TW202304482A - Methods of increasing nkg2d-positive lymphocytes in a subject and uses thereof - Google Patents

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

Method for increasing NKG2D positive lymphocytes in an individual and use thereof

Cross References to Related Applications

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

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

This application contains a Sequence Listing which has been filed electronically in an ASCII text file named 47472.0083WO1_ST25.txt. The ASCII text archive was created on March 1, 2022 and is 106 kilobytes in size. The material in the ASCII text archive is incorporated herein by reference in its entirety.

Engineered enucleated erythroid cells are being developed as therapeutic agents that carry or present exogenous proteins to patients in need.

The present invention relates to the use of MHC class I polypeptide-associated sequence A (MICA) and/or MHC class I polypeptide-related sequence B (MICB) as biomarkers for the identification of individuals treated with enucleated erythrocytes, which The erythroid cell includes a first exogenous fusion polypeptide, and the first exogenous fusion polypeptide includes (i) IL-15 or its functional fragment, and (ii) IL-15 receptor α or its functional fragment. The present invention is based, at least in part, on the discovery that administration of enucleated erythroid blood cells comprising a first exogenous fusion polypeptide and The second exogenous polypeptide on its extracellular surface, the first exogenous fusion polypeptide includes (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor α or a functional fragment thereof, the Two exogenous polypeptides include 4-1BBL or functional fragments thereof. Therefore, determining the MICA positivity and/or MICB positivity of a cancer in an individual is particularly useful for identifying patients who are likely to respond to therapy with these cells. In light of this discovery, provided herein are methods of increasing the number of NKG2D-positive lymphocytes (e.g., NKG2D-positive NK cells) in an individual previously identified or diagnosed as having a MICA-positive cancer, an MICB-positive cancer, or a MICA/MICB-positive cancer; treatment Method for an individual previously identified or diagnosed as having a MICA-positive cancer, an MICB-positive cancer, or a MICA/MICB-positive cancer; reducing the number and/or proliferation of MICA-positive cancer cells in an individual previously identified or diagnosed as having a MICA-positive cancer A method for reducing the number and/or proliferation of MICB-positive cancer cells in an individual previously identified or diagnosed as having a MICB-positive cancer; reducing MICA/MICB in an individual previously identified or diagnosed as having a MICA/MICB-positive cancer Methods of Amount and/or Proliferation of MICB Positive Cancer Cells; Methods of Killing MICA Positive Cancer Cells in Individuals Previously Identified or Diagnosed as Having MICA Positive Cancers; In Individuals Previously Identified or Diagnosed as Having MICB Positive Cancers Methods of Killing MICB Positive Cancer Cells; Methods of Killing MICA/MICB Positive Cancer Cells in Individuals Previously Identified or Diagnosed as Having MICA/MICB Positive Cancers; A method of reducing solid tumor volume in individuals with positive cancer or MICA/MICB positive cancer. Also provided herein are methods of selecting enucleated erythrocytes comprising a first exogenous fusion polypeptide on their extracellular surface in an individual previously identified or diagnosed as having a MICA-positive cancer, an MICB-positive cancer, or a MICA/MICB-positive cancer , the first exogenous fusion polypeptide includes (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof.

Provided herein are methods of increasing the number of NKG2D-positive lymphocytes in an individual previously identified or diagnosed with a MICA-positive cancer comprising administering to the individual a therapeutically effective amount of a pharmaceutical composition comprising an enucleated erythroid population, The enucleated erythroid cells include on their extracellular surface a first exogenous fusion polypeptide comprising (i) IL-15 or a functional fragment thereof, and (ii) an IL-15 receptor α or functional fragments thereof. Some embodiments of any of the methods described herein further comprise identifying or diagnosing that the individual has a MICA-positive cancer. In some embodiments, the MICA-positive cancer is selected from the group consisting of adrenocortical carcinoma, cholangiocarcinoma, pancreatic cancer, kidney cancer, thyroid cancer, mesothelioma, cutaneous melanoma, colorectal cancer, cervical squamous 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.

Provided herein are methods of increasing the number of NKG2D-positive lymphocytes in an individual previously identified or diagnosed with an MICB-positive cancer comprising administering to the individual a therapeutically effective amount of a pharmaceutical composition comprising an enucleated erythroid population, The enucleated erythroid cells include on their extracellular surface a first exogenous fusion polypeptide comprising (i) IL-15 or a functional fragment thereof, and (ii) an IL-15 receptor α or functional fragments thereof. In some embodiments of any of the methods described herein, the method further comprises identifying or diagnosing that the individual has an MICB-positive cancer. In some embodiments, the MICB-positive cancer is selected from the group consisting of acute myelogenous leukemia, lymphoid neoplasm diffuse large B-cell lymphoma, testicular germ cell tumor, gastric adenocarcinoma, ovarian serous cystadenocarcinoma, esophageal cancer , and lung cancer. In some embodiments of any of the methods described herein, the MICB positive cancer is an MICB positive/HLA-E negative cancer.

Provided herein are methods of increasing the number of NKG2D-positive lymphocytes in an individual previously identified or diagnosed with a MICA/MICB-positive cancer comprising administering to the individual a therapeutically effective amount of a pharmaceutical composition comprising enucleated erythrocytes A population of enucleated erythroid cells comprising on their extracellular surface a first exogenous fusion polypeptide comprising (i) IL-15 or a functional fragment thereof, and (ii) IL-15 Receptor alpha or functional fragments thereof. In some embodiments of any of the methods described herein, the method further comprises identifying or diagnosing that the individual has 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 myelogenous leukemia, pancreatic cancer, bile duct cancer, kidney cancer, squamous cell carcinoma of the cervix, Endocervical cancer, colorectal cancer, mesothelioma, 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 individual by at least 5% compared to the number of NKG2D-positive lymphocytes in the individual prior to administration. In some embodiments of any of the methods described herein, the administering results in an increase in the number of NKG2D-positive lymphocytes in the individual by at least 10% compared to the number of NKG2D-positive lymphocytes in the individual prior to administration.

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

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

Also provided herein is a method of treating an individual previously identified or diagnosed as having a MICA-positive cancer comprising administering to the individual previously identified or diagnosed as having a MICA-positive cancer a therapeutically effective amount of a pharmaceutical composition comprising A population of nucleated erythroid cells comprising on their extracellular surface a first exogenous polypeptide comprising (i) IL-15 or a functional fragment thereof, and (ii) IL-15 15 receptor alpha or functional fragments thereof. In some embodiments of any of the methods described herein, the method further comprises identifying or diagnosing that the individual has 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 cancer, kidney cancer, thyroid cancer, mesothelioma, cutaneous epidermal melanoma, colon rectal cancer, squamous cell carcinoma of the cervix, 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 an individual previously identified or diagnosed with an MICB-positive cancer comprising administering to the individual previously identified or diagnosed with an MICB-positive cancer a therapeutically effective amount of a pharmaceutical composition comprising A population of nucleated erythroid cells comprising on their extracellular surface a first exogenous polypeptide comprising (i) IL-15 or a functional fragment thereof, and (ii) IL-15 15 receptor alpha or functional fragments thereof. Some embodiments of any of the methods described herein further comprise identifying or diagnosing that the individual has an 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 myelogenous leukemia, lymphoid neoplasm diffuse large B-cell lymphoma, testicular germ cell tumor, gastric adenocarcinoma, ovarian serous Cystadenocarcinoma, esophageal cancer, and lung cancer. In some embodiments of any of the methods described herein, the MICB positive cancer is an MICB positive/HLA-E negative cancer.

Also provided herein are methods of treating an individual previously identified or diagnosed as having a MICA/MICB positive cancer comprising administering to the individual previously identified or diagnosed as having a MICA/MICB positive cancer a therapeutically effective amount of a pharmaceutical composition, the pharmaceutical The composition comprises a population of enucleated erythroid cells comprising on their extracellular surface a first exogenous polypeptide comprising (i) IL-15 or a functional fragment thereof, and ( ii) IL-15 receptor alpha or a functional fragment thereof. Some embodiments of any of the methods described herein further comprise identifying or diagnosing the individual 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 myelogenous leukemia, pancreatic cancer, bile duct cancer, kidney cancer, squamous cell carcinoma of the cervix , endocervical cancer, colorectal cancer, mesothelioma, 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 administration results in an increase in the number of NKG2D-positive lymphocytes in the individual and/or an increase in the number of NKG2D-positive/NKG2A-negative lymphocytes in the individual. 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 comprise administering to the individual an NKG2A inhibitor. In some embodiments of any of the methods described herein, the NKG2A inhibitor is an antagonist antibody that specifically binds to NKG2A.

Also provided herein are methods of reducing the number and/or proliferation of MICA-positive cancer cells in an individual previously identified or diagnosed with MICA-positive cancer comprising administering a therapeutically effective amount of a pharmaceutical composition comprising enucleation A population of erythroid cells comprising on their extracellular surface a first exogenous polypeptide comprising (i) IL-15 or a functional fragment thereof, and (ii) IL-15 Receptor alpha or functional fragments thereof.

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

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

Some embodiments of any of the methods described herein further comprise identifying or diagnosing that the individual has 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 cancer, kidney cancer, thyroid cancer, mesothelioma, cutaneous epidermal melanoma, colon rectal cancer, squamous cell carcinoma of the cervix, 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 individual. In some embodiments of any of the methods described herein, the administering results in a reduction in the number of MICA-positive/HLA-E-negative cancer cells in the individual compared to the number of MICA-positive/HLA-E-negative cancer cells in the individual prior to administration by at least 5%. In some embodiments of any of the methods described herein, the administering results in a reduction in the number of MICA-positive/HLA-E-negative cancer cells in the individual compared to the number of MICA-positive/HLA-E-negative cancer cells in the individual prior to administration by at least 10%.

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

Also provided herein are methods of reducing the number and/or proliferation of MICB-positive cancer cells in an individual previously identified or diagnosed with MICB-positive cancer comprising administering a therapeutically effective amount of a pharmaceutical composition comprising enucleated A population of erythroid cells comprising on their extracellular surface a first exogenous polypeptide comprising (i) IL-15 or a functional fragment thereof, and (ii) IL-15 Receptor alpha or functional fragments thereof.

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

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

Some embodiments of any of the methods described herein further comprise identifying or diagnosing that the individual has an 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 myelogenous leukemia, lymphoid neoplasm diffuse large B-cell lymphoma, testicular germ cell tumor, gastric adenocarcinoma, ovarian serous Cystadenocarcinoma, esophageal cancer, and lung cancer. In some embodiments of any of the methods described herein, the MICB positive cancer is an 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 individual. In some embodiments of any of the methods described herein, the administering results in a reduction in the number of MICB positive/HLA-E negative cancer cells in the individual compared to the number of MICB positive/HLA-E negative cancer cells in the individual prior to administration by at least 5%. In some embodiments of any of the methods described herein, the administering results in a reduction in the number of MICB positive/HLA-E negative cancer cells in the individual compared to the number of MICB positive/HLA-E negative cancer cells in the individual prior to administration by at least 10%.

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

Also provided herein is a method of reducing the number and/or proliferation of MICA/MICB positive cancer cells in an individual previously identified or diagnosed with a MICA/MICB positive cancer comprising administering a therapeutically effective amount of a pharmaceutical composition, the pharmaceutical composition The substance comprises a population of enucleated erythroid cells comprising on their extracellular surface a first exogenous polypeptide comprising (i) IL-15 or a functional fragment thereof, and (ii ) IL-15 receptor alpha or a functional fragment thereof.

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

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

Some embodiments of any of the methods described herein further comprise identifying or diagnosing the individual 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 myelogenous leukemia, pancreatic cancer, bile duct cancer, kidney cancer, squamous cell carcinoma of the cervix , endocervical cancer, colorectal cancer, mesothelioma, 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 individual. In some embodiments of any of the methods described herein, the administration results in MICA/MICB positive/HLA-E negative cancer cells in the individual compared to the number of MICA/MICB positive/HLA-E negative cancer cells in the individual prior to administration decrease by at least 5%. In some embodiments of any of the methods described herein, the administration results in MICA/MICB positive/HLA-E negative cancer cells in the individual compared to the number of MICA/MICB positive/HLA-E negative cancer cells in the individual prior to administration decrease by at least 10%.

In some embodiments of any of the methods described herein, the administering results in decreased proliferation of MICA/MICB positive/HLA-E negative cancer cells in the individual. In some embodiments of any of the methods described herein, the administering results in the proliferation of MICA/MICB positive/HLA-E negative cancer cells in the individual compared to the proliferation of MICA/MICB positive/HLA-E negative cancer cells in the individual prior to the administration The hyperplasia is reduced by at least 5%. In some embodiments of any of the methods described herein, the administering results in the proliferation of MICA/MICB positive/HLA-E negative cancer cells in the individual compared to the proliferation of MICA/MICB positive/HLA-E negative cancer cells in the individual prior to the administration The hyperplasia is reduced by at least 10%. In some embodiments of any of the methods described herein, the administering results in the proliferation of MICA/MICB positive/HLA-E negative cancer cells in the individual compared to the proliferation of MICA/MICB positive/HLA-E negative cancer cells in the individual prior to the administration The hyperplasia is reduced by at least 15%.

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

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

In some embodiments of any of the methods described herein, the MICA-positive cancer cells are cancer cells selected from the group consisting of adrenocortical cancer cells, cholangiocarcinoma cells, pancreatic cancer cells, kidney cancer cells, thyroid cancer cells, mesenchymal dermatoma cells, skin epidermal melanoma cells, colorectal cells, cervical squamous cell cells, and endocervical adenocarcinoma cells.

Some embodiments of any of the methods described herein further comprise identifying or diagnosing that the individual has 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 cancer, kidney cancer, thyroid cancer, mesothelioma, cutaneous epidermal melanoma, colon rectal cancer, squamous cell carcinoma of the cervix, 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 cells are MICA-positive and HLA-E-negative cancer cells.

Also provided herein is a method of inducing killing of MICB-positive cancer cells in an individual previously identified or diagnosed with MICB-positive cancer comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an enucleated erythroid population, The enucleated erythroid cells include on their extracellular surface a first exogenous fusion polypeptide comprising (i) IL-15 or a functional fragment thereof, and (ii) an IL-15 receptor α or functional fragments thereof.

Some embodiments of any of the methods described herein further comprise identifying or diagnosing that the individual has an MICB-positive cancer. In some embodiments of any of the methods described herein, the MICB-positive cancer cells are cancer cells selected from the group consisting of acute myeloid leukemia cancer cells, lymphoid neoplasms diffuse large B-cell lymphoma cancer cells, testicular germ cells Tumor cancer cells, gastric adenocarcinoma cells, ovarian serous cystadenocarcinoma cells, esophageal cancer cells, and lung cancer cells.

In some embodiments of any of the methods described herein, the MICB-positive cancer is selected from the group consisting of acute myelogenous leukemia, lymphoid neoplasm diffuse large B-cell lymphoma, testicular germ cell tumor, gastric adenocarcinoma, ovarian serous Cystadenocarcinoma, esophageal cancer, and lung cancer.

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

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

In some embodiments of any of the methods described herein, the MICA/MICB positive cancer cells are cancer cells selected from the group consisting of: adrenocortical cancer cells, acute myeloid leukemia cancer cells, pancreatic cancer cells, cholangiocarcinoma cells, Renal cancer cells, cervical squamous cell cancer cells, endocervical cancer cells, colorectal cancer cells, mesothelioma cancer cells, skin epidermal melanoma cancer cells, and lung cancer cells.

Some embodiments of any of the methods described herein further comprise identifying or diagnosing the individual 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 myelogenous leukemia, pancreatic cancer, bile duct cancer, kidney cancer, squamous cell carcinoma of the cervix , endocervical cancer, colorectal cancer, mesothelioma, 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 cells are MICA/MICB positive and HLA-E negative cancer cells.

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

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

Also provided herein is a method of reducing the volume of a solid tumor in an individual previously identified or diagnosed with a MICA-positive cancer comprising administering a therapeutically effective amount of a population of enucleated erythroid cells on their extracellular surface A first exogenous fusion polypeptide is included, and the first exogenous fusion polypeptide includes (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof.

Some embodiments of the methods described herein further comprise identifying or diagnosing the individual 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 cancer, kidney cancer, thyroid cancer, mesothelioma, cutaneous epidermal melanoma, colon rectal cancer, squamous cell carcinoma of the cervix, 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 is a method of reducing the volume of a solid tumor in an individual previously identified or diagnosed with an MICB-positive cancer comprising administering a therapeutically effective amount of a population of enucleated erythroid cells on their extracellular surface A first exogenous fusion polypeptide is included, and the first exogenous fusion polypeptide includes (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof.

Some embodiments of any of the methods described herein further comprise identifying or diagnosing that the individual has an 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 myelogenous leukemia, lymphoid neoplasm diffuse large B-cell lymphoma, testicular germ cell tumor, gastric adenocarcinoma, ovarian serous Cystadenocarcinoma, esophageal cancer, and lung cancer. In some embodiments of any of the methods described herein, the MICB positive cancer is an MICB positive/HLA-E negative cancer.

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

Some embodiments of any of the methods described herein further comprise identifying or diagnosing the individual 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 myelogenous leukemia, pancreatic cancer, bile duct cancer, kidney cancer, squamous cell carcinoma of the cervix , endocervical cancer, colorectal cancer, mesothelioma, 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% reduction in the volume of the solid tumor as compared to the volume of the solid tumor in the individual prior to administration. In some embodiments of any of the methods described herein, the administering results in at least a 10% reduction in the volume of the solid tumor as compared to the volume of the solid tumor in the individual prior to administration. In some embodiments of any of the methods described herein, the administering results in at least a 15% reduction in the volume of the solid tumor as compared to the volume of the solid tumor in the individual prior to administration.

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

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

In some embodiments of any of the methods described herein, enucleated erythroid cells are produced by a method comprising: introducing a nucleic acid encoding a first exogenous fusion polypeptide into a nucleated erythroid precursor cell; and Nucleated erythroid precursor cells are cultured under the condition of exogenous fusion polypeptide and enucleation of nucleated erythroid precursor cells.

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

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

In some embodiments of any of the methods described herein, the enucleated erythroid cells are not hypotonic cells. In some embodiments of any of the methods described herein, the enucleated erythroid blood cells do not include a sortase-transfer signature.

In some embodiments of any of the methods described herein, the individual is a human and the enucleated erythroid cells are human cells.

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

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

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

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

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

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

Also provided herein are methods of selecting a pharmaceutical composition comprising a population of enucleated erythroid cells comprising on their extracellular surface a first exogenous fusion polypeptide, the first exogenous fusion Polypeptides include (i) IL-15 or functional fragments thereof, and (ii) IL-15 receptor alpha or functional fragments thereof, for use in individuals previously identified or diagnosed with a MICA-positive cancer.

In some embodiments of any of the methods described herein, further comprising identifying or diagnosing the individual 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 cancer, kidney cancer, thyroid cancer, mesothelioma, cutaneous epidermal melanoma, colon rectal cancer, squamous cell carcinoma of the cervix, 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 comprising a population of enucleated erythroid cells comprising on their extracellular surface a first exogenous fusion polypeptide, the first exogenous fusion Polypeptides include (i) IL-15 or functional fragments thereof, and (ii) IL-15 receptor alpha or functional fragments thereof, for use in individuals previously identified or diagnosed with MICB-positive cancer.

