVLLIDGPL S WYSDP GL AGV SLTGGL S YKEDTKELV V AKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPP AS SEARNS AF GFQGRLLHLS AGQRLGVHLHTEARARHAW QLTQGATVLGLF RVTPEIPAGLPSPRSEGGSGGSGGGPEDEPGSGSGGGSGGGSLSTTEVAMHTS TS S S VTKS YIS SQTNDTHKRDTY AATPRAHEV SEIS VRTVYPPEEET GERVQLA HHFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDTDVPLSSVE IENPETSDQ (SEQ ID NO: 58). In some embodiments, a 4-1BBL polypeptide includes a sequence of SEQ ID NO: 58.

In some embodiments, an exogenous fusion polypeptide is encoded by a nucleic acid 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

ATGTATGGAAAAATAATCTTTGTATTACTATTGTCAGAAATTGTGAGCATA

TCAGCAGCCTGCCCCTGGGCCGTGTCCGGGGCTCGCGCCTCGCCCGGCTC

CGCGGCCAGCCCGAGACTCCGCGAGGGTCCCGAGCTTTCGCCCGACGATC

CCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCC

CAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGG

CCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACG

AAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACT AGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTG

CGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTT

TGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTC

GGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGT

CCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGG

GCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATCCCAGCCGGA

CTCCCTTCACCGAGGTCGGAAGGAGGATCTGGCGGGTCTGGAGGCGGCCC

CGAGGACGAGCCCGGCAGCGGCAGCGGCGGAGGGTCTGGAGGCGGTTCC

TTAAGTACCACTGAGGTGGCAATGCACACTTCAACCTCTTCTTCAGTCACA

AAGAGTTACATCTCATCACAGACAAATGATACGCACAAACGGGACACATA

TGCAGCCACTCCTAGAGCTCATGAAGTTTCAGAAATTTCTGTTAGAACTGT

TTACCCTCCAGAAGAGGAAACCGGAGAAAGGGTACAACTTGCCCATCATT

TCTCTGAACCAGAGATAACACTCATTATTTTTGGGGTGATGGCTGGTGTTA

TTGGAACGATCCTCTTAATTTCTTACGGTATTCGCCGACTGATAAAGAAAA

GCCCATCTGATGTAAAACCTCTCCCCTCACCTGACACAGACGTGCCTTTAA

GTTCTGTTGAAATAGAAAATCCAGAGACAAGTGATCAA (SEQ ID NO: 59).

In some embodiments, the second exogenous polypeptide includes a leader (signal) sequence. In some embodiments, the 4-1BBL or functional fragment thereof is fused to the leader (signal) sequence. Non-limiting examples of a leader (signal) sequence include the amino acid sequences provided 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: 35. In some embodiments, the second exogenous polypeptide comprises a 4- 1BBL or a functional fragment thereof and a leader (signal) sequence of the amino acid sequence of SEQ ID NO: 35. In some embodiments, the second exogenous polypeptide comprises a leader (signal) sequence that includes the amino acid sequence of SEQ ID NO: 35, and a 4-1BBL having an amino acid sequence of SEQ ID NO: 56.

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

In some embodiments, the second exogenous polypeptide includes one or more linkers (e.g., any of the exemplary linkers described herein). For example, the second exogenous polypeptide can includes one or more linkers provided in Table 2. In some embodiments, a linker is disposed between the 4-1BBL or a functional fragment thereof and an anchor or transmembrane domain in the second exogenous polypeptide.

In some embodiments, the second exogenous polypeptide can further include a signal peptide (e.g., 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 second exogenous polypeptide includes a signal peptide, the 4-1BBL or a functional fragment thereof, and an anchor. In some embodiments, the second exogenous polypeptide includes a signal peptide, the 4- 1BBL or functional fragment thereof, a linker, and an anchor. In some embodiments, the second exogenous polypeptide comprises (e.g., from N-terminus to C-terminus) a signal peptide that includes the amino acid sequence of SEQ ID NO: 35, the 4-1BBL or a functional fragment thereof that includes the amino acid sequence of SEQ ID NO: 54, a linker that includes the amino acid sequence of SEQ ID NO: 60 (e.g., disposed between the 4-1BBL or the functional fragment thereof and the anchor), and an anchor that includes the amino acid sequence of SEQ ID NO: 44. In some embodiments, the second exogenous polypeptide comprises the 4-1BBL or a functional fragment thereof, a linker, and an anchor. In some embodiments, the second exogenous polypeptide comprises (e.g., from N-terminus to C-terminus) the 4- 1BBL or functional fragment thereof that includes the amino acid sequence of SEQ ID NO: 56, a linker that includes the amino acid sequence of SEQ ID NO: 60 (e.g., disposed between the 4-1BBL or the functional fragment thereof and the anchor), and an anchor that includes the amino acid sequence of SEQ ID NO: 44. In some embodiments, the second exogenous polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 58.

In some embodiments, the first and/or second exogenous polypeptides may have post-translational modifications characteristic of eukaryotic cells, e.g., mammalian cells, e.g., human cells. In some embodiments, one or more (e.g., 2, 3, 4, 5, or more) of the exogenous polypeptides are glycosylated, phosphorylated, or both. In vitro detection of glycoproteins is routinely accomplished on SDS-PAGE gels and Western Blots using a modification of Periodic acid-Schiff (PAS) methods. Cellular localization of glycoproteins may be accomplished utilizing lectin fluorescent conjugates known in the art. Phosphorylation may be assessed by Western blot using phospho-specific antibodies.

Post-translation modifications also include conjugation to a hydrophobic group (e.g., myristoylation, palmitoylation, isoprenylation, prenylation, or glypiation), conjugation to a cofactor (e.g., lipoylation, flavin moiety (e.g., FMN or FAD), heme C attachment, phosphopantetheinylation, or retinylidene Schiff base formation), diphthamide formation, ethanolamine phosphoglycerol attachment, hypusine formation, acylation (e.g. O-acylation, N-acylation, or S-acylation), formylation, acetylation, alkylation (e.g., methylation or ethylation), amidation, butyrylation, gamma-carboxylation, malonylation, hydroxylation, iodination, nucleotide addition such as ADP- ribosylation, oxidation, phosphate ester (O-linked) or phosphoramidate (N-linked) formation, (e.g., phosphorylation or adenylylation), propionylation, pyroglutamate formation, S-glutathionylation, S-nitrosylation, succinylation, sulfation, ISGylation, SUMOylation, ubiquitination, Neddylation, or a chemical modification of an amino acid (e.g., citrullination, deamidation, eliminylation, or carbamylation), formation of a disulfide bridge, racemization (e.g., of proline, serine, alanine, or methionine). In embodiments, glycosylation includes the addition of a glycosyl group to arginine, asparagine, cysteine, hydroxylysine, serine, threonine, tyrosine, or tryptophan, resulting in a glycoprotein. In embodiments, the glycosylation comprises, e.g., O- linked glycosylation or N-linked glycosylation.

In some embodiments, the first and second exogenous polypeptides are fusion polypeptides, e.g., is a fusion with an endogenous red blood cell polypeptide or fragment thereof, e.g., a transmembrane polypeptide, e.g., GPA or a transmembrane fragment thereof. In some embodiments, one or more of the exogenous polypeptides is fused with a domain that promotes dimerization or multimerization, e.g., with a second fusion exogenous polypeptide, which optionally comprises a dimerization domain. In some embodiments, the dimerization domain comprises a portion of an antibody molecule, e.g., an Fc domain or CH3 domain. In some embodiments, the first and second dimerization domains comprise knob-in-hole mutations (e.g., a T366Y knob and a Y407T hole) to promote heterodimerization.

Anchor/Transmembrane domains

In some embodiments, the transmembrane domain comprises or consists of a transmembrane domain of a type 1 membrane polypeptide. In some embodiments, the type 1 membrane polypeptide 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; Erythroblast Membrane Associated Protein (ERMAP); junctional adhesion molecule A (JAM- A); neuroplastin (NPTN);

AMIG02; 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 polypeptide. In some embodiments, the type 2 membrane polypeptide 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 erythroid cell membrane comprises, consists of, or is derived from (e.g., a fragment of) a GPI-linked membrane polypeptide. In some embodiments, the GPI-linked membrane polypeptide is selected from the group consisting of CD59; CD55; and Semaphorin 7A (SEMA7A).

In particular embodiments, the transmembrane domain comprises GPA or a transmembrane portion thereof. Without being bound by theory, in certain embodiments, GPA is preferred because it has a cytoplasmic domain that interacts with the reticulocyte cytoskeleton that has a role in retaining the GPA as the cell differentiates and matures. 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 an amino acid sequence listed in Table 1.

Table 1. Anchor Sequences

In some embodiments where the enucleated erythroid cells include a first and second exogenous polyepptide, the first exogenous polypeptide (e.g., any of the exogenous fusion polypeptides described herein) or the second exogenous polypeptide (e.g., any of the exogenous polypeptides described herein) comprises a endogenous red blood cell polypeptide or fragment thereof, e.g., a transmembrane polypeptide, e.g., GPA or a transmembrane fragment thereof. In some embodiments, the exogenous fusion polypeptide or one or more of the exogenous polypeptides (e.g., any of the exogenous polypeptides described herein) is fused with a domain that promotes dimerization or multimerization, e.g., with a second exogenous polypeptide, which optionally comprises a dimerization domain. In some embodiments, the dimerization domain comprises a portion of an antibody molecule, e.g., an Fc domain or CH3 domain. In some embodiments, the first and second dimerization domains comprise knob-in-hole mutations (e.g., a T366Y knob and a Y407T hole) to promote heterodimerization.

Linkers

In some embodiments where the enucleated erythroid cells include a first and second exogenous polyepptide, the first exogenous fusion polypeptide (e.g., any of the fusion polypeptides described herein) and/or the second exogenous polypeptide (e.g., any of the exemplary polypeptides described herein) may include one or more linkers. For example, a linker may be disposed between a cytokine polypeptide sequence (e.g., 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 may be disposed between a 4-1BBL polypeptide, of a functional fragment thereof, and a transmembrane domain sequence.

In some embodiments, the linker comprises or consists of 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 length. In some embodiments, the linker comprises or consists of between about 5 and about 25 amino acids in length, between about 5 and about 20 amino acids in length, between about 10 and about 25 amino acids in length, or between about 10 and about 20 amino acids in length. In some embodiments, the linker useful in the disclosure comprises or consists 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 an amino acid sequence presented in Table 2.

Table 2. Linker Sequences

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 the (GGGGS)_n linker (SEQ ID NO: 38), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, the linker comprises the amino acid sequence GGGGS GGGGS GGGGS (SEQ ID NO: 37). In some embodiments, the linker consists of the amino acid sequence of SEQ ID NO: 37. In some embodiments, the linker comprises the amino acid sequence SEQ ID NO: 65.

In some embodiments, the linker consists of the amino acid sequence of SEQ ID NO: 65. In some embodiments, the linker comprises the amino acid sequence SEQ ID

NO: 43. In some embodiments, the linker consists of the amino acid sequence of SEQ ID NO: 43. In some embodiments, the linker comprises the amino acid sequence SEQ ID NO: 60. In some embodiments, the linker consists of the amino acid sequence of SEQ ID NO: 60. Other suitable linkers that can be used in the exogenous fusion polypeptide or the exogenous polypeptide (e.g., any of the exogenous polypeptides described herein) are known in the art.

Leader (signal) sequences

In some embodiments where the enucleated erythroid cells include a first and second exogenous polyepptide, the first exogenous fusion polypeptide or the exogenous polypeptide (e.g., any of the exemplary exogenous polypeptides described herein) comprises a leader (signal) sequence. In some embodiments, the leader sequence is selected from the sequences set forth in Table 3.

Table 3. Leader (Signal) Sequences

Exemplary Enucleated Erythroid Cells

In some embodiments, the enucleated erythroid cell comprises a combination of: a first exogenous fusion polypeptide comprising IL-15, or a fragment thereof, linked to an extracellular portion of IL-15 receptor alpha (IL-15Ra), or a fragment thereof (e.g., an IL-15Ra sushi-binding domain), linked to a transmembrane polypeptide (e.g., GPA, or a transmembrane fragment thereof), and a second exogenous polypeptide comprising 4-1BBL, or a fragment thereof, linked to a transmembrane polypeptide (e.g., GPA, or a transmembrane fragment thereof); e.g., as described in U.S. Patent Application Publication No. 2019/0298769, incorporated herein by reference).

In some embodiments, the enucleated erythroid cells (e.g., human enucleated erythroid cells) described herein are negative for (i.e. , do not include) one or more minor blood group antigens, e.g., Le(a b ) (for Lewis antigen system), Fy(ab ) (for Duffy system), Jk(ab ) (for Kidd system), MN (for MNS system), Kk (for Kell system), Lu(ab ) (for 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 groups are described, e.g., in Agarwal et al., “Blood group phenotype frequencies in blood donors from a tertiary care hospital in north India,” Blood Res. 48(l):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 (e.g., human enucleated erythroid cells) described herein exhibit substantially the same osmotic membrane fragility as an isolated, uncultured enucleated erythroid cell that does not comprise an exogenous polypeptide (e.g., any of the exogenous polypeptides described herein or known in the art). 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. Osmotic fragility is determined, in some embodiments, 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 (e.g., human enucleated erythroid cells) have approximately the same diameter or volume as a wild-type, untreated enucleated erythroid cell. In some embodiments, the population of enucleated erythroid cells (e.g., human enucleated erythroid cells) have an average 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, 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, or about 11.0 to about 11.5 microns, and optionally the standard deviation of the population is less than 1, 2, or 3 microns. Enucleated erythroid cell diameter can be measured, e.g., using an Advia 120 hematology system, or a Moxi Z cell counter (Orflo).

In some embodiment the volume of the mean corpuscular volume of the enucleated erythroid cell is about 10 fL to about 175 fL, about 10 fL to about 160 fL, about 10 fL to about 140 fL, about 10 fL to about 120 fL, about 10 fL to about 100 fL, about 10 fL to about 95 fL, about 10 fL to about 90 fL, about 10 fL to about 85 fL, about 10 fL to about 80 fL, about 10 fL to about 75 fL, about 10 fL to about 70 fL, about 10 fL to about 65 fL, about 10 fL to about 60 fL, about 10 fL to about 55 fL, about 10 fL to about 50 fL, about 10 fL to about 45 fL, about 10 fL to about 40 fL, about 10 fL to about 35 fL, about 10 fL to about 30 fL, about 10 fL to about 25 fL, about 10 fL to about 20 fL, about 10 fL to about 15 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 95 fL, about 20 fL to about 90 fL, about 20 fL to about 85 fL, about 20 fL to about 80 fL, about 20 fL to about 75 fL, about 20 fL to about 70 fL, about 20 fL to about 65 fL, about 20 fL to about 60 fL, about 20 fL to about 55 fL, about 20 fL to about 50 fL, about 20 fL to about 45 fL, about 20 fL to about 40 fL, about 20 fL to about 35 fL, about 20 fL to about 30 fL, about 20 fL to about 25 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 95 fL, about 30 fL to about 90 fL, about 30 fL to about 85 fL, about 30 fL to about 80 fL, about 30 fL to about 75 fL, about 30 fL to about 70 fL, about 30 fL to about 65 fL, about 30 fL to about 60 fL, about 30 fL to about 55 fL, about 30 fL to about 50 fL, about 30 fL to about 45 fL, about 30 fL to about 40 fL, about 30 fL to about 35 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 95 fL, about 40 fL to about 90 fL, about 40 fL to about 85 fL, about 40 fL to about 80 fL, about 40 fL to about 75 fL, about 40 fL to about 70 fL, about 40 fL to about 65 fL, about 40 fL to about 60 fL, about 40 fL to about 55 fL, about 40 fL to about 50 fL, about 40 fL to about 45 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 95 fL, about 50 fL to about 90 fL, about 50 fL to about 85 fL, about 50 fL to about 80 fL, about 50 fL to about 75 fL, about 50 fL to about 70 fL, about 50 fL to about 65 fL, about 50 fL to about 60 fL, about 50 fL to about 55 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 95 fL, about 60 fL to about 90 fL, about 60 fL to about 85 fL, about 60 fL to about 80 fL, about 60 fL to about 75 fL, about 60 fL to about 70 fL, about 60 fL to about 65 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, about 70 fL to about 100 fL, about 70 fL to about 95 fL, about 70 fL to about 90 fL, about 70 fL to about 85 fL, about 70 fL to about 80 fL, about 70 fL to about 75 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 120 fL, about 80 fL to about 100 fL, about 80 fL to about 95 fL, about 80 fL to about 90 fL, about 80 fL to about 85 fL, about 100 fL to 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 optionally, the standard deviation of the population is less than 50, 40, 30, 20, 10, 5, or 2 fL. The mean corpuscular volume can be measured, e.g., using a hematological analysis instrument, e.g., 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) physical characteristics described herein, e.g., osmotic fragility, cell size, hemoglobin concentration, or phosphatidylserine content. While not wishing to be bound by theory, in some embodiments an enucleated erythroid cell that expresses an exogenous polypeptide has physical characteristics that resemble a wild-type, untreated enucleated erythroid cell. In contrast, a hypotonically loaded enucleated erythroid cell sometimes displays aberrant physical characteristics such as increased osmotic fragility, altered cell size, reduced hemoglobin concentration, or increased phosphatidylserine levels on the outer leaflet of the cell membrane.

In some embodiments, the enucleated erythroid cell comprises an exogenous polypeptide that was encoded by an exogenous nucleic acid that was not retained by the cell, has not been purified, or has not existed fully outside an enucleated erythroid cell. In some embodiments, the enucleated erythroid cell is in a composition that lacks a stabilizer.

In some embodiments, the enucleated erythroid cells has a hemoglobin content similar to a wild-type, untreated enucleated erythroid cell. In some embodiments, the enucleated erythroid cells comprise greater than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or greater than 10% fetal hemoglobin. 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. Hemoglobin levels are determined, in some embodiments, using the Drabkin’s reagent method of Example 33 of WO2015/073587.

In some embodiments, the enucleated enucleated erythroid cells has approximately the same phosphatidylserine content on the outer leaflet of its cell membrane as a wild-type, untreated enucleated erythroid cell. Phosphatidylserine is predominantly on the inner leaflet of the cell membrane of wild-type, untreated enucleated erythroid cells, and hypotonic loading can cause the phosphatidylserine to distribute to the outer leaflet where it can trigger 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% of cells that are positive for Annexin V staining. Phosphatidylserine exposure is assessed, in some embodiments, by staining for Annexin-V-FITC, which binds preferentially to PS, and measuring FITC fluorescence by flow cytometry, e.g., using the method of Example 54 of WO2015/073587.

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%) of cells that are positive for GPA. The presence of GPA is detected, in some embodiments, using FACS.

In some embodiments, the enucleated erythroid cells have a half-life of at least 30, 45, or 90 days in a subject. In some embodiments, a population of cells comprising enucleated erythroid cells comprises less than about 10, 5, 4, 3, 2, or 1% echinocytes.

In some embodiments, a composition including a plurality of enucleated erythroid cells can be administered to a subject (e.g., any of the subjects described herein. In such embodiments, greater than 50%, 60%, 70%, 80%, or 90% of the cells in the composition can be enucleated. In some embodiments, a cell, e.g., an enucleated erythroid cell, contains a nucleus that is non-functional, e.g., has been inactivated.

In some embodiments of any of the compositions described herein, the enucleated erythroid cells are human (e.g., derived from a human donor erythroid cell precursor) 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 polypeptide. In other examples, the engineered enucleated erythroid cells comprise two or more exogenous polypeptides (e.g., any of the exemplary exogenous polypeptides described herein).