Some embodiments of any of the methods described herein further comprise identifying or diagnosing that the individual has an 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 myelogenous leukemia, lymphoid neoplasm diffuse large B-cell lymphoma, testicular germ cell tumor, gastric adenocarcinoma, ovarian serous Cystadenocarcinoma, esophageal cancer, and lung cancer. In some embodiments of any of the methods described herein, the MICB positive cancer is an MICB positive/HLA-E negative cancer.

Also provided herein are methods of selecting a pharmaceutical composition comprising a population of enucleated erythroid cells comprising on their extracellular surface a first exogenous fusion polypeptide, the first exogenous fusion Polypeptides include (i) IL-15 or functional fragments thereof, and (ii) IL-15 receptor alpha or functional fragments thereof, for use in individuals previously identified or diagnosed with MICA/MICB positive cancer.

Some embodiments of any of the methods described herein further comprise identifying or diagnosing the individual 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 myelogenous leukemia, pancreatic cancer, bile duct cancer, kidney cancer, squamous cell carcinoma of the cervix , endocervical cancer, colorectal cancer, mesothelioma, 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 "modified enucleated erythroid cells" refers to enucleated erythroid cells (e.g., human enucleated erythroid cells) that comprise one or more (e.g., two, three, four, five, or six) Exogenous polypeptides (eg, any combination of the exemplary exogenous polypeptides described herein or known in the art). For example, an engineered enucleated erythroid cell may have one or more exogenous polypeptides present in its cytoplasm. In some examples, engineered enucleated erythroid cells can have one or more exogenous polypeptides present on their extracellular surface. In some examples, engineered enucleated erythroid cells may have (i) one or more exogenous polypeptides present in their cytoplasm, and (ii) one or more exogenous polypeptides present on their extracellular surface sex peptides. Non-limiting examples of engineered enucleated erythroid cells include click-engaged enucleated erythroid cells, enucleated erythroid cells that have been hypotonically loaded, and enucleated erythroid cells that have been modified by physical manipulation (e.g., as described herein or known in the art). Any exemplary type of physical manipulation) loaded enucleated erythroid cells. Other non-limiting aspects of engineered enucleated erythroid cells are described herein.

The term "conjugated enucleated erythroid cell" means an engineered enucleated erythroid cell having at least one exogenous polypeptide conjugated to another exogenous polypeptide present in A polypeptide on the extracellular surface of an engineered enucleated erythroid cell (eg, an endogenous polypeptide or a different exogenous polypeptide of the enucleated erythroid cell).

"Hypotonically loaded enucleated erythroid cell" means an enucleated erythroid cell engineered, at least in part, by exposing an enucleated erythroid cell or erythroid precursor cell comprising one or more exogenous polypeptides to a low ionic strength buffer (eg, any of the exemplary low ionic strength buffers described herein). Non-limiting examples of methods that can be used to generate hypotonically loaded enucleated erythroid cells are described herein. Additional methods of generating hypotonically loaded enucleated erythroid cells are known in the art.

The term "enucleated erythroid cell loaded by physical manipulation" refers to an enucleated erythroid cell at least a portion of which has been loaded by encoding one or more exogenous polypeptides (such as described herein or known in the art) Any exemplary exogenous polypeptide) and/or nucleic acid of the exogenous polypeptide are introduced into the erythroid precursor cells to generate erythroid precursor cells by physical manipulation. Non-limiting examples of physical manipulations that can be used to introduce nucleic acids encoding one or more exogenous polypeptides into erythroid precursor cells include electroporation and particle-mediated transfection. Additional examples of physical manipulations that can be used to introduce nucleic acids encoding one or more exogenous polypeptides into erythroid precursor cells are known in the art.

The term "exogenous polypeptide" refers to a polypeptide that is introduced into or onto a cell, or is expressed by a 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 introduced into a cell or precursor to a cell, optionally not retained by the cell. In some embodiments, the exogenous polypeptide is a polypeptide bound to the cell surface 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, immunosuppressive molecules, MHC molecules, APC binding molecules, autologous Antigens, allergens, toxins, targeting agents, receptor ligands (eg, receptor agonists or receptor antagonists), and antibodies or antibody fragments.

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

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

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

As used herein, "treating" refers to 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 MICA mRNA, including wild-type human MICA polypeptide and wild-type human MICA mRNA, and variants (eg, truncated or mutated forms) thereof. The term "MICA" excludes soluble MICA polypeptides. Non-limiting examples of methods of detecting MICA levels are described herein.

As used herein, the term "MICA-positive cancer" refers to a cancer that includes MICA-positive cancer cells. Non-limiting examples of MICA positive cancers are described herein.

As used herein, the term "MICA-positive cancer cells" refers to cancer cells whose MICA content (MICA polypeptide or MICA mRNA) is higher than the MICA reference content. 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 MICB polypeptide or MICB mRNA, including wild-type human MICB polypeptide and wild-type human MICB mRNA, and variants (eg, truncated or mutated forms) thereof. The term "MICB" excludes soluble MICB polypeptides. Non-limiting examples of methods of detecting MICB levels are described herein.

As used herein, the term "MICB-positive cancer" refers to a cancer that includes MICB-positive cancer cells. Non-limiting examples of MICB positive cancers are described herein.

As used herein, the term "MICB-positive cancer cells" refers to cancer cells whose MICB content (MICB polypeptide or MICB mRNA) is higher than the MICB reference level. Non-limiting examples of MICB reference levels are described herein. Non-limiting examples of MICB-positive cancer cells are also described herein.

As used herein, the term "MICA/MICB positive cancer" refers to a cancer comprising MICA/MICB positive cancer cells, or a cancer comprising MICA/MICB positive cancer cells. Non-limiting examples of MICA/MICB positive cancers are described herein.

As used herein, the term "MICA/MICB positive cancer cells" refers to cancer cells whose MICA content (MICA polypeptide or MICA mRNA) is higher than the MICA reference content, and MICB content (MICB polypeptide or MICB mRNA) is higher than the MICB reference content. Non-limiting examples of MICA/MICB positive cancer cells are also described herein.

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

As used herein, the term "HLA-E negative cancer" refers to a cancer with HLA-E negative cancer cells.

As used herein, the term "HLA-E negative cancer cells" refers to cancer cells whose HLA-E content (HLA-E polypeptide or HLA-E mRNA) is lower than the HLA-E reference level. Non-limiting examples of HLA-E reference levels are described herein.

As used herein, the term "NKG2D" refers to NKG2D polypeptide or NKG2D mRNA, including wild-type human NKG2D polypeptide and mRNA encoding wild-type human NKG2D, and variants (eg, truncated or mutated forms) thereof. The term "NKG2D" also includes all known isoforms of NKG2D polypeptides and NKG2D mRNA. Non-limiting examples of methods of detecting NKG2D levels are described herein.

As used herein, the term "NKG2D-positive lymphocytes" refers to lymphocytes whose NKG2D content (NKG2D polypeptide or NKG2D mRNA) is higher than the NKG2D reference content. Non-limiting examples of reference amounts of NKG2D are described herein.

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

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

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

As used herein, the term "NKG2A-positive lymphocyte" means a lymphocyte with an NKG2A content (NKG2A polypeptide or NKG2A mRNA) higher than a reference NKG2A content. Non-limiting examples of reference amounts of NKG2A are described herein.

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

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

As used herein, the term "trafficking" refers to NK cells in the blood of an individual. For example, "increased 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, "increased 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" refers to an exogenous polypeptide comprising a sequence that can be produced by a sortase reaction. Non-limiting examples of polypeptides lacking the sortase transfer feature are described in WO 2017/123646, which is incorporated by reference in its entirety.

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

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

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

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

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

Some embodiments of the methods described herein result in an increase in trafficking of NKG2D-positive lymphocytes (e.g., NKG2D-positive NK cells) in an individual by at least 1%, by at least 2%, by at least 3%, by at least 4%, by at least 5%, by At least 6%, increase by at least 7%, increase by at least 8%, increase by at least 9%, increase by at least 10%, increase by at least 15%, increase by at least 20%, increase by at least 25%, increase by at least 30%, increase by at least 35%, increase At least 40%, increase by at least 45%, increase by at least 50%, increase by at least 55%, increase by at least 60%, increase by at least 65%, increase by at least 70%, increase by at least 75%, increase by at least 80%, increase by at least 85%, increase At least 90%, increase by at least 95%, increase by at least 100%, increase by at least 200%, increase by at least 250%, increase by at least 300%, increase by at least 350%, increase by at least 400%, increase by at least 450%, increase by at least 500%, increase At least 550%, an increase of at least 600%, an increase of at least 650%, an increase of at least 700%, an increase of at least 750%, an increase of at least 800%, an increase of at least 850%, an increase of at least 900%, an increase of at least 950%, or an increase of at least 1000% ( For example, compared to the trafficking of NKG2D-positive lymphocytes (eg, NKG2D-positive NK cells) in an individual prior to administration). Some embodiments of any of the methods described herein result in an increase in trafficking of NKG2D-positive lymphocytes (e.g., NKG2D-positive NK cells) in an individual by about 1% to about 1000% (or any exemplary subrange of this range described herein) (e.g., Compared to the trafficking of NKG2D-positive lymphocytes (eg, NKG2D-positive NK cells) in individuals prior to administration).

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

Some embodiments of the methods described herein result in a maximum fold change increase of at least 0.1-fold, an increase of at least 0.2-fold 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 individual's blood , increased by at least 0.3 times, increased by at least 0.4 times, increased by at least 0.5 times, increased by at least 0.6 times, increased by at least 0.7 times, increased by at least 0.8 times, increased by at least 0.9 times, increased by at least 1.0 times, increased by at least 1.5 times, increased by at least 2.0 times , increase by at least 2.5 times, increase by at least 3.0 times, increase by at least 3.5 times, increase by at least 4.0 times, increase by at least 4.5 times, increase by at least 5.0 times, increase by at least 5.5 times, increase by at least 6.0 times, increase by at least 6.5 times, increase by at least 7.0 times , increased by at least 7.5-fold, increased by at least 8.0-fold, increased by at least 8.5-fold, increased by at least 9.0-fold, increased by at least 9.5-fold, or increased by at least 10-fold (for example, compared with NKG2D-positive lymphocytes in the individual's blood before administration (such as NKG2D-positive NK cells) compared to the ratio of NKG2A-positive lymphocytes (eg, NKG2A-positive NK cells)). Some embodiments of the methods described herein result in a maximum fold change of about 0.01-fold to about a 10-fold increase 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 individual's blood , increased by about 0.01 times to about 9 times, increased by about 0.01 times to about 8 times, increased by about 0.01 times to about 7 times, increased by about 0.01 times to about 6 times, increased by about 0.01 times to about 5 times , increased by about 0.01 times to about 4 times, increased by about 0.01 times to about 3 times, increased by about 0.01 times to about 2 times, increased by about 0.01 times to about 1 times, increased by about 0.01 times to about 0.5 times , increased by about 0.01 times to about 0.1 times, increased by about 0.1 times to about 10 times, increased by about 0.1 times to about 9 times, increased by about 0.1 times to about 8 times, increased by about 0.1 times to about 7 times , increased by about 0.1 times to about 6 times, increased by about 0.1 times to about 5 times, increased by about 0.1 times to about 4 times, increased by about 0.1 times to about 3 times, increased by about 0.1 times to about 2 times , increased by about 0.1 times to about 1 times, increased by about 0.1 times to about 0.5 times, increased by about 0.5 times to about 10 times, increased by about 0.5 times to about 9 times, increased by about 0.5 times to about 8 times , increased by about 0.5 times to about 7 times, increased by about 0.5 times to about 6 times, increased by about 0.5 times to about 5 times, increased by about 0.5 times to about 4 times, increased by about 0.5 times to about 3 times , increased by about 0.5 times to about 2 times, increased by about 0.5 times to about 1 times, increased by about 1 time to about 10 times, increased by about 1 time to about 9 times, increased by about 1 time to about 8 times , about 1-fold increase to about 7-fold increase, about 1-fold increase to about 6-fold increase, about 1-fold increase to about 5-fold increase, about 1-fold increase to about 4-fold increase, about 1-fold increase to about 3-fold increase , about 1-fold increase to about 2-fold increase, about 2-fold increase to about 10-fold increase, about 2-fold increase to about 9-fold increase, about 2-fold increase to about 8-fold increase, about 2-fold increase to about 7-fold increase , increased by about 2 times to about 6 times, increased by about 2 times to about 5 times, increased by about 2 times to about 4 times, increased by about 2 times to about 3 times, increased by about 3 times to about 10 times , increased by about 3 times to about 9 times, increased by about 3 times to about 8 times, increased by about 3 times to about 7 times, increased by about 3 times to about 6 times, increased by about 3 times to about 5 times , increased by about 3 times to about 4 times, increased by about 4 times to about 10 times, increased by about 4 times to about 9 times, increased by about 4 times to about 8 times, increased by about 4 times to about 7 times , increased by about 4 times to about 6 times, increased by about 4 times to about 5 times, increased by about 5 times to about 10 times, increased by about 5 times to about 9 times, increased by about 5 times to about 8 times , increased by about 5 times to about 7 times, increased by about 5 times to about 6 times, increased by about 6 times to about 10 times , increased by about 6 times to about 9 times, increased by about 6 times to about 8 times, increased by about 6 times to about 7 times, increased by about 7 times to about 10 times, increased by about 7 times to about 9 times , about 7-fold increase to about 8-fold increase, about 8-fold increase to about 10-fold increase, about 8-fold increase to about 9-fold increase, or about 9-fold increase to about 10-fold increase (e.g. Compared with the ratio of NKG2D positive lymphocytes (eg NKG2D positive NK cells) to NKG2A positive lymphocytes (eg NKG2A positive NK cells) in the middle.

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

Some embodiments of the methods described herein result in a maximum fold increase in the absolute number of NK cells in the individual's blood, 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 times, increase by at least 0.7 times, increase by at least 0.8 times, increase by at least 0.9 times, increase by at least 1.0 times, increase by at least 1.5 times, increase by at least 2.0 times, increase by at least 2.5 times, increase by at least 3.0 times, increase by at least 3.5 times, increase by at least 4.0 times fold, increased by at least 4.5 fold, or increased by at least 5.0 fold (eg, compared to the maximum fold increase in absolute number of NK cells in the individual's blood prior to administration). Some embodiments of the methods described herein result in a maximum fold increase in the absolute number of NK cells in the individual's blood, from about 0.01-fold increase to about 5-fold increase, from about 0.01-fold increase to about 4.5-fold increase, from about 0.01-fold increase to about 4-fold increase times, increased by about 0.01 times to about 3.5 times, increased by about 0.01 times to about 3 times, increased by about 0.01 times to about 2.5 times, increased by about 0.01 times to about 2 times, increased by about 0.01 times to about 1.5 times times, from about 0.01 times to about 1 times, from about 0.01 times to about 0.8 times, from about 0.01 times to about 0.6 times, from about 0.01 times to about 0.4 times, from about 0.01 times to about 0.2 times times, increase about 0.01 times to about 0.1 times, increase about 0.01 times to about 0.05 times, increase about 0.05 times to about 5 times, increase about 0.05 times to about 4.5 times, increase about 0.05 times to about 4 times times, increased by about 0.05 times to about 3.5 times, increased by about 0.05 times to about 3 times, increased by about 0.05 times to about 2.5 times, increased by about 0.05 times to about 2 times, increased by about 0.05 times to about 1.5 times times, about 0.05 times to about 1 times, about 0.05 times to about 0.8 times, about 0.05 times to about 0.6 times, about 0.05 times to about 0.4 times, about 0.05 times to about 0.2 times times, increased by about 0.05 times to about 0.1 times, increased by about 0.1 times to about 5 times, increased by about 0.1 times to about 4.5 times, increased by about 0.1 times to about 4 times, increased by about 0.1 times to about 3.5 times times, increased by about 0.1 times to about 3 times, increased by about 0.1 times to about 2.5 times, increased by about 0.1 times to about 2 times, increased by about 0.1 times to about 1.5 times, increased by about 0.1 times to about 1 times, from about 0.1 times to about 0.8 times, from about 0.1 times to about 0.6 times, from about 0.1 times to about 0.4 times, from about 0.1 times to about 0.2 times, from about 0.2 times to about 5 times times, about 0.2 times to about 4.5 times, about 0.2 times to about 4 times, about 0.2 times to about 3.5 times, about 0.2 times to about 3 times, about 0.2 times to about 2.5 times times, about 0.2 times to about 2 times, about 0.2 times to about 1.5 times, about 0.2 times to about 1 times, about 0.2 times to about 0.8 times, about 0.2 times to about 0.6 times times, about 0.2 times to about 0.4 times, about 0.4 times to about 5 times, about 0.4 times to about 4.5 times, about 0.4 times to about 4 times, about 0.4 times to about 3.5 times times, increase about 0.4 times to about 3 times, increase about 0.4 times to about 2.5 times, increase about 0.4 times to about 2 times, increase about 0.4 times From about 1.5 times to about 1.5 times, from about 0.4 times to about 1 times, from about 0.4 times to about 0.8 times, from about 0.4 times to about 0.6 times, from about 0.6 times to about 5 times, from about 0.6 times From about 4.5 times to about 4.5 times, from about 0.6 times to about 4 times, from about 0.6 times to about 3.5 times, from about 0.6 times to about 3 times, from about 0.6 times to about 2.5 times, from about 0.6 times From about 2 times to about 2 times, from about 0.6 times to about 1.5 times, from about 0.6 times to about 1 times, from about 0.6 times to about 0.8 times, from about 0.8 times to about 5 times, from about 0.8 times From about 4.5 times to about 4.5 times, from about 0.8 times to about 4 times, from about 0.8 times to about 3.5 times, from about 0.8 times to about 3 times, from about 0.8 times to about 2.5 times, from about 0.8 times From about 2 times to about 2 times, from about 0.8 times to about 1.5 times, from about 0.8 times to about 1 times, from about 1 times to about 5 times, from about 1 times to about 4.5 times, about 1 times 1 to 4 times, 1 to 3.5, 1 to 3, 1 to 2.5, 1 to 2, 1 From about 1.5 times to about 1.5 times, from about 1.5 times to about 5 times, from about 1.5 times to about 4.5 times, from about 1.5 times to about 4 times, from about 1.5 times to about 3.5 times, about 1.5 times From about 3 times to about 3 times, from about 1.5 times to about 2.5 times, from about 1.5 times to about 2 times, from about 2 times to about 5 times, from about 2 times to about 4.5 times, about 2 times From about 4 times to about 4 times, from about 2 times to about 3.5 times, from about 2 times to about 3 times, from about 2 times to about 2.5 times, from about 2.5 times to about 5 times, about 2.5 times From about 4.5 times to about 4.5 times, from about 2.5 times to about 4 times, from about 2.5 times to about 3.5 times, from about 2.5 times to about 3 times, from about 3 times to about 5 times, from about 3 times From about 4.5 times to about 4.5 times, from about 3 times to about 4 times, from about 3 times to about 3.5 times, from about 3.5 times to about 5 times, from about 3.5 times to about 4.5 times, about 3.5 times From about 4-fold increase, from about 4-fold increase to about 5-fold increase, from about 4-fold increase to about 4.5-fold increase, or from about 4.5-fold increase to about 5-fold increase (e.g., compared to the amount of NK cells in the individual's blood prior to administration compared to the largest multiple increase in absolute quantity).

Some embodiments of the methods described herein result in a maximum fold increase in the absolute number of CD56 ^bright lymphocytes (e.g., CD56 ^bright NK cells) in the individual's blood by at least 0.1-fold, at least 0.2-fold, at least 0.3-fold, at least 0.4-fold , increase by at least 0.5 times, increase by at least 0.6 times, increase by at least 0.7 times, increase by at least 0.8 times, increase by at least 0.9 times, increase by at least 1.0 times, increase by at least 1.5 times, increase by at least 2.0 times, increase by at least 2.5 times, increase by at least 3.0 times , an increase of at least 3.5-fold, an increase of at least 4.0-fold, an increase of at least 4.5-fold, or an increase of at least 5.0-fold (e.g., compared to the greatest fold increase in the absolute number of CD56 ^bright lymphocytes (e.g., CD56 ^bright NK cells) in the individual's blood prior to administration Compare). Some embodiments of the methods described herein result in a maximum fold increase in the absolute number of CD56 ^bright lymphocytes (e.g., CD56 ^bright NK cells) in the individual's blood, from about a 0.01-fold increase to about a 5-fold increase (or any exemplification of this range described herein range) (eg, compared to the maximum fold increase in the absolute number of CD56 ^bright lymphocytes (eg, CD56 ^bright NK cells) in the individual's blood prior to administration).

Some embodiments of the methods described herein result in a maximum fold increase in the absolute number of CD16 ⁺ CD56 ^dim lymphocytes (e.g., CD16 ⁺ CD56 ^dim NK cells) in the individual's blood, at least a 0.1-fold increase, at least a 0.2-fold increase, at least a 0.3-fold increase, Increased by at least 0.4 times, increased by at least 0.5 times, increased by at least 0.6 times, increased by at least 0.7 times, increased by at least 0.8 times, increased by at least 0.9 times, increased by at least 1.0 times, increased by at least 1.5 times, increased by at least 2.0 times, increased by at least 2.5 times, Increased by at least 3.0-fold, increased by at least 3.5-fold, increased by at least 4.0-fold, increased by at least 4.5-fold, or increased by at least 5.0-fold (e.g., compared to CD16 ⁺ CD56 ^dim lymphocytes (e.g., CD16 ⁺ CD56 ^dim NK cells) in the individual's blood prior to administration compared to the largest multiple increase in absolute quantity). Some embodiments of the methods described herein result in a maximum fold increase in the absolute number of CD16 ⁺ CD56 ^dim lymphocytes (e.g., CD16 ⁺ CD56 ^dim NK cells) in the individual's blood, from about a 0.01-fold increase to about a 5-fold increase (or such as described herein Any exemplary subrange of the range) (eg, compared to the maximum fold increase in the absolute number of CD16 ⁺ CD56 ^dim lymphocytes (eg, CD16 ⁺ CD56 ^dim NK cells) in the individual's blood prior to administration).

Some embodiments of the methods described herein result in a maximum fold increase in the absolute number of HLA-DR ⁺ lymphocytes (e.g., HLA-DR ⁺ NK cells) in the individual's blood, at least a 0.1-fold increase, at least a 0.2-fold increase, at least a 0.3-fold increase, Increased by at least 0.4 times, increased by at least 0.5 times, increased by at least 0.6 times, increased by at least 0.7 times, increased by at least 0.8 times, increased by at least 0.9 times, increased by at least 1.0 times, increased by at least 1.5 times, increased by at least 2.0 times, increased by at least 2.5 times, Increased by at least 3.0-fold, increased by at least 3.5-fold, increased by at least 4.0-fold, increased by at least 4.5-fold, or increased by at least 5.0-fold (e.g., compared to HLA-DR ⁺ lymphocytes (e.g., HLA-DR ⁺ NK cells) in the individual's blood before administration compared to the largest multiple increase in absolute quantity). Some embodiments of the methods described herein result in a maximum fold increase in the absolute number of HLA-DR ⁺ lymphocytes (e.g., HLA-DR ⁺ NK cells) in the individual's blood, from about a 0.01-fold increase to about a 5-fold increase (or such as described herein). Any exemplary subrange of the range) (eg, compared to the maximum fold increase in the absolute number of HLA-DR ⁺ lymphocytes (eg, HLA-DR ⁺ NK cells) in the individual's blood prior to administration).

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

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

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

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 in the number of MICA-positive/HLA-E-negative cancer cells in the individual, At least 7% reduction, at least 8% reduction, at least 9% reduction, at least 10% reduction, at least 15% reduction, at least 20% reduction, at least 25% reduction, at least 30% reduction, at least 35% reduction, at least 40% reduction, At least 45% reduction, at least 50% reduction, at least 55% reduction, at least 60% reduction, at least 65% reduction, at least 70% reduction, at least 75% reduction, at least 80% reduction, at least 85% reduction, at least 90% reduction, A reduction of at least 95%, or a reduction of at least 99% (eg, compared to the number of MICA-positive/HLA-E-negative cancer cells in the individual prior to administration). Some embodiments of these methods result in a reduction in the number of MICA-positive/HLA-E-negative cancer cells in an individual from about 1% to about 99% (or any exemplary subrange of this range described herein) (e.g., compared to the individual prior to administration compared with the number of MICA-positive/HLA-E-negative cancer cells).

Some embodiments of these methods result in a reduction of at least 1%, a reduction of at least 2%, a reduction of at least 3%, a reduction of at least 4%, a reduction of at least 5%, a reduction of at least 6%, a reduction of at least 7%, in the individual, At least 8% reduction, at least 9% reduction, at least 10% reduction, at least 15% reduction, at least 20% reduction, at least 25% reduction, at least 30% reduction, at least 35% reduction, at least 40% reduction, at least 45% reduction, At least 50% reduction, at least 55% reduction, at least 60% reduction, at least 65% reduction, at least 70% reduction, at least 75% reduction, at least 80% reduction, at least 85% reduction, at least 90% reduction, at least 95% reduction, Or a reduction of at least 99% (eg, compared to the proliferation of MICA-positive cancer cells in the subject prior to administration). Some embodiments of these methods result in about 1% to about 99% reduction in proliferation of MICA-positive cancer cells in the individual (or any exemplary subrange of this range described herein) (e.g., compared to MICA-positive cancer cells in the individual prior to administration growth compared to).

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 in the proliferation of MICA-positive/HLA-E-negative cancer cells in the individual, At least 7% reduction, at least 8% reduction, at least 9% reduction, at least 10% reduction, at least 15% reduction, at least 20% reduction, at least 25% reduction, at least 30% reduction, at least 35% reduction, at least 40% reduction, At least 45% reduction, at least 50% reduction, at least 55% reduction, at least 60% reduction, at least 65% reduction, at least 70% reduction, at least 75% reduction, at least 80% reduction, at least 85% reduction, at least 90% reduction, A reduction of at least 95%, or a reduction of at least 99% (eg, compared to the proliferation of MICA-positive/HLA-E-negative cancer cells in the subject prior to administration). Some embodiments of these methods result in a reduction in proliferation of MICA-positive/HLA-E-negative cancer cells in an individual from about 1% to about 99% (or any exemplary subrange of this range described herein) (e.g., compared to pre-administration to the individual Compared with the proliferation of MICA-positive and HLA-E-negative cancer cells).

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

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

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

Some embodiments of these methods result in a reduction in the number of MICB positive cancer cells in the individual by at least 1%, by at least 2%, by at least 3%, by at least 4%, by at least 5%, by at least 6%, by at least 7%, At least 8% reduction, at least 9% reduction, at least 10% reduction, at least 15% reduction, at least 20% reduction, at least 25% reduction, at least 30% reduction, at least 35% reduction, at least 40% reduction, at least 45% reduction, At least 50% reduction, at least 55% reduction, at least 60% reduction, at least 65% reduction, at least 70% reduction, at least 75% reduction, at least 80% reduction, at least 85% reduction, at least 90% reduction, at least 95% reduction, Or a reduction of at least 99% (eg, compared to the number of MICB-positive cancer cells in the individual prior to administration). Some embodiments of these methods result in a reduction in the number of MICB-positive cancer cells in the individual from about 1% to about 99% (or any exemplary subrange of this range described herein) (e.g., compared to MICB-positive cancer cells in the individual prior to administration compared to the number).

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 in the number of MICB positive/HLA-E negative cancer cells in the individual, At least 7% reduction, at least 8% reduction, at least 9% reduction, at least 10% reduction, at least 15% reduction, at least 20% reduction, at least 25% reduction, at least 30% reduction, at least 35% reduction, at least 40% reduction, At least 45% reduction, at least 50% reduction, at least 55% reduction, at least 60% reduction, at least 65% reduction, at least 70% reduction, at least 75% reduction, at least 80% reduction, at least 85% reduction, at least 90% reduction, A reduction of at least 95%, or a reduction of at least 99% (eg, compared to the number of MICB-positive/HLA-E-negative cancer cells in the individual prior to administration). Some embodiments of these methods result in a reduction in the number of MICB-positive/HLA-E-negative cancer cells in an individual from about 1% to about 99% (or any exemplary subrange of this range described herein) (e.g., compared to the individual prior to administration compared with the number of MICB-positive/HLA-E-negative cancer cells).

Some embodiments of these methods result in a reduction of at least 1%, a reduction of at least 2%, a reduction of at least 3%, a reduction of at least 4%, a reduction of at least 5%, a reduction of at least 6%, a reduction of at least 7%, in the individual, At least 8% reduction, at least 9% reduction, at least 10% reduction, at least 15% reduction, at least 20% reduction, at least 25% reduction, at least 30% reduction, at least 35% reduction, at least 40% reduction, at least 45% reduction, At least 50% reduction, at least 55% reduction, at least 60% reduction, at least 65% reduction, at least 70% reduction, at least 75% reduction, at least 80% reduction, at least 85% reduction, at least 90% reduction, at least 95% reduction, Or a reduction of at least 99% (eg, compared to proliferation of MICB-positive cancer cells in an individual prior to administration). Some embodiments of these methods result in a reduction in the proliferation of MICB-positive cancer cells in the individual by about 1% to about 99% (or any exemplary subrange of this range described herein) (e.g., compared to MICB-positive cancer cells in the individual prior to administration growth compared to).

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 in the proliferation of MICB positive/HLA-E negative cancer cells in the individual, At least 7% reduction, at least 8% reduction, at least 9% reduction, at least 10% reduction, at least 15% reduction, at least 20% reduction, at least 25% reduction, at least 30% reduction, at least 35% reduction, at least 40% reduction, At least 45% reduction, at least 50% reduction, at least 55% reduction, at least 60% reduction, at least 65% reduction, at least 70% reduction, at least 75% reduction, at least 80% reduction, at least 85% reduction, at least 90% reduction, A reduction of at least 95%, or a reduction of at least 99% (eg, compared to proliferation of MICB-positive/HLA-E-negative cancer cells in an individual prior to administration). Some embodiments of these methods result in a reduction in proliferation of MICB-positive/HLA-E-negative cancer cells in an individual from about 1% to about 99% (or any exemplary subrange of this range described herein) (e.g., compared to pre-administration to the individual Compared with the proliferation of MICB-positive/HLA-E-negative cancer cells).

Provided herein are methods of inducing killing of MICB-positive cancer cells in an individual previously identified or diagnosed as having MICB-positive cancer comprising administering to the individual a therapeutically effective amount of a pharmaceutical composition comprising an enucleated erythroid population , the enucleated erythroid cells comprise a first exogenous fusion polypeptide on their extracellular surface, the first exogenous fusion polypeptide comprising (i) IL-15 or a functional fragment thereof, and (ii) an IL-15 receptor body α or its functional fragments. Also provided herein is a method of killing MICB-positive cancer cells in an individual previously identified or diagnosed with MICB-positive cancer comprising administering to the individual a therapeutically effective amount of a pharmaceutical composition comprising an enucleated erythroid population , the enucleated erythroid cells comprise a first exogenous fusion polypeptide on their extracellular surface, the first exogenous fusion polypeptide comprising (i) IL-15 or a functional fragment thereof, and (ii) an IL-15 receptor body α or a functional fragment thereof; and a second exogenous polypeptide on the extracellular surface thereof, the second exogenous polypeptide includes 4-1BBL or a functional fragment thereof.

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

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

Some embodiments of these methods result in a reduction in the number of MICA/MICB positive cancer cells in an individual by at least 1%, by at least 2%, by at least 3%, by at least 4%, by at least 5%, by at least 6%, by at least 7% %, at least 8% reduction, at least 9% reduction, at least 10% reduction, at least 15% reduction, at least 20% reduction, at least 25% reduction, at least 30% reduction, at least 35% reduction, at least 40% reduction, at least 45% reduction %, at least 50% reduction, at least 55% reduction, at least 60% reduction, at least 65% reduction, at least 70% reduction, at least 75% reduction, at least 80% reduction, at least 85% reduction, at least 90% reduction, at least 95% reduction %, or at least a 99% reduction (eg, compared to the number of MICA/MICB positive cancer cells in the individual prior to administration). Some embodiments of these methods result in about a 1% to about 99% reduction in the number of MICA/MICB positive cancer cells in an individual (or any exemplary subrange of this range described herein) (e.g., compared to MICA/MICB in an individual prior to administration). compared with the number of MICB-positive cancer cells).

Some embodiments of these methods result in a reduction in the number of MICA/MICB positive/HLA-E negative cancer cells in the individual by at least 1%, by at least 2%, by at least 3%, by at least 4%, by at least 5%, by at least 6 %, at least 7% reduction, at least 8% reduction, at least 9% reduction, at least 10% reduction, at least 15% reduction, at least 20% reduction, at least 25% reduction, at least 30% reduction, at least 35% reduction, at least 40% reduction %, at least 45% reduction, at least 50% reduction, at least 55% reduction, at least 60% reduction, at least 65% reduction, at least 70% reduction, at least 75% reduction, at least 80% reduction, at least 85% reduction, at least 90% reduction %, a reduction of at least 95%, or a reduction of at least 99% (eg, compared to the number of MICA/MICB positive/HLA-E negative cancer cells in an individual prior to administration). Some embodiments of these methods result in a reduction in the number of MICA/MICB positive/HLA-E negative cancer cells in an individual from about 1% to about 99% (or any exemplary subrange of this range described herein) (e.g., in combination with administering compared to the number of MICA/MICB-positive/HLA-E-negative cancer cells in previous individuals).

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

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 in the proliferation of MICA/MICB positive/HLA-E negative cancer cells in the individual %, at least 7% reduction, at least 8% reduction, at least 9% reduction, at least 10% reduction, at least 15% reduction, at least 20% reduction, at least 25% reduction, at least 30% reduction, at least 35% reduction, at least 40% reduction %, at least 45% reduction, at least 50% reduction, at least 55% reduction, at least 60% reduction, at least 65% reduction, at least 70% reduction, at least 75% reduction, at least 80% reduction, at least 85% reduction, at least 90% reduction %, a reduction of at least 95%, or a reduction of at least 99% (eg, compared to the proliferation of MICA/MICB positive/HLA-E negative cancer cells in an individual prior to administration). Some embodiments of these methods result in a reduction in the proliferation of MICA/MICB positive/HLA-E negative cancer cells in an individual from about 1% to about 99% (or any exemplary subrange of this range described herein) (e.g., in combination with administration of hyperplasia of MICA/MICB-positive/HLA-E-negative cancer cells in former individuals).

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

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

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 an 8% reduction in the solid tumor volume in the individual %, at least 9% reduction, at least 10% reduction, at least 15% reduction, at least 20% reduction, at least 25% reduction, at least 30% reduction, at least 35% reduction, at least 40% reduction, at least 45% reduction, at least 50% reduction %, at least 55% reduction, at least 60% reduction, at least 65% reduction, at least 70% reduction, at least 75% reduction, at least 80% reduction, at least 85% reduction, at least 90% reduction, at least 95% reduction, or at least 99% (eg, compared to pre-administration solid tumor volume).

Some embodiments of these methods result in a reduction in solid tumor volume in the individual of about 1% to about 99% (or any exemplary subrange of this range described herein) (eg, compared to the volume of the solid tumor prior to administration).

Also provided herein are methods of increasing the ratio of NKG2D concentration to NKG2A concentration on the surface of lymphocytes in an individual previously identified or diagnosed as having a MICA-positive, MICB-positive, or MICA/MICB-positive cancer, comprising administering to the individual a therapeutically effective A quantity of a pharmaceutical composition comprising a population of enucleated erythrocytes comprising on their extracellular surface a first exogenous fusion polypeptide comprising (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof. Also provided herein are methods for increasing the concentration of NKG2D-positive lymphocytes (e.g., NKG2D-positive NK cells) in association with NKG2A-positive lymphocytes (e.g., NKG2A-positive NK cells) in individuals previously identified or diagnosed as having MICA-positive, MICB-positive, or MICA/MICB-positive cancers. cells) comprising administering to an individual a pharmaceutical composition comprising a population of enucleated erythroid cells comprising a first exogenous fusion polypeptide on their extracellular surface, the The first exogenous fusion polypeptide comprises (i) IL-15 or its functional fragment, and (ii) IL-15 receptor α or its functional fragment; and the second exogenous polypeptide on its extracellular surface, the The second exogenous polypeptide includes 4-1BBL or a functional fragment thereof.

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 in the ratio of NKG2D concentration to NKG2A concentration on the cell surface of lymphocytes (e.g., NK cells) in an individual , increase by at least 0.6 times, increase by at least 0.7 times, increase by at least 0.8 times, increase by at least 0.9 times, increase by at least 1.0 times, increase by at least 1.5 times, increase by at least 2.0 times, increase by at least 2.5 times, increase by at least 3.0 times, increase by at least 3.5 times , increase by at least 4.0 times, increase by at least 4.5 times, increase by at least 5.0 times, increase by at least 5.5 times, increase by at least 6.0 times, increase by at least 6.5 times, increase by at least 7.0 times, increase by at least 7.5 times, increase by at least 8.0 times, increase by at least 8.5 times , an increase of at least 9.0-fold, an increase of at least 9.5-fold, or an increase of at least 10-fold (eg, compared to the ratio of the concentration of NKG2D to the concentration of NKG2A on the cell surface of lymphocytes (eg, NK cells) in an individual prior to administration). Some embodiments of these methods result in an about 0.01-fold to about 10-fold increase (or any exemplary subrange of this range described herein) in the ratio of NKG2D concentration to NKG2A concentration on the cell surface of lymphocytes (e.g., NK cells) in an individual (e.g. , compared with the ratio of NKG2D concentration to NKG2A concentration on the cell surface of lymphocytes (eg, NK cells) in the individual before administration).

Some embodiments of the methods described herein result in a maximum fold change increase of at least 0.1-fold, an increase of at least 0.2-fold 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 individual's blood , increased by at least 0.3 times, increased by at least 0.4 times, increased by at least 0.5 times, increased by at least 0.6 times, increased by at least 0.7 times, increased by at least 0.8 times, increased by at least 0.9 times, increased by at least 1.0 times, increased by at least 1.5 times, increased by at least 2.0 times , increase by at least 2.5 times, increase by at least 3.0 times, increase by at least 3.5 times, increase by at least 4.0 times, increase by at least 4.5 times, increase by at least 5.0 times, increase by at least 5.5 times, increase by at least 6.0 times, increase by at least 6.5 times, increase by at least 7.0 times , increased by at least 7.5-fold, increased by at least 8.0-fold, increased by at least 8.5-fold, increased by at least 9.0-fold, increased by at least 9.5-fold, or increased by at least 10-fold (for example, compared with NKG2D-positive lymphocytes in the individual's blood before administration (such as NKG2D-positive NK cells) compared to the ratio of NKG2A-positive lymphocytes (eg, NKG2A-positive NK cells)). Some embodiments of the methods described herein result in a maximum fold change of about 0.01-fold to about a 10-fold increase 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 individual's blood (or any exemplary subrange of this range described herein) (e.g., 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 individual's blood prior to administration ).