In some embodiments, an exogenous polypeptide present on the membrane of the engineered enucleated erythroid cell can be a product of a click chemistry reaction (e.g., the exogenous polypeptide may be conjugated to a polypeptide present on the membrane of the cell (e.g., a second exogenous polypeptide or an endogenous polypeptide) using any of the methods described herein). In some embodiments, an exogenous polypeptide present on the membrane of the engineered enucleated erythroid cell can the a product of a conjugation reaction using a sortase enzyme (e.g., the exogenous polypeptide may be conjugated to a polypeptide present on the membrane of the cell (e.g., a second exogenous polypeptide or an endogenous polypeptide) using any of the methods described herein). Non-limiting examples of a conjugation reaction using a sortase enzyme can be found in U.S. Pat. No. 10,260,038 and U.S. Pat. Pub. No. 2016/0082046 Al. In some embodiments, an exogenous polypeptide present on the membrane of the engineered enucleated erythroid cell can be a lipid-anchored polypeptide, e.g., a GPI-anchor, an N-myristolyated polypeptide, or a S-palmitoylated polypeptide. In some embodiments an exogenous polypeptide present on the membrane of the engineered enucleated erythroid cell can be a transmembrane polypeptide (e.g., a single-pass or multi-pass transmembrane polypeptide) or a peripheral membrane polypeptide. In some embodiments, an exogenous polypeptide present on the membrane of the engineered enucleated erythroid cell can be a fusion polypeptide comprising a transmembrane domain (e.g., a fusion polypeptide comprising the transmembrane domain of small integral membrane protein 1 (SMIM1) or glycophorin A (GPA)). In some embodiments, an exogenous polypeptide present on the membrane of the engineered enucleated erythroid cell does not have any amino acids protruding into the extracellular space.

In some embodiments, an exogenous polypeptide present on the membrane of the engineered enucleated erythroid cell does not have any amino acids protruding into the cytosol of the engineered enucleated erythroid cell. In some embodiments, an exogenous polypeptide present on the membrane of the engineered enucleated erythroid cell has amino acids protruding into the extracellular space and amino acids protruding into the cytosol of the engineered enucleated erythroid cells. Exemplary methods of producing enucleated erythroid cells using sortagging are described in W02014/183071 or WO2014/183066, each of which is incorporated by reference in its entirety.

The engineered enucleated erythroid cells can be produced by introducing one or more nucleic acids (e.g., DNA expression vectors or mRNA) encoding one or more exogenous polypeptides (e.g., any of the exogenous polypeptides described herein or known in the art) into an erythroid cell precursor (e.g., any of the erythroid cell precursors described herein or known in the art). Exemplary methods for introducing DNA expression vectors into erthyroid cell precursor include, but are not limited to, liposome-mediated transfer, transformation, gene guns, transfection, and transduction, e.g., viral -mediated gene transfer (e.g., performed using viral vectors including adenovirus vectors, adeno-associated viral vectors, lentiviral vectors, herpes viral vectors, and retroviral-based vectors). Additional exemplary methods for introducing DNA expression vectors into erythroid cell precursor include the use of, e.g., naked DNA, CaPCri precipitation, DEAE dextran, electroporation, protoplast fusion, lipofection, and cell microinjection.

An erythroid cell precursor can optionally be cultured, e.g., before and/or after introduction of one or more nucleic acids encoding one or more exogenous polypeptides, under suitable conditions allowing for differentiation into engineered enucleated erythroid cells. In some embodiments, the resulting engineered enucleated erythroid cells comprise polypeptides associated with mature erythrocytes, e.g., hemoglobin (e.g., adult hemoglobin and/or fetal hemoglobin), glycophorin A, and exogenous polypeptides which can be validated and quantified by standard methods (e.g. Western blotting or FACS analysis).

In some embodiments, the two or more exogenous polypeptides are encoded in a single nucleic acid, e.g. a single vector. In embodiments, the single vector has a separate promoter for each gene, has two polypeptides that are initially transcribed into a single polypeptide having a protease cleavage site in the middle, so that subsequent proteolytic processing yields two exogenous polypeptides, or any other suitable configuration. In some embodiments, the two or more polypeptides are encoded in two or more nucleic acids, e.g., each vector encodes one of the exogenous polypeptides.

The nucleic acid may be, e.g., DNA or RNA. A number of viruses may be used as gene transfer vehicles including retroviruses, Moloney murine leukemia virus (MMLV), adenovirus, adeno-associated virus (AAV), herpes simplex virus (HSV), lentiviruses such as human immunodeficiency virus 1 (HIV 1), and spumaviruses such as foamy viruses, for example.

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 up to 100,000, 200,000, or 500,000 fold). Number of cells is measured, in some embodiments, using an automated cell counter.

In some examples, enucleated erythroid cells or erythroid cell precursors can be transfected with mRNA encoding an exogenous polypeptide to generate engineered enucleated erythroid cells. Messenger RNA can be derived from in vitro transcription of a cDNA plasmid construct containing a sequence encoding an exogenous polypeptide. For example, the cDNA sequence encoding an exogenous polypeptide may be inserted into a cloning vector containing a promoter sequence compatible with specific RNA polymerases. For example, the cloning vector ZAP Express® pBK- CMV (Stratagene, La Jolla, Calif., USA) contains T3 and T7 promoter sequences compatible with the T3 and T7 RNA polymerases, respectively. For in vitro transcription of sense mRNA, the plasmid is linearized at a restriction site downstream of the stop codon(s) corresponding to the end of the sequence encoding the exogenous polypeptide. The mRNA is transcribed from the linear DNA template using a commercially available kit such as, for example, the RNAMaxx® High Yield Transcription Kit (from Stratagene, La Jolla, Calif., USA). In some instances, it may be desirable to generate 5'-m7GpppG-capped mRNA. As such, transcription of a linearized cDNA template may be carried out using, for example, the mMESSAGE mMACHINE High Yield Capped RNA Transcription Kit from Ambion (Austin, Tex., USA). Transcription may be carried out in a reaction volume of 20-100 pL at 37 °C for 30 min to 4 h. The transcribed mRNA is purified from the reaction mix by a brief treatment with DNase I to eliminate the linearized DNA template followed by precipitation in 70% ethanol in the presence of lithium chloride, sodium acetate, or ammonium acetate. The integrity of the transcribed mRNA may be assessed using electrophoresis with an agarose-formaldehyde gel or commercially available Novex pre-cast TBE gels (Novex, Invitrogen, Carlsbad, Calif., USA).

Messenger RNA encoding an exogenous polypeptide may be introduced into enucleated erythroid cells or erythroid cell precursors using a variety of approaches including, for example, lipofection and electroporation (van Tandeloo et al., Blood 98:49-56, 2001). For lipofection, for example, 5 pg of in vitro transcribed mRNA in Opti-MEM (Invitrogen, Carlsbad, Calif., USA) is incubated for 5-15 min at a 1:4 ratio with the cationic lipid DMRIE-C (Invitrogen).

Alternatively, a variety of other cationic lipids or cationic polymers may be used to transfect erythroid cell precursors with mRNA including, for example,

DOTAP, various forms of polyethylenimine, and polyL-lysine (Sigma-Aldrich, Saint Louis, Mo., USA), and Superfect (Qiagen, Inc., Valencia, Calif., USA; See, e.g., Bettinger et al., Nucleic Acids Res. 29:3882-3891, 2001). The resulting mRNA/lipid complexes are incubated with cells (1 - 2 x 10⁶ cells/mL) for 2 hours at 37° C, washed, and returned to culture. For electroporation, for example, about 5 to 20 x 10⁶ cells in 500 pL of Opti-MEM (Invitrogen, Carlsbad, Calif., USA) are mixed with about 20 pg of in vitro transcribed mRNA and electroporated in a 0.4-cm cuvette using, for example, an Easyject Plus device (EquiBio, Kent, United Kingdom). In some instances, it may be necessary to test various voltages, capacitances, and electroporation volumes to determine the useful conditions for transfection of a particular mRNA into an erythroid cell precursor. In general, the electroporation parameters required to efficiently transfect cells with mRNA appear to be less detrimental to cells than those required for electroporation of DNA (van Tandeloo et al., Blood 98:49-56, 2001).

Alternatively, mRNA may be transfected into enucleated erythroid cells or erythroid cell precursors using a peptide-mediated RNA delivery strategy (See, e.g., Bettinger et al., Nucleic Acids Res. 29:3882-3891, 2001). For example, the cationic lipid polyethylenimine 2 kDA (Sigma-Aldrich, Saint Louis, Mo., USA) may be combined with the melittin peptide (Alta Biosciences, Birmingham, UK) to increase the efficiency of mRNA transfection, particularly in postmitotic primary cells. The mellitin peptide may be conjugated to the PEI using a disulfide cross-linker such as, for example, the hetero-bifunctional cross-linker succinimidyl 3-(2-pyridyldithio) propionate. In vitro transcribed mRNA is preincubated for 5 to 15 min with the mellitin-PEI to form an RNA/peptide/lipid complex. This complex is then added to cells in serum-free culture medium for 2 to 4 h at 37 °C in a 5% CO2 humidified environment, then removed, and the transfected cells further cultured.

In some embodiments, the engineered enucleated erythroid cells are generated by introducing a nucleic acid (e.g., any of the exemplary nucleic acids described herein) encoding one or more exogenous polypeptide(s) (e.g., any exogenous polypeptide or any combination of exogenous polypeptides described herein) into an erythroid cell precursor. In some embodiments, the exogenous polypeptide is encoded by a DNA, which is introduced into an erythroid cell precursor. In some embodiments, the exogenous polypeptide is encoded by an RNA, which is introduced into an erythroid cell precursor.

Nucleic acid encoding one or more exogenous polypeptide(s) may be introduced into an erythroid cell precursor prior to terminal differentiation into an enucleated erythroid cell using a variety of DNA techniques, including, e.g., transient or stable transfections and gene therapy approaches.

Viral gene transfer may be used to transfect the cells with a nucleic acid encoding one or more exogenous polypeptide(s). A number of viruses may be used as gene transfer vehicles including Moloney murine leukemia virus (MMLV), adenovirus, adeno-associated virus (AAV), herpes simplex virus (HSV), lentiviruses such as human immunodeficiency virus 1 (HIV1), and spumaviruses such as foamy viruses (see, e.g., Osten et al., HEP 178:177-202, 2007). Retroviruses, for example, efficiently transduce mammalian cells including human cells and integrate into chromosomes, conferring stable gene transfer.

A nucleic acid encoding one or more exogenous polypeptide(s) can be transfected into an erythroid cell precursor. A suitable vector is the Moloney murine leukemia virus (MMLV) vector (Malik et al., Blood 91:2664-2671, 1998). Vectors based on MMLV, 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 the cDNA encoding an exogenous polypeptide can be generated in the MMLV vector backbone using standard molecular biology techniques. The construct is transfected into a packaging cell line such as, for example, PA317 cells and the viral supernatant is used to transfect producer cells such as, for example,

PG13 cells. The PG13 viral supernatant is incubated with an erythroid cell precursor. The expression of the exogenous polypeptide may be monitored using FACS analysis (fluorescence-activated cell sorting), for example, with a fluorescently labeled antibody directed against the exogenous polypeptide, if it is present on the membrane of the engineered human enucleated erythroid cell. Similar methods may be used such that an exogenous polypeptide is present in the cytosol of an engineered human enucleated erythroid cell.

Optionally, a nucleic acid encoding a fluorescent tracking molecule such as, for example, green fluorescent protein (GFP), can be transfected into an erythroid cell precursor using a viral-based approach (Tao et al., Stem Cells 25:670-678, 2007). Ecotopic retroviral vectors containing DNA encoding the enhanced green fluorescent protein (EGFP) or a red fluorescent protein (e.g., DsRed-Express) are packaged using a packaging cell such as, for example, the Phoenix-Eco cell line (distributed by Orbigen, San Diego, Calif.). Packaging cell lines stably express viral polypeptides needed for proper viral packaging including, for example, gag, pol, and env. Supernatants from the Phoenix-Eco cells into which viral particles have been shed are used to transduce erythroid cell precursors. In some instances, transduction may be performed on a specially coated surface such as, for example, fragments of recombinant fibronectin to improve the efficiency of retroviral mediated gene transfer (e.g., RetroNectin, Takara Bio USA, Madison, Wis.). Cells are incubated in RetroNectin-coated plates with retroviral Phoenix-Eco supernatants plus suitable co factors. Transduction may be repeated the next day. In this instance, the percentage of erythroid precursor cells expressing EGFP or DsRed-Express may be assessed by FACS. Other reporter genes that may be used to assess transduction efficiency include, for example, beta-galactosidase, chloramphenicol acetyltransferase, and luciferase, as well as low-afifmity nerve growth factor receptor (LNGFR), and the human cell surface CD24 antigen (Bierhuizen et al., Leukemia 13:605-613, 1999).

Nonviral vectors may be used to introduce a nucleic acid encoding one or more exogenous polypeptide(s) into an erythroid precursor cell to generate engineered enucleated erythroid cells. A number of delivery methods can be used to introduce nonviral vectors into erythroid cell precursors including chemical and physical methods.

A nonviral vector encoding an exogenous polypeptide may be introduced into an erythroid cell precursor using synthetic macromolecules, such as cationic lipids and polymers (Papapetrou et al., Gene Therapy 12:S118-S130, 2005). Cationic liposomes, for example form complexes with DNA through charge interactions. The positively charged DNA/lipid complexes bind to the negative cell surface and are taken up by the cell by endocytosis. This approach may be used, for example, to transfect hematopoietic cells (see, e.g., Keller et al., Gene Therapy 6:931-938, 1999). For erythroid cell precursors, the plasmid DNA (in a serum-free medium, such as, for example, OptiMEM (Invitrogen, Carlsbad, Calif.)) is mixed with a cationic liposome (in serum free medium), such as the commercially available transfection reagent Lipofectamine™ (Invitrogen, Carlsbad, Calif.), and allowed to incubate for at least 20 minutes to form complexes. The DNA/liposome complex is added to erythroid cell precursors and allowed to incubate for 5-24 h, after which time transgene expression of the exogenous polypeptide(s) may be assayed. Alternatively, other commercially available liposome transfection agents may be used (e.g., In vivo GeneSHUTTLE™, Qbiogene, Carlsbad, Calif.).

Optionally, a cationic polymer such as, for example, polyethylenimine (PEI) may be used to efficiently transfect erythroid cell precursors, for example hematopoietic and umbilical cord blood-derived CD34⁺ cells (see, e.g., Shin et al., Biochim. Biophys. Acta 1725:377-384, 2005). Human CD34⁺ cells are 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. Plasmid DNA encoding the exogenous polypeptide(s) is incubated with branched or linear PEIs varying in size from 0.8 K to 750 K (Sigma Aldrich, Saint Louis, Mo., USA; Fermetas, Hanover, Md., USA). PEI is prepared as a stock solution at 4.2 mg/mL distilled water and slightly acidified to pH 5.0 using HC1. The DNA may be combined with the PEI for 30 min at room temperature at various nitrogen/phosphate ratios based on the calculation that 1 pg of DNA contains 3 nmol phosphate and 1 pL of PEI stock solution contains 10 nmol amine nitrogen. The isolated CD34⁺ cells are seeded with the DNA/cationic complex, centrifuged at 280 xg for 5 minutes and incubated in culture medium for 4 or more hours until expression of the exogenous polypeptide(s) is/are assessed.

A plasmid vector may be introduced into suitable erythroid cell precursors using a physical method such as particle-mediated transfection, “gene gun,” biolistics, or particle bombardment technology (Papapetrou, et al., Gene Therapy 12:S118-S130, 2005). In this instance, DNA encoding the exogenous polypeptide is absorbed onto gold particles and administered to cells by a particle gun. This approach may be used, for example, to transfect erythroid cell precursors, e.g., hematopoietic stem cells derived from umbilical cord blood (See, e.g., Verma et al., Gene Therapy 5:692-699, 1998). As such, umbilical cord blood is isolated and diluted three-fold in phosphate buffered saline. CD34⁺ cells are purified using an anti-CD34 monoclonal antibody in combination with magnetic microbeads coated with a secondary antibody and a magnetic isolation system (e.g., Miltenyi MiniMac System, Auburn, Calif., USA).

The CD34⁺ enriched cells may be cultured as described herein. For transfection, plasmid DNA encoding the exogenous polypeptide(s) is precipitated onto a particle, e.g., gold beads, by treatment with calcium chloride and spermidine. Following washing of the DNA-coated beads with ethanol, the beads may be delivered into the cultured cells using, for example, aBiolistic PDS-1000/He System (Bio-Rad, Hercules, Calif., USA). A reporter gene such as, for example, beta-galactosidase, chloramphenicol acetyltransferase, luciferase, or green fluorescent protein may be used to assess efficiency of transfection.

Optionally, electroporation methods may be used to introduce a plasmid vector into erythroid cell precursors. Electroporation creates transient pores in the cell membrane, allowing for the introduction of various molecules into the cells including, for example, DNA and RNA. As such, CD34⁺ cells are isolated and cultured as described herein. Immediately prior to electroporation, the cells are isolated by centrifugation for 10 min at 250xg at room temperature and resuspended at 0.2- lOxlO⁶ viable cells/ml in an electroporation buffer such as, for example, X-VIVO 10 supplemented with 1.0% human serum albumin (HSA). The plasmid DNA (1-50 pg) is added to an appropriate electroporation cuvette along with 500 pL of cell suspension.

Electroporation may be done using, for example, an ECM 600 electroporator (Genetronics, San Diego, Calif., USA) with 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 may be used for this purpose (e.g., Gene Pulser Xcell™, BioRad, Hercules, Calif.; Cellject Duo, Thermo Science, Milford, Mass.). Alternatively, efficient electroporation of isolated CD34⁺ cells may be performed using the following parameters: 4 mm cuvette, 1600 pE, 550 V/cm, and 10 pg of DNA per 500 pL of cells at lxlO⁵ cells/mL (Oldak et al., Acta Biochim. Polonica 49:625-632, 2002).

Nucleofection, a form of electroporation, may also be used to transfect erythroid cell precursors. In this instance, transfection is performed using electrical parameters in cell-type specific solutions that enable DNA (or other reagents) to be directly transported to the nucleus, thus reducing the risk of possible degradation in the cytoplasm. For example, a Human CD34 Cell Nucleofector™ Kit (from Amaxa Inc.) may be used to transfect erythroid cell precursors. In this instance, l-5xl0⁶ cells in Human CD34 Cell Nucleofector™ Solution are mixed with 1-5 pg of DNA and transfected in the Nucleofector™ instrument using preprogrammed settings as determined by the manufacturer.

Erythroid cell precursors may be non-virally transfected with a conventional expression vector which is unable to self-replicate in mammalian cells unless it is integrated in the genome. Alternatively, erythroid cell precursors may be transfected with an episomal vector which may persist in the host nucleus as autonomously replicating genetic units without integration into chromosomes (Papapetrou et al., Gene Therapy 12:S118-S130, 2005). These vectors exploit genetic elements derived from viruses that are normally extrachromosomally replicating in cells upon latent infection such as, for example, EBV, human polyomavirus BK, bovine papilloma virus-1 (BPV-1), herpes simplex virus- 1 (HSV), and Simian virus 40 (SV40). Mammalian artificial chromosomes may also be used for nonviral gene transfer (Vanderbyl et al., Exp. Hematol. 33:1470-1476, 2005).

Exogenous nucleic acid encoding one or more exogenous polypeptide(s) can be assembled into expression vectors by standard molecular biology methods known in the art, e.g., restriction digestion, overlap-extension PCR, and Gibson assembly.

Exogenous nucleic acids can comprise a gene encoding an exogenous polypeptide that is not normally present on the cell surface, e.g., of an enucleated erythroid cell, fused to a gene that encodes an endogenous or native membrane polypeptide, such that the exogenous polypeptide is expressed on the cell surface. For example, an exogenous gene encoding an exogenous polypeptide can be cloned at the N terminus following the leader sequence of a type 1 membrane polypeptide, at the C terminus of a type 2 membrane polypeptide, or upstream of the GPI attachment site of a GPI-linked membrane polypeptide.

Standard cloning methods can be used to introduce flexible amino acid linkers between two fused genes. For example, the flexible linker is a poly-glycine poly serine linker such as [Gly₄Ser]₃ (SEQ ID NO: 37) commonly used in generating 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 polypeptides such as those used to generate single-chain Arc repressors (Robinson & Sauer, Proc. Nat’l. Acad. Sci. U.S.A. 95: 5929-34, 1998). In some embodiments, the flexible linker provides the exogenous polypeptide with more flexibility and steric freedom than the equivalent construct without the flexible linker. This added flexibility is useful in applications that require binding to a target, e.g., an antibody or polypeptide, or an enzymatic reaction of the polypeptide for which the active site must be accessible to the substrate (e.g., the target).

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

Conditions for the electroporation of cell lines have been described in the literature, e.g. by Van Tendeloo et al., Blood 98(l):49-56, 2001. Suitable electroporation conditions for the methods described herein include for a Life Technologies Neon Transfection System: a pulse voltage ranging from about 500 to about 2000 V, from about 800 to about 1800 V, or from about 850 to about 1700 V; a pulse width ranging from about 5 to about 50 msec, or from about 10 to about 40 msec; and a pulse number ranging from 1 to 2 pulses, 1 to 3 pulses, 1 to 4 pulses, or 1 to 5 pulses.