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

Some embodiments of the methods described herein result in a maximum fold increase in the absolute number of NK cells in the individual's blood, 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 times, increase by at least 0.7 times, increase by at least 0.8 times, increase by at least 0.9 times, increase by at least 1.0 times, increase by at least 1.5 times, increase by at least 2.0 times, increase by at least 2.5 times, increase by at least 3.0 times, increase by at least 3.5 times, increase by at least 4.0 times fold, increased by at least 4.5 fold, or increased by at least 5.0 fold (eg, compared to the maximum fold increase in absolute number of NK cells in the individual's blood prior to administration). Some embodiments of the methods described herein result in a maximum fold increase in the absolute number of NK cells in the individual's blood, from about a 0.01-fold increase to about a 5-fold increase (or any exemplary subrange of this range described herein) (e.g., compared to administering largest fold increase in absolute number of NK cells in the blood of the former individual).

Some embodiments of the methods described herein result in a maximum fold increase in the absolute number of CD56 ^bright lymphocytes (e.g., CD56 ^bright NK cells) in the individual's blood by at least 0.1-fold, at least 0.2-fold, at least 0.3-fold, at least 0.4-fold , increase by at least 0.5 times, increase by at least 0.6 times, increase by at least 0.7 times, increase by at least 0.8 times, increase by at least 0.9 times, increase by at least 1.0 times, increase by at least 1.5 times, increase by at least 2.0 times, increase by at least 2.5 times, increase by at least 3.0 times , an increase of at least 3.5-fold, an increase of at least 4.0-fold, an increase of at least 4.5-fold, or an increase of at least 5.0-fold (e.g., compared to the greatest fold increase in the absolute number of CD56 ^bright lymphocytes (e.g., CD56 ^bright NK cells) in the individual's blood prior to administration Compare). Some embodiments of the methods described herein result in a maximum fold increase in the absolute number of CD56 ^bright lymphocytes (e.g., CD56 ^bright NK cells) in the individual's blood, from about a 0.01-fold increase to about a 5-fold increase (or any exemplification of this range described herein range) (eg, compared to the maximum fold increase in the absolute number of CD56 ^bright lymphocytes (eg, CD56 ^bright NK cells) in the individual's blood prior to administration).

Some embodiments of the methods described herein result in a maximum fold increase in the absolute number of CD16 ⁺ CD56 ^dim lymphocytes (e.g., CD16 ⁺ CD56 ^dim NK cells) in the individual's blood, at least a 0.1-fold increase, at least a 0.2-fold increase, at least a 0.3-fold increase, Increased by at least 0.4 times, increased by at least 0.5 times, increased by at least 0.6 times, increased by at least 0.7 times, increased by at least 0.8 times, increased by at least 0.9 times, increased by at least 1.0 times, increased by at least 1.5 times, increased by at least 2.0 times, increased by at least 2.5 times, Increased by at least 3.0-fold, increased by at least 3.5-fold, increased by at least 4.0-fold, increased by at least 4.5-fold, or increased by at least 5.0-fold (e.g., compared to CD16 ⁺ CD56 ^dim lymphocytes (e.g., CD16 ⁺ CD56 ^dim NK cells) in the individual's blood prior to administration compared to the largest multiple increase in absolute quantity). Some embodiments of the methods described herein result in a maximum fold increase in the absolute number of CD16 ⁺ CD56 ^dim lymphocytes (e.g., CD16 ⁺ CD56 ^dim NK cells) in the individual's blood, from about a 0.01-fold increase to about a 5-fold increase (or such as described herein Any exemplary subrange of the range) (eg, compared to the maximum fold increase in the absolute number of CD16 ⁺ CD56 ^dim lymphocytes (eg, CD16 ⁺ CD56 ^dim NK cells) in the individual's blood prior to administration).

Some embodiments of the methods described herein result in a maximum fold increase in the absolute number of HLA-DR ⁺ lymphocytes (e.g., HLA-DR ⁺ NK cells) in the individual's blood, at least a 0.1-fold increase, at least a 0.2-fold increase, at least a 0.3-fold increase, Increased by at least 0.4 times, increased by at least 0.5 times, increased by at least 0.6 times, increased by at least 0.7 times, increased by at least 0.8 times, increased by at least 0.9 times, increased by at least 1.0 times, increased by at least 1.5 times, increased by at least 2.0 times, increased by at least 2.5 times, Increased by at least 3.0-fold, increased by at least 3.5-fold, increased by at least 4.0-fold, increased by at least 4.5-fold, or increased by at least 5.0-fold (e.g., compared to HLA-DR ⁺ lymphocytes (e.g., HLA-DR ⁺ NK cells) in the individual's blood prior to administration compared to the largest multiple increase in absolute quantity). Some embodiments of the methods described herein result in a maximum fold increase in the absolute number of HLA-DR ⁺ lymphocytes (e.g., HLA-DR ⁺ NK cells) in the individual's blood, from about a 0.01-fold increase to about a 5-fold increase (or such as described herein). Any exemplary subrange of the range) (e.g., compared to the maximum fold increase in the absolute number of HLA-DR ⁺ lymphocytes (eg, HLA-DR ⁺ NK cells) in the individual's blood prior to administration).

Non-limiting aspects of these compositions and methods are described below. The exemplary aspects listed below can be used in any combination and combined with other aspects known in the art, as will be appreciated by those of ordinary skill in the art. NK2D

As used herein, NKG2D polypeptide refers to the amino acid sequence encoded by the killer lectin-like receptor K1 (KLRK1 ) gene. The KLRK1 gene is also known as CD314, NKG2D, and NKG2-D. Exemplary NKG2D polypeptides include, but are not limited to, the amino acid sequence corresponding to NCBI Reference Sequence: NP_031386.2.

In some embodiments, the NKG2D polypeptide comprises a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) % consistent) amino acid sequence: MGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCRENASPFFFCCFIAVAMGIRFIIMVTIWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV (SEQ ID NO：1) (具有或不具有訊號序列)。

In some embodiments, the NKG2D polypeptide consists of a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to a sequence % identical) encoded by the nucleic acid sequence: ATGGGGTGGATTCGTGGTCGGAGGTCTCGACACAGCTGGGAGATGAGTGAATTTCATAATTATAACTTGGATCTGAAGAAGAGTGATTTTTCAACACGATGGCAAAAGCAAAGATGTCCAGTAGTCAAAAGCAAATGTAGAGAAAATGCATCTCCATTTTTTTTCTGCTGCTTCATCGCTGTAGCCATGGGAATCCGTTTCATTATTATGGTAACAATATGGAGTGCTGTATTCCTAAACTCATTATTCAACCAAGAAGTTCAAATTCCCTTGACCGAAAGTTACTGTGGCCCATGTCCTAAAAACTGGATATGTTACAAAAATAACTGCTACCAATTTTTTGATGAGAGTAAAAACTGGTATGAGAGCCAGGCTTCTTGTATGTCTCAAAATGCCAGCCTTCTGAAAGTATACAGCAAAGAGGACCAGGATTTACTTAAACTGGTGAAGTCATATCATTGGATGGGACTAGTACACATTCCAACAAATGGATCTTGGCAGTGGGAAGATGGCTCCATTCTCTCACCCAACCTACTAACAATAATTGAAATGCAGAAGGGAGACTGTGCACTCTATGCCTCGAGCTTTAAAGGCTATATAGAAAACTGTTCAACTCCAAATACGTACATCTGCATGCAAAGGACTGTGTAA (SEQ ID NO：2)。

In some embodiments, the methods comprise detecting the number of NKG2D-positive lymphocytes (eg, NKG2D-positive NK cells) in the individual. Any suitable sample containing NKG2D-positive lymphocytes (eg, NKG2D-positive NK cells) can be obtained from an individual. In some embodiments, whole blood samples, plasma samples, serum samples, samples containing cell fractions (PBMCs) of whole blood samples, tissue samples, tumor samples, sliced samples, and laser capture dissected biological samples can be obtained and evaluated. samples (eg, tumor or tissue samples) to determine the number of NKG2D-positive lymphocytes (eg, NKG2D-positive NK cells). In some embodiments, the number of NKG2D-positive lymphocytes (e.g., NKG2D-positive NK cells) is detected before, simultaneously with, or after administration of a therapeutically effective amount of a pharmaceutical composition comprising an enucleated erythroid population (e.g., herein any of the exemplary enucleated erythroid cells described). In some embodiments, NKG2D can be assayed using any antibody or antigen-binding fragment thereof that specifically binds to NKG2D (e.g., anti-NKG2D antibody strain 149810 (R&D Systems) and anti-NKG2D mAb strain 1D11 (BD Biosciences)) Positive cells (eg NKG2D positive NK cells). 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 by other immunological-based methods, including Without limitation, Western blot analysis, immunohistochemical staining, ELISA, tissue array analysis, in situ hybridization, and immunofluorescence, using, for example, antibodies or antigen-binding fragments thereof that specifically bind to NKG2D. In some embodiments, NKG2D positive lymphocytes (eg, NKG2D positive NK cells) can be determined by measuring NKG2D mRNA. Non-limiting methods for quantifying RNA include: qRT-PCR, RNA sequencing, fluorescence in situ hybridization combined with flow cytometry, microfluidic capillary electrophoresis, and in situ hybridization.

As described herein, by comparing the NKG2D content in lymphocytes (e.g., NK cells) with reference levels (e.g., NKG2D content in control non-activated or resting lymphocytes (e.g., NK cells), or not in lymphocytes (e.g., not NKG2D content in cells) to determine NKG2D-positive lymphocytes (such as NKG2D-positive NK cells). In some embodiments, the reference level of NKG2D can be one transcript per million transcripts. In some embodiments, NKG2D-positive lymphocytes (eg, NKG2D-positive NK cells) are identified based on a flow cytometry banding strategy using fluorescence minus a control sample, fluorescence compensation, and quantitative experience. MICA

As used herein, a MICA polypeptide refers to the amino acid sequence encoded by the MHC class I polypeptide-associated sequence A (MICA) gene. Exemplary MICA polypeptides include, but are not limited to, the amino acid sequences corresponding to NCBI reference sequences: NP_000238.1, NP_001170990.1, NP_001276081.1, and NP_001276083.1. MICA, a member of major histocompatibility complex class I, is the cell surface ligand of NKG2D.

In some embodiments, a MICA polypeptide may comprise a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100%一致)的序列： MGLGPVFLLLAGIFPFAPPGAAA EPHSLRYNLTVLSWDGSVQSGFLTEVHLDGQPFLRCDRQKCRAKPQGQWAEDVLGNKTWDRETRDLTGNGKDLRMTLAHIKDQKEGLHSLQEIRVCEIHEDNSTRSSQHFYYDGELFLSQNLETKEWTMPQSSRAQTLAMNVRNFLKEDAMKTKTHYHAMHADCLQELRRYLKSGVVLRRTVPPMVNVTRSEASEGNITVTCRASGFYPWNITLSWRQDGVSLSHDTQQWGDVLPDGNGTYQTWVATRICQGEEQRFTCYMEHSGNHSTHPVPSGKVLVLQSHWQTFHVSAVAAAAIFVIIIFYVRCCKKKTSAAEGPELVSLQVLDQHPVGTSDHRDATQLGFQPLMSDLGSTGSTEGA (SEQ ID NO：3) (具有或不具有訊號序列)。 The underlined part of SEQ ID NO: 3 above represents the signal peptide.

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

In some embodiments, a MICA polypeptide may comprise a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100%一致)的序列： MGLGPVFLLLAGIFPFAPPGAAA EPHSLRYNLTVLSWDGSVQSGFLAEVHLDGQPFLRYDRQKCRAKPQGQWAEDVLGNKTWDRETRDLTGNGKDLRMTLAHIKDQKEGLHSLQEIRVCEIHEDNSTRSSQHFYYDGELFLSQNLETEEWTVPQSSRAQTLAMNVRNFLKEDAMKTKTHYHAMHADCLQELRRYLESGVVLRRTVPPMVNVTRSEASEGNITVTCRASSFYPRNIILTWRQDGVSLSHDTQQWGDVLPDGNGTYQTWVATRICRGEEQRFTCYMEHSGNHSTHPVPSGKVLVLQSHWQTFHVSAVAAGCCYFCYYYFLCPLL (SEQ ID NO：5) (具有或不具有訊號序列)。 The underlined portion of SEQ ID NO: 5 above represents the signal peptide.

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

In some embodiments, a MICA polypeptide may comprise a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% consistent) sequence: MTLAHIKDQKEGLHSLQEIRVCEIHEDNSTRSSQHFYYDGELFLSQNLETEEWTVPQSSRAQTLAMNVRNFLKEDAMKTKTHYHAMHADCLQELRRYLESGVVLRRTVPPMVNVTRSEASEGNITVTCRASSFYPRNIILTWRQDGVSLSHDTQQWGDVLPDGNGTYQTWVATRICRGEEQRFTCYMEHSGNHSTHPVPSGKVLVLQSHWQTFHVSAVAAGCCYFCYYYFLCPLL (SEQ ID NO：7) (具有或不具有訊號序列)。

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

In some embodiments, a MICA polypeptide may comprise a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% consistent) sequence: MGQRDQGLDRERKGPQDDPGSYQGPERRNFLKEDAMKTKTHYHAMHADCLQELRRYLESGVVLRRTVPPMVNVTRSEASEGNITVTCRASSFYPRNIILTWRQDGVSLSHDTQQWGDVLPDGNGTYQTWVATRICRGEEQRFTCYMEHSGNHSTHPVPSGKVLVLQSHWQTFHVSAVAAGCCYFCYYYFLCPLL  (SEQ ID NO：9) (具有或不具有訊號肽)。

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

In some embodiments, the methods comprise detecting the presence of MICA-positive cancer cells in the individual. Any suitable sample containing MICA-positive cancer cells can be obtained from an individual. In some embodiments, whole blood samples, plasma samples, serum samples, samples containing cell fractions (PBMC) of whole blood samples, tissue samples, tumor samples, sliced samples, and laser capture dissected biological samples can be obtained and evaluated. samples (eg, tumor or tissue samples) to determine the number of MICA-positive cancer cells. In some embodiments, MICA-positive cancer cells are detected before, concurrently with, or after administration of enucleated erythroid cells. In some embodiments, antibodies or antigen-binding fragments thereof that specifically bind to MICA (e.g., on the cell surface of cancer cells) (e.g., anti-MICA antibodies ab62540 (Abcam), LS-B9176 (LSBio), and 1E2C8 (ThermoFisher Scientific)) to detect MICA-positive cancer cells. MICA polypeptide expression can be assessed using fluorescence-assisted cell sorting (FACS) or by other immunological-based methods including, but not limited to, western blotting, immunohistochemical staining, ELISA, and immunological fluorescent. In some embodiments, MICA positive cells can be determined by measuring MICA mRNA. Non-limiting methods for quantifying RNA include: qRT-PCR, RNA sequencing, fluorescence in situ hybridization combined with flow cytometry, microfluidic capillary electrophoresis, and in situ hybridization.

Exemplary MICA reference levels that can be used to identify MICA-positive cancer cells or MICA-positive cancers can be the level of MICA in non-cancerous cells, the median level of MICA in multiple tumor section samples, the level of MICA that is higher than that of the same type of cancer, or the level of multiple different cancers. One standard deviation of the median content of MICA in tumor slice samples, one-quarter standard deviation higher than the median content of MICA in multiple tumor slice samples of the same type of cancer or multiple different cancers, higher than the same type One-half standard deviation of the median content of MICA in multiple tumor slice samples of cancer or multiple different cancers, higher than the median MICA content in multiple tumor slice samples of the same type of cancer or multiple different cancers Three-quarters standard deviations higher, one and one-half standard deviations higher than the median MICA content in multiple tumor section samples of the same type of cancer or multiple different cancers, or higher than the same type of cancer or multiple Two standard deviations of the median MICA content in multiple tumor section samples from different cancers. In some embodiments, the reference level of MICA may be one transcript per million transcripts.

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

As used herein, MICB polypeptide refers to the amino acid sequence encoded by the MHC class I polypeptide-related sequence B (MICB) gene. Exemplary MICB polypeptides include, but are not limited to, the amino acid sequences corresponding to NCBI reference sequences: NP_001276089.1, NP_001276090.1, and NP_005922.2. MICB, a member of major histocompatibility complex class I, is the cell surface ligand of NKG2D.

In some embodiments, the MICB polypeptide may comprise a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% consistent) sequence: MVLSQDGSVQSGFLAEGHLDGQPFLRYDRQKRRAKPQGQWAENVLGAKTWDTETEDLTENGQDLRRTLTHIKDQKGGLHSLQEIRVCEIHEDSSTRGSRHFYYDGELFLSQNLETQESTVPQSSRAQTLAMNVTNFWKEDAMKTKTHYRAMQADCLQKLQRYLKSGVAIRRTVPPMVNVTCSEVSEGNITVTCRASSFYPRNITLTWRQDGVSLSHNTQQWGDVLPDGNGTYQTWVATRIRQGEEQRFTCYMEHSGNHGTHPVPSGKALVLQSQRTDFPYVSAAMPCFVIIIILCVPCCKKKTSAAEGPELVSLQVLDQHPVGTGDHRDAAQLGFQPLMSATGSTGSTEGT (SEQ ID NO：11) (具有或不具有訊號肽)。

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

In some embodiments, the MICB polypeptide may comprise a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100%一致)的序列： MGLGRVLLFLAVAFPFAPPAAA AEPHSLRYNLMVLSQDGSVQSGFLAEGHLDGQPFLRYDRQKRRAKPQGQWAENVLGAKTWDTETEDLTENGQDLRRTLTHIKDQKGVPQSSRAQTLAMNVTNFWKEDAMKTKTHYRAMQADCLQKLQRYLKSGVAIRRTVPPMVNVTCSEVSEGNITVTCRASSFYPRNITLTWRQDGVSLSHNTQQWGDVLPDGNGTYQTWVATRIRQGEEQRFTCYMEHSGNHGTHPVPSGKALVLQSQRTDFPYVSAAMPCFVIIIILCVPCCKKKTSAAEGPELVSLQVLDQHPVGTGDHRDAAQLGFQPLMSATGSTGSTEGT (SEQ ID NO：13) (具有或不具有訊號肽)。 The underlined portion of SEQ ID NO: 13 above represents the signal peptide.

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

In some embodiments, the MICB polypeptide may comprise a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100%一致)的序列： MGLGRVLLFLAVAFPFAPPAAA AEPHSLRYNLMVLSQDGSVQSGFLAEGHLDGQPFLRYDRQKRRAKPQGQWAENVLGAKTWDTETEDLTENGQDLRRTLTHIKDQKGGLHSLQEIRVCEIHEDSSTRGSRHFYYDGELFLSQNLETQESTVPQSSRAQTLAMNVTNFWKEDAMKTKTHYRAMQADCLQKLQRYLKSGVAIRRTVPPMVNVTCSEVSEGNITVTCRASSFYPRNITLTWRQDGVSLSHNTQQWGDVLPDGNGTYQTWVATRIRQGEEQRFTCYMEHSGNHGTHPVPSGKALVLQSQRTDFPYVSAAMPCFVIIIILCVPCCKKKTSAAEGPELVSLQVLDQHPVGTGDHRDAAQLGFQPLMSATGSTGSTEGT (SEQ ID NO：15) (具有或不具有訊號肽)。 The underlined portion of SEQ ID NO: 15 above represents the signal peptide.