Particularly suitable conditions for electroporation of erythroid cell precursors include, e.g., for 4 days: a) pulse voltage 1300-1400, pulse width: 10-20 msec, number of pulses: 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.

Particularly suitable conditions for electroporation of erythroid cell precursors include, e.g., for 8-9 days: a) pulse voltage: 1400-1600, pulse width: 20, number of pulses: 1 ; b) pulse voltage: 1100-1300, pulse width: 30, number of pulses: 1; c) pulse voltage: 1000-1200, pulse width: 40, number of pulses: 1 ; d) pulse voltage: 1100- 1400, pulse width: 20, number of pulses: 2; e) pulse voltage: 950-1150, pulse width: 30, number of pulses: 2; f) pulse voltage: 1300-1600, pulse width: 10, number of pulses: 3. These conditions generally lead to transfections efficiencies 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 cell viability of at least about 70% or more (e.g. at least about 75%, 80%, 85%, 90%, 95% or at least about 97%, or more).

Particularly suitable conditions for electroporation of erythroid cell precursors in culture under differentiation conditions include, e.g. for 12-13 days: a) pulse voltage: 1500-1700, pulse width: 20, number of pulses: 1; and b) pulse voltage: 1500- 1600, pulse width: 10, number of pulses: 3. These conditions generally lead to transfections efficiencies 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 cell viability of 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 with reference to the Life Technologies Neon system can easily be adjusted by one of ordinary skill in the art to fit a different electroporator and/or different electroporation set-ups with only routine experimentation and the specific electroporator described herein is not limiting for the methods disclosed.

In some embodiments, using the electroporation conditions described herein cultured erythroid cell precursors are electroporated for a first time, then cultured for a desired period of time (optionally under differentiation conditions) and then re-electroporated a second time. In some embodiments, cultured erythroid cell precursors are electroporated for a first time, then cultured for a desired period of time (optionally under differentiation conditions) and then re-electroporated a second, third, fourth, fifth, or sixth time. Optionally, the culturing period in between the first and second, the second and third, etc. electroporation can be varied. For example, the period in between electroporations may be adjusted as desired, e.g. the period may be 30 minutes, 1 hour, 6 hours, 12, hours, 18 hours, 24 hours, 30 hours, 36 hours, 48 hours,

3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days 12 days, 13 days 14 days, or 21 days. For example, erythroid cell precursors may be electroporated on day 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 and 13, 1 and 14, 1 and 15, or 1 and 16. In another example, cells may be electroporated on day 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 and 13, 2 and 14, 2 and 15, or 2 and 16. In yet another example, erythroid cell precursors may be electroporated on day 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, 3 and 14, 3 and 15, or 3 and 16. In yet another example, cells may be electroporated on day 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, 4 and 15, or 4 and 16. In yet another example, cells may be electroporated on day 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 5 and 16. In yet another example, erythroid cell precursors may be electroporated on day 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. In yet another example, erythroid cell precursors may be electroporated on day 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. In yet another example, erythroid cell precursors may be electroporated on day 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. In yet another example, erythroid cell precursors may be electroporated on day 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, erythroid cell precursors may be electroporated on day 10 and 11, 10 and 12, 10 and 13, 10 and 14, 10 and 15, or 10 and 16. In yet another example, erythroid cell precursors may be electroporated on day 11 and 12, 11 and 13, 11 and 14, 11 and 15, or 11 and 16. In yet another example, erythroid cell precursors may be electroporated on day 12 and 13,

12 and 14, 12 and 15, or 12 and 16. In yet another example, erythroid cell precursors may be electroporated on day 13 and 14, 13 and 15, or 13 and 16. In yet another example, erythroid cell precursors may be electroporated on day 14 and 15, or 14 and 16. Optionally, the erythroid cell precursors may be electroporated more than twice, e.g., three times, four times, five times, or six times and the interval may be selected as desired at any points of the differentiation process of the cells.

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

In some embodiments, the engineered enucleated erythroid cells can be click- conjugated engineered enucleated erythroid cells. A catalytic bond-forming polypeptide domain can be expressed on or in, e.g., an erythroid cell precursor, present in the cytosol or present on the membrane. Many catalytic bond-forming polypeptides exist, including transpeptidases, sortases, and isopeptidases, including those derived from Spy0128, a polypeptide isolated from Streptococcus pyogenes. It has been demonstrated that splitting the autocatalytic isopeptide bond-forming subunit (CnaB2 domain) of Spy0128 results in two distinct polypeptides that retain catalytic activity with specificity for each other. The polypeptides in this system are termed Spy Tag and SpyCatcher. Upon mixing, Spy Tag and SpyCatcher undergo isopeptide bond formation between Aspl 17 on Spy Tag and Lys31 on SpyCatcher (Zakeri and Howarth, JACS 132:4526, 2010). The reaction is compatible with the cellular environment and highly specific for polypeptide/peptide conjugation (Zakeri et ak, Proc. Natl. Acad. Sci. U.S.A. 109:E690-E697, 2012). Spy Tag and SpyCatcher have been shown to direct post-translational topological modification in elastin-like protein. For example, placement of Spy Tag at the N-terminus and SpyCatcher at the C -terminus directs formation of circular elastin-like proteins (Zhang et al, J. Am. Chem. Soc. 2013).

The components Spy Tag and SpyCatcher can be interchanged such that a system in which molecule A is fused to Spy Tag 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 Spy Tag. For the purposes of this disclsoure, when Spy Tag and SpyCatcher are used, it is to be understood that the complementary molecule could be substituted in its place.

A catalytic bond-forming polypeptide, such as a SpyTag/SpyCatcher system, can be used to attach the exogenous polypeptide to the surface of, e.g., an erythroid cell precursor or an enucleated erythroid cell. The Spy Tag polypeptide sequence can be expressed on the extracellular surface of the erytroid cell precursor or the enucleated erythroid cell. The SpyTag polypeptide can be, for example, fused to the N terminus of a type- 1 or type-3 transmembrane polypeptide, e.g., glycophorin A, fused to the C terminus of a type-2 transmembrane polypeptide, e.g., Kell, inserted in-frame at the extracellular terminus or in an extracellular loop of a multi-pass transmembrane polypeptide, e.g., Band 3, fused to a GPI-acceptor polypeptide, e.g., CD55 or CD59, fused to a lipid-chain-anchored polypeptide, or fused to a peripheral membrane polypeptide. An exogenous polypeptide can be fused to SpyCatcher. The nucleic acid encoding the SpyCatcher fusion can be expressed and secreted from the same erythroid cell precursor or enucleated erythroid cell that expresses the SpyTag fusion. Alternatively, the nucleic acid sequence encoding the SpyCatcher fusion can be produced exogenously, for example in a bacterial, fungal, insect, mammalian, or cell- free production system. Upon reaction of the SpyTag and SpyCatcher polypeptides, a covalent bond will be formed that attaches the exogenous polypeptide to the surface of the erythroid cell precursor or the enucleated erythroid cell.

In one embodiment, the SpyTag polypeptide may be expressed as a fusion to the N terminus of glycophorin A under the control of the Gatal promoter in an erythroid cell. An exogenous polypeptide fused to the SpyCatcher polypeptide sequence can be expressed under the control of the Gatal promoter in the same erythroid cell. Upon expression of both fusion polypeptides, an isopeptide bond will be formed between the SpyTag and SpyCatcher polypeptides, forming a covalent bond between the erythroid cell surface and the exogenous polypeptide. In another embodiment, the SpyTag polypeptide may be expressed as a fusion to the N terminus of glycophorin A under the control of the Gatal promoter in an erythroid cell precursor or an enucleated erythroid cell. An exogenous polypeptide fused to the SpyCatcher polypeptide sequence can be expressed in a suitable mammalian cell expression system, for example HEK293 cells. Upon expression of the SpyTag fusion polypeptide on the erythroid cell precursor or enucleated erythroid cell, the SpyCatcher fusion polypeptide can be brought in contact with the cell. Under suitable reaction conditions, an isopeptide bond will be formed between the SpyTag and SpyCatcher polypeptides, forming a covalent bond between the erythroid cell precursor surface or enucleated erythroid cell surface and the exogenous polypeptide.

A catalytic bond-forming polypeptide, such as a SpyTag/SpyCatcher system, can be used to anchor an exogenous polypeptide to the intracellular space of an erythroid cell precursor or enucleated erythroid cell. The SpyTag polypeptide sequence can be expressed in the intracellular space of the erythroid cell precursor or enucleated erythroid cell by a number of methods, including direct expression of the transgene, fusion to an endogenous intracellular polypeptide such as, e.g., hemoglobin, fusion to the intracellular domain of endogenous cell surface polypeptides such as, e.g., Band 3, glycophorin A, Kell, or fusion to a structural component of the cytoskeleton. The SpyTag sequence is not limited to a polypeptide terminus and may be integrated within the interior sequence of an endogenous polypeptide such that polypeptide translation and localization is not perturbed. An exogenous polypeptide can be fused to SpyCatcher. The nucleic acid sequence encoding the SpyCatcher fusion can be expressed within the same erythroid cell precursor or enucleated erythroid cell that expresses the SpyTag fusion. Upon reaction of the SpyTag and SpyCatcher polypeptides, a covalent bond will be formed that acts to anchor the exogenous polypeptide in the intracellular space of the erythroid cell precursor or enucleated erythroid cell.

In one embodiment, an erythroid cell precursor or an enucleated erythroid cell may express SpyTag fused to hemoglobin beta intracellularly. The erythroid cell precursor or enucleated erythroid cell may be genetically modified with a gene sequence that includes a hemoglobin promoter, beta globin gene, and a SpyTag sequence such that upon translation, intracellular beta globin is fused to SpyTag at is C terminus. In addition, the erythroid cell precursor or enucleated erythroid cell expresses a Gatal promoter-led gene that codes for SpyCatcher driving polypeptide expression (e.g., phenylalanine hydroxylase (PAH) expression) such that upon translation, intracellular polypeptide (e.g., PAH) is fused to SpyCatcher at its N terminus. Upon expression of both fusion polypeptides the SpyTag bound beta globin is linked through an isopeptide bond to the SpyCatcher bound polypeptide (e.g., PAH) in the intracellular space, allowing the polypeptide (e.g., PAH) to be anchored to beta globin and retained during maturation.

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

Other molecular fusions may be formed between polypeptides and include direct or indirect conjugation. The polypeptides may be directly conjugated to each other or indirectly through a linker. The linker may be a peptide, a polymer, an aptamer, or a nucleic acid. The polymer may be, e.g., natural, synthetic, linear, or branched. Exogenous polypeptides can comprise a heterologous fusion polypeptide that comprises a first polypeptide and a second polypeptide with the fusion polypeptide comprising the polypeptides directly joined to each other or with intervening linker sequences and/or further sequences at one or both ends. The conjugation to the linker may be through covalent bonds or ionic bonds.

In some embodiments, the engineered enucleated erythroid cells are human enucleated erythroid cells that have been hypotonically loaded. For hypotonic loading/lysis, erythroid cell precursors or enucleated erythroid cells are exposed to low ionic strength buffer, causing them to burst. The exogenous polypeptide distributes within the cells. Enucleated erythroid cells or erythroid cell precursors may be hypotonically lysed by adding 30-50 fold volume excess of 5 mM phosphate buffer (pH 8) to a pellet of isolated enucleated erythroid cells. The resulting lysed cell membranes are isolated by centrifugation. The pellet of lysed cell membranes is resuspended and incubated in the presence of the exogenous polypeptide in a low ionic strength buffer, e.g., for 30 min. Alternatively, the lysed cell membranes may be incubated with the exogenous polypeptide for as little as one minute or as long as several days, depending upon the best conditions determined to efficiently load the enucleated erythroid cells or erythroid cell precursors. For hypotonic loading of a nucleic acid encoding one or more exogenous polypeptide(s) (e.g., any of the exemplary exogenous polypeptides described herein or known in the art), a nucleic acid can be suspended in a hypotonic Tris-HCl solution (pH 7.0) and injected into erythroid cell precursors. The concentration of Tris-HCl can be from about 20 mmol/1 to about 150 mmol/1, depending upon the best conditions determined to efficiently load the enucleated erythroid cells.

Alternatively, erythroid cell precursors or enucleated erythroid cells may be loaded with an exogenous polypeptide using controlled dialysis against a hypotonic solution to swell the cells and create pores in the cell membrane (See, e.g., U.S. Pat. Nos. 4,327,710; 5,753,221; 6,495,351, and 10,046,009). For example, a pellet of cells is resuspended in 10 mM HEPES, 140 mM NaCl, 5 mM glucose pH 7.4 and dialyzed against a low ionic strength buffer containing 10 mM Na¾P04, 10 mM NaHCCh, 20 mM glucose, and 4 mM MgC'h. pH 7.4. After 30-60 min, the cells are further dialyzed against 16 mM NaH2P04, pH 7.4 solution containing the exogenous polypeptide for an additional 30-60 min. All of these procedures may be advantageously performed at a temperature of 4 °C. In some instances, it may be beneficial to load a large quantity of erythroid cell precursors or enucleated erythroid cells by a dialysis approach and a specific apparatus designed for this purpose may be used (See, e.g., U.S. Pat. Nos. 4,327,710, 6,139,836 and 6,495,351).

Exemplary methods of manufacturing enucleated erythroid cells (e.g., reticulocytes or erythrocytes) comprising exogenous polypeptides are described, e.g., in WO2015/073587, W02015/153102, W02020/243006, and W02020/219909 each of which is incorporated by reference in its entirety.

In some embodiments, a population of enucleated erythroid cells contains less than 1% live enucleated cells, e.g., contains no detectable live enucleated cells. Enucleation is measured, in some embodiments, by FACS using a nuclear stain. 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 enucleated erythroid cells in the population comprise one or more (e.g., 2, 3, 4 or more) of the exogenous polypeptides. Expression of the exogenous polypeptide can be measured, in some embodiments, by FACS using labeled antibodies 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 cells in the population are enucleated and comprise one or more (e.g., 2, 3, 4, or more) of the exogenous polypeptides. In some embodiments, the population of enucleated erythroid cells comprises about lxl 0⁹ - 2x10⁹, 2xl0⁹ - 5xl0⁹, 5xl0⁹ - lxlO¹⁰, lxlO¹⁰ - 2xl0¹⁰, 2xl0¹⁰ - 5xl0¹⁰, 5xl0¹⁰ - lxlO¹¹, lxlO¹¹ - 2xlOⁿ, 2xlOⁿ - 5xl0ⁿ, 5xl0ⁿ - lxlO¹², lxlO¹² - 2xl0¹², 2xl0¹² - 5xl0¹², or 5xl0¹² - lxlO¹³ cells.

Any of the enucleated erythroid cells described herein can be formulated as described in, e.g., WO 2020/219909 (incorporated herein by reference).

Methods of Increasing the Number of NKG2D-positive Lymphocytes in a Subject Previously Identified or Diagnosed as Having a MICA-Positive, a MICB- Positive, or MICA/MICB-Positive Cancer

Also provided herein are methods of increasing the number of NKG2D- positive lymphocytes (e.g., NKG2D-positive NK cells) in a subject previously identified or diagnosed as having a MICA-positive cancer, a MICB-positive cancer, or a MICA/MICB-positive cancer that include administering to the subject a therapeutically effective number of enucleated erythroid cells comprising 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, on its extracellular surface (e.g., any of the exemplary enucleated erythroid cells described herein).

Also provided herein are methods of increasing the number of NKG2D- positive lymphocytes (e.g., NKG2D-positive NK cells) in a subject previously identified or diagnosed as having a MICA-positive cancer, a MICB-positive cancer, or a MICA/MICB-positive cancer comprising administering to the subject a therapeutically effective number of enucleated erythroid cells comprising 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, on its extracellular surface; and a second exogenous polypeptide comprising a 4-1BBL polypeptide or functional fragment thereof (e.g., any of the exemplary enucleated erythroid cells described herein). Some embodiments of these methods further include: administering to the subject an NKG2A inhibitor (e.g., any of the exemplary NKG2A inhibitors described herein).

In some embodiments of these methods, the administration comprises intravenous administration to the subject.

Some embodiments of any of the methods described herein further include administering one or more additional therapeutic agents to the subject. In some embodiments, the one or more additional therapeutic agents is a cancer therapeutic agent. In some embodiments, the cancer therapeutic agent is selected from the group of: an inhibitor of an immune checkpoint molecule (e.g., any of the exemplary inhibitors of an immune checkpoint molecule described herein), a chemotherapeutic agent, a therapeutic antibody, a chimeric antigen receptor-T cell, a kinase inhibitor, or a soluble cytokine. In some embodiments, the one or more additional therapeutic agents can be administered to the subject at the substantially the same time as any of the compositions provided herein.

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

Some embodiments of these methods can further include identifying or diagnosing a subject as having a MICA-positive cancer, a MICB-positive cancer, or a MICA/MICB-positive cancer. Some embodiments of these methods can further include identifying or diagnosing a subject as having a MICA-positive and HLA-E- negative cancer (MICA-positive/HLA-E-negative cancer), a MICB-positive and HLA-E-negative cancer (MICB-positive/HLA-E-negative cancer), or a MICA/MICB- positive and HLA-E-negative cancer (MICA/MICB-positive cancer/HLA-E negative cancer).

For example, in some embodiments, the one or more additional therapeutic agents include an agent that blocks, reduces and/or inhibits PD-1 and either PD-L1 or PD-L2, and/or blocks, reduces and/or inhibits the binding of PD-1 with PD-L1 or PD- L2 (non-limiting examples include one or more of nivolumab (ONO-4538/BMS- 936558, MDX1106, OPDIVO, BRISTOL MYERS SQUIBB), pembrolizumab (KEYTRUDA, MERCK), pidilizumab (CT-011, CURE TECH), MK-3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), and MPDL3280A (ROCHE)). In some embodiments, the one or more additional therapeutic agents include an agent that blocks, reduces and/or inhibits the activity of CTLA-4 and/or the binding of CTLA-4 with one or more of its receptors (e.g., CD80, CD86, AP2M1, SHP-2, and PPP2R5A). For example, in some embodiments, the agent that inhibits the activity of CTLA-4 is an antibody such as ipilimumab (MDX-010, MDX-101, Yervoy, BRISTOL MYERS SQUIBB) and/or tremelimumab (PFIZER). Further, in some embodiments, the one or more additional therapeutic agents include blocking antibodies targeted to an immune checkpoint molecule such as, for example, BTLA, HVEM, TIM3, GALS, LAG3, VISTA, KIR, 2B4, CD160 (also referred to as BY55), CGEN-15049, CHK 1 and CHK2 kinases, A2aR, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), GITR, GITRL, galectin-9, CD244, CD 160, TIGIT, SIRPa, ICOS, CD172a, TMIGD2 and various B-7 family ligands (including, but are 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, the one or more additional therapeutic agents include one or more molecules (e.g., small molecules) that block, reduce, and/or inhibit the activity of PD-1, PD-L1, or PD-L2 (non-limiting examples include one or more of BMS-1166 (BRISTOL MYERS SQUIBB), BMS-103 (BRISTOL MYERS SQUIBB), BMS-142 (BRISTOL MYERS SQUIBB), BMS-202 (BRISTOL MYERS SQUIBB), BMS-1001 (BRISTOL MYERS SQUIBB), BMS-242 (BRISTOL MYERS SQUIBB), BMS-200 (BRISTOL MYERS SQUIBB), BMS-986189 (BRISTOL MYERS SQUIBB), INCB086550 (INCYTE), Aurigene-1 (AURIGENE), AUNP12 (AURIGENE), CA-170 (CURIS), CCX4503 (CHEMOCENTRYX), AMP-224 (ASTRAZENECA/MEDIMMUNE, GSK), and AMP-512 (ASTRAZENECA)). In some embodiments, the one or more additional therapuetic agents include one or more molecules (e.g., small molecules) that block, reduce, and/or inhibit the activity of CTLA-4 and/or the binding of CTLA-4 with one or more of its receptors (e.g., CD80, CD86, AP2M1, SHP-2, and PPP2R5A).

In some embodiments of the methods, the methods result in an increase (e.g., at least a 5% increase, at least 10%, at least a 15% increase, at least a 20% increase, at least a 25% increase, at least a 30% increase, at least a 35% increase, at least a 40% increase, at least a 45% increase, at least a 50% increase, at least a 55% increase, at least a 60% increase, at least a 65% increase, at least a 70% increase, at least a 75% increase, at least a 80% increase, at least a 85% increase, at least a 90% increase, at least a 95% increase, at least a 100% increase, at least a 120% increase, at least a 140% increase, at least a 160% increase, at least a 180% increase, at least a 200% increase, at least a 220% increase, at least a 240% increase, at least a 260% increase, at least a 280% increase, at least a 300% increase, at least a 350% increase, at least a 400% increase, at least a 450% increase, at least 500% increase, at least a 550% increase, at least a 600% increase, at least a 650% increase, at least a 700% increase, at least a 750% increase, at least a 800% increase, at least a 850% increase, at least a 900% increase, at least a 950% increase, or at least a 1,000% increase) in the number of NKG2D-positive lymphocyte (e.g., NKG2D-positive NK cells) in the subject (e.g., as compared to the number of NKG2D-positive lymphocytes (e.g., NKG2D-positive NK cells) in the subject prior to the administering).

In some embodiments of the methods, the methods result in an increase (e.g., at least a 5% increase, at least 10%, at least a 15% increase, at least a 20% increase, at least a 25% increase, at least a 30% increase, at least a 35% increase, at least a 40% increase, at least a 45% increase, at least a 50% increase, at least a 55% increase, at least a 60% increase, at least a 65% increase, at least a 70% increase, at least a 75% increase, at least a 80% increase, at least a 85% increase, at least a 90% increase, at least a 95% increase, at least a 100% increase, at least a 120% increase, at least a 140% increase, at least a 160% increase, at least a 180% increase, at least a 200% increase, at least a 220% increase, at least a 240% increase, at least a 260% increase, at least a 280% increase, at least a 300% increase, at least a 350% increase, at least a 400% increase, at least a 450% increase, at least a 500% increase, at least a 550% increase, at least a 600% increase, at least a 650% increase, at least a 700% increase, at least a 750% increase, at least a 800% increase, at least a 850% increase, at least a 900% increase, at least a 950% increase, or at least a 1,000%) in the number of NKG2D-positive/NKG2A-negative lymphocytes (e.g., NKG2D-positive/NKG2A- negative NK cells) in the subject (e.g., as compared to the number of NKG2D- positive/NKG2A-negative positive cells (e.g., NKG2D-positive/NKG2A-negative NK cells) in the subject prior to administering).

In some embodiments of these methods, the methods result in an increase (e.g., at least a 5% increase, at least 10%, at least a 15% increase, at least a 20% increase, at least a 25% increase, at least a 30% increase, at least a 35% increase, at least a 40% increase, at least a 45% increase, at least a 50% increase, at least a 55% increase, at least a 60% increase, at least a 65% increase, at least a 70% increase, at least a 75% increase, at least a 80% increase, at least a 85% increase, at least a 90% increase, at least a 95% increase, at least a 100% increase, at least a 120% increase, at least a 140% increase, at least a 160% increase, at least a 180% increase, at least a 200% increase, at least a 220% increase, at least a 240% increase, at least a 260% increase, at least a 280% increase, or at least a 300% increase) in the number of trafficking NKG2D-positive lymphocytes (e.g., NKG2D-positive NK cells) in the subject (e.g., as compared to the number of trafficking NKG2D-positive lymphocytes (e.g., NKG2D-positive NK cells) in the subject prior to the administering).

In some embodiments of these methods, the methods result in an increase (e.g., at least a 5% increase, at least 10%, at least a 15% increase, at least a 20% increase, at least a 25% increase, at least a 30% increase, at least a 35% increase, at least a 40% increase, at least a 45% increase, at least a 50% increase, at least a 55% increase, at least a 60% increase, at least a 65% increase, at least a 70% increase, at least a 75% increase, at least a 80% increase, at least a 85% increase, at least a 90% increase, at least a 95% increase, at least a 100% increase, at least a 120% increase, at least a 140% increase, at least a 160% increase, at least a 180% increase, at least a 200% increase, at least a 220% increase, at least a 240% increase, at least a 260% increase, at least a 280% increase, or at least a 300% increase) in the number of trafficking NKG2D-positive/NKG2A-negative lymphocytes (e.g., NKG2D- positive/NKG2A-negative NK cells) in the subject (e.g., as compared to the number of trafficking NKG2D-positive/NKG2A-negative lymphocytes (e.g., NKG2D- positive/NKG2A-negative NK cells) in the subject prior to the administering).

Some embodiments of the methods described herein result in the at least a 0.1- fold increase, at least a 0.2-fold increase, at least a 0.3-fold increase, at least a 0.4-fold increase, at least a 0.5-fold increase, at least a 0.6-fold increase, at least a 0.7-fold increase, at least a 0.8-fold increase, at least a 0.9-fold increase, at least a 1.0-fold increase, at least a 1.5-fold increase, at least a 2.0-fold increase, at least a 2.5-fold increase, at least a 3.0-fold increase, at least a 3.5-fold increase, at least a 4.0-fold increase, at least a 4.5-fold increase, at least a 5.0-fold increase, at least a 5.5-fold increase, at least a 6.0-fold increase, at least a 6.5-fold increase, at least a 7.0-fold increase, at least a 7.5-fold increase, at least a 8.0-fold incrase, at least a 8.5-fold increase, at least a 9.0-fold increase, at least a 9.5-fold increase, or at least a 10-fold increase in the maximum fold change in the ratio of NKG2D-positive lymphocytes (e.g., NKG2D-positive NK cells) to NKG2A-positive lymphocytes (e.g., NKG2A- positive NK cells) in the blood of a subject (e.g., as compared to the ratio of NKG2D- positive lymphocytes (e.g., NKG2D-positive NK cells) to NKG2A-positive lymphocytes (e.g., NKG2A-positive NK cells) in the blood of the subject prior to the administering).

Some embodiments of the methods described herein result in the at least a 0.1- fold increase, at least a 0.2-fold increase, at least a 0.3-fold increase, at least a 0.4-fold increase, at least a 0.5-fold increase, at least a 0.6-fold increase, at least a 0.7-fold increase, at least a 0.8-fold increase, at least a 0.9-fold increase, at least a 1.0-fold increase, at least a 1.5-fold increase, at least a 2.0-fold increase, at least a 2.5-fold increase, at least a 3.0-fold increase, at least a 3.5-fold increase, at least a 4.0-fold increase, at least a 4.5-fold increase, or at least a 5.0-fold increase in the maximum fold increase in the absolute number of NK cells in the blood of the subject (e.g., as compared to the maximum fold increase in the absolute number of NK cells in the blood of the subject prior to the administering).

Some embodiments of the methods described herein result in the at least a 0.1- fold increase, at least a 0.2-fold increase, at least a 0.3-fold increase, at least a 0.4-fold increase, at least a 0.5-fold increase, at least a 0.6-fold increase, at least a 0.7-fold increase, at least a 0.8-fold increase, at least a 0.9-fold increase, at least a 1.0-fold increase, at least a 1.5-fold increase, at least a 2.0-fold increase, at least a 2.5-fold increase, at least a 3.0-fold increase, at least a 3.5-fold increase, at least a 4.0-fold increase, at least a 4.5-fold increase, or at least a 5.0-fold increase in the maximum fold increase in the absolute number of CD56^bnght lymphocytes (e.g., CD56^bnght NK cells) in the blood of the subject (e.g., as compared to the maximum fold increase in the absolute number of CD56^bnght lymphocytes (e.g., CD56^bnght NK cells) in the blood of the subject prior to the administering).

CD56^dim NK cells) in the blood of the subject prior to the administering).

Some embodiments of the methods described herein result in the at least a 0.1- fold increase, at least a 0.2-fold increase, at least a 0.3-fold increase, at least a 0.4-fold increase, at least a 0.5-fold increase, at least a 0.6-fold increase, at least a 0.7-fold increase, at least a 0.8-fold increase, at least a 0.9-fold increase, at least a 1.0-fold increase, at least a 1.5-fold increase, at least a 2.0-fold increase, at least a 2.5-fold increase, at least a 3.0-fold increase, at least a 3.5-fold increase, at least a 4.0-fold increase, at least a 4.5-fold increase, or at least a 5.0-fold increase in the maximum fold increase in the absolute number of HLA-DR⁺ lymphocytes (e.g., HLA-DR ⁺ NK cells) in the blood of the subject (e.g., as compared to the maximum fold increase in the absolute number of circulating HLA-DR lymphocytes (e.g., HLA-DR NK cells) in the subject prior to the administering).

In some embodiments, the subject has previously been diagnosed or identified as having a MICA-positive cancer (e.g., any of the exemplary MICA-positive cancers described herein). In some embodiments, the subject has previously been diagnosed or identified as having a MICA-positive/HLA-E-negative cancer. Some embodiments of these methods can further include identifying or diagnosing a subject as having a MICA-positive cancer. Some embodiments of these methods can further include identifying or diagnosing a subject as having a MICA-positive/HLA-E-negative cancer.

Non-limiting examples of MIC A-positive cancers include: adrenocortical carcinoma, cholangiocarcinoma, pancreatic adenocarcinoma, kidney cancer, thyroid carcinoma, mesothelioma, skin cutaneous melanoma, colorectal cancer, cervical squamous cell carcinoma and endocervical adenocarcinoma. In some embodiments, a MICA-positive cancer includes, without limitation: adrenocortical carcinoma, anal cancer, bile duct cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, acute myeloid leukemia, lymphoid neoplasm 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 corpus endometrial carcinoma, uterine carcinosarcoma, and uveal melanoma.

In some embodiments, the subject has previously been diagnosed or identified as having a MICB-positive cancer (e.g., any of the exemplary MICB-positive cancers described herein). In some embodiments, the subject has previously been diagnosed or identified as having a MICB-positive/HLA-E -negative cancer. Some embodiments of these methods can further include identifying or diagnosing a subject as having a MICB-positive cancer. Some embodiments of these methods can further include identifying or diagnosing a subject as having a MICB-positive/HLA-E-negative cancer.

Non-limiting examples of MICB-positive cancers include: acute myeloid leukemia, lymphoid neoplasm diffuse large B-cell lymphoma, testicular germ cell tumors, stomach adenocarcinoma, ovarian serous cystadenocarcinoma, esophageal carcinoma and lung cancer. In some embodiments, a MICB-positive cancer includes, without limitation: adrenocortical carcinoma, anal cancer, bile duct cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, acute myeloid leukemia, lymphoid neoplasm 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 corpus endometrial carcinoma, uterine carcinosarcoma, and uveal melanoma.

In some embodiments, the subject has previously been diagnosed or identified as having a MICA/MICB-positive cancer (e.g., any of the exemplary MICA/MICB- positive cancers described herein). In some embodiments, the subject has previously been diagnosed or identified as having a MICA/MICB-positive/HLA-E -negative cancer. Some embodiments of these methods can further include identifying or diagnosing a subject as having a MICA/MICB-positive cancer. Some embodiments of these methods can further include identifying or diagnosing a subject as having a MICA/MICB-positive/HLA-E-negative cancer. Non-limiting examples of MIC A/MICB-positive cancers include: adrenocortical carcinoma, acute myeloid leukemia, pancreatic adenomcarcinoma, cholangiocarcinoma, kidney cancer, cervical squamous cell carcinoma, endocervical carcinoma, colorectal cancer, mesothelioma, skin cutaneous melanoma and lung cancer. In some embodiments, a MICA/MICB-positive cancer includes, without limitation: adrenocortical carcinoma, anal cancer, bile duct cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, acute myeloid leukemia, lymphoid neoplasm 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 corpus endometrial carcinoma, uterine carcinosarcoma, and uveal melanoma.

The enucleated erythroid cells can be administered to the subject using any of the doses and at any of the frequencies described herein.

Methods of Increasing the Ratio of the Concentration of NKG2D to the Concentration ofNKG2A on the Cell Surface of Lymphocytes

Also provided herein are methods of increasing the ratio of the concentration ofNKG2D to the concentration ofNKG2A on the cell surface of lymphocytes (e.g., NK cells) in a subject previously identified or diagnosed as having a MICA-positive, a MICB-positive, or a MICA/MICB-positive cancer that include adminsitering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a population of enucleated erythroid cells including an first exogenous polypeptide that includes (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof, on its extracellular surface. Also provided herein are methods of increasing the ratio of the concentration of NKG2D to the concentration of NKG2A on the cell surface of lymphocytes (e.g., NK cells) in a subject previously identified or diagnosed as having a MICA-positive, a MICB- positive, or a MICA/MICB-positive cancer that include adminsitering to the subject a pharmaceutical composition comprising a population of enucleated erythroid cells including a first exogenous fusion polypeptide including (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof, on its extracellular surface; and a second exogenous polypeptide including 4-1BBL or a functional fragment thereof, on its extracellular surface.

Some embodiments of these methods result in at least a 1% increase, at least a 5% increase, at least a 10% increase, at least a 15% increase, at least a 20% increase, at least a 30% increase, at least a 40% increase, at least a 50% increase, at least a 60% increase, at least a 70% increase, at least a 80% increase, at least a 90% increase, at least 100% increase, at least a 110% increase, at least a 120% increase, at least a 130% increase, at least a 140% increase, at least a 150% increase, at least a 160% increase, at least a 170% increase, at least a 180% increase, at least a 190% increase, at least a 200% increase, at least a 250% increase, at least a 300% increase, at least a 350% increase, at least a 400% increase, at least a 450% increase, at least a 500% increase, at least a 550% increase, at least a 600% increase, at least a 650% increase, at least a 700% increase, at least a 750% increase, at least a 800% increase, at least a 850% increase, at least a 900% increase, at least a 950% increase, or at least a 1,000% increase, in the ratio of the concentration of NKG2D to the concentration of NKG2A on the cell surface of lymphocytes (e.g., NK cells) in the subject (e.g., as compared to the ratio of the concentration of NKG2D to the concentration of NKG2A on the cell surface of lymphocytes (e.g., NK cells) in the subject prior to the administering).

Some embodiments of these methods result in about a 0.01 -fold increase to about a 10-fold increase (or any of the exemplary subranges of this range described herein) in the ratio of the concentration of NKG2D to the concentration of NKG2A on the cell surface of lymphocytes (e.g., NK cells) in the subject (e.g., as compared to the ratio of the concentration of NKG2D to the concentration of NKG2A on the cell surface of lymphocytes (e.g., NK cells) in the subject prior to the administering).

Some embodiments of these methods further include: administering to the subject an NKG2A inhibitor (e.g., any of the exemplary NKG2A inhibitors described herein).

In some embodiments, the one or more additional therapeutic agents can be administered to the subject before or after the administration of any of the compositions described herein to the subject. Some embodiments of these methods can further include identifying or diagnosing a subject as having a MICA-positive cancer, a MICB-positive cancer, or a MICA/MICB positive cancer. Some embodiments of these methods can further include identifying or diagnosing a subject as having a MICA-positive/HLA-E- negative cancer, a MICB-positive/HLA-E-negative cancer, or a a MICA/MICB- positive/HLA-E-negative cancer.

For example, in some embodiments, the one or more additional therapeutic agents include an agent that blocks, reduces and/or inhibits PD-1 and either PD-L1 or PD-L2, and/or blocks, reduces and/or inhibits the binding of PD-1 with PD-L1 or PD- L2 (non-limiting examples include one or more of nivolumab (ONO-4538/BMS- 936558, MDX1106, OPDIVO, BRISTOL MYERS SQUIBB), pembrolizumab (KEYTRUDA, MERCK), pidilizumab (CT-011, CURE TECH), MK-3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), and MPDL3280A (ROCHE)). In some embodiments, the one or more additional therapeutic agents include an agent that blocks, reduces and/or inhibits the activity of CTLA-4 and/or the binding of CTLA-4 with one or more of its receptors (e.g., CD80, CD86, AP2M1, SHP-2, and PPP2R5A). For example, in some embodiments, the agent that inhibits the activity of CTLA-4 is an antibody such as ipilimumab (MDX-010, MDX-101, Yervoy, BRISTOL MYERS SQUIBB) and/or tremelimumab (PFIZER). Further, in some embodiments, the one or more additional therapeutic agents include blocking antibodies targeted to an immune checkpoint molecule such as, for example, BTLA, HVEM, TIM3, GALS, LAG3, VISTA, KIR, 2B4, CD160 (also referred to as BY55), CGEN-15049, CHK 1 and CHK2 kinases, A2aR, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), GITR, GITRL, galectin-9, CD244, CD 160, TIGIT, SIRPa, ICOS, CD172a, TMIGD2 and various B-7 family ligands (including, but are 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, the one or more additional therapeutic agents include one or more molecules (e.g., small molecules) that block, reduce, and/or inhibit the activity of PD-1, PD-L1 or PD-L2 (non-limiting examples include one or more of BMS-1166 (BRISTOL MYERS SQUIBB), BMS-103 (BRISTOL MYERS SQUIBB), BMS-142 (BRISTOL MYERS SQUIBB), BMS-202 (BRISTOL MYERS SQUIBB), BMS-1001 (BRISTOL MYERS SQUIBB), BMS-242 (BRISTOL MYERS SQUIBB), BMS-200 (BRISTOL MYERS SQUIBB), BMS-986189 (BRISTOL MYERS SQUIBB), INCB086550 (INCYTE), Aurigene-1 (AURIGENE), AUNP12 (AURIGENE), CA-170 (CURIS), CCX4503 (CHEMOCENTRYX), AMP-224 (ASTRAZENECA/MEDIMMUNE, GSK), and AMP-512 (ASTRAZENECA)). In some embodiments, the one or more additional therapuetic agents include one or more molecules (e.g., small molecules) that block, reduce, and/or inhibit the activity of CTLA-4 and/or the binding of CTLA-4 with one or more of its receptors (e.g., CD80, CD86, AP2M1, SHP-2, and PPP2R5A).

Non-limiting examples of MIC A-positive cancers include: adrenocortical carcinoma, cholangiocarcinoma, pancreatic adenocarcinoma, kidney cancer, thyroid carcinoma, mesothelioma, skin cutaneous melanoma, colorectal cancer, cervical squamous cell carcinoma and endocervical adenocarcinoma. In some embodiments, a MICA-positive cancer include, without limitation: adrenocortical carcinoma, anal cancer, bile duct cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, acute myeloid leukemia, lymphoid neoplasm 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 corpus endometrial carcinoma, uterine carcinosarcoma, and uveal melanoma.

Non-limiting examples of MICB-positive cancers include: acute myeloid leukemia, lymphoid neoplasm diffuse large B-cell lymphoma, testicular germ cell tumors, stomach adenocarcinoma, ovarian serous cystadenocarcinoma, esophageal carcinoma and lung cancer. In some embodiments, a MICB-positive cancer include, without limitation: adrenocortical carcinoma, anal cancer, bile duct cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, acute myeloid leukemia, lymphoid neoplasm 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 corpus endometrial carcinoma, uterine carcinosarcoma, and uveal melanoma.

In some embodiments, the subject has previously been diagnosed or identified as having a MICA/MICB-positive cancer (e.g., any of the exemplary MICA/MICB- positive cancers described herein). In some embodiments, the subject has previously been diagnosed or identified as having a MICA/MICB-positive/HLA-E -negative cancer. Some embodiments of these methods can further include identifying or diagnosing a subject as having a MICA/MICB-positive cancer. Some embodiments of these methods can further include identifying or diagnosing a subject as having a MICA/MICB-positive/HLA-E-negative cancer.

Non-limiting examples of MIC A/MICB-positive cancers include: adrenocortical carcinoma, acute myeloid leukemia, pancreatic adenomcarcinoma, cholangiocarcinoma, kidney cancer, cervical squamous cell carcinoma, endocervical carcinoma, colorectal cancer, mesothelioma, skin cutaneous melanoma and lung cancer. In some embodiments, a MICA/MICB-positive cancer include, without limitation: adrenocortical carcinoma, anal cancer, bile duct cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, acute myeloid leukemia, lymphoid neoplasm 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 corpus endometrial carcinoma, uterine carcinosarcoma, and uveal melanoma.

Methods of Treating a Subject having a MICA-Positive Cancer

Also provided herein are methods of treating a subject previously identified or diagnosed as having a MICA-positive cancer (e.g., any of the exemplary MICA- positive cancers described herein) that include administering to the subject a therapeutically effective number of enucleated erythroid cells comprising 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, on its extracellular surface (e.g., any of the exemplary enucleated erythroid cells described herein).