In some embodiments, the MICB polypeptide consists of a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to a sequence % identical) encoded by the nucleic acid sequence: ATGGGGCTGGGCCGGGTCCTGCTGTTTCTGGCCGTCGCCTTCCCTTTTGCACCCCCGGCAGCCGCCGCTGAGCCCCACAGTCTTCGTTACAACCTCATGGTGCTGTCCCAGGATGGATCTGTGCAGTCAGGGTTTCTCGCTGAGGGACATCTGGATGGTCAGCCCTTCCTGCGCTATGACAGGCAGAAACGCAGGGCAAAGCCCCAGGGACAGTGGGCAGAAAATGTCCTGGGAGCTAAGACCTGGGACACAGAGACCGAGGACTTGACAGAGAATGGGCAAGACCTCAGGAGGACCCTGACTCATATCAAGGACCAGAAAGGAGGCTTGCATTCCCTCCAGGAGATTAGGGTCTGTGAGATCCATGAAGACAGCAGCACCAGGGGCTCCCGGCATTTCTACTACGATGGGGAGCTCTTCCTCTCCCAAAACCTGGAGACTCAAGAATCGACAGTGCCCCAGTCCTCCAGAGCTCAGACCTTGGCTATGAACGTCACAAATTTCTGGAAGGAAGATGCCATGAAGACCAAGACACACTATCGCGCTATGCAGGCAGACTGCCTGCAGAAACTACAGCGATATCTGAAATCCGGGGTGGCCATCAGGAGAACAGTGCCCCCCATGGTGAATGTCACCTGCAGCGAGGTCTCAGAGGGCAACATCACCGTGACATGCAGGGCTTCCAGCTTCTATCCCCGGAATATCACACTGACCTGGCGTCAGGATGGGGTATCTTTGAGCCACAACACCCAGCAGTGGGGGGATGTCCTGCCTGATGGGAATGGAACCTACCAGACCTGGGTGGCCACCAGGATTCGCCAAGGAGAGGAGCAGAGGTTCACCTGCTACATGGAACACAGCGGGAATCACGGCACTCACCCTGTGCCCTCTGGGAAGGCGCTGGTGCTTCAGAGTCAACGGACAGACTTTCCATATGTTTCTGCTGCTATGCCATGTTTTGTTATTATTATTATTCTCTGTGTCCCTTGTTGCAAGAAGA AAACATCAGCGGCAGAGGGTCCAGAGCTTGTGAGCCTGCAGGTCCTGGATCAACACCCAGTTGGGACAGGAGACCACAGGGATGCAGCACAGCTGGGATTTCAGCCTCTGATGTCAGCTACTGGGTCCACTGGTTCCACTGAGGGCACCTAG (SEQ ID NO: 16).

In some embodiments, the methods comprise detecting the presence of MICB-positive cancer cells in the individual. Any suitable sample containing MICB-positive cancer cells can be obtained from an individual. In some embodiments, whole blood samples, plasma samples, serum samples, samples containing cell fractions (PBMC) of whole blood samples, tissue samples, tumor samples, sliced samples, and laser capture dissected biological samples can be obtained and evaluated. samples (eg, tumor or tissue samples) to determine the number of MICB-positive cancer cells. In some embodiments, MICB-positive cancer cells are detected prior to, concurrently with, or after administration of enucleated erythroid cells. In some embodiments, antibodies or antigen-binding fragments thereof that specifically bind to MICB (e.g., on the cell surface of cancer cells) can be used (e.g., anti-MICB antibody MM0473-3C37 (Abcam), anti-MICB antibody 3C37 (Abcam) , with anti-MICB antibody ARG56879 (Arigo Biolaboratories)) to detect MICB-positive cancer cells. MICB polypeptide expression can be assessed using fluorescence-assisted cell sorting (FACS) or by other immunological-based methods including, but not limited to, western blotting, immunohistochemical staining, ELISA, and immunoassay fluorescent. In some embodiments, MICB positive cells can be determined by measuring MICB mRNA. Non-limiting methods for quantifying RNA include: qRT-PCR, RNA sequencing, fluorescence in situ hybridization combined with flow cytometry, microfluidic capillary electrophoresis, and in situ hybridization.

Exemplary MICB reference levels that can be used to identify MICB-positive cancer cells or MICB-positive cancers can be MICB levels in non-cancerous cells, median MICB levels in multiple tumor section samples, high levels in the same type of cancer, or multiples in multiple different cancers. One standard deviation of the median content of MICB in tumor slice samples, one-quarter standard deviation higher than the median content of MICB in multiple tumor slice samples of the same type of cancer or multiple different cancers, higher than the same type One-half standard deviation of the median content of MICB in multiple tumor section samples of cancer or multiple different cancers, higher than the median content of MICB in multiple tumor section samples of the same type of cancer or multiple different cancers Three-quarters standard deviations higher, one and one-half standard deviations higher than the median MICB content in multiple tumor section samples from the same type of cancer or multiple different cancers, or higher than the same type of cancer or multiple Two standard deviations of the median MICB content in multiple tumor section samples from different cancers. In some embodiments, the reference level of MICB may be one transcript per million transcripts.

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

As used herein, NKG2A polypeptide refers to the amino acid sequence encoded by the killer lectin-like receptor C1 (KLRC1 ) gene. NKG2A forms a complex with KLRD1/CD94. Exemplary KLRCl polypeptides include, but are not limited to, the amino acid sequences corresponding to NCBI reference sequences: NP_001291377.1, NP_002250.2, and NP_998823.1.

In some embodiments, the NKG2A polypeptide can comprise a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% consistent) sequence: MDNQGVIYSDLNLPPNPKRQQRKPKGNKNSILATEQEITYAELNLQKASQDFQGNDKTYHCKDLPSAPEKLIVGILGIICLILMASVVTIVVIPSTLIQRHNNSSLNTRTQKARHCGHCPEEWITYSNSCYYIGKERRTWEESLLACTSKNSSLLSIDNEEEMKFLSIISPSSWIGVFRNSSHHPWVTMNGLAFKHEIKDSDNAELNCAVLQVNRLKSAQCGSSIIYHCKHKL (SEQ ID NO：17)。

In some embodiments, the NKG2A polypeptide consists of a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to a sequence % identical) encoded by the nucleic acid sequence: ATGGATAACCAAGGAGTAATCTACTCAGACCTGAATCTGCCCCCAAACCCAAAGAGGCAGCAACGAAAACCTAAAGGCAATAAAAACTCCATTTTAGCAACTGAACAGGAAATAACCTATGCGGAATTAAACCTTCAAAAAGCTTCTCAGGATTTTCAAGGGAATGACAAAACCTATCACTGCAAAGATTTACCATCAGCTCCAGAGAAGCTCATTGTTGGGATCCTGGGAATTATCTGTCTTATCTTAATGGCCTCTGTGGTAACGATAGTTGTTATTCCCTCTACATTAATACAGAGGCACAACAATTCTTCCCTGAATACAAGAACTCAGAAAGCACGTCATTGTGGCCATTGTCCTGAGGAGTGGATTACATATTCCAACAGTTGTTACTACATTGGTAAGGAAAGAAGAACTTGGGAAGAGAGTTTGCTGGCCTGTACTTCGAAGAACTCCAGTCTGCTTTCTATAGATAATGAAGAAGAAATGAAATTTCTGTCCATCATTTCACCATCCTCATGGATTGGTGTGTTTCGTAACAGCAGTCATCATCCATGGGTGACAATGAATGGTTTGGCTTTCAAACATGAGATAAAAGACTCAGATAATGCTGAACTTAACTGTGCAGTGCTACAAGTAAATCGACTTAAATCAGCCCAGTGTGGATCTTCAATAATATATCATTGTAAGCATAAGCTTTAG (SEQ ID NO：18)。

In some embodiments, the NKG2A polypeptide can comprise a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% consistent) sequence: MDNQGVIYSDLNLPPNPKRQQRKPKGNKSSILATEQEITYAELNLQKASQDFQGNDKTYHCKDLPSAPEKLIVGILGIICLILMASVVTIVVIPSTLIQRHNNSSLNTRTQKARHCGHCPEEWITYSNSCYYIGKERRTWEESLLACTSKNSSLLSIDNEEEMKFLSIISPSSWIGVFRNSSHHPWVTMNGLAFKHEIKDSDNAELNCAVLQVNRLKSAQCGSSIIYHCKHKL (SEQ ID NO：19)。

In some embodiments, the NKG2A polypeptide consists of a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to a sequence % identical) encoded by the nucleic acid sequence: ATGGATAACCAAGGAGTAATCTACTCAGACCTGAATCTGCCCCCAAACCCAAAGAGGCAGCAACGAAAACCTAAAGGCAATAAAAACTCCATTTTAGCAACTGAACAGGAAATAACCTATGCGGAATTAAACCTTCAAAAAGCTTCTCAGGATTTTCAAGGGAATGACAAAACCTATCACTGCAAAGATTTACCATCAGCTCCAGAGAAGCTCATTGTTGGGATCCTGGGAATTATCTGTCTTATCTTAATGGCCTCTGTGGTAACGATAGTTGTTATTCCCTCTACATTAATACAGAGGCACAACAATTCTTCCCTGAATACAAGAACTCAGAAAGCACGTCATTGTGGCCATTGTCCTGAGGAGTGGATTACATATTCCAACAGTTGTTACTACATTGGTAAGGAAAGAAGAACTTGGGAAGAGAGTTTGCTGGCCTGTACTTCGAAGAACTCCAGTCTGCTTTCTATAGATAATGAAGAAGAAATGAAATTTCTGTCCATCATTTCACCATCCTCATGGATTGGTGTGTTTCGTAACAGCAGTCATCATCCATGGGTGACAATGAATGGTTTGGCTTTCAAACATGAGATAAAAGACTCAGATAATGCTGAACTTAACTGTGCAGTGCTACAAGTAAATCGACTTAAATCAGCCCAGTGTGGATCTTCAATAATATATCATTGTAAGCATAAGCTTTAG (SEQ ID NO：20)。

In some embodiments, the methods comprise detecting NKG2A levels in lymphocytes (eg, NK cells) in an individual. Any suitable sample containing NKG2A-negative or NKG2D-positive/NKG2A-negative lymphocytes (eg, NKG2A-negative or NKG2D-positive/NKG2A-negative NK cells) can be obtained from an individual. In some embodiments, whole blood samples, plasma samples, serum samples, samples containing cell fractions (PBMCs) of whole blood samples, tissue samples, tumor samples, sliced samples, and laser capture dissected biological samples can be obtained and evaluated. samples (eg, tumor or tissue samples) to determine the number of NKG2A-negative or NKG2D-positive/NKG2A-negative lymphocytes (eg, NKG2A-negative or NKG2D-positive/NKG2A-negative NK cells). In some embodiments, the level of NKG2A in lymphocytes (eg, NK cells) is detected before, simultaneously with, or after administration of enucleated erythroid cells. In some embodiments, NKG2A-negative lymphocytes can be assayed using antibodies or antigen-binding fragments thereof that specifically bind to NKG2A (e.g., Beckman Coulter, strain Z199.1, and anti-NKG2A mAb strain 131411 (BD Biosciences)). (eg NKG2A negative NK cells). In some embodiments, NKG2A levels in lymphocytes (eg, NK cells) can be assessed using fluorescence-assisted cell sorting (FACS). NKG2A content in lymphocytes (eg, NK cells) can be assessed by other immunological-based methods including, but not limited to, Western blotting, immunohistochemical staining, ELISA, and immunofluorescence. In some embodiments, NKG2A-positive lymphocytes (eg, NKG2A-positive NK cells) can be determined by measuring NKG2A mRNA. Non-limiting methods for quantifying RNA include: qRT-PCR, RNA sequencing, fluorescence in situ hybridization combined with flow cytometry, microfluidic capillary electrophoresis, and in situ hybridization.

Exemplary NKG2A reference levels for identifying NKG2A negative lymphocytes (eg, NKG2A negative NK cells) are known in the art. For example, NKG2A-positive lymphocytes (eg, NKG2A-positive NK cells) are identified based on a flow cytometry banding strategy using fluorescence minus one control samples, fluorescence compensation, and quantitative experience.

In some embodiments, the methods comprise modulating the activity of an NKG2A polypeptide. In some embodiments, the method further comprises administering to the individual an NKG2A inhibitor (eg, an antagonist antibody). For example, the method can comprise administering to the individual monalizumab (see, Andre et al., Cell 175(7):1731- 1743; 2018), dasatinib (Chang et al., Front. Immunol. 9:3152l; 2019), HY-0102, or S095029 (NCT05162755). HLA-E

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

In some embodiments, the HLA-E polypeptide may comprise a sequence at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical ，或100%一致)的胺基酸序列： MVDGTLLLLLSEALALTQTWA GSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRFDNDAASPRMVPRAPWMEQEGSEYWDRETRSARDTAQIFRVNLRTLRGYYNQSEAGSHTLQWMHGCELGPDGRFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDASEAEHQRAYLEDTCVEWLHKYLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQQDGEGHTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPVTLRWKPASQPTIPIVGIIAGLVLLGSVVSGAVVAAVIWRKKSSGGKGGSYSKAEWSDSAQGSESHSL (SEQ ID NO：21) (具有或不具有訊號肽)。 The underlined part in SEQ ID NO: 21 represents the signal peptide.

In some embodiments, the HLA-E polypeptide consists of a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) nucleic acid sequence encoded by: ATGGTAGATGGAACCCTCCTTTTACTCCTCTCGGAGGCCCTGGCCCTTACCCAGACCTGGGCGGGCTCCCACTCCTTGAAGTATTTCCACACTTCCGTGTCCCGGCCCGGCCGCGGGGAGCCCCGCTTCATCTCTGTGGGCTACGTGGACGACACCCAGTTCGTGCGCTTCGACAACGACGCCGCGAGTCCGAGGATGGTGCCGCGGGCGCCGTGGATGGAGCAGGAGGGGTCAGAGTATTGGGACCGGGAGACACGGAGCGCCAGGGACACCGCACAGATTTTCCGAGTGAATCTGCGGACGCTGCGCGGCTACTACAATCAGAGCGAGGCCGGGTCTCACACCCTGCAGTGGATGCATGGCTGCGAGCTGGGGCCCGACGGGCGCTTCCTCCGCGGGTATGAACAGTTCGCCTACGACGGCAAGGATTATCTCACCCTGAATGAGGACCTGCGCTCCTGGACCGCGGTGGACACGGCGGCTCAGATCTCCGAGCAAAAGTCAAATGATGCCTCTGAGGCGGAGCACCAGAGAGCCTACCTGGAAGACACATGCGTGGAGTGGCTCCACAAATACCTGGAGAAGGGGAAGGAGACGCTGCTTCACCTGGAGCCCCCAAAGACACACGTGACTCACCACCCCATCTCTGACCATGAGGCCACCCTGAGGTGCTGGGCCCTGGGCTTCTACCCTGCGGAGATCACACTGACCTGGCAGCAGGATGGGGAGGGCCATACCCAGGACACGGAGCTCGTGGAGACCAGGCCTGCAGGGGATGGAACCTTCCAGAAGTGGGCAGCTGTGGTGGTGCCTTCTGGAGAGGAGCAGAGATACACGTGCCATGTGCAGCATGAGGGGCTACCCGAGCCCGTCACCCTGAGATGGAAGCCGGCTTCCCAGCCCACCATCCCCATCGTGGGCATCATTGCTGGCCTGGTTCTCCTTGGATCTGTGGTCTCTGGAGCTGTGGTTGCTGCTGTGATATGGAGGAAGAAGAGCT CAGGTGGAAAAGGAGGGAGCTACTCTAAGGCTGAGTGGAGCGACAGTGCCCAGGGGTCTGAGTCTCACAGCTTGTAA (SEQ ID NO: 22).

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

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

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

In some embodiments, the IL-15 comprises an immature form of wild-type human IL-15. In some embodiments, the IL-15 comprises a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) amino acid sequence: MRISKPHLRSISIQCYLCLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 23), or a functional fragment thereof. In some embodiments, IL-15 comprises the sequence of SEQ ID NO:23.

In some embodiments, the IL-15 comprises a mature form of wild-type human IL-15 or a functional fragment thereof. In some embodiments, IL-15 comprises at least 70% identity (e.g., at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 99% identity, or 100% identical) amino acid sequence: NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 16), or a functional fragment thereof. In some embodiments, IL-15 comprises the sequence of SEQ ID NO:24.

In some embodiments, IL-15 consists of a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) encoded by the nucleic acid sequence: AACTGGGTGAACGTTATTAGTGACCTTAAAAAGATCGAAGATTTGATACAGTCAATGCACATAGACGCGACGCTTTATACAGAATCTGATGTACATCCTTCATGCAAGGTTACTGCTATGAAGTGTTTTCTTCTCGAACTCCAAGTAATAAGTCTTGAGAGCGGAGATGCGAGCATTCATGACACCGTTGAGAATCTTATTATATTGGCTAACAACTCTCTGTCCAGCAATGGTAATGTGACAGAAAGCGGGTGTAAGGAGTGCGAGGAACTCGAGGAGAAGAACATCAAAGAGTTCTTGCAGTCTTTCGTCCATATTGTCCAGATGTTCATAAATACTAGC (SEQ ID NO：25)。

In some embodiments, the IL-15 polypeptide consists of a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical to, or 100% identical) nucleic acid sequence encoded by: AACTGGGTAAACGTCATAAGCGACCTCAAAAAGATCGAGGACCTTATACAGTCTATGCACATAGATGCGACACTTTACACGGAATCAGACGTGCACCCGTCCTGCAAAGTCACAGCCATGAAGTGCTTTCTTCTCGAACTGCAGGTAATTTCTCTCGAATCAGGTGACGCATCTATCCACGACACAGTTGAAAATCTTATTATCCTGGCTAATAACTCCCTTAGCTCAAACGGCAATGTCACCGAGAGTGGATGTAAAGAATGTGAGGAACTTGAAGAGAAAAACATAAAGGAATTCTTGCAGAGTTTCGTTCATATTGTGCAAATGTTCATCAATACTAGT (SEQ ID NO：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 less than 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, or 160 amino acids. In some embodiments, the functional fragment of IL-15 retains at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% of the ability of wild-type human IL-15 polypeptide to bind IL-15RA polypeptide %, 90%, 95%, 98% or 99%, as measured by assays well known in the art, such as ELISA and surface plasmon resonance (SPR) binding assays, or co-immunoprecipitation. In some embodiments, the functional fragment of the IL-15 polypeptide retains at least 50%, 55%, 60%, 65%, 70%, 75% of the ability of the wild-type human IL-15 polypeptide to induce IL-15-mediated signaling , 80%, 85%, 90%, 95%, 98% or 99%, as measured by assays well known in the art, such as electromobility shift assays, ELISAs, and other immunoassays. In some embodiments, the functional fragment of an IL-15 polypeptide can be an IL-15 receptor binding fragment of an IL-15 polypeptide.

In some embodiments, the interleukin-15 receptor alpha (IL-15RA) or functional fragment thereof may comprise an immature form of wild-type human IL-15RA. In some embodiments, the IL-15RA can comprise at least 70% identity (e.g., at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 99% identity) to the following sequence, or 100% identical) sequence: MAPRRARGCRTLGLPALLLLLLLRPPATRGITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL (SEQ ID NO：27)或其功能片段。 In some embodiments, the IL-15RA polypeptide comprises the sequence of SEQ ID NO:27.