Also provided herein are methods of treating a subject previously identified or diagnosed as having a MICA-positive cancer (e.g., any of the exemplary MICA- positive cancers described herein) that include administering to the subject a therapeutically effective number of enucleated erythroid cells comprising 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, on its extracellular surface; and a second exogenous polypeptide comprising a 4-1BBL polypeptide or a functional fragment thereof (e.g., any of the exemplary enucleated erythroid cells described herein).

In some embodiments of these methods, the administration is intravenous administration to the subject.

Some embodiments of these methods further include administering to the subject an NKG2A inhibitor (e.g., any of the exemplary NKG2A inhibitors described herein).

Some embodiments of any of the methods described herein further include administering one or more additional therapeutic agents to the subject. In some embodiments, the one or more additional therapeutic agents is a cancer therapeutic agent. In some embodiments, the cancer therapeutic agent is selected from the group of: an inhibitor of an immune checkpoint molecule (e.g., any of the exemplary inhibitors of an immune checkpoint molecule described herein), a chemotherapeutic agent, a therapeutic antibody, a chimeric antigen receptor-T cell, a kinase inhibitor, or a soluble cytokine. In some embodiments, the one or more additional therapeutic agents can be administered to the subject at the substantially the same time as any of the compositions provided herein. In some embodiments, the one or more additional therapeutic agents can be administered to the subject before or after the administration of any of the compositions described herein to the subject.

For example, in some embodiments, the one or more additional therapeutic agents include an agent that blocks, reduces and/or inhibits PD-1 and either PD-L1 or PD-L2, and/or blocks, reduces and/or inhibits the binding of PD-1 with PD-L1 or PD- L2 (non-limiting examples include one or more of nivolumab (ONO-4538/BMS- 936558, MDX1106, OPDIVO, BRISTOL MYERS SQUIBB), pembrolizumab (KEYTRUDA, MERCK), pidilizumab (CT-011, CURE TECH), MK-3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), and MPDL3280A (ROCHE)). In some embodiments, the one or more additional therapeutic agents include an agent that blocks, reduces and/or inhibits the activity of CTLA-4 and/or the binding of CTLA-4 with one or more of its receptors (e.g., CD80, CD86, AP2M1, SHP-2, and PPP2R5A). For example, in some embodiments, the agent that inhibits the activity of CTLA-4 is an antibody such as ipilimumab (MDX-010, MDX-101, Yervoy, BRISTOL MYERS SQUIBB) and/or tremelimumab (PFIZER). Further, in some embodiments, the one or more additional therapeutic agents include blocking antibodies targeted to an immune checkpoint molecule such as, for example, BTLA, HVEM, TIM3, GALS, LAG3, VISTA, KIR, 2B4, CD160 (also referred to as BY55), CGEN-15049, CHK 1 and CHK2 kinases, A2aR, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), GITR, GITRL, galectin-9, CD244, CD 160, TIGIT, SIRPa, ICOS, CD172a, TMIGD2 and various B-7 family ligands (including, but are 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, the one or more additional therapeutic agents include one or more molecules (e.g., small molecules) that block, reduce, and/or inhibit the activity of PD- 1, PD-L1 or PD-L2 (non-limiting examples include one or more of BMS- 1166 (BRISTOL MYERS SQUIBB), BMS-103 (BRISTOL MYERS SQUIBB), BMS-142 (BRISTOL MYERS SQUIBB), BMS-202 (BRISTOL MYERS SQUIBB), BMS-1001 (BRISTOL MYERS SQUIBB), BMS-242 (BRISTOL MYERS SQUIBB), BMS-200 (BRISTOL MYERS SQUIBB), BMS-986189 (BRISTOL MYERS SQUIBB), INCB086550 (INCYTE), Aurigene-1 (AURIGENE), AUNP12 (AURIGENE), CA- 170 (CURIS), CCX4503 (CHEMOCENTRYX), AMP-224 (ASTRAZENECA/MEDIMMUNE, GSK), and AMP-512 (ASTRAZENECA)). In some embodiments, the one or more additional therapuetic agents include one or more molecules (e.g., small molecules) that block, reduce, and/or inhibit the activity of CTLA-4 and/or the binding of CTLA-4 with one or more of its receptors (e.g., CD80, CD86, AP2M1, SHP-2, and PPP2R5A).

Some embodiments of these methods can further include identifying or diagnosing a subject as having a MICA-positive cancer. Some embodiments of these methods can further include identifying or diagnosing a subject as having a MICA- positive/HLA-E-negative cancer.

Methods of Treating a Subject having a MICB-Positive Cancer

Also provided herein are methods of treating a subject previously identified or diagnosed as having a MICB-positive cancer (e.g., any of the exemplary MICB- positive cancers described herein) that include administering to the subject a therapeutically effective number of enucleated erythroid cells comprising 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, on its extracellular surface (e.g., any of the exemplary enucleated erythroid cells described herein).

Also provided herein are methods of treating a subject previously identified or diagnosed as having a MICB-positive cancer (e.g., any of the exemplary MICB- positive cancers described herein) that include administering to the subject previously identified or diagnosed as having a MICB-positive cancer a therapeutically effective number of enucleated erythroid cells comprising 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, on its extracellular surface; and a second exogenous polypeptide comprising a 4-1BBL polypeptide or a functional fragment thereof (e.g., any of the exemplary enucleated erythroid cells described herein).

For example, in some embodiments, the one or more additional therapeutic agents include an agent that blocks, reduces and/or inhibits PD-1 and either PD-L1 or PD-L2, and/or blocks, reduces and/or inhibits the binding of PD-1 with PD-L1 or PD- L2 (non-limiting examples include one or more of nivolumab (ONO-4538/BMS- 936558, MDX1106, OPDIVO, BRISTOL MYERS SQUIBB), pembrolizumab (KEYTRUDA, MERCK), pidilizumab (CT-011, CURE TECH), MK-3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), and MPDL3280A (ROCHE)). In some embodiments, the one or more additional therapeutic agents include an agent that blocks, reduces and/or inhibits the activity of CTLA-4 and/or the binding of CTLA-4 with one or more of its receptors (e.g., CD80, CD86, AP2M1, SHP-2, and PPP2R5A). For example, in some embodiments, the agent that inhibits the activity of CTLA-4 is an antibody such as ipilimumab (MDX-010, MDX-101, Yervoy, BRISTOL MYERS SQUIBB) and/or tremelimumab (PFIZER). Further, in some embodiments, the one or more additional therapeutic agents include blocking antibodies targeted to an immune checkpoint molecule such as, for example, BTLA, HVEM, TIM3, GALS, LAG3, VISTA, KIR, 2B4, CD160 (also referred to as BY55), CGEN-15049, CHK 1 and CHK2 kinases, A2aR, CEACAM (e.g, CEACAM-1, CEACAM-3 and/or CEACAM- 5), GITR, GITRL, galectin-9, CD244, CD 160, TIGIT, SIRPa, ICOS, CD 172a, TMIGD2 and various B-7 family ligands (including, but are 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, the one or more additional therapeutic agents include one or more molecules (e.g., small molecules) that block, reduce, and/or inhibit the activity of PD- 1, PD-L1 or PD-L2 (non-limiting examples include one or more of BMS- 1166 (BRISTOL MYERS SQUIBB), BMS-103 (BRISTOL MYERS SQUIBB), BMS-142 (BRISTOL MYERS SQUIBB), BMS-202 (BRISTOL MYERS SQUIBB), BMS-1001 (BRISTOL MYERS SQUIBB), BMS-242 (BRISTOL MYERS SQUIBB), BMS-200 (BRISTOL MYERS SQUIBB), BMS-986189 (BRISTOL MYERS SQUIBB), INCB086550 (INCYTE), Aurigene-1 (AURIGENE), AUNP12 (AURIGENE), CA- 170 (CURIS), CCX4503 (CHEMOCENTRYX), AMP-224 (ASTRAZENECA/MEDIMMUNE, GSK), and AMP-512 (ASTRAZENECA)). In some embodiments, the one or more additional therapuetic agents include one or more molecules (e.g., small molecules) that block, reduce, and/or inhibit the activity of CTLA-4 and/or the binding of CTLA-4 with one or more of its receptors (e.g., CD80, CD86, AP2M1, SHP-2, and PPP2R5A).

Some embodiments of these methods can further include identifying or diagnosing a subject as having a MICB-positive cancer. Some embodiments of these methods can further include identifying or diagnosing a subject as having a MICB- positive/HLA-E-negative cancer.

Non-limiting examples of MICB-positive cancers include: acute myeloid leukemia, lymphoid neoplasm diffuse large B-cell lymphoma, testicular germ cell tumors, stomach adenocarcinoma, ovarian serous cystadenocarcinoma, esophageal carcinoma and lung cancer. In some embodiments, a MICB-positive cancer includes, without limitation: adrenocortical carcinoma, bile duct cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, acute myeloid leukemia, lymphoid neoplasm 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 corpus endometrial carcinoma, uterine carcinosarcoma, and uveal melanoma.

Methods of Treating a Subject Having a MICA/MICB-Positive Cancer

Also provided herein are methods of treating a subject previously identified or diagnosed as having a MICA/MICB-positive cancer (e.g., any of the exemplary MICA/MICB-positive cancers described herein) that include administering to the subject a therapeutically effective number of enucleated erythroid cells comprising 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, on its extracellular surface (e.g., any of the exemplary enucleated erythroid cells described herein).

Also provided herein are methods of treating a subject previously identified or diagnosed as having a MICA/MICB-positive cancer (e.g., any of the exemplary MICA/MICB-positive cancers described herein) that include administering to the subject a therapeutically effective number of enucleated erythroid cells comprising 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, on its extracellular surface; and a second exogenous polypeptide comprising a 4-1BBL polypeptide or a functional fragment thereof (e.g., any of the exemplary enucleated erythroid cells described herein).

For example, in some embodiments, the one or more additional therapeutic agents include an agent that blocks, reduces and/or inhibits PD-1 and either PD-L1 or PD-L2, and/or blocks, reduces and/or inhibits the binding of PD-1 with PD-L1 or PD- L2 (non-limiting examples include one or more of nivolumab (ONO-4538/BMS- 936558, MDX1106, OPDIVO, BRISTOL MYERS SQUIBB), pembrolizumab (KEYTRUDA, MERCK), pidilizumab (CT-011, CURE TECH), MK-3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), and MPDL3280A (ROCHE)). In some embodiments, the one or more additional therapeutic agents include an agent that blocks, reduces and/or inhibits the activity of CTLA-4 and/or the binding of CTLA-4 with one or more of its receptors (e.g., CD80, CD86, AP2M1, SHP-2, and PPP2R5A). For example, in some embodiments, the agent that inhibits the activity of CTLA-4 is an antibody such as ipilimumab (MDX-010, MDX-101, Yervoy, BRISTOL MYERS SQUIBB) and/or tremelimumab (PFIZER). Further, in some embodiments, the one or more additional therapeutic agents include blocking antibodies targeted to an immune checkpoint molecule such as, for example, BTLA, HVEM, TIM3, GALS, LAG3, VISTA, KIR, 2B4, CD160 (also referred to as BY55), CGEN-15049, CHK 1 and CHK2 kinases, A2aR, CEACAM (e g., CEACAM-1, CEACAM-3 and/or CEACAM- 5), GITR, GITRL, galectin-9, CD244, CD 160, TIGIT, SIRPa, ICOS, CD 172a, TMIGD2 and various B-7 family ligands (including, but are 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, the one or more additional therapeutic agents include one or more molecules (e.g., small molecules) that block, reduce, and/or inhibit the activity of PD- 1, PD-L1 or PD-L2 (non-limiting examples include one or more of BMS-1166 (BRISTOL MYERS SQUIBB), BMS-103 (BRISTOL MYERS SQUIBB), BMS-142 (BRISTOL MYERS SQUIBB), BMS-202 (BRISTOL MYERS SQUIBB), BMS-1001 (BRISTOL MYERS SQUIBB), BMS-242 (BRISTOL MYERS SQUIBB), BMS-200 (BRISTOL MYERS SQUIBB), BMS-986189 (BRISTOL MYERS SQUIBB), INCB086550 (INCYTE), Aurigene-1 (AURIGENE), AUNP12 (AURIGENE), CA- 170 (CURIS), CCX4503 (CHEMOCENTRYX), AMP-224 (ASTRAZENECA/MEDIMMUNE, GSK), and AMP-512 (ASTRAZENECA)). In some embodiments, the one or more additional therapuetic agents include one or more molecules (e.g., small molecules) that block, reduce, and/or inhibit the activity of CTLA-4 and/or the binding of CTLA-4 with one or more of its receptors (e.g., CD80, CD86, AP2M1, SHP-2, and PPP2R5A).

Some embodiments of these methods can further include identifying or diagnosing a subject as having a MICA/MICB-positive cancer. Some embodiments of these methods can further include identifying or diagnosing a subject as having a MICA/MICB-positive/HLA-E-negative cancer.

Non-limiting examples of MIC A/MICB-positive cancers include: adrenocortical carcinoma, acute myeloid leukemia, pancreatic adenomcarcinoma, cholangiocarcinoma, kidney cancer, cervical squamous cell carcinoma, endocervical carcinoma, colorectal cancer, mesothelioma, skin cutaneous melanoma and lung cancer. In some embodiments, a MICA/MICB-positive cancer includes, without limitation: adrenocortical carcinoma, anal cancer, bile duct cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, acute myeloid leukemia, lymphoid neoplasm 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 corpus endometrial carcinoma, uterine carcinosarcoma, and uveal melanoma.

Methods of Decreasing the Number and/or Proliferation of MICA-Positive Cancer Cells in a Subjet Previously Identified or Diagnosed as Having a MICA- Positive Cancer

Also provided herein are methods of decreasing the number and/or proliferation of MICA-positive cancer cells (e.g., any of the exemplary MICA- positive cancer cells described herein or known in the art) in a subject previously identified or diagnosed as having a MICA-positive cancer (e.g., any of the exemplary MICA-positive cancers described herein or known in the art) that include administering to the subject a therapeutically effective number of enucleated erythroid cells comprising 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, on its extracellular surface (e.g., any of the exemplary enucleated erythroid cells described herein).

Also provided herein are method of decreasing the number and/or proliferation of MICA-positive cancer cells (e.g., any of the exemplary MICA-positive cancer cells described herein or known in the art) in a subject previously identified or diagnosed as having a MICA-positive cancer (e.g., any of the exemplary MICA-positive cancers described herein) that include administering to the subject a therapeutically effective number of enucleated erythroid cells comprising 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, on its extracellular surface; and a second exogenous polypeptide comprising a 4-1BBL polypeptide or a functional fragment thereof (e.g., any of the exemplary enucleated erythroid cells described herein). In some embodiments of these methods, the administration is intravenous administration to the subject.

In some embodiments, the MICA-positive cancer cell is a MICA- positive/HLA-E-negative cancer cell.

In some embodiments of these methods, the method results in a decrease (e.g., at least a 5% decrease, at least a 10% decrease, at least a 15% decrease, at least a 20% decrease, at least a 25% decrease, at least a 30% decrease, at least a 35% decrease, at least a 40% decrease, at least a 45% decrease, at least a 50% decrease, at least a 55% decrease, at least a 60% decrease, at least a 65% decrease, at least a 70% decrease, at least a 75% decrease, at least a 80% decrease, at least a 85% decrease, at least a 90% decrease, or at least a 95% decrease, or about a 5% decrease to about a 99% decrease (or any of the exemplary subranges of this range described herein)) in the number of MICA-positive cancer cells in the subject (e.g., as compared to the number of MIC A- positive cancer cells in the subject prior to the administering).

In some embodiments of these methods, the method results in a decrease (e.g., at least a 5% decrease, at least a 10% decrease, at least a 15% decrease, at least a 20% decrease, at least a 25% decrease, at least a 30% decrease, at least a 35% decrease, at least a 40% decrease, at least a 45% decrease, at least a 50% decrease, at least a 55% decrease, at least a 60% decrease, at least a 65% decrease, at least a 70% decrease, at least a 75% decrease, at least a 80% decrease, at least a 85% decrease, at least a 90% decrease, or at least a 95% decrease, or about a 5% decrease to about a 99% decrease (or any of the exemplary subranges of this range described herein)) in the number of MICA-positive/ HLA-E-negative cancer cells in the subject (e.g., as compared to the number of MIC A-positive/HLA-E-negative cancer cells in the subject prior to the administering).

In some embodiments of these methods, the method results in a decrease (e.g., at least a 5% decrease, at least a 10% decrease, at least a 15% decrease, at least a 20% decrease, at least a 25% decrease, at least a 30% decrease, at least a 35% decrease, at least a 40% decrease, at least a 45% decrease, at least a 50% decrease, at least a 55% decrease, at least a 60% decrease, at least a 65% decrease, at least a 70% decrease, at least a 75% decrease, at least a 80% decrease, at least a 85% decrease, at least a 90% decrease, or at least a 95% decrease, or about a 5% decrease to about a 99% decrease (or any of the exemplary subranges of this range described herein)) in the proliferation of MICA-positive cancer cells in the subject (e.g., as compared to the proliferation of MICA-positive cancer cells in the subject prior to the administering).

In some embodiments of these methods, the method results in a decrease (e.g., at least a 5% decrease, at least a 10% decrease, at least a 15% decrease, at least a 20% decrease, at least a 25% decrease, at least a 30% decrease, at least a 35% decrease, at least a 40% decrease, at least a 45% decrease, at least a 50% decrease, at least a 55% decrease, at least a 60% decrease, at least a 65% decrease, at least a 70% decrease, at least a 75% decrease, at least a 80% decrease, at least a 85% decrease, at least a 90% decrease, or at least a 95% decrease, or about a 5% decrease to about a 99% decrease (or any of the exemplary subranges of this range described herein)) in the proliferation of MIC A-positive/HLA-E-negative cancer cells in the subject (e.g., as compared to the proliferation of MIC A-positive/HLA-E-negative cancer cells in the subject prior to the administering).

Non-limiting examples of MIC A-positive cancer cells include: an adrenocortical carcinoma cancer cell, a cholangiocarcinoma cancer cell, a pancreatic adenocarcinoma cancer cell, a kidney a cancer cancer cell, a thyroid carcinoma cancer cell, a mesothelioma cancer cell, a skin a cutaneous melanoma cancer cell, a colorectal cancer cancer cell, a cervical squamous cell carcinoma cancer cell and an endocervical adenocarcinoma cancer cell. In some embodiments, a MICA-positive cancer includes, without limitation: adrenocortical carcinoma, anal cancer, bile duct cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, acute myeloid leukemia, lymphoid neoplasm 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 corpus endometrial carcinoma, uterine carcinosarcoma, and uveal melanoma.

Methods of Decreasing the Number and/or Proliferation of MICB-Positive Cancer Cells in a Subjet Previously Identified or Diagnosed as Having a MICB- Positive Cancer

Also provided herein are methods of decreasing the number and/or proliferation of MICB-positive cancer cells (e.g., any of the exemplary MICB- positive cancer cells described herein or known in the art) in a subject previously identified or diagnosed as having a MICB-positive cancer (e.g., any of the exemplary MICB-positive cancers described herein or known in the art) that include administering to the subject a therapeutically effective number of enucleated erythroid cells comprising 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, on its extracellular surface (e.g., any of the exemplary enucleated erythroid cells described herein).

Also provided herein are method of decreasing the number and/or proliferation of MICB-positive cancer cells (e.g., any of the exemplary MICB-positive cancer cells described herein or known in the art) in a subject previously identified or diagnosed as having a MICB-positive cancer (e.g., any of the exemplary MICB-positive cancers described herein) that include administering to the subject a therapeutically effective number of enucleated erythroid cells comprising 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, on its extracellular surface; and a second exogenous polypeptide comprising a 4-1BBL polypeptide or a functional fragment thereof (e.g., any of the exemplary enucleated erythroid cells described herein).

In some embodiments, the MICB-positive cancer cell is a MICB- positive/HLA-E-negative cancer cell.

Some embodiments of any of the methods described herein further include administering one or more additional therapeutic agents to the subject. In some embodiments, the one or more additional therapeutic agents is a cancer therapeutic agent. In some embodiments, the cancer therapeutic agent is selected from the group of: an inhibitor of an immune checkpoint molecule (e.g., any of the exemplary inhibitors of an immune checkpoint molecule described herein), a chemotherapeutic agent, a therapeutic antibody, a chimeric antigen receptor-T cell, a kinase inhibitor, or a soluble cytokine. In some embodiments, the one or more additional therapeutic agents can be administered to the subject at the substantially the same time as any of the compositions provided herein. In some embodiments, the one or more additional therapeutic agents can be administered to the subject before or after the administration of any of the compositions described herein to the subject. For example, in some embodiments, the one or more additional therapeutic agents include an agent that blocks, reduces and/or inhibits PD-1 and either PD-L1 or PD-L2, and/or blocks, reduces and/or inhibits the binding of PD-1 with PD-L1 or PD- L2 (non-limiting examples include one or more of nivolumab (ONO-4538/BMS- 936558, MDX1106, OPDIVO, BRISTOL MYERS SQUIBB), pembrolizumab (KEYTRUDA, MERCK), pidilizumab (CT-011, CURE TECH), MK-3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), and MPDL3280A (ROCHE)). In some embodiments, the one or more additional therapeutic agents include an agent that blocks, reduces and/or inhibits the activity of CTLA-4 and/or the binding of CTLA-4 with one or more of its receptors (e.g., CD80, CD86, AP2M1, SHP-2, and PPP2R5A). For example, in some embodiments, the agent that inhibits the activity of CTLA-4 is an antibody such as ipilimumab (MDX-010, MDX-101, Yervoy, BRISTOL MYERS SQUIBB) and/or tremelimumab (PFIZER). Further, in some embodiments, the one or more additional therapeutic agents include blocking antibodies targeted to an immune checkpoint molecule such as, for example, BTLA, HVEM, TIM3, GALS, LAG3, VISTA, KIR, 2B4, CD160 (also referred to as BY55), CGEN-15049, CHK 1 and CHK2 kinases, A2aR, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), GITR, GITRL, galectin-9, CD244, CD 160, TIGIT, SIRPa, ICOS, CD172a, TMIGD2 and various B-7 family ligands (including, but are 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, the one or more additional therapeutic agents include one or more molecules (e.g., small molecules) that block, reduce, and/or inhibit the activity of PD-1, PD-L1 or PD-L2 (non-limiting examples include one or more of BMS-1166 (BRISTOL MYERS SQUIBB), BMS-103 (BRISTOL MYERS SQUIBB), BMS-142 (BRISTOL MYERS SQUIBB), BMS-202 (BRISTOL MYERS SQUIBB), BMS-1001 (BRISTOL MYERS SQUIBB), BMS-242 (BRISTOL MYERS SQUIBB), BMS-200 (BRISTOL MYERS SQUIBB), BMS-986189 (BRISTOL MYERS SQUIBB), INCB086550 (INCYTE), Aurigene-1 (AURIGENE), AUNP12 (AURIGENE), CA-170 (CURIS), CCX4503 (CHEMOCENTRYX), AMP-224 (ASTRAZENECA/MEDIMMUNE, GSK), and AMP-512 (ASTRAZENECA)). In some embodiments, the one or more additional therapuetic agents include one or more molecules (e.g., small molecules) that block, reduce, and/or inhibit the activity of CTLA-4 and/or the binding of CTLA-4 with one or more of its receptors (e.g., CD80, CD86, AP2M1, SHP-2, and PPP2R5A).

In some embodiments of these methods, the method results in a decrease (e.g., at least a 5% decrease, at least a 10% decrease, at least a 15% decrease, at least a 20% decrease, at least a 25% decrease, at least a 30% decrease, at least a 35% decrease, at least a 40% decrease, at least a 45% decrease, at least a 50% decrease, at least a 55% decrease, at least a 60% decrease, at least a 65% decrease, at least a 70% decrease, at least a 75% decrease, at least a 80% decrease, at least a 85% decrease, at least a 90% decrease, or at least a 95% decrease, or about a 5% decrease to about a 99% decrease (or any of the exemplary subranges of this range described herein)) in the number of MICB-positive cancer cells in the subject (e.g., as compared to the number of MICB- positive cancer cells in the subject prior to the administering).

In some embodiments of these methods, the method results in a decrease (e.g., at least a 5% decrease, at least a 10% decrease, at least a 15% decrease, at least a 20% decrease, at least a 25% decrease, at least a 30% decrease, at least a 35% decrease, at least a 40% decrease, at least a 45% decrease, at least a 50% decrease, at least a 55% decrease, at least a 60% decrease, at least a 65% decrease, at least a 70% decrease, at least a 75% decrease, at least a 80% decrease, at least a 85% decrease, at least a 90% decrease, or at least a 95% decrease, or about a 5% decrease to about a 99% decrease (or any of the exemplary subranges of this range described herein)) in the number of MICB-positive/ HLA-E-negative cancer cells in the subject (e.g., as compared to the number of MICB-positive/HLA-E-negative cancer cells in the subject prior to the administering).

In some embodiments of these methods, the method results in a decrease (e.g., at least a 5% decrease, at least a 10% decrease, at least a 15% decrease, at least a 20% decrease, at least a 25% decrease, at least a 30% decrease, at least a 35% decrease, at least a 40% decrease, at least a 45% decrease, at least a 50% decrease, at least a 55% decrease, at least a 60% decrease, at least a 65% decrease, at least a 70% decrease, at least a 75% decrease, at least a 80% decrease, at least a 85% decrease, at least a 90% decrease, or at least a 95% decrease, or about a 5% decrease to about a 99% decrease (or any of the exemplary subranges of this range described herein)) in the proliferation of MICB-positive cancer cells in the subject (e.g., as compared to the proliferation of MICB-positive cancer cells in the subject prior to the administering).

In some embodiments of these methods, the method results in a decrease (e.g., at least a 5% decrease, at least a 10% decrease, at least a 15% decrease, at least a 20% decrease, at least a 25% decrease, at least a 30% decrease, at least a 35% decrease, at least a 40% decrease, at least a 45% decrease, at least a 50% decrease, at least a 55% decrease, at least a 60% decrease, at least a 65% decrease, at least a 70% decrease, at least a 75% decrease, at least a 80% decrease, at least a 85% decrease, at least a 90% decrease, or at least a 95% decrease, or about a 5% decrease to about a 99% decrease (or any of the exemplary subranges of this range described herein)) in the proliferation of MICB-positive/HLA-E-negative cancer cells in the subject (e.g., as compared to the proliferation of MICB-positive/HLA-E-negative cancer cells in the subject prior to the administering).

Non-limiting examples of MICB-positive cancer cells include: an acute myeloid leukemia cancer cell, a lymphoid neoplasm diffuse large B-cell lymphoma cancer cell, a testicular germ cell tumor cancer cell, a stomach adenocarcinoma cancer cell, a ovarian serous cystadenocarcinoma cancer cell, a esophageal carcinoma cancer cell and a lung cancer cancer cell. In some embodiments, a MICB-positive cancer includes, without limitation: adrenocortical carcinoma, anal cancer, bile duct cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, acute myeloid leukemia, lymphoid neoplasm 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 corpus endometrial carcinoma, uterine carcinosarcoma, and uveal melanoma. The enucleated erythroid cells can be administered to the subject using any of the doses and at any of the frequencies described herein.

Methods of Decreasing the Number and/or Proliferation of MICA/MICB- Positive Cancer Cells in a Subjet Previously Identified or Diagnosed as Having a MICA/MICB-Positive Cancer

Also provided herein are methods of decreasing the number and/or proliferation of MICA/MICB-positive cancer cells (e.g., any of the exemplary MICA/MICB-positive cancer cells described herein or known in the art) in a subject previously identified or diagnosed as having a MICA/MICB-positive cancer (e.g., any of the exemplary MICA/MICB-positive cancers described herein or known in the art) that include administering to the subject a therapeutically effective number of enucleated erythroid cells comprising 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, on its extracellular surface (e.g., any of the exemplary enucleated erythroid cells described herein).

Also provided herein are method of decreasing the number and/or proliferation of MICA/MICB-positive cancer cells (e.g., any of the exemplary MICA/MICB- positive cancer cells described herein or known in the art) in a subject previously identified or diagnosed as having a MICA/MICB-positive cancer (e.g., any of the exemplary MICA/MICB-positive cancers described herein) that include administering to the subject a therapeutically effective number of enucleated erythroid cells comprising 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, on its extracellular surface; and a second exogenous polypeptide comprising a 4-1BBL polypeptide or a functional fragment thereof (e.g., any of the exemplary enucleated erythroid cells described herein).

Some embodiments of these methods further include administering to the subject an NKG2A inhibitor (e.g., any of the exemplary NKG2A inhibitors described herein). In some embodiments, the MICA/MICB-positive cancer cell is a MICA/MICB-positive/HLA-E-negative cancer cell.

In some embodiments of these methods, the method results in a decrease (e.g., at least a 5% decrease, at least a 10% decrease, at least a 15% decrease, at least a 20% decrease, at least a 25% decrease, at least a 30% decrease, at least a 35% decrease, at least a 40% decrease, at least a 45% decrease, at least a 50% decrease, at least a 55% decrease, at least a 60% decrease, at least a 65% decrease, at least a 70% decrease, at least a 75% decrease, at least a 80% decrease, at least a 85% decrease, at least a 90% decrease, or at least a 95% decrease, or about a 5% decrease to about a 99% decrease (or any of the exemplary subranges of this range described herein)) in the number of MICA/MICB-positive cancer cells in the subject (e.g., as compared to the number of MICA/MICB-positive cancer cells in the subject prior to the administering).

In some embodiments of these methods, the method results in a decrease (e.g., at least a 5% decrease, at least a 10% decrease, at least a 15% decrease, at least a 20% decrease, at least a 25% decrease, at least a 30% decrease, at least a 35% decrease, at least a 40% decrease, at least a 45% decrease, at least a 50% decrease, at least a 55% decrease, at least a 60% decrease, at least a 65% decrease, at least a 70% decrease, at least a 75% decrease, at least a 80% decrease, at least a 85% decrease, at least a 90% decrease, or at least a 95% decrease, or about a 5% decrease to about a 99% decrease (or any of the exemplary subranges of this range described herein)) in the number of MICA/MICB-positive/ HLA-E-negative cancer cells in the subject (e.g., as compared to the number of MICA/MICB-positive/HLA-E-negative cancer cells in the subject prior to the administering).

In some embodiments of these methods, the method results in a decrease (e.g., at least a 5% decrease, at least a 10% decrease, at least a 15% decrease, at least a 20% decrease, at least a 25% decrease, at least a 30% decrease, at least a 35% decrease, at least a 40% decrease, at least a 45% decrease, at least a 50% decrease, at least a 55% decrease, at least a 60% decrease, at least a 65% decrease, at least a 70% decrease, at least a 75% decrease, at least a 80% decrease, at least a 85% decrease, at least a 90% decrease, or at least a 95% decrease, or about a 5% decrease to about a 99% decrease (or any of the exemplary subranges of this range described herein)) in the proliferation of MICA/MICB-positive cancer cells in the subject (e.g., as compared to the proliferation of MICA/MICB-positive cancer cells in the subject prior to the administering).

In some embodiments of these methods, the method results in a decrease (e.g., at least a 5% decrease, at least a 10% decrease, at least a 15% decrease, at least a 20% decrease, at least a 25% decrease, at least a 30% decrease, at least a 35% decrease, at least a 40% decrease, at least a 45% decrease, at least a 50% decrease, at least a 55% decrease, at least a 60% decrease, at least a 65% decrease, at least a 70% decrease, at least a 75% decrease, at least a 80% decrease, at least a 85% decrease, at least a 90% decrease, or at least a 95% decrease, or about a 5% decrease to about a 99% decrease (or any of the exemplary subranges of this range described herein)) in the proliferation of MICA/MICB-positive/HLA-E-negative cancer cells in the subject (e.g., as compared to the proliferation of MICA/MICB-positive/HLA-E-negative cancer cells in the subject prior to the administering).

Some embodiments of these methods can further include identifying or diagnosing a subject as having a MICA/MICB-positive cancer. Some embodiments of these methods can further include identifying or diagnosing a subject as having a MICA/MICB-positive/HLA-E-negative cancer. Non-limiting examples of MIC A/MICB-positive cancer cells include: an adrenocortical carcinoma cancer cell, an acute myeloid leukemia cancer cell, a pancreatic adenomcarcinoma cancer cell, a cholangiocarcinoma cancer cell, a kidney cancer cancer cell, a cervical squamous cell carcinoma cancer cell, an endocervical carcinoma cancer cell, a colorectal cancer cell, a mesothelioma cancer cell, a skin cutaneous melanoma cancer cell and a lung cancer cancer cell. In some embodiments, a MICA/MICB-positive cancer includes, without limitation: adrenocortical carcinoma, anal cancer, bile duct cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, acute myeloid leukemia, lymphoid neoplasm 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 corpus endometrial carcinoma, uterine carcinosarcoma, and uveal melanoma.

Methods of Inducing Killing a MICA-Positive Cancer Cell in a Subject Previously Identified or Diagnosed as Having a MICA-Positive Cancer

Also provided herein are methods of inducing killing of a MICA-positive cancer cell (e.g., any of the exemplary MICA-positive cancer cells described herein) in a subject previously identified or diagnosed as having a MICA-positive cancer (e.g., any of the exemplary MICA-positive cancers described herein) that include administering to the subject a therapeutically effective number of enucleated erythroid cells comprising 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, on its extracellular surface (e.g., any of the exemplary enucleated erythroid cells described herein).

Also provided herein are methods of inducing killing of a MICA-positive cancer cell (e.g., any of the exemplary MICA-positive cancer cells described herein) in a subject previously identified or diagnosed as having a MICA-positive cancer (e.g., any of the exemplary MICA-positive cancers described herein) that include administering to the subject a therapeutically effective number of enucleated erythroid cells comprising 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, on its extracellular surface; and a second exogenous polypeptide comprising a 4-1BBL polypeptide or a functional fragment thereof (e.g., any of the exemplary enucleated erythroid cells described herein).

In some embodiments, inducing killing of a MICA-positive cancer cell also results in the induced killing of a non-MICA positive cancer cell in the subject (e.g., a non-MICA positive cancer cell in a tumor also comprising a MICA-positive cancer cell).

In some embodiments, the killing of the MICA-positive cancer cell is necrosis. In some embodiments, the killing of the MICA-positive cancer cell is apoptosis. In some embodiments, the kill is NK cell-mediated cytolysis or NK cell-mediated cytoxicity.

In some embodiments of these methods, the administering includes intravenous administration to the subject.

Methods of Inducing Killing a MICB-Positive Cancer Cell in a Subject Previously Identified or Diagnosed as Having a MICB-Positive Cancer

Also provided herein are methods of inducing killing of a MICB-positive cancer cell (e.g., any of the exemplary MICB-positive cancer cells described herein) in a subject previously identified or diagnosed as having a MICB-positive cancer (e.g., any of the exemplary MICB-positive cancers described herein) that include administering to the subject a therapeutically effective number of enucleated erythroid cells comprising 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, on its extracellular surface (e.g., any of the exemplary enucleated erythroid cells described herein).

Also provided herein are methods of inducing killing of a MICB-positive cancer cell (e.g., any of the exemplary MICB-positive cancer cells described herein) in a subject previously identified or diagnosed as having a MICB-positive cancer (e.g., any of the exemplary MICB-positive cancers described herein) that include administering to the subject a therapeutically effective number of enucleated erythroid cells comprising 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, on its extracellular surface; and a second exogenous polypeptide comprising a 4-1BBL polypeptide or a functional fragment thereof (e.g., any of the exemplary enucleated erythroid cells described herein).

In some embodiments, inducing killing of a MICB-positive cancer cell also results in the induced killing of a non-MICB positive cancer cell in the subject (e.g., a non-MICB positive cancer cell in a tumor also comprising a MICB-positive cancer cell).

In some embodiments, the killing of the MICB-positive cancer cell is necrosis. In some embodiments, the killing of the MICB-positive cancer cell is apoptosis. In some embodiments, the kill is NK cell-mediated cytolysis or NK cell-mediated cytoxicity.

Non-limiting examples of MICB-positive cancer cells include: an acute myeloid leukemia cancer cell, a lymphoid neoplasm diffuse large B-cell lymphoma cancer cell, a testicular germ cell tumor cancer cell, a stomach adenocarcinoma cancer cell, a ovarian serous cystadenocarcinoma cancer cell, a esophageal carcinoma cancer cell and a lung cancer cancer cell. In some embodiments, a MICB-positive cancer includes, without limitation: adrenocortical carcinoma, anal cancer, bile duct cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, acute myeloid leukemia, lymphoid neoplasm 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 corpus endometrial carcinoma, uterine carcinosarcoma, and uveal melanoma.

Methods of Inducing Killing a MICA/MICB-Positive Cancer Cell in a Subject Previously Identified or Diagnosed as Having a MICA/MICB-Positive Cancer

Also provided herein are methods of inducing killing of a MICA/MICB- positive cancer cell (e.g., any of the exemplary MICA/MICB-positive cancer cells described herein) in a subject previously identified or diagnosed as having a MICA/MICB-positive cancer (e.g., any of the exemplary MICA/MICB-positive cancers described herein) that include administering to the subject a therapeutically effective number of enucleated erythroid cells comprising 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, on its extracellular surface (e.g., any of the exemplary enucleated erythroid cells described herein).

Also provided herein are methods of inducing killing of a MICA/MICB- positive cancer cell (e.g., any of the exemplary MICA/MICB-positive cancer cells described herein) in a subject previously identified or diagnosed as having a MICA/MICB-positive cancer (e.g., any of the exemplary MICA/MICB-positive cancers described herein) that include administering to the subject a therapeutically effective number of enucleated erythroid cells comprising 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, on its extracellular surface; and a second exogenous polypeptide comprising a 4-1BBL polypeptide or a functional fragment thereof (e.g., any of the exemplary enucleated erythroid cells described herein).

In some embodiments, inducing killing of a MICA/MICB-positive cancer cell also results in the induced killing of a non-MICA/MICB-positive cancer cell in the subject (e.g., a non-MICA/MICB-positive cancer cell in a tumor also comprising a MICA/MICB-positive cancer cell).

In some embodiments, the killing of the MICA/MICB-positive cancer cell is necrosis. In some embodiments, the killing of the MICA/MICB-positive cancer cell is apoptosis. In some embodiments, the kill is NK cell-mediated cytolysis or NK cell- mediated cytoxicity.

Non-limiting examples of MIC A/MICB-positive cancer cells include: an adrenocortical carcinoma cancer cell, an acute myeloid leukemia cancer cell, a pancreatic adenomcarcinoma cancer cell, a cholangiocarcinoma cancer cell, a kidney cancer cancer cell, a cervical squamous cell carcinoma cancer cell, an endocervical carcinoma cancer cell, a colorectal cancer cell, a mesothelioma cancer cell, a skin cutaneous melanoma cancer cell and a lung cancer cancer cell. In some embodiments, a MICA/MICB-positive cancer includes, without limitation: adrenocortical carcinoma, anal cancer, bile duct cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, acute myeloid leukemia, lymphoid neoplasm 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 corpus endometrial carcinoma, uterine carcinosarcoma, and uveal melanoma.

The enucleated erythroid cells can be administered to the subject using any of the doses and at any of the frequencies described herein. Methods of Reducing the Volume of a Solid Tumor in a Subject Previously Identified or Diagnosed as Having a MICA-Positive Cancer, a MICB-Positive Cancer, or a MICA/MICB-Positive Cancer

Also provided herein are methods of reducing the volume of a solid tumor in a subject previously identified or diagnosed as having a MICA-positive cancer (e.g., any of the exemplary MICA cancers described herein or known in the art), a MICB- positive cancer (e.g., any of the exemplary MICB cancers described herein or known in the art), or a MICA/MICB-positive cancer (e.g., any of the exemplary MICA/MICB cancers described herein or known in the art) that include administering to the subject a therapeutically effective number of enucleated erythroid cells comprising 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, on its extracellular surface (e.g., any of the exemplary enucleated erythroid cells described herein).

Also provided herein are methods of reducing the volume of a solid tumor in a subject previously identified or diagnosed as having a MICA-positive cancer (e.g., any of the exemplary MICA positive cancers described herein), a MICB-positive cancer (e.g., any of the exemplary MICB cancers described herein or known in the art), or a MICA/MICB-positive cancer (e.g., any of the exemplary MICA/MICB cancers described herein or known in the art) that include administering to the subject a therapeutically effective number of enucleated erythroid cells comprising 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, on its extracellular surface; and a second exogenous polypeptide comprising a 4-1BBL polypeptide, or a functional fragment thereof (e.g., any of the exemplary enucleated erythroid cells described herein).