In some embodiments, the IL-15RA comprises a mature form of wild-type human IL-15RA. In some embodiments, the IL-15RA comprises a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) amino acid sequence: ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL  (SEQ ID NO：28)或其功能片段。 In some embodiments, the IL-15RA comprises the sequence of SEQ ID NO:28.

In some embodiments, IL-15RA comprises an extracellular portion of an IL-15RA polypeptide. For example, an IL-15RA polypeptide may lack the transmembrane domain of wild-type IL-15RA, and optionally lack the intracellular domain of wild-type IL-15RA. In some embodiments, the IL-15RA comprises an immature form of extracellular wild-type human IL-15RA. In some embodiments, the IL-15RA polypeptide may comprise a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical , or 100% identical) sequence: MAPRRARGCRTLGLPALLLLLLRPPATRGITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTT (SEQ ID) or a functional fragment thereof. In some embodiments, the IL-15RA polypeptide comprises the sequence of SEQ ID NO:29.

In some embodiments, the IL-15RA comprises a mature form of extracellular wild-type human IL-15RA. In some embodiments, the IL-15RA can comprise at least 70% identity (e.g., at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 99% identity) to the following sequence, or 100% identical) sequence: ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTT (SEQ ID NO: 30) or a functional fragment thereof. In some embodiments, the IL-15RA polypeptide comprises the sequence of SEQ ID NO:30.

In some embodiments, the IL-15RA consists of a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) encoded by the nucleic acid sequence: ATCACATGCCCACCGCCCATGTCTGTTGAACACGCAGACATTTGGGTTAAAAGTTACTCACTTTACTCACGCGAGAGATATATATGCAACAGCGGCTTCAAGCGCAAAGCAGGCACTAGTAGTCTTACAGAGTGCGTGCTCAATAAAGCTACAAATGTAGCTCATTGGACTACTCCTAGTCTCAAATGCATTCGGGACCCCGCGCTTGTGCACCAGAGACCTGCGCCGCCGTCCACAGTGACGACAGCTGGTGTAACCCCCCAACCTGAATCCCTTAGTCCGTCTGGTAAAGAACCGGCGGCGTCTTCACCTTCCAGCAATAATACTGCGGCGACAACAGCCGCGATAGTTCCTGGATCCCAACTCATGCCGTCAAAGTCTCCTTCAACGGGAACGACAGAGATCTCTTCACATGAAAGTTCTCATGGAACACCGAGCCAAACTACGGCAAAGAACTGGGAACTGACTGCCTCAGCAAGCCACCAGCCGCCAGGGGTGTACCCGCAAGGGCACTCAGATACTACT  (SEQ ID NO：31)。

In some embodiments, IL-15RA or a functional fragment thereof includes a "sushi domain" in exon 2 of the receptor extracellular domain (Wei et al., J Immunol. 2001; 167:277-282). In some embodiments, the IL-15RA or functional fragment thereof comprising the sushi domain of wild-type human IL-15RA comprises at least 70% identity (e.g., at least 75% identity, at least 80% identity, at least 85% identity, Amino acid sequence of at least 90% identity, at least 95% identity, at least 99% identity, or 100% identity): ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLECVLNKATNVAHWTTPSLKCIR (SEQ ID NO: 32), or a functional fragment thereof. In some embodiments, IL-15RA or a functional fragment thereof comprises the sushi domain of wild-type human IL-15RA comprising the sequence of SEQ ID NO:32.

In some embodiments, the IL-15RA comprises the sushi domain of wild-type human IL15RA or a functional fragment thereof and additionally at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,30,35,40,45,50,60,70,80,90 or 100 amino acids. In some embodiments, IL-15RA or a functional fragment thereof comprises the sushi domain of wild-type human IL15RA and an additional 13 amino acids of wild-type human IL-15RA. In some embodiments, the IL-15RA comprises a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) amino acid sequence: ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSLLECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPS (SEQ ID NO: 33) or a functional fragment thereof. In some embodiments, the IL-15RA polypeptide comprises the sequence of SEQ ID NO:33.

In some embodiments, the IL-15RA consists of a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) encoded by the nucleic acid sequence: ATCACCTGCCCGCCTCCCATGAGCGTGGAACACGCGGACATTTGGGTTAAGAGCTACAGTCTTTACAGCCGGGAGCGCTATATCTGCAACTCAGGGTTTAAGCGGAAAGCAGGGACATCAAGTTTGACAGAATGTGTGTTGAACAAGGCTACAAATGTTGCTCACTGGACCACGCCATCTTTGAAGTGTATCCQGAGATCCCGCGCTTGTCCATCAGC3GCCCAGCCT.(CCCAGCCT4)

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

In some embodiments, the first exogenous polypeptide further includes a signal peptide. In some embodiments, the first exogenous polypeptide comprises a signal peptide comprising the amino acid sequence listed in Table 3. In some embodiments, the first exogenous polypeptide includes a signal peptide that includes a GPA signal peptide. In some embodiments, the first exogenous polypeptide includes a signal peptide having the 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 comprises a leader (signal) sequence comprising the amino acid sequence of SEQ ID NO: 35, a mature human IL-15 polypeptide comprising the amino acid sequence of SEQ ID NO: 24, and An IL-15RA polypeptide comprising the amino acid sequence of SEQ ID NO:30. In some embodiments, the mature human IL-15 polypeptide and the IL-15RA polypeptide are linked by a flexible linker having the amino acid sequence of SEQ ID NO:37.

In some embodiments, one or more linkers are disposed between IL-15 or a functional fragment thereof and IL-15RA or a functional fragment thereof. Any linker provided herein can be used. In some embodiments, the linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 in length peptides of one or more amino acids. In some embodiments, the length of the linker is sufficient to retain the ability of IL-15 to bind to IL-15RA. In other embodiments, the linker is of sufficient length to retain the ability of the IL-15/IL-15RA complex to bind to the βγ IL-15 receptor complex and act as an agonist that mediates IL-15 signaling. In some embodiments, the linker comprises the amino acid sequences 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 (GGGGS) _n linker (SEQ ID NO: 30), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, the linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 37).

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

In some embodiments, the first exogenous polypeptide comprises an IL-15 polypeptide or a functional fragment thereof, and an extracellular region of an IL-15Ra polypeptide. In some embodiments, the first exogenous polypeptide comprises the amino acid sequence of SEQ ID NO:39. In some embodiments, the first exogenous polypeptide comprises a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical) consistent, or 100% consistent) sequences: NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGGSGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTT  (SEQ ID NO：39)。

In some embodiments, the first exogenous polypeptide consists of a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical) Consistent, or 100% identical) nucleic acid sequence encoded by: AACTGGGTGAACGTTATTAGTGACCTTAAAAAGATCGAAGATTTGATACAGTCAATGCACATAGACGCGACGCTTTATACAGAATCTGATGTACATCCTTCATGCAAGGTTACTGCTATGAAGTGTTTTCTTCTCGAACTCCAAGTAATAAGTCTTGAGAGCGGAGATGCGAGCATTCATGACACCGTTGAGAATCTTATTATATTGGCTAACAACTCTCTGTCCAGCAATGGTAATGTGACAGAAAGCGGGTGTAAGGAGTGCGAGGAACTCGAGGAGAAGAACATCAAAGAGTTCTTGCAGTCTTTCGTCCATATTGTCCAGATGTTCATAAATACTAGCGGGGGAGGTGGCTCTGGTGGAGGCGGGAGTGGCGGGGGCGGCTCAATCACATGCCCACCGCCCATGTCTGTTGAACACGCAGACATTTGGGTTAAAAGTTACTCACTTTACTCACGCGAGAGATATATATGCAACAGCGGCTTCAAGCGCAAAGCAGGCACTAGTAGTCTTACAGAGTGCGTGCTCAATAAAGCTACAAATGTAGCTCATTGGACTACTCCTAGTCTCAAATGCATTCGGGACCCCGCGCTTGTGCACCAGAGACCTGCGCCGCCGTCCACAGTGACGACAGCTGGTGTAACCCCCCAACCTGAATCCCTTAGTCCGTCTGGTAAAGAACCGGCGGCGTCTTCACCTTCCAGCAATAATACTGCGGCGACAACAGCCGCGATAGTTCCTGGATCCCAACTCATGCCGTCAAAGTCTCCTTCAACGGGAACGACAGAGATCTCTTCACATGAAAGTTCTCATGGAACACCGAGCCAAACTACGGCAAAGAACTGGGAACTGACTGCCTCAGCAAGCCACCAGCCGCCAGGGGTGTACCCGCAAGGGCACTCAGATACTACT  (SEQ ID NO：40)。

In some embodiments, the first exogenous polypeptide comprises an IL-15 polypeptide and a sushi domain of an IL-15RA polypeptide. In some embodiments, the first exogenous polypeptide comprises the amino acid sequence of SEQ ID NO:41. In some embodiments, the first exogenous polypeptide comprises a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical) consistent, or 100% consistent) sequences: NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGGSGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAPPSLTECVLNKATNVAHWTTPSHLK:

In some embodiments, the first exogenous polypeptide is composed of a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical) % identical, or 100% identical) nucleic acid sequence encoded by: AACTGGGTAAACGTCATAAGCGACCTCAAAAAGATCGAGGACCTTATACAGTCTATGCACATAGATGCGACACTTTACACGGAATCAGACGTGCACCCGTCCTGCAAAGTCACAGCCATGAAGTGCTTTCTTCTCGAACTGCAGGTAATTTCTCTCGAATCAGGTGACGCATCTATCCACGACACAGTTGAAAATCTTATTATCCTGGCTAATAACTCCCTTAGCTCAAACGGCAATGTCACCGAGAGTGGATGTAAAGAATGTGAGGAACTTGAAGAGAAAAACATAAAGGAATTCTTGCAGAGTTTCGTTCATATTGTGCAAATGTTCATCAATACTAGTGGCGGGGGGGGAAGCGGTGGTGGAGGGAGCGGGGGTGGTGGATCCATCACCTGCCCGCCTCCCATGAGCGTGGAACACGCGGACATTTGGGTTAAGAGCTACAGTCTTTACAGCCGGGAGCGCTATATCTGCAACTCAGGGTTTAAGCGGAAAGCAGGGACATCAAGTTTGACAGAATGTGTGTTGAACAAGGCTACAAATGTTGCTCACTGGACCACGCCATCTTTGAAGTGTATCCGAGATCCCGCGCTTGTCCATCAGCGCCCAGCGCCTCCCTCC  (SEQ ID NO：42)。

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

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

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

In some embodiments, an anchor or transmembrane domain may comprise 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 I membrane polypeptide or a transmembrane portion thereof selected from the group consisting of: glycophorin A (GPA); Glycophorin B (GPB); basal immunoglobulin (basigin) (also known as CD147); CD44; CD58 (also known as LFA3); intercellular adhesion molecule 4 (ICAM4); basal cell adhesion molecule (BCAM); CR1 ; CD99; erythrocyte blastocyte membrane-associated protein (ERMAP); junctional adhesion molecule A (JAM-A); neuroplastin (NPTN); AMIGO2; and DS cell adhesion molecule-like 1 (DSCAML1). In some embodiments, the anchoring or transmembrane domain comprises or consists of a type 2 membrane 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 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 may comprise small integral membrane protein 1 (SMIM1) or a transmembrane portion thereof. In some embodiments, the anchor or transmembrane domain comprises glycophorin A (GPA), or a fragment thereof (eg, the transmembrane portion thereof). In some embodiments, the anchor or transmembrane domain comprises the amino acid sequences provided in Table 1.

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

In some embodiments, a linker is disposed between the anchor or transmembrane domain and the IL-15 polypeptide, IL-15RA polypeptide or IL-15/IL-15RA polypeptide. Suitable linkers include, but are not limited to, any of the linker amino acid sequences provided in Table 2. In some embodiments, the linker between the anchor or transmembrane domain (e.g., GPA) and the IL-15 polypeptide, IL-15RA polypeptide, or IL-15/IL-15RA fusion polypeptide comprises or consists of an HA linker . In some embodiments, the linker comprises or consists of the amino acid sequence of SEQ ID NO: 41.

In some embodiments, the first exogenous polypeptide can further include an anchor. In some embodiments, the first exogenous polypeptide may include the amino acid sequence of SEQ ID NO: 44 (which is encoded by the nucleic acid sequence of SEQ ID NO: 45), an interleukin-15 (IL-15) polypeptide, and an interleukin-15 (IL-15) polypeptide. The extracellular portion of the interleukin 15 receptor alpha (IL-15RA) polypeptide. In some embodiments, the first exogenous polypeptide can include an anchor comprising the amino acid sequence of SEQ ID NO: 44, mature human IL-15 comprising the amino acid sequence of SEQ ID NO: 24, and comprising the amino acid sequence The mature human extracellular IL-15RA of the sequence SEQ ID NO: 30, thereby the mature human IL-15 amino acid sequence and the mature human extracellular IL-15 RA amino acid sequence by including the amino acid sequence of SEQ ID NO: 37 elastic linker connections.

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

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

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

In some embodiments, the first exogenous polypeptide comprises a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical) Consistent, or 100% identical) amino acid sequence: MYGKIIFVLLLSEIVSISANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGGSGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTGGSGGSGGYPYDVPDYAGGGSGGGSLSTTEVAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRAHEVSEISVRTVYPPEEETGERVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDTDVPLSSVEIENPETSDQ (SEQ ID NO：48)或其功能片段。

In some embodiments, the first exogenous polypeptide consists of a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical) Consistent, or 100% identical) nucleic acid sequence encoded by: ATGTATGGAAAAATAATCTTTGTATTACTATTGTCAGAAATTGTGAGCATATCAGCAAACTGGGTGAACGTTATTAGTGACCTTAAAAAGATCGAAGATTTGATACAGTCAATGCACATAGACGCGACGCTTTATACAGAATCTGATGTACATCCTTCATGCAAGGTTACTGCTATGAAGTGTTTTCTTCTCGAACTCCAAGTAATAAGTCTTGAGAGCGGAGATGCGAGCATTCATGACACCGTTGAGAATCTTATTATATTGGCTAACAACTCTCTGTCCAGCAATGGTAATGTGACAGAAAGCGGGTGTAAGGAGTGCGAGGAACTCGAGGAGAAGAACATCAAAGAGTTCTTGCAGTCTTTCGTCCATATTGTCCAGATGTTCATAAATACTAGCGGGGGAGGTGGCTCTGGTGGAGGCGGGAGTGGCGGGGGCGGCTCAATCACATGCCCACCGCCCATGTCTGTTGAACACGCAGACATTTGGGTTAAAAGTTACTCACTTTACTCACGCGAGAGATATATATGCAACAGCGGCTTCAAGCGCAAAGCAGGCACTAGTAGTCTTACAGAGTGCGTGCTCAATAAAGCTACAAATGTAGCTCATTGGACTACTCCTAGTCTCAAATGCATTCGGGACCCCGCGCTTGTGCACCAGAGACCTGCGCCGCCGTCCACAGTGACGACAGCTGGTGTAACCCCCCAACCTGAATCCCTTAGTCCGTCTGGTAAAGAACCGGCGGCGTCTTCACCTTCCAGCAATAATACTGCGGCGACAACAGCCGCGATAGTTCCTGGATCCCAACTCATGCCGTCAAAGTCTCCTTCAACGGGAACGACAGAGATCTCTTCACATGAAAGTTCTCATGGAACACCGAGCCAAACTACGGCAAAGAACTGGGAACTGACTGCCTCAGCAAGCCACCAGCCGCCAGGGGTGTACCCGCAAGGGCACTCAGATACTACTGGAGGATCTGGCGGGTCTGGAGGCTACCCCT ATGACGTGCCCGACTATGCCGGCGGAGGGTCTGGAGGCGGTTCCTTAAGTACCACTGAGGTGGCAATGCACACTTCAACCTCTTCTTCAGTCACAAAGAGTTACATCTCATCACAGACAAATGATACGCACAAACGGGACACATATGCAGCCACTCCTAGAGCTCATGAAGTTTCAGAAATTTCTGTTAGAACTGTTTACCCTCCAGAAGAGGAAACCGGAGAAAGGGTACAACTTGCCCATCATTTCTCTGAACCAGAGATAACACTCATTATTTTTGGGGTGATGGCTGGTGTTATTGGAACGATCCTCTTAATTTCTTACGGTATTCGCCGACTGATAAAGAAAAGCCCATCTGATGTAAAACCTCTCCCCTCACCTGACACAGACGTGCCTTTAAGTTCTGTTGAAATAGAAAATCCAGAGACAAGTGATCAA (SEQ ID NO：49)。

In some embodiments, the first exogenous polypeptide comprises a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical) Consistent, or 100% identical) amino acid sequence: MYGKIIFVLLLSEIVSISANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGGSGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSGGSGGSGGYPYDVPDYAGGGSGGGSLSTTEVAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRAHEVSEISVRTVYPPEEETGERVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDTDVPLSSVEIENPETSDQ (SEQ ID NO：50)或其功能片段。

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

In some embodiments, the first exogenous polypeptide comprises a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical) Consistent, or 100% identical) amino acid sequence: MYGKIIFVLLLSEIVSISANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGSGGSGGGGGSGGGSGGGSGGGSLSTTEVAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRAHEVSEISVRTVYPPEEETGERVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDTDVPLSSVEIENPETSDQ (SEQ ID NO：52)或其功能片段。

In some embodiments, the first exogenous polypeptide consists of a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical) Consistent, or 100% identical) nucleic acid sequence encoded by: ATGTATGGAAAAATAATCTTTGTATTACTATTGTCAGAAATTGTGAGCATATCAGCAAACTGGGTAAATGTGATTTCAGACCTGAAAAAAATCGAAGACCTTATTCAATCCATGCACATCGACGCGACACTTTATACTGAATCAGACGTACACCCGTCCTGTAAGGTTACTGCGATGAAGTGCTTTCTGTTGGAATTGCAAGTGATCTCCCTCGAATCAGGGGATGCATCCATTCATGATACCGTCGAGAATTTGATCATTCTGGCAAATAACTCCCTCAGTAGTAACGGGAATGTGACCGAGTCTGGGTGTAAGGAGTGCGAAGAGTTGGAGGAAAAGAATATCAAAGAATTCCTTCAGTCCTTTGTTCACATCGTGCAAATGTTTATTAATACATCTGGAGGATCTGGCGGGTCTGGAGGCGGCGGCGGCAGCGGCGGCGGCAGCGGCGGAGGGTCTGGAGGCGGTTCCTTAAGTACCACTGAGGTGGCAATGCACACTTCAACCTCTTCTTCAGTCACAAAGAGTTACATCTCATCACAGACAAATGATACGCACAAACGGGACACATATGCAGCCACTCCTAGAGCTCATGAAGTTTCAGAAATTTCTGTTAGAACTGTTTACCCTCCAGAAGAGGAAACCGGAGAAAGGGTACAACTTGCCCATCATTTCTCTGAACCAGAGATAACACTCATTATTTTTGGGGTGATGGCTGGTGTTATTGGAACGATCCTCTTAATTTCTTACGGTATTCGCCGACTGATAAAGAAAAGCCCATCTGATGTAAAACCTCTCCCCTCACCTGACACAGACGTGCCTTTAAGTTCTGTTGAAATAGAAAATCCAGAGACAAGTGATCAA (SEQ ID NO：53)。

In some embodiments, the first exogenous polypeptide comprises a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical) Consistent, or 100% identical) amino acid sequence: MYGKIIFVLLLSEIVSISANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGGSGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTGGSGGSGGGGGSGGGSGGGSGGGSLSTTEVAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRAHEVSEISVRTVYPPEEETGERVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDTDVPLSSVEIENPETSDQ (SEQ ID NO：54)或其功能片段。