Some embodiments of these methods can further include identifying or diagnosing a subject as having a MICA-positive cancer, a MICB-positive cancer, or a MICA/MICB-positive cancer. Some embodiments of these methods can further include identifying or diagnosing a subject as having a MICA-positive/HLA-E- negative cancer, a MICB-positive/HLA-E-negative cancer, or a MICA/MICB- positive/HLA-E-negative cancer.

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

Doses and Administration

Methods of administering enucleated red blood cells (e.g., reticulocytes) comprising (e.g., expressing) exogenous agent (e.g., polypeptides) are described, e.g., in WO2015/073587, W02015/153102, and WO 2019/173798 each of which is incorporated by reference in its entirety.

In embodiments, the enucleated red blood cells described herein are administered to a subject, e.g., a mammal, e.g., a human. Exemplary mammals that can be treated include without limitation, humans, domestic animals (e.g., dogs, cats and the like), farm animals (e.g., cows, sheep, pigs, horses and the like) and laboratory animals (e.g., monkey, rats, mice, rabbits, guinea pigs and the like). The methods described herein are applicable to both human therapy and veterinary applications.

In some embodiments, a dose of enucleated erythroid cells comprises about lxlO⁹ - 2x10⁹, 2xl0⁹ - 5xl0⁹, 5xl0⁹ - lxlO¹⁰, lxlO¹⁰ - 2xl0¹⁰, 2xl0¹⁰ - 5xl0¹⁰, 5xl0¹⁰ - lxlO¹¹, lxlO¹¹ - 2xlOⁿ, 2xlOⁿ - 5xl0ⁿ, 5xl0ⁿ - lxlO¹², lxlO¹² - 2xl0¹², 2xl0¹² - 5xl0¹², or 5xl0¹² - lxlO¹³ cells.

The engineered erythroid cell can be administered to the subject in a formulation suitable for parenteral, intral-lesional, intravenous, intra-organ, or another route of administration.

The engineered erythroid cell can be administered to a subject as frequently as several times daily, or it can be administered less frequently, such as once a day, once a week, once every two weeks, once every three weeks, once a month, or even less frequently, such as one every several months or even once a year or less. The frequency of the dose will be readily apparent to the skilled artisan and will depend upon any number of factors, such as, but not limited to, the type and severity of the disease being treated, the type and age of the subject, etc.

In some embodiments, the MICA-positive cancer is a cancer selected from the group consisting of: adrenocortical carcinoma, cholangiocarcinoma, pancreatic adenocarcinoma, kidney cancer, thyroid carcinoma, mesothelioma, skin cutaneous melanoma, colorectal cancer, cervical squamous cell carcinoma and endocervical adenocarcinoma.

In some embodiments, the MICB-positive cancer is a cancer selected from the group consisting of: acute myeloid leukemia, lymphoid neoplasm diffuse large B-cell lymphoma, testicular germ cell tumors, stomach adenocarcinoma, ovarian serous cystadenocarcinoma, esophageal carcinoma and lung cancer.

In some embodiments, the MICA/MICB-positive cancer is a cancer selected from the group consisting of: adrenocortical carcinoma, acute myeloid leukemia, pancreatic adenomcarcinoma, cholangiocarcinoma, kidney cancer, cervical squamous cell carcinoma, endocervical carcinoma, colorectal cancer, mesothelioma, skin cutaneous melanoma and lung cancer.

Kits

Also provided herein are kits that include any of the compositions provided herein. For example, provided herein is a kit that includes a pharmaceutical composition comprising an enucleated erythroid cell comprising 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, on its extracellular surface (e.g., any of the exemplary enucleated erythroid cells described herein); and instructions for performing the any of the methods described herein. In some embodiments, the enucleated erythroid cell comprises at least 1,000 copies, at least 10,000 copies, or at least 15,000 copies of the first exogenous fusion polypeptide. In some embodiments, the enucleated erythroid cell comprises 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 first exogenous fusion polypeptide.

In another example, provided herein is a kit that includes a pharmaceutical composition comprising an enucleated erythroid cell comprising 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, on its extracellular surface; and a second exogenous polypeptide comprising a 4-1BBL polypeptide or a functional fragment thereof, where the exogenous polypeptide is present on the surface of the enucleated erythroid cell (e.g., any of the exemplary enucleated erythroid cells described herein); and instructions for performing the any of the methods described herein.

In some embodiments, the composition is formulated as described in any of the enucleated erythroid cells described herein can be formulated for administration to a subject as described in, e.g., WO 2020/219909 (incorporated herein by reference).

Also provided herein are kits that include one or more sterile vessels containing any of the compositions described herein (e.g., a sterile conical tube, a sterile petri dish, a sterile vial (e.g., a borosilicate glass vial), and sterile plastic bags (a di-2-ethylhexyl phthalate (DEHP)-plasticized polyvinyl chloride (PVC) bag, or n- butyryl-tri(n-hexyl)-citrate (BTHC)-plasticized PVC bag). In some embodiments, any of the kits provided herein can further include instructions for administration of any of the compositions to a subject in need thereof.

Some embodiments of the kits described herein include a suitable single dosage form of any of the compositions described herein. For example, a single dosage form of any of the compositions described herein can have a volume of, e.g., about 0.5 mL to about 2 L, about 0.5 mL to about 1500 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 150 mL, about 0.5 mL to about 100 mL, about 0.5 mL to about 50 mL, about 1.0 mL to about 2 L, about 1.0 mL to about 1500 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 150 mL, about 1.0 mL to about 100 mL, about 1.0 mL to about 50 mL, about 5 mL to about 2 L, about 5 mL to about 1500 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 150 mL, about 5 mL to about 100 mL, about 5 mL to about 50 mL, about 10 mL to about 2 L, about 10 mL to about 1500 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 150 mL, about 10 mL to about 100 mL, about 10 mL to about 50 mL, about 20 mL to about 2 L, about 20 mL to about 1500 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 150 mL, about 20 mL to about 100 mL, about 20 mL to about 50 mL, about 50 mL to about 2 L, about 50 mL to about 1500 mL, about 50 mL to about 1000 mL, about 50 mL to about 800 mL, about 50 mL to about 600 mL, about 50 mL to about 400 mL, about 50 mL to about 200 mL, about 50 mL to about 150 mL, about 50 mL to about 100 mL, about 100 mL to about 2 L, about 100 mL to about 1500 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 150 mL, about 150 mL to about 2 L, about 150 mL to about 1500 mL, about 150 mL to about 1000 mL, about 150 mL to about 800 mL, about 150 mL to about 600 mL, about 150 mL to about 400 mL, about 150 mL to about 200 mL, about 200 mL to about 2 L, about 200 mL to about 1500 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 400 mL, about 400 mL to about 2 L, about 400 mL to about 1500 mL, about 400 mL to about 1000 mL, about 400 mL to about 800 mL, about 400 mL to about 600 mL, about 600 mL to about 2 L, about 600 mL to about 1500 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 1500 mL, about 800 mL to about 1000 mL, about 1000 mL to about 2 L, about 1000 mL to about 1500 mL, or about 1500 mL to about 2 L.

EXAMPLES

Example 1. Generation of erythroid cells genetically engineered to express the first fusion polypeptide

IL-15 and IL-15/IL-l 5RA 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 NOs. refer to amino acid sequences.

The DNA constructs were cloned into the multiple cloning site of a lentivirus vector under the control of a MSCV promoter sequence for expression in erythroid cells, as described below.

Production of Lentiviral Vector

The constructs were cloned into the multiple cloning site of the lentivirus vector pCDH under the control of the MSCV promoter sequence (System Biosciences). Lentivirus was produced in 293T cells by transfecting the cells with pPACKHl (System Biosciences) and pCDH lentivirus vector containing constructs. Cells were placed in fresh culturing medium. The virus supernatant was collected 48 hours post-medium change by centrifugation at 1,500 rpm for 5 minutes. The supernatant was collected, filtered, and frozen in aliquots at -80°C. Expansion and differentiation of erythroid cells

Human CD34⁺ cells derived from mobilized peripheral blood cells from normal human donors were used. The expansion/differentiation procedure comprises 3 stages. In the first stage, thawed CD34⁺ erythroid cell precursors were cultured in Iscove’s MDM (IMDM) medium comprising 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. The cultures were maintained at 37°C in 5% C02 incubator.

Transduction of erythroid precursor cells

Erythroid precursor cells were transduced during stage 1 of the culture process described above. Erythroid precursor cells in culturing medium were combined with lentiviral supernatant and polybrene. Infection was achieved by spinoculation, spinning the plate at 2,000 rpm for 90 minutes at room temperature. After spinoculation, the cells were incubated at 37°C overnight.

Antibody Binding Binding of a PE-labelled anti-IL-15-RA antibody (e.g., anti-IL-15RA antibody

(JM7A4) (ab91270), AbCam) was used to validate expression of the the first exogenous polypeptide in the engineered erythroid cells. Binding of the antibody was measured by flow cytometry for APC fluorescence or PE fluorescence. A gate was set based on stained untransduced cells.

Example 2. Generation of erythroid cells genetically engineered to express 4- 1BBL.

4-1BBL constructs DNA constructs were prepared for expression in erythroid cells as shown in

Table 6 below:

Table 6. 4-1BBL construct. SEQ ID NOs. refer to amino acid sequences.

Production of Lentiviral Vector

4-1BBL gene construct was constructed as shown in Table 6. Genes were cloned into the multiple cloning site of lentivirus vector pCDH with the MSCV promoter sequence from System Biosciences. Lentivirus was produced in 293T cells by transfecting the cells with pPACKHl (System Biosciences) and pCDH lentivirus vector containing 4-1BBL genes. Cells were placed in fresh culturing medium. The virus supernatant was collected 48 hours post-medium change by centrifugation at 1,500 rpm for 5 minutes. The supernatant was collected, filtered, and frozen in aliquots at -80°C. Expansion and differentiation of erythroid cells

Human CD34⁺ cells derived from mobilized peripheral blood cells from normal human donors were purchased frozen from AllCells Inc. The expansion/differentiation procedure comprises 3 stages. In the first stage, thawed CD34⁺ erythroid precursors were cultured in Iscove’s MDM medium comprising recombinant human insulin, human transferrin, recombinant human recombinant human 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. The cultures were maintained at 37°C in 5% C02 incubator.

Transduction of erythroid precursor cells

Erythroid precursor cells were transduced during step 1 of the culture process described above. Erythroid cells in culturing medium were combined with lentiviral supernatant and polybrene. Infection was achieved by spinoculation, spinning the plate at 2000 rpm for 90 minutes at room temperature. After spinoculation, the cells were incubated at 37°C overnight.

Antibody Binding

Binding of a PE-labelled anti-4-lBBL antibody (e.g., purified anti-human 4- 1BB Ligand (CD137L) antibody, BioLegend) was used to validate expression of 4- 1BBL in the engineered erythroid cells. Binding of the antibody was measured by flow cytometry for PE fluorescence. A gate was set based on stained untransduced cells. Example 3. Generation of erythroid cells genetically engineered to express the first and second exogenous polypeptides

Production of Lentiviral Vectors

Genes encoding IL-15/IL-15-RA fusion polypeptide and 4-1BBL were constructed. Each gene was cloned into the multiple cloning site of lentivirus vector pCDH under the control of the MSCV promoter sequence (System Biosciences), such that one vector comprised the gene for IL-15/IL-15RA and another vector comprised the gene for 4-1BBL. Lentivirus was produced in 293T cells by co transfecting the cells with pPACKHl (System Biosciences) and pCDH lentivirus vector containing IL-15/IL-15-RA gene and pCDH lentivirus vector containing 4- 1BBL gene. Cells were placed in fresh culturing medium. The virus supernatant was collected 48 hours post-medium change by centrifugation at 1,500 rpm for 5 minutes. The supernatant was collected, filtered, and frozen in aliquots at -80°C.

Alternatively, genes encoding IL-15/IL-15-RA fusion polypeptide and 4- 1BBL were constructed and cloned into the multiple cloning site of lentivirus vector pCDH, under the control of the MSCV promoter sequence (System Biosciences), such that a single vector comprised the genes for IL-15/IL-15RA and the gene for 4- 1BBL. Lentivirus was produced in 293T cells by co-transfecting the cells with pPACKHl (System Biosciences) and pCDH lentivirus vector containing both IL- 15/IL-15-RA gene and 4-1BBL gene. Cells were placed in fresh culturing medium. The virus supernatant was collected 48 hours post-medium change by centrifugation at 1,500 rpm for 5 minutes. The supernatant was collected, filtered, and frozen in aliquots at -80°C.

Expansion and differentiation of erythroid cells

Human CD34+ cells derived from mobilized peripheral blood cells from normal human donors were purchased frozen from AllCells Inc. The expansion/differentiation procedure comprises 3 stages. In the first stage, thawed CD34+ erythroid precursors were cultured in Iscove’s MDM medium comprising recombinant human insulin, human transferrin, recombinant human recombinant human 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. The cultures were maintained at 37°C in 5% C02 incubator.

Transduction of erythroid precursor cells

Antibody Binding

Binding of a PE-labelled anti-IL-15-RA antibody (e.g., anti-IL-15RA antibody (JM7A4) (ab91270), AbCam) was used to validate expression of the IL-15/IL-15-RA in the engineered erythroid cells. Binding of a PE-labelled anti-4-lBBL antibody (e.g., purified anti-human 4-1BB Ligand (CD137L) antibody, BioLegend) was used to validate expression of 4-1BBL in the engineered erythroid cells. Binding of the antibody was measured by flow cytometry for PE fluorescence. A gate was set based on stained untransduced cells.

Example 4. Administration of enucleated erythroid cells genetically engineered to express IL-15/IL-15RA and 4-1BBL results an increase in the ratio of NKG2D-positive lymphocytes to NKG2A-positive lymphocytes in vivo

To assess the effect of administration of the human enucleated erythroid cells genetically engineered to express IL-15/IL-15RA and 4-1BBL on NKG2D-positive lymphocytes and NKG2A-positive lymphocytes in human subjects, flow cytometry was used to measure levels of NKG2D-positive and NKG2A-positive lymphocytes in whole blood samples obtained from the subjects prior to and at various timepoints following the administration.

Flow cytometry was performed on whole blood samples using the following antibodies: APC-H7 mouse anti-human CD45, clone 2D1 (BD Pharmingen), PE-Cy7 mouse anti-human CD16 clone B73.1 (BD Biosciences), PE mouse anti-human CD56, clone NCAM16.2(BD Biosciences), APC mouse anti-human NKG2D, cloned 1D11 (BD Biosciences), BV421 mouse anti -human NKG2 A, clone 131411 (BD Biosciences) and viability with 7-amino-actinomycin D (7-AAD) (BD Biosciences). Lymphocyteswere defined by scatter/Singlet/CD45⁺(lymphocytes)/viability/CD16⁺ or CD56⁺. NKG2D-positive lymphocytes and NKG2A-positive lymphocytes were identified as being positive based on gates established through fluorescence minus one control samples, fluorescence compensation, and qualification experience. The ratio of NKG2D-positive/NKG2A-positive was defined as the percentage of NKG2D- positive lymphocytes divided by the percentage of NKG2A-positive lymphocytes. Maximum fold-change was the greatest result of the ratio of NKG2D- positive/NKG2D-positive across all available timepoints following administration of the enucleated human erythroid cells comprising IL-15/IL-15RA and 4-1BBL divided by the baseline (pre-administration) value.

As shown in FIG. 1, administration of the human enucleated erythroid cells genetically engineered to express IL-15/IL-15RA and 4-1BBL resulted in an increase in the ratio of NKG2D-positive lymphocytes to NKG2A-positive lymphocytes in the blood of the human subjects.

Example 5. Administration of enucleated erythroid cells genetically engineered to express IL-15/IL-15RA and 4-1BBL results in an increase in NKG2D-positive lymphocytes, NK cells, and CD8+ Memory T Cells

Cell surface markers in fresh whole blood were evaluated by flow cytometry at multiple timepoints in patients prior to treatment and during treatment with greater than or equal to 1 x 10¹⁰ human enucleated erythroid cells genetically engineered to express IL-15/IL-15RA and 4-1BBL.

Whole blood was collected from patients at baseline prior to treatment and post-treatment at 4 hours, 1 day, 2 days, 7 days, and 14 days for the first dosing cycle and 2 days, 7 days, and 14 days post-treatment after subsequent dosing cycles. Whole blood was stained with fluorophore-conjugated antibodies using standard techniques for analysis by flow cytometry. Multiple antibody panels were used, with separate tubes for each panel and flow cytometric analysis. The panels included markers to identify lymphocyte, NK cell, and T cell populations along with markers of activation state. The described data were obtained with the following panels: Panel 1--CD3, CD8, CD16/CD56, CD45, CD45RA, CCR7, HLA-DR, and live/dead stain; Panel 2- CD3, CD4, Foxp3, CD25, CD127, Ki67, live/dead stain; Panel 3--CD45, CD16, CD56, NKG2D, NKp30, and live/dead stain. The data were analyzed for frequency of population as a percent of the established parent gate (e.g., %NKG2D⁺ of CD45⁺CD56⁺ live lymphocytes). In the case of Panel 1, data were also analyzed for quantitative cell population numbers per volume of blood on the basis of an included quantitative count control. Data are reported as maximum fold change observed relative to baseline for each parameter of interest (cells per pL whole blood, % of parent population).

The data in FIG. 2A show that administration of human enucleated erythroid cells genetically engineered to express IL-15/IL-15RA and 4-1BBL results in an increase in the percentage of CD56⁺ lymphocytes expressing NKG2D.

The data in FIG. 2B show that administration of human enucleated erythroid cells genetically engineered to express IL-15/IL-15RA and 4-1BBL results in an increase in the percentage of CD56^dim CD16⁺ lymphocytes expressing NKG2D.

The data in FIG. 3 A show that administration of human enucleated erythroid cells genetically engineered to express IL-15/IL-15RA and 4-1BBL results in an increase in the absolute number of circulating NK cells.

The data in FIG. 3B show that administration of human enucleated erythroid cells genetically engineered to express IL-15/IL-15RA and 4-1BBL results in an increase in the percentage of CD8⁺ memory T cells expressing granzyme B.

The data in FIG. 4A show that there is no significant change in the percentage of CD4⁺ T cells of total CD3⁺ T cells.

The data in FIG. 4B show that there is no significant change in the percentage of CD8⁺ T cells of total CD3⁺ T cells.

SEQUENCE APPENDIX

Claims

WHAT IS CLAIMED IS:

1. A method of increasing the number of NKG2D-positive lymphocytes in a subject previously identified or diagnosed as having a MICA-positive cancer comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a population of enucleated erythroid cells comprising 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, on its extracellular surface.

2. The method of claim 1, wherein the method further comprises identifying or diagnosing a subject as having a MICA-positive cancer.

3. The method of claim 1 or 2, wherein the MICA-positive cancer is selected from the group consisting of: adrenocortical carcinoma, cholangiocarcinoma, pancreatic adenocarcinoma, kidney cancer, thyroid carcinoma, mesothelioma, skin cutaneous melanoma, colorectal cancer, cervical squamous cell carcinoma and endocervical adenocarcinoma.

4. The method of any one of claims 1-3, wherein the MICA-positive cancer is a MICA-positive/HLA-E-negative cancer.

5. A method of increasing the number of NKG2D-positive lymphocytes in a subject previously identified or diagnosed as having a MICB-positive cancer comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a population of enucleated erythroid cells comprising 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, on its extracellular surface.

6. The method of claim 5, wherein the method further comprises identifying or diagnosing a subject as having a MICB-positive cancer.

7. The method of claim 5 or 6, wherein the MICB-positive cancer is selected from the group consisting of: acute myeloid leukemia, lymphoid neoplasm diffuse large B-cell lymphoma, testicular germ cell tumors, stomach adenocarcinoma, ovarian serous cystadenocarcinoma, esophageal carcinoma and lung cancer.

8. The method of any one of claims 5-7, wherein the MICB-positive cancer is a MICB-positive/HLA-E-negative cancer.

9. The method of claim 1-8, wherein the administering results in at least a 5% increase in the number of NKG2D-positive lymphocytes in the subject as compared to the number of NKG2D-positive lymphocytes in the subject prior to the administering.

10. The method of claim 9, wherein the administering results in at least a 10% increase in the number of NKG2D-positive lymphocytes in the subject as compared to the number of NKG2D-positive lymphocytes in the subject prior to the administering.

11. The method of any one of claims 1-10, wherein the NKG2D-positive lymphocytes are NKG2D-positive NK cells.

12. The method of any one of claims 1-11, wherein the method further results in an increase in the number of NKG2D-positive/NKG2A-negative lymphocytes in the subject.

13. The method of claim 12, wherein the administering step results in at least a 5% increase in the number of NKG2D-positive/NKG2A-negative lymphocytes in the subject.

14. The method of claim 13, wherein the administering step results in at least a 10% increase in the number of NKG2D-positive/NKG2A-negative lymphocytes in the subject.

15. The method of claim 14, wherein the administering results in at least a 15% increase in the number of NKG2D-positive/NKG2A-negative lymphocytes in the subject.

16. The method of any one of claims 12-15, wherein the NKG2D- positive/NKG2A-negative lymphocytes are NKG2D-positive/NKG2A-negative NK cells.

17. The method of any one of claims 1-16, wherein the method further comprises administering to the subject an NKG2A inhibitor.

18. The method of claim 17, wherein the NKG2A inhibitor is an antagonistic antibody that binds specifically to NKG2A.

19. A method of treating a subject previously identified or diagnosed as having a MICA-positive cancer, the method comprising administering to a subject previously identified or diagnosed as having a MICA-positive cancer a therapeutically effective amount of a pharmaceutical composition comprising a population of enucleated erythroid cells comprising a first exogenous polypeptide comprising (i) IL- 15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof, on its extracellular surface.

20. The method of claim 19, wherein the method further comprises identifying or diagnosing a subject as having a MICA-positive cancer.

21. The method of claim 19 or 20, wherein the MICA-positive cancer is selected from the group consisting of: adrenocortical carcinoma, cholangiocarcinoma, pancreatic adenocarcinoma, kidney cancer, thyroid carcinoma, mesothelioma, skin cutaneous melanoma, colorectal cancer, cervical squamous cell carcinoma and endocervical adenocarcinoma.

22. The method of any one of claims 19-21, wherein the MICA-positive cancer is a MICA-positive/HLA-E-negative cancer.

23. A method of treating a subject previously identified or diagnosed as having a MICB-positive cancer, the method comprising administering to a subject previously identified or diagnosed as having a MICB-positive cancer a therapeutically effective amount of a pharmaceutical composition comprising a population of enucleated erythroid cells comprising a first exogenous polypeptide comprising (i) IL- 15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof, on its extracellular surface.

24. The method of claim 23, wherein the method further comprises identifying or diagnosing a subject as having a MICB-positive cancer.

25. The method of claim 23 or 24, wherein the MICB-positive cancer is selected from the group consisting of: acute myeloid leukemia, lymphoid neoplasm diffuse large B-cell lymphoma, testicular germ cell tumors, stomach adenocarcinoma, ovarian serous cystadenocarcinoma, esophageal carcinoma and lung cancer.

26. The method of any one of claims 23-25, wherein the MICB-positive cancer is a MICB-positive/HLA-E-negative cancer.

27. The method of any one of claims 19-26, wherein the administering results in an increase in the number of NKG2D-positive lymphocytes in the subject and/or an increase in the number of NKG2D-positive/NKG2A-negative lymphocytes in the subject.

28. The method of claim 27, wherein the NKG2D-positive lymphocytes are NKG2D-positive NK cells.

29. The method of claim 27 or 28, wherein the NKG2D-positive/NKG2A- negative lymphocytes are NKG2D-positive/NKG2A-negative NK cells.

30. The method of any one of claims 19-29, wherein the method further comprises administering to the subject an NKG2A inhibitor.

31. The method of claim 30, wherein the NKG2A inhibitor is an antagonistic antibody that binds specifically to NKG2A.

32. A method of decreasing the number and/or proliferation of MIC A-positive cancer cells in a subject previously identified or diagnosed as having a MICA-positive cancer, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising a population of enucleated erythroid cells comprising a first exogenous polypeptide comprising (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof, on its extracellular surface.

33. The method of claim 32, wherein the administering results in a decrease in the number of MIC A-positive cancer cells in the subject.

34. The method of claim 33, wherein the administering results in at least a 5% decrease in the number of MIC A-positive cancer cells in the subject as compared to the number of MIC A-positive cancer cells in the subject prior to the administering.

35. The method of claim 34, wherein the administering results in at least a 10% decrease in the number of MIC A-positive cancer cells in the subject as compared to the number of MICA-positive cancer cells in the subject prior to the administering.

36. The method of claim 32, wherein the administering results in a decrease in the proliferation of MICA-positive cancer cells in the subject.

37. The method of claim 36, wherein the administering results in at least a 5% decrease in the proliferation of MICA-positive cancer cells in the subject as compared to the proliferation of MICA-positive cancer cells in the subject prior to the administering.

38. The method of claim 37, wherein the administering results in at least a 10% decrease in the proliferation of MIC A-positive cancer cells in the subject as compared to the proliferation of MIC A-positive cancer cells in the subject prior to the administering.

39. The method of any one of claims 32-38, wherein the method further comprises identifying or diagnosing a subject as having a MIC A-positive cancer.

40. The method of any one of claims 32-39, wherein the MICA-positive cancer is selected from the group consisting of: adrenocortical carcinoma, cholangiocarcinoma, pancreatic adenocarcinoma, kidney cancer, thyroid carcinoma, mesothelioma, skin cutaneous melanoma, colorectal cancer, cervical squamous cell carcinoma and endocervical adenocarcinoma.

41. The method of any one of claims 32-40, wherein the MICA-positive cancer is a MICA-positive/HLA-E-negative cancer.

42. The method of claim 41, wherein the administering results in a decrease in the number of MIC A-positive/HLA-E -negative cancer cells in the subject.

43. The method of claim 42, wherein the administering results in at least a 5% decrease in the number of MIC A-positive/HLA-E-negative cancer cells in the subject as compared to the number of MICA-positive/HLA-E-negative cancer cells in the subject prior to the administering.

44. The method of claim 43, wherein the administering results in at least a 10% decrease in the number of MIC A-positive/HLA-E-negative cancer cells in the subject as compared to the number of MIC A-positive/HLA-E-negative cancer cells in the subject prior to the administering.

45. The method of claim 41, wherein the administering results in a decrease in the proliferation of MICA-positive/HLA-E-negative cancer cells in the subject.

46. The method of claim 45, wherein the administering results in at least a 5% decrease in the proliferation of MICA-positive/HLA-E-negative cancer cells in the subject as compared to the proliferation of MICA-positive/HLA-E-negative cancer cells in the subject prior to the administering.

47. The method of claim 46, wherein the administering results in at least a 10% decrease in the proliferation of MIC A-positive/HLA-E-negative cancer cells in the subject as compared to the proliferation of MIC A-positive/HLA-E-negative cancer cells in the subject prior to the administering.

48. A method of decreasing the number and/or proliferation of MICB-positive cancer cells in a subject previously identified or diagnosed as having a MICB-positive cancer, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising a population of enucleated erythroid cells comprising a first exogenous polypeptide comprising (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof, on its extracellular surface.

49. The method of claim 48, wherein the administering results in a decrease in the number of MICB-positive cancer cells in the subject.

50. The method of claim 49, wherein the administering results in at least a 5% decrease in the number of MICB-positive cancer cells in the subject as compared to the number of MICB-positive cancer cells in the subject prior to the administering.

51. The method of claim 50, wherein the administering results in at least a 10% decrease in the number of MICB-positive cancer cells in the subject as compared to the number of MICB-positive cancer cells in the subject prior to the administering.

52. The method of claim 48, wherein the administering results in a decrease in the proliferation of MICB-positive cancer cells in the subject.

53. The method of claim 52, wherein the administering results in at least a 5% decrease in the proliferation of MICB-positive cancer cells in the subject as compared to the proliferation of MICB-positive cancer cells in the subject prior to the administering.

54. The method of claim 53, wherein the administering results in at least a 10% decrease in the proliferation of MICB-positive cancer cells in the subject as compared to the proliferation of MICB-positive cancer cells in the subject prior to the administering.

55. The method of any one of claims 48-54, wherein the method further comprises identifying or diagnosing a subject as having a MICB-positive cancer.

56. The method of any one of claims 48-55, wherein the MICB-positive cancer is selected from the group consisting of: acute myeloid leukemia, lymphoid neoplasm diffuse large B-cell lymphoma, testicular germ cell tumors, stomach adenocarcinoma, ovarian serous cystadenocarcinoma, esophageal carcinoma and lung cancer.

57. The method of any one of claims 48-56, wherein the MICB-positive cancer is a MICB-positive/HLA-E-negative cancer.

58. The method of claim 57, wherein the administering results in a decrease in the number of MICB-positive/HLA-E-negative cancer cells in the subject.

59. The method of claim 58, wherein the administering results in at least a 5% decrease in the number of MICB-positive/HLA-E-negative cancer cells in the subject as compared to the number of MICB-positive/HLA-E-negative cancer cells in the subject prior to the administering.

60. The method of claim 59, wherein the administering results in at least a 10% decrease in the number of MICB-positive/HLA-E-negative cancer cells in the subject as compared to the number of MICB-positive/HLA-E-negative cancer cells in the subject prior to the administering.

61. The method of claim 57, wherein the administering results in a decrease in the proliferation of MICB-positive/HLA-E-negative cancer cells in the subject.

62. The method of claim 61, wherein the administering results in at least a 5% decrease in the proliferation of MICB-positive/HLA-E-negative cancer cells in the subject as compared to the proliferation of MICB-positive/HLA-E-negative cancer cells in the subject prior to the administering.

63. The method of claim 62, wherein the administering results in at least a 10% decrease in the proliferation of MICB-positive/HLA-E-negative cancer cells in the subject as compared to the proliferation of MICB-positive/HLA-E-negative cancer cells in the subject prior to the administering.

64. The method of any one of claims 32-63, wherein the method further comprises administering to the subject an NKG2A inhibitor.

65. The method of claim 64, wherein the NKG2A inhibitor is an antagonistic antibody that binds specifically to NKG2A.

66. A method of inducing killing a MICA-positive cancer cell in a subject previously identified or diagnosed as having a MICA-positive cancer, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising a population of enucleated erythroid cells comprising 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, on its extracellular surface.

67. The method of claim 66, wherein the MICA-positive cancer cell is a cancer cell selected from the group consisting of: an adrenocortical carcinoma cancer cell, a cholangiocarcinoma cancer cell, a pancreatic adenocarcinoma cancer cell, a kidney cancer cancer cell, a thyroid carcinoma cancer cell, a mesothelioma cancer cell, a skin cutaneous melanoma cancer cell, a colorectal cancer cancer cell, a cervical squamous cell carcinoma cancer cell and an endocervical adenocarcinoma cancer cell.

68. The method of claim 66 or 67, wherein the method further comprises identifying or diagnosing the subject as having a MICA-positive cancer.

69. The method of claim 68, wherein the MICA-positive cancer is selected from the group consisting of: adrenocortical carcinoma, cholangiocarcinoma, pancreatic adenocarcinoma, kidney cancer, thyroid carcinoma, mesothelioma, skin cutaneous melanoma, colorectal cancer, cervical squamous cell carcinoma and endocervical adenocarcinoma.

70. The method of claim 68 or 69, wherein the MICA-positive cancer is a MICA-positive/HLA-E-negative cancer.

71. The method of claim 70, wherein the MICA-positive cancer cell is a MICA-positive and HLA-E-negative cancer cell.

72. A method of inducing killing a MICB-positive cancer cell in a subject previously identified or diagnosed as having a MICB-positive cancer, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising a population of enucleated erythroid cells comprising 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, on its extracellular surface.

73. The method of claim 72, wherein the MICB-positive cancer cell is a cancer cell selected from the group consisting of: an acute myeloid leukemia cancer cell, a lymphoid neoplasm diffuse large B-cell lymphoma cancer cell, a testicular germ cell tumor cancer cell, a stomach adenocarcinoma cancer cell, a ovarian serous cystadenocarcinoma cancer cell, an esophageal carcinoma cancer cell and a lung cancer cancer cell.

74. The method of claim 72 or 73, wherein the method further comprises identifying or diagnosing the subject as having a MICB-positive cancer.

75. The method of claim 74, wherein the MICB-positive cancer is selected from the group consisting of: acute myeloid leukemia, lymphoid neoplasm diffuse large B-cell lymphoma, testicular germ cell tumors, stomach adenocarcinoma, ovarian serous cystadenocarcinoma, esophageal carcinoma and lung cancer.

76. The method of claim 74 or 75, wherein the MICB-positive cancer is a MICB-positive/HLA-E-negative cancer.

77. The method of claim 76, wherein the MICB-positive cancer cell is a MICB-positive and HLA-E-negative cancer cell.

78. The method of any one of claims 66-77, wherein the killing comprises necrosis.

79. The method of any one of claims 66-77, wherein the killing comprises apoptosis.

80. The method of any one of claims 66-77, wherein the killing is mediated via NK-cell mediated cytolysis.

81. The method of any one of claims 66-80, wherein the administering also results in the killing of non-MICA-positive and non-MICB-positive cancer cells within the subject.

82. The method of any one of claims 66-81, wherein the method further comprises administering to the subject an NKG2A inhibitor.

83. The method of claim 82, wherein the NKG2A inhibitor is an antagonistic antibody that binds specifically to NKG2A.

84. A method of reducing the volume of a solid tumor in a subject previously identified or diagnosed as having a MICA-positive cancer, the method comprising administering a therapeutically effective amount of a population of enucleated erythroid cells comprising 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, on its extracellular surface.

85. The method of claim 84, wherein the method further comprises identifying or diagnosing a subject as having a MICA-positive cancer.

86. The method of claim 84 or 85, wherein the MICA-positive cancer is selected from the group consisting of: adrenocortical carcinoma, cholangiocarcinoma, pancreatic adenocarcinoma, kidney cancer, thyroid carcinoma, mesothelioma, skin cutaneous melanoma, colorectal cancer, cervical squamous cell carcinoma and endocervical adenocarcinoma.

87. The method of any one of claims 84-86, wherein the MICA-positive cancer is a MICA-positive/HLA-E-negative cancer.

88. A method of reducing the volume of a solid tumor in a subject previously identified or diagnosed as having a MICB-positive cancer, the method comprising administering a therapeutically effective amount of a population of enucleated erythroid cells comprising 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, on its extracellular surface.

89. The method of claim 88, wherein the method further comprises identifying or diagnosing a subject as having a MICB-positive cancer.

90. The method of claim 88 or 89, wherein the MICB-positive cancer is selected from the group consisting of: acute myeloid leukemia, lymphoid neoplasm diffuse large B-cell lymphoma, testicular germ cell tumors, stomach adenocarcinoma, ovarian serous cystadenocarcinoma, esophageal carcinoma and lung cancer.

91. The method of any one of claims 88-90, wherein the MICB-positive cancer is a MICB-positive/HLA-E-negative cancer.

92. The method of any one of claims 84-91, wherein the administering results in at least a 5% decrease in the volume of the solid tumor in the subject as compared to the volume of the solid tumor prior to the administering.

93. The method of claim 92, wherein the administering results in at least a 10% decrease in the volume of the solid tumor in the subject as compared to the volume of the solid tumor prior to the administering.

94. The method of claim 93, wherein the administering results in at least a 15% decrease in the volume of the solid tumor in the subject as compared to the volume of the solid tumor prior to the administering.

95. The method of any one of claims 84-94, wherein the method further comprises administering to the subject an NKG2A inhibitor.

96. The method of claim 95, wherein the NKG2A inhibitor is an antagonistic antibody that binds specifically to NKG2A.

97. The method of any one of claims 1-96, wherein the enucleated erythroid cell comprises at least 1,000 copies of the first exogenous fusion polypeptide.

98. The method of any one of claims 1-97, wherein the enucleated erythroid cell is made by a process comprising: introducing into a nucleated erythroid cell precursor a nucleic acid encoding the first exogenous fusion polypeptide; and culturing the nucleated erythroid cell precursor under conditions sufficient for expression of the first exogenous fusion polypeptide and enucleation of the nucleated erythroid cell precursor.

99. The method of any one of claims 1-98, wherein the enucleated erythroid cell further comprises a second exogenous polypeptide comprising 4-1BBL or a functional fragment thereof, wherein the second exogenous polypeptide is present on the extracellular surface of the enucleated erythroid cell.

100. The method of claim 99, wherein the enucleated erythroid cell comprises at least 1,000 copies of the second exogenous polypeptide.

101. The method of claim 99 or 100, wherein the enucleated erythroid cell is made by a process comprising: introducing into a nucleated erythroid cell precursor a nucleic acid encoding the first exogenous fusion polypeptide or a functional fragment thereof, and a nucleic acid encoding the second exogenous polypeptide; and culturing the nucleated erythroid cell precursor under conditions sufficient for expression of the first exogenous fusion polypeptide and the second exogenous polypeptide, and enucleation of the nucleated erythroid cell precursor.

102. The method of any one of claims 1-101, wherein the enucleated erythroid cell is not a hypotonically-dialyzed cell.

103. The method of any one of claims 1-102, wherein the enucleated erythroid cell does not comprise a sortase-transfer signature.

104. The method of claim 103, wherein the subject is a human and the enucleated erythroid cell is a human cell.

105. A kit comprising: a pharmaceutical composition comprising an enucleated erythroid cell comprising 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, on its extracellular surface; and instructions for performing the method of any one of claims 1-135.

106. The kit of claim 105, wherein the enucleated erythroid cell comprises at least 1,000 copies of the first exogenous fusion polypeptide.

107. The kit of claim 105 or 106, wherein the enucleated erythroid cell is made by a process comprising: introducing into a nucleated erythroid cell precursor a nucleic acid encoding the first exogenous fusion polypeptide; and culturing the nucleated erythroid cell precursor under conditions sufficient for expression of the first exogenous fusion polypeptide and enucleation of the nucleated erythroid cell precursor.

108. The kit of any one of claims 105-107, wherein the enucleated erythroid cell further comprises a second exogenous polypeptide comprising 4-1BBL or a functional fragment thereof, wherein the second exogenous polypeptide is present on the extracellular surface of the enucleated erythroid cell.

109. The kit of claim 108, wherein the enucleated erythroid cell comprises at least 1,000 copies of the second exogenous polypeptide.

110. The kit of claim 108 or 109, wherein the enucleated erythroid cell is made by a process comprising: introducing into a nucleated erythroid cell precursor a nucleic acid encoding the first exogenous fusion polypeptide and a nucleic acid encoding the second exogenous polypeptide; and culturing the nucleated erythroid cell precursor under conditions sufficient for expression of the first exogenous fusion polypeptide and the second exogenous polypeptide, and enucleation of the nucleated erythroid cell precursor.

111. The kit of any one of claims 105-110, wherein the enucleated erythroid cell is not a hypotonically-dialyzed cell.

112. The kit of any one of claims 105-111, wherein the enucleated erythroid cell does not comprise a sortase-transfer signature.

113. The kit of any one of claims 105-112, wherein the enucleated erythroid cell is a human cell.

114. A method of selecting a pharmaceutical composition comprising a population of enucleated erythroid cells comprising 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, on its extracellular surface, for a subject previously identified or diagnosed as having a MICA-positive cancer.

115. The method of claim 114, wherein the method further comprises identifying or diagnosing the subject as having a MICA-positive cancer.

116. The method of claim 114 or 115, wherein the MICA-positive cancer is selected from the group consisting of: adrenocortical carcinoma, cholangiocarcinoma, pancreatic adenocarcinoma, kidney cancer, thyroid carcinoma, mesothelioma, skin cutaneous melanoma, colorectal cancer, cervical squamous cell carcinoma and endocervical adenocarcinoma.

117. The method of any one of claims 114-116, wherein the MICA-positive cancer is a MICA-positive/HLA-E-negative cancer.

118. A method of selecting a pharmaceutical composition comprising a population of enucleated erythroid cells comprising 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, on its extracellular surface, for a subject previously identified or diagnosed as having a MICB-positive cancer.

119. The method of claim 118, wherein the method further comprises identifying or diagnosing the subject as having a MICB-positive cancer.

120. The method of claim 118 or 119, wherein the MICB-positive cancer is selected from the group consisting of: acute myeloid leukemia, lymphoid neoplasm diffuse large B-cell lymphoma, testicular germ cell tumors, stomach adenocarcinoma, ovarian serous cystadenocarcinoma, esophageal carcinoma and lung cancer.

121. The method of any one of claims 118-120, wherein the MICB-positive cancer is a MICB-positive/HLA-E-negative cancer.