In some embodiments, the first exogenous polypeptide consists of a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical) Consistent, or 100% identical) nucleic acid sequence encoded by: ATGTATGGAAAAATAATCTTTGTATTACTATTGTCAGAAATTGTGAGCATATCAGCAAACTGGGTGAACGTTATTAGTGACCTTAAAAAGATCGAAGATTTGATACAGTCAATGCACATAGACGCGACGCTTTATACAGAATCTGATGTACATCCTTCATGCAAGGTTACTGCTATGAAGTGTTTTCTTCTCGAACTCCAAGTAATAAGTCTTGAGAGCGGAGATGCGAGCATTCATGACACCGTTGAGAATCTTATTATATTGGCTAACAACTCTCTGTCCAGCAATGGTAATGTGACAGAAAGCGGGTGTAAGGAGTGCGAGGAACTCGAGGAGAAGAACATCAAAGAGTTCTTGCAGTCTTTCGTCCATATTGTCCAGATGTTCATAAATACTAGCGGGGGAGGTGGCTCTGGTGGAGGCGGGAGTGGCGGGGGCGGCTCAATCACATGCCCACCGCCCATGTCTGTTGAACACGCAGACATTTGGGTTAAAAGTTACTCACTTTACTCACGCGAGAGATATATATGCAACAGCGGCTTCAAGCGCAAAGCAGGCACTAGTAGTCTTACAGAGTGCGTGCTCAATAAAGCTACAAATGTAGCTCATTGGACTACTCCTAGTCTCAAATGCATTCGGGACCCCGCGCTTGTGCACCAGAGACCTGCGCCGCCGTCCACAGTGACGACAGCTGGTGTAACCCCCCAACCTGAATCCCTTAGTCCGTCTGGTAAAGAACCGGCGGCGTCTTCACCTTCCAGCAATAATACTGCGGCGACAACAGCCGCGATAGTTCCTGGATCCCAACTCATGCCGTCAAAGTCTCCTTCAACGGGAACGACAGAGATCTCTTCACATGAAAGTTCTCATGGAACACCGAGCCAAACTACGGCAAAGAACTGGGAACTGACTGCCTCAGCAAGCCACCAGCCGCCAGGGGTGTACCCGCAAGGGCACTCAGATACTACTGGAGGATCTGGCGGGTCTGGAGGCGGCGGCG GCAGCGGCGGCGGCAGCGGCGGAGGGTCTGGAGGCGGTTCCTTAAGTACCACTGAGGTGGCAATGCACACTTCAACCTCTTCTTCAGTCACAAAGAGTTACATCTCATCACAGACAAATGATACGCACAAACGGGACACATATGCAGCCACTCCTAGAGCTCATGAAGTTTCAGAAATTTCTGTTAGAACTGTTTACCCTCCAGAAGAGGAAACCGGAGAAAGGGTACAACTTGCCCATCATTTCTCTGAACCAGAGATAACACTCATTATTTTTGGGGTGATGGCTGGTGTTATTGGAACGATCCTCTTAATTTCTTACGGTATTCGCCGACTGATAAAGAAAAGCCCATCTGATGTAAAACCTCTCCCCTCACCTGACACAGACGTGCCTTTAAGTTCTGTTGAAATAGAAAATCCAGAGACAAGTGATCAA (SEQ ID NO：55)。

In some embodiments, the exogenous fusion polypeptide includes the amino acid sequence of SEQ ID NO:46. In some embodiments, the exogenous fusion polypeptide includes the amino acid sequence of SEQ ID NO:48. In some embodiments, the exogenous fusion polypeptide comprises the amino acid sequence of SEQ ID NO:50. In some embodiments, the exogenous fusion polypeptide includes the amino acid sequence of SEQ ID NO:52. In some embodiments, the exogenous fusion polypeptide includes the 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 incorporated herein by reference in its entirety. second exogenous polypeptide

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

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

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

In some embodiments, 4-1BBL comprises at least 70% identity (e.g., at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 99% identity, or 100% identical) amino acid sequence: ACPWAVSGARASPPGSASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQPRSETVLGLFRVTPPEIPA function or its fragment.:GL In some embodiments, 4-1BBL comprises the sequence of SEQ ID NO:56.

In some embodiments, 4-1BBL consists of a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) encoded by the nucleic acid sequence: GCCTGCCCCTGGGCCGTGTCCGGGGCTCGCGCCTCGCCCGGCTCCGCGGCCAGCCCGAGACTCCGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA  (SEQ ID NO：57)。

In some embodiments, the second exogenous polypeptide comprises a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical) Consistent, or 100% identical) amino acid sequence: MYGKIIFVLLLSEIVSISAACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSEGGSGGSGGGPEDEPGSGSGGGSGGGSLSTTEVAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRAHEVSEISVRTVYPPEEETGERVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDTDVPLSSVEIENPETSDQ  (SEQ ID NO：58)。 In some embodiments, the 4-1BBL polypeptide comprises the sequence of SEQ ID NO:58.

In some embodiments, the second exogenous polypeptide consists of a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical) Consistent, or 100% identical) nucleic acid sequence encoded by: ATGTATGGAAAAATAATCTTTGTATTACTATTGTCAGAAATTGTGAGCATATCAGCAGCCTGCCCCTGGGCCGTGTCCGGGGCTCGCGCCTCGCCCGGCTCCGCGGCCAGCCCGAGACTCCGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAAGGAGGATCTGGCGGGTCTGGAGGCGGCCCCGAGGACGAGCCCGGCAGCGGCAGCGGCGGAGGGTCTGGAGGCGGTTCCTTAAGTACCACTGAGGTGGCAATGCACACTTCAACCTCTTCTTCAGTCACAAAGAGTTACATCTCATCACAGACAAATGATACGCACAAACGGGACACATATGCAGCCACTCCTAGAGCTCATGAAGTTTCAGAAATTTCTGTTAGAACTGTTTACCCTCCAGAAGAGGAAACCGGAGAAAGGGTACAACTTGCCCATCATTTCTCTGAACCAGAGATAACACTCATTATTTTTGGGGTGATGGCTGGTG TTATTGGAACGATCCTCTTAATTTCTTACGGTATTCGCCGACTGATAAAGAAAAGCCCATCTGATGTAAAACCTCTCCCCTCACCTGACACAGACGTGCCTTTAAGTTCTGTTGAAATAGAAAATCCAGGACAAGTGATCAA (SEQ ID NO: 59).

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

In some embodiments, the second exogenous polypeptide is attached to the erythroid membrane (eg, the membrane of an enucleated erythroid). 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 anchoring or transmembrane domain that anchors the second exogenous polypeptide to the erythroid membrane (eg, the membrane of an enucleated erythroid). In some embodiments, the anchoring or transmembrane domain is heterologous to a second exogenous polypeptide (eg, 4-1BBL). In some embodiments, the anchor or transmembrane domain comprises an endogenous erythrocyte transmembrane polypeptide or a fragment or transmembrane portion thereof. In certain embodiments, the anchor or transmembrane domain comprises GPA or a transmembrane portion thereof. In some embodiments, the anchor or transmembrane domain comprises Small Integral Membrane Protein 1 (SMIM1), Transferrin Receptor, Fas Ligand (FasL), Kellor Band 3, or a transmembrane portion thereof (eg, transmembrane domain). In some embodiments, the second exogenous polypeptide can include any of the anchoring or transmembrane domains described herein. In some embodiments, the second exogenous polypeptide includes an anchor or transmembrane domain listed in Table 1.

In some embodiments, the second exogenous polypeptide includes one or more linkers (eg, any of the exemplary linkers described herein). For example, the second exogenous polypeptide can include one or more linkers provided in Table 2. In some embodiments, a linker is placed between 4-1BBL or a functional fragment thereof and the anchoring domain or transmembrane domain in the second exogenous polypeptide. In some embodiments, the second exogenous polypeptide can further include a signal peptide (eg, any of the exemplary signal peptides described herein). For example, the second exogenous polypeptide can include a signal peptide provided in Table 3.

In some embodiments, the second exogenous polypeptide includes a signal peptide, 4-1BBL or a functional fragment thereof, and an anchor. In some embodiments, the second exogenous polypeptide includes a signal peptide, 4-1BBL or a functional fragment thereof, a linker, and an anchor. In some embodiments, the second exogenous polypeptide comprises (eg, from N-terminus to C-terminus) a signal peptide comprising the amino acid sequence of SEQ ID NO: 35, a 4-peptide comprising the amino acid sequence of SEQ ID NO: 54. 1BBL or a functional fragment thereof, a linker comprising the amino acid sequence of SEQ ID NO: 60 (for example, placed between 4-1BBL or a functional fragment thereof and the anchor), and an anchor comprising the amino acid sequence of SEQ ID NO: 44 Certainly. In some embodiments, the second exogenous polypeptide comprises 4-1BBL or a functional fragment thereof, a linker and an anchor. In some embodiments, the second exogenous polypeptide comprises (eg, from N-terminus to C-terminus) 4-1BBL comprising the amino acid sequence of SEQ ID NO: 56 or a functional fragment thereof, comprising the amino acid sequence of SEQ ID NO :60 linker (eg, positioned between 4-1BBL or a functional fragment thereof and the anchor), and an anchor comprising 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 (eg, mammalian cells, eg, human cells). In some embodiments, one or more (eg, 2, 3, 4, 5 or more) exogenous polypeptides are glycosylated, phosphorylated, or both. In vitro detection of glycoproteins is usually done on SDS-PAGE gels and Western blotting using a modified Periodic acid-Schiff (PAS) method. Cellular localization of glycoproteins can be accomplished using lectin fluorescent conjugates known in the art. Phosphorylation can be assessed by Western blotting using phospho-specific antibodies. Post-translational modifications also include attachment to hydrophobic groups (e.g., myristylation, palmitoylation, prenylation, polyprenylation, or glycosylation), attachment to cofactors (e.g., aliphatic flavin moiety (e.g., FMN or FAD), heme C attachment, phosphopanthiolation or retinylidene Schiff base formation), dibenzamide formation, ethanolamine phosphoglycerol attachment, putrefaction Amine hypusine formation, acylation (eg, O-acylation, N-acylation, or S-acylation), formylation, acetylation, alkylation (eg, methylation or ethylation) , amidation, butylation, γ-carboxylation, malonylation, hydroxylation, iodination, nucleotide addition (such as ADP-ribosylation), oxidation, phosphate (O-linkage) or amine Phosphate (N-linked) formation, (e.g., phosphorylation or adenylation), propylation, pyroglutamate formation, S-glutathionylation, S-nitrosylation, succinylation, Sulfation, ISGylation, SUMOylation, ubiquitination, neddylation, or chemical modification of amino acids (such as citrullination, deamidation, deimidation, or carbamylation), disulfide bond formation, racemization (for example of proline, serine, alanine or methionine). In embodiments, glycosylation comprises adding a sugar group to arginine, asparagine, cysteine, hydroxylysine, serine, threonine, tyrosine, or tryptophan to produce glycoprotein. In embodiments, glycosylation includes, for example, O-linked glycosylation or N-linked glycosylation.

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

In some embodiments, the transmembrane domain comprises or consists of a transmembrane domain of a Type 1 membrane polypeptide. In some embodiments, the type 1 membrane polypeptide is selected from the group consisting of: glycophorin A (GPA); glycophorin B (GPB); basal immunoglobulin (also known as CD147); CD44; CD58 ( Also known as LFA3); intercellular adhesion molecule 4 (ICAM4); basal cell adhesion molecule (BCAM); CR1; CD99; erythrocyte blastocyte membrane-associated protein (ERMAP); NPTN); AMIGO2; 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 erythrocyte membrane comprises, consists of, or is derived from a GPI-linked membrane protein (eg, a fragment thereof). 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, GPA is preferred in certain embodiments because it has a cytoplasmic domain that interacts with the reticulocyte cytoskeleton, which has the ability to retain GPA during cell differentiation and maturation. effect. In some embodiments, the transmembrane domain comprises small integral membrane protein 1 (SMIM1) or a transmembrane portion thereof. In some embodiments, the anchor is selected from the amino acid sequences listed in Table 1. Table 1. Anchor sequences SEQ ID NO : sequence name sequence description amino acid sequence 62 GPA full-length GPA MYGKIIFVLLLSAIVSISALSTTEVAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRAHEVSEISVRTVYPPEEETGERVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDDVPLSSVEIEENPETSDQ 44 GPA Fragment of GPA including transmembrane domain LSTTEVAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRAHEVSEISVRTVYPPEEETGE RVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDTDVPLSS VEIENPETSDQ 63 SMIM1 SMIM1 MQPQESHVHYSRWEDGSRDGVSLGAVSSTEEASRCRRISQRLCTGKLGIAMKVLGGVALFWIIFILGYLTGYYVHKCK

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

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

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

In some embodiments, the linker is selected from the amino acid sequences shown in Table 2. Table 2. Linker sequences SEQ ID NO. sequence description amino acid sequence 64 G4S linker GGGGS 37 (G4S) ₃ linker GGGGSGGGGSGGGGS 65 Linker-HA-Linker GGSGGSGGYPYDVPDYAGGGSGGGS 43 Linker GGSGGSGGGGGSGGGSGGGSGGGS 60 Linker GGSGGSGGGPEDEPGSGSGGGSGGGS 66 Linker GSGSGSGSGSEDEDEDEDEDGSGSGSGSGS 67 Linker GGGGSGGGGSGGGGSGGGGS 68 Linker GSGSGSGSEDGSGSGSGS 69 Linker GSGSGSGSGSGSGSGSGSGS 70 Linker GCGGSGGGGSGGGGS 71 Linker SGRGGGGSGGGGSGGGGSGGGGSSPA 72 Linker GGGGSGGGGSGGGGSGGGGSGGGG 73 Snorkel linker SGRGASSGSSGSGSQKKPRYEIRWKVVVISAILALVVLTVISLIILIMLWGSGMQSPA

In some embodiments, the linker comprises the amino acid sequence (GGGGS) _n (SEQ ID NO: 30), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, the linker consists of (GGGGS) _n linker (SEQ ID NO: 38), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, the linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 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 of 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 of 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 of 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 with exogenous fusion polypeptides or exogenous polypeptides (such as any of the exogenous polypeptides described herein) are known in the art. preamble ( signal ) sequence

In some embodiments where the enucleated erythroid cells include first and second exogenous polypeptides, the first exogenous fusion polypeptide or exogenous polypeptide (such as any of the exemplary exogenous polypeptides described herein) includes a leader (signal )sequence. In some embodiments, the leader sequence is selected from the sequences listed in Table 3. Table 3. Preamble (signal) sequence SEQ ID NO. sequence description amino acid sequence 35 GPA signal peptide MYGKIIFVLLLSEIVSISA 74 Ig heavy chain V region 3 signal sequence MGWSCIILFLVATATGVHS 75 light chain leader MRVPAQLLGLLLLWLPGARC Exemplary Enucleated Erythroid Cells

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

In some embodiments, the enucleated erythroid cells described herein (eg, human enucleated erythroid cells) are negative for (i.e., do not include) one or more minor blood group antigens, such as Le(a ^- b ^- ) (Lewis antigen system (Lewis antigen system)), Fy(a ^- b ^- ) (Duffy system), JK(a ^- b ^- ) (Kidd system), M ^- N ^- (MNS system), K ^- k ^- (Kell system), Lu(a ^- b ^- ) (Lutheran system) and H antigen negative (Bombay phenotype) or any combination thereof. In some embodiments, the enucleated erythroid cells are also type O and/or Rh ⁻ . Minor blood groups are described, for example, in Agarwal et al. "Blood group phenotype frequencies in blood donors from a tertiary care hospital in north India", Blood Res. 48(1):51-54, 2013, and Mitra et al. "Blood groups systems ”, Indian J. Anaesth. 58(5):524-528, 2014, the description of which is incorporated herein by reference. In some embodiments, the enucleated erythroid cells described herein (e.g., human enucleated erythroid cells) exhibit and do not include an exogenous polypeptide (e.g., any exogenous polypeptide described herein or known in the art) The osmotic membrane fragility of isolated, uncultured, enucleated erythroid cells is essentially the same. In some embodiments, the enucleated erythroid population has an osmotic fragility of less than 50% cell lysis at 0.3%, 0.35%, 0.4%, 0.45%, or 0.5% NaCl. In some embodiments, osmotic fragility is determined using the method described in Example 59 of WO 2015/073587, the description of which is incorporated herein by reference.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Nucleic acids encoding one or more exogenous polypeptides can be introduced into erythroid precursor cells prior to final differentiation into enucleated erythroid cells using a variety of DNA techniques including, for example, transient or stable transfection and gene therapy approaches.

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

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

Nucleic acids encoding fluorescent tracking molecules such as, for example, green fluorescent protein (GFP) can optionally be transfected into erythroid precursor cells using virus-based methods (Tao et al., Stem Cells 25:670-678 , 2007). Encapsulating cells containing DNA encoding enhanced green fluorescent protein (EGFP) or red fluorescent protein (e.g., DsRed-Express) are encapsulated using encapsulating cells such as, for example, the Phoenix-Eco cell line (distributed by Orbigen, San Diego, Calif.) ectopic retroviral vector. Encapsulating cell lines stably express the viral polypeptides required for proper viral encapsulation, including eg gag, pol and env. Phoenix-Eco cell supernatants from shed virus particles can be used to transduce erythroid precursor cells. In some cases, transduction can be performed on specially coated surfaces (such as, for example, fragments of recombinant fibronectin) to enhance the efficiency of retrovirus-mediated gene transfer (e.g., RetroNectin, Takara Bio USA, Madison, Wis. .). Cells were grown in RetroNectin-coated dishes with retroviral Phoenix-Eco supernatant and appropriate cofactors. Transduction can be repeated the next day. In this case, the percentage of erythroid precursors expressing EGFP or DsRed-Express can be assessed by FACS. Other reporter genes that can be used to assess transduction efficiency include, for example, β-galactosidase, chloramphenicol acetyltransferase, and luciferase, as well as low-affinity nerve growth factor receptor (LNGFR), as well as human cell surface CD24 antigen (Bierhuizen et al., Leukemia 13:605-613, 1999).

Non-viral vectors can be used to introduce nucleic acid encoding one or more exogenous polypeptides into erythroid precursor cells to generate engineered enucleated erythroid cells. A variety of delivery methods are available for introducing non-viral vectors into erythroid precursor cells, including chemical and physical methods. Non-viral vectors encoding exogenous polypeptides can be introduced into erythroid precursor cells using synthetic macromolecules such as cationic lipids and polymers (Papapetrou et al., Gene Therapy 12:S118-S130, 2005). Cationic liposomes form complexes with DNA, for example, through charge interactions. Positively charged DNA/lipid complexes bind to negatively charged cell surfaces and are taken up by cells through endocytosis. This approach can be used, for example, to transfect hematopoietic cells (see, eg, Keller et al., Gene Therapy 6:931-938, 1999). For erythroid precursor cells, plastid DNA (in serum-free medium, such as, for example, OptiMEM (Invitrogen, Carlsbad, CA)) was mixed with cationic liposomes (in serum-free medium) such as the commercially available transfection reagent Lipofectamine™ ( Invitrogen, Carlsbad, Calif.)) and incubated for at least 20 minutes to form complexes. The DNA/liposome complexes are added to erythroid precursor cells and allowed to incubate for 5 to 24 hours before analysis of transgenic expression of exogenous polypeptides. Alternatively, other commercially available liposome transfection reagents (eg, In vivo GeneSHUTTLE™, Qbiogene, Carlsbad, Calif.) can be used.

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

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

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

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

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

Erythroid precursor cells can be transfected non-virally with conventional expression vectors that cannot self-replicate in mammalian cells unless they are integrated into the gene body. Alternatively, erythroid precursor cells can be transfected with an episome vector that acts as a self-replicating genetic unit in the host nucleus without integration into the chromosome (Papapetrou et al., Gene Therapy 12:S118-S130, 2005). These vectors utilize genetic elements derived from viruses that normally replicate extrachromosomally in cells following latent infection, such as, for example, EBV, human polyomavirus BK, bovine papillomavirus-1 (BPV-1), herpes simplex Herpes virus-1 (HSV), and Simian virus 40 (SV40). Mammalian artificial chromosomes can also be used for non-viral gene transfer (Vanderbyl et al., Exp. Hematol. 33:1470-1476, 2005).

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

Exogenous nucleic acid may include a gene encoding an exogenous polypeptide not normally present on the cell surface (e.g., the surface of an enucleated erythroid cell) fused to a gene encoding an endogenous or native membrane polypeptide so that the cell Exogenous polypeptides are expressed on the surface. For example, an exogenous gene encoding an exogenous polypeptide can be colonized 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 at the GPI appendage of a GPI-linked membrane polypeptide. upstream of the junction site.

An elastic amino acid linker can be introduced between the two fusion genes using standard breeding methods. For example, a flexible linker is a polyglycine-polyserine linker, such as [Gly ₄ Ser] ₃ (SEQ ID NO: 37), commonly used to generate single-chain antibody fragments from full-length antibodies (Antibody Engineering: Methods & Protocols, B. Lo, ed., Humana Press, 2004, 576 pp.), or Ala-Gly-Ser-Thr polypeptides, such as those used to generate single-chain Arc suppressors (Robinson & Sauer, Proc. Nat' l. Acad. Sci. USA 95: 5929-34, 1998). In some embodiments, the flexible linker provides greater flexibility and steric freedom to the exogenous polypeptide than an equivalent construct without the flexible linker. This adds flexibility and can be used in applications requiring binding to a target (eg, an antibody or polypeptide, or an enzymatic reaction of a polypeptide whose active site must be accessible to a substrate (eg, target)).

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

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

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

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

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

The conditions disclosed herein can be easily adjusted by those skilled in the art with reference to the Life Technologies Neon system, so that different electroporators and/or different electroporation settings can be used with only routine experiments, and in terms of the disclosed methods, The particular electroporator described herein is not limited. In some embodiments, using the electroporation conditions described herein, cultured erythroid precursor cells are subjected to a first electroporation, then cultured for a desired period of time (optionally under differentiation conditions), and then subjected to a second re-poration. Electroporation. In some embodiments, cultured erythroid precursor cells are subjected to a first electroporation, then cultured for a desired period of time (optionally under differentiating conditions), and then subjected to a second, third, fourth, fifth, or second electroporation. Electroporation was performed six times. Depending on the situation, the culture period between the first and second, second and third electroporation, etc. may be changed. For example, the period between electroporations can be adjusted as desired, for example, the period can be 30 minutes, 1 hour, 6 hours, 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 48 hours, 3 days, 4 hours. days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days or 21 days. For example, on day 1 and day 2, day 1 and day 3, day 1 and day 4, day 1 and day 5, day 1 and day 6, day 1 and day 7 , Day 1 and Day 8, Day 1 and Day 9, Day 1 and Day 10, Day 1 and Day 11, Day 1 and Day 12, Day 1 and Day 13, Day 1 Erythroid precursor cells were electroporated on day 1 and day 14, day 1 and day 15, or day 1 and day 16. In another example, day 2 and day 3, day 2 and day 4, day 2 and day 5, day 2 and day 6, day 2 and day 7, day 2 day and day 8, day 2 and day 9, day 2 and day 10, day 2 and day 11, day 2 and day 12, day 2 and day 13, day 2 and day Cells were electroporated on day 14, day 2 and day 15, or day 2 and day 16. In yet another example, on days 3 and 4, days 3 and 5, days 3 and 6, days 3 and 7, days 3 and 8, days 3 and 9 day, 3 and 10 days, 3 and 11 days, 3 and 12 days, 3 and 13 days, 3 and 14 days, 3 and 15 days, or 3 and 16 days Electroporation of erythroid precursor cells was performed on the following day. In yet another example, on days 4 and 5, days 4 and 6, days 4 and 7, days 4 and 8, days 4 and 9, days Day 4 and Day 10, Day 4 and Day 11, Day 4 and Day 12, Day 4 and Day 13, Day 4 and Day 14, Day 4 and Day 15, or Day 4 Cells were electroporated on days 1 and 16. In yet another example, on days 5 and 6, days 5 and 7, days 5 and 8, days 5 and 9, days 5 and 10, days Cells were processed on days 5 and 11, days 5 and 12, days 5 and 13, days 5 and 14, days 5 and 15, or days 5 and 16 Electroporation. In yet another example, on days 6 and 7, days 6 and 8, days 6 and 9, days 6 and 10, days 6 and 11, days 6 Erythroid precursor cells were electroporated on days and 12, days 6 and 13, days 6 and 14, days 6 and 15, or days 6 and 16. In yet another example, on day 7 and day 8, day 7 and day 9, day 7 and day 10, day 7 and day 11, day 7 and day 12, day Erythroid precursor cells were electroporated on days 7 and 13, days 7 and 14, days 7 and 15, or days 7 and 16. In yet another example, on days 8 and 9, days 8 and 10, days 8 and 11, days 8 and 12, days 8 and 13, days Erythroid precursor cells were electroporated on days 8 and 14, days 8 and 15, or days 8 and 16. In yet another example, on days 9 and 10, days 9 and 11, days 9 and 12, days 9 and 13, days 9 and 14, days Erythroid precursor cells were electroporated on days 9 and 15, or days 9 and 16. In yet another example, on day 10 and day 11, day 10 and day 12, day 10 and day 13, day 10 and day 14, day 10 and day 15, or Erythroid precursor cells were electroporated on days 10 and 16. In yet another example, on day 11 and day 12, day 11 and day 13, day 11 and day 14, day 11 and day 15, or day 11 and day 16 Erythroid precursor cells were electroporated. In yet another example, the erythroid precursor cells can be electroporated on days 12 and 13, days 12 and 14, days 12 and 15, or days 12 and 16. In yet another example, the erythroid precursor cells can be electroporated on days 13 and 14, days 13 and 15, or days 13 and 16. In yet another example, the erythroid precursor cells can be electroporated on days 14 and 15, or days 14 and 16. Optionally, the erythroid precursor cells may be electroporated more than two times, such as three, four, five or six times, and the intervals may be selected as desired at any point in the cell differentiation process.

In some embodiments, using the electroporation conditions described herein, at differentiation stage 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 The cultured erythroid precursor cells were electroporated on the following day.

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

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

Polypeptides that form catalytic bonds (such as the SpyTag/SpyCatcher system) can be used to attach exogenous polypeptides to surfaces (eg, erythroid precursor cells or enucleated erythroid cells). The SpyTag polypeptide sequence can be expressed on the extracellular surface of erythroid precursor cells or enucleated erythroid cells. For example, a SpyTag polypeptide can be fused to the N-terminus of a type 1 or type 3 transmembrane polypeptide (eg, glycophorin A), to the C-terminus of a type 2 transmembrane polypeptide (eg, Kell), in frame Inserted within the extracellular terminus or extracellular loop of multiple membrane-penetrating polypeptides (such as band 3), fused to GPI-receptor polypeptides (such as CD55 or CD59), fused to lipid chain-anchored polypeptides, or fused to Peripheral Membrane Polypeptides. Exogenous polypeptides can be fused to SpyCatcher. Nucleic acids encoding SpyCatcher fusions can be expressed and secreted by the same erythroid precursor cells or enucleated erythroid cells that express SpyTag fusions. Alternatively, nucleic acid sequences encoding SpyCatcher fusions can be produced exogenously, eg, in bacterial, fungal, insect, mammalian or cell-free production systems. Following the reaction of the SpyTag and SpyCatcher polypeptides, a covalent bond will be formed which attaches the exogenous polypeptide to the surface of the erythroid precursor or enucleated erythroid cells. In one embodiment, the SpyTag polypeptide can be expressed as fused to the N-terminus of glycophorin A under the control of the Gatal promoter in erythroid cells. The exogenous polypeptide fused to the SpyCatcher polypeptide sequence can be expressed in the same erythrocyte under the control of the Gatal promoter. After expression of both fusion polypeptides, an isopeptide bond will be formed between the SpyTag and SpyCatcher polypeptides, thereby forming a covalent bond between the erythroid surface and the exogenous polypeptide.

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

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

In one embodiment, erythroid precursor cells or enucleated erythroid cells can express SpyTag fused to heme beta intracellularly. Erythroid precursor cells or enucleated erythroid cells can be genetically modified with a gene sequence including a heme promoter, a β-globin gene, and a SpyTag sequence such that after translation, the intracellular β-globin is fused to the SpyTag at the C-terminus. Additionally, erythroid precursor cells or enucleated erythroid cells express a gene directed by the Gatal promoter that encodes SpyCatcher-driven polypeptide expression (eg, phenylalanine hydroxylase (PAH) expression), thereby rendering intracellular A polypeptide (eg, PAH) is fused to SpyCatcher at the N-terminus. After expression of both fusion polypeptides, the SpyTag-bound β-globin binds to the SpyCatcher-bound polypeptide (e.g., PAH) in the intracellular space via an isopeptide bond, thereby anchoring the polypeptide (e.g., PAH) to the β-globin and is preserved during maturity.

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

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

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

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

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

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

Any of the enucleated erythroid cells described herein may be formulated as described, eg, in WO 2020/219909 (incorporated herein by reference). Method of increasing the number of NKG2D positive lymphocytes in an individual previously identified or diagnosed with a MICA positive, 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 an individual previously identified or diagnosed as having a MICA-positive, MICB-positive, or MICA/MICB-positive cancer, comprising administering to the individual An effective amount of enucleated erythroid cells (such as any of the exemplary enucleated erythroid cells described herein) comprising on their extracellular surface a first exogenous fusion polypeptide, the first exogenous fusion polypeptide Including (i) IL-15 or its functional fragments, and (ii) IL-15 receptor alpha or its functional fragments.

Also provided herein are methods of increasing the number of NKG2D-positive lymphocytes (e.g., NKG2D-positive NK cells) in an individual previously identified or diagnosed as having a MICA-positive, MICB-positive, or MICA/MICB-positive cancer, comprising administering to the individual An effective amount of enucleated erythroid cells (such as any of the exemplary enucleated erythroid cells described herein) comprising on their extracellular surface a first exogenous fusion polypeptide, the first exogenous fusion polypeptide Including (i) IL-15 or its functional fragments, and (ii) IL-15 receptor α or its functional fragments; and a second exogenous polypeptide comprising 4-1BBL polypeptide or its functional fragment.

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

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

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

Some embodiments of these methods may further comprise identifying or diagnosing that the individual has a MICA-positive cancer, an MICB-positive cancer, or a MICA/MICB-positive cancer. Some embodiments of these methods may further include identifying or diagnosing an individual with MICA-positive and HLA-E-negative cancers (MICA-positive/HLA-E-negative cancers), MICB-positive and HLA-E-negative cancers (MICB-positive/HLA-E-negative cancers). cancer), or MICA/MICB positive and HLA-E negative cancer (MICA/MICB positive cancer/HLA-E negative cancer).

For example, in some embodiments, one or more additional therapeutic agents include blocking, reducing and/or inhibiting PD-1 and PD-L1 or PD-L2, and/or blocking, reducing and/or inhibiting PD-1 and PD-L1 PD-L1 or PD-L2 binding agents (non-limiting examples include 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 MPDL328OA (ROCHE) one or more). In some embodiments, one or more additional therapeutic agents include blocking, reducing and/or inhibiting the activity of CTLA-4 and/or inhibiting the interaction of CTLA-4 with one or more of its receptors (e.g., CD80, CD86, AP2M1, SHP -2 and PPP2R5A) binding agents. For example, in some embodiments, the agent that inhibits CTLA-4 activity is an antibody, such as ipilimumab (MDX-010, MDX-101, Yervoy, BRISTOL MYERS SQUIBB) and/or tremelimumab anti (tremelimumab) (PFIZER). Furthermore, in some embodiments, the one or more additional therapeutic agents include blocking antibodies targeting immune checkpoint molecules such as, for example, BTLA, HVEM, TIM3, GALS, LAG3, VISTA, KIR, 2B4, CD160 (also known as BY55 ), CGEN-15049, CHK1 and CHK2 kinases, A2aR, CEACAM (eg, CEACAM-1, CEACAM-3 and/or CEACAM-5), GITR, GITRL, Galectin-9, CD244, CD160, TIGIT, SIRPα , ICOS, CD172a, TMIGD2 and various B-7 family ligands (including but not limited to B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H5 , B7-H6 and B7-H7). In some embodiments, 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 example Includes 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 therapeutic agents include one or more molecules (e.g., small molecules) that block, reduce and/or inhibit the activity of CTLA-4 and/or the interaction of CTLA-4 with one or more of its regulated Binding of antibodies (eg, CD80, CD86, AP2M1, SHP-2, and PPP2R5A).

In some embodiments of the methods, the methods result in an increase (e.g., at least 5%, at least 10%, at least 15%, at least 20% increase) in the number of NKG2D-positive lymphocytes (e.g., NKG2D-positive NK cells) in the individual %, increase by at least 25%, increase by at least 30%, increase by at least 35%, increase by at least 40%, increase by at least 45%, increase by at least 50%, increase by at least 55%, increase by at least 60%, increase by at least 65%, increase by at least 70% %, increase by at least 75%, increase by at least 80%, increase by at least 85%, increase by at least 90%, increase by at least 95%, increase by at least 100%, increase by at least 120%, increase by at least 140%, increase by at least 160%, increase by at least 180 %, increase by at least 200%, increase by at least 220%, increase by at least 240%, increase by at least 260%, increase by at least 280%, increase by at least 300%, increase by at least 350%, increase by at least 400%, increase by at least 450%, increase by at least 500 %, increase by at least 550%, increase by at least 600%, increase by at least 650%, increase by at least 700%, increase by at least 750%, increase by at least 800%, increase by at least 850%, increase by at least 900%, increase by at least 950%, or increase by at least 1000%) (eg, compared to the number of NKG2D-positive lymphocytes (eg, NKG2D-positive NK cells) in the subject prior to administration).

In some embodiments of the methods, the methods result in an increase (e.g., an increase of at least 5%, an increase of at least 10%, an increase of at least 15%, increase by at least 20%, increase by at least 25%, increase by at least 30%, increase by at least 35%, increase by at least 40%, increase by at least 45%, increase by at least 50%, increase by at least 55%, increase by at least 60%, increase by at least 65%, increase by at least 70%, increase by at least 75%, increase by at least 80%, increase by at least 85%, increase by at least 90%, increase by at least 95%, increase by at least 100%, increase by at least 120%, increase by at least 140%, increase by at least 160%, increase by at least 180%, increase by at least 200%, increase by at least 220%, increase by at least 240%, increase by at least 260%, increase by at least 280%, increase by at least 300%, increase by at least 350%, increase by at least 400%, increase by at least 450%, increase by at least 500%, increase by at least 550%, increase by at least 600%, increase by at least 650%, increase by at least 700%, increase by at least 750%, increase by at least 800%, increase by at least 850%, increase by at least 900%, increase by at least 950%, or an increase of at least 1000%) (eg, compared to the number of NKG2D-positive/NKG2A-negative cells (eg, NKG2D-positive/NKG2A-negative NK cells) in the subject prior to administration).

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

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

Some embodiments of the methods described herein result in a maximum fold change increase of at least 0.1-fold, an increase of at least 0.2-fold 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 individual's blood , increased by at least 0.3 times, increased by at least 0.4 times, increased by at least 0.5 times, increased by at least 0.6 times, increased by at least 0.7 times, increased by at least 0.8 times, increased by at least 0.9 times, increased by at least 1.0 times, increased by at least 1.5 times, increased by at least 2.0 times , increase by at least 2.5 times, increase by at least 3.0 times, increase by at least 3.5 times, increase by at least 4.0 times, increase by at least 4.5 times, increase by at least 5.0 times, increase by at least 5.5 times, increase by at least 6.0 times, increase by at least 6.5 times, increase by at least 7.0 times , increased by at least 7.5-fold, increased by at least 8.0-fold, increased by at least 8.5-fold, increased by at least 9.0-fold, increased by at least 9.5-fold, or increased by at least 10-fold (for example, compared with NKG2D-positive lymphocytes in the individual's blood before administration (such as NKG2D-positive NK cells) compared to the ratio of NKG2A-positive lymphocytes (eg, NKG2A-positive NK cells)).

Some embodiments of the methods described herein result in a maximum fold increase in the absolute number of NK cells in the individual's blood, 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 times, increase by at least 0.7 times, increase by at least 0.8 times, increase by at least 0.9 times, increase by at least 1.0 times, increase by at least 1.5 times, increase by at least 2.0 times, increase by at least 2.5 times, increase by at least 3.0 times, increase by at least 3.5 times, increase by at least 4.0 times fold, increased by at least 4.5 fold, or increased by at least 5.0 fold (eg, compared to the maximum fold increase in absolute number of NK cells in the individual's blood prior to administration).

Some embodiments of the methods described herein result in a maximum fold increase in the absolute number of CD56 ^bright lymphocytes (e.g., CD56 ^bright NK cells) in the individual's blood, 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 , increase by at least 0.5 times, increase by at least 0.6 times, increase by at least 0.7 times, increase by at least 0.8 times, increase by at least 0.9 times, increase by at least 1.0 times, increase by at least 1.5 times, increase by at least 2.0 times, increase by at least 2.5 times, increase by at least 3.0 times , an increase of at least 3.5-fold, an increase of at least 4.0-fold, an increase of at least 4.5-fold, or an increase of at least 5.0-fold (e.g., compared to the greatest fold increase in the absolute number of CD56 ^bright lymphocytes (e.g., CD56 ^bright NK cells) in the individual's blood prior to administration Compare).

Some embodiments of the methods described herein result in a maximum fold increase in the absolute number of CD16 ⁺ CD56 ^dim lymphocytes (e.g., CD16 ⁺ CD56 ^dim NK cells) in the individual's blood, at least a 0.1-fold increase, at least a 0.2-fold increase, at least a 0.3-fold increase, Increased by at least 0.4 times, increased by at least 0.5 times, increased by at least 0.6 times, increased by at least 0.7 times, increased by at least 0.8 times, increased by at least 0.9 times, increased by at least 1.0 times, increased by at least 1.5 times, increased by at least 2.0 times, increased by at least 2.5 times, Increased by at least 3.0-fold, increased by at least 3.5-fold, increased by at least 4.0-fold, increased by at least 4.5-fold, or increased by at least 5.0-fold (e.g., compared to CD16 ⁺ CD56 ^dim lymphocytes (e.g., CD16 ⁺ CD56 ^dim NK cells) in the individual's blood prior to administration compared to the largest multiple increase in absolute quantity).

Some embodiments of the methods described herein result in a maximum fold increase in the absolute number of HLA-DR ⁺ lymphocytes (e.g., HLA-DR ⁺ NK cells) in the individual's blood, at least a 0.1-fold increase, at least a 0.2-fold increase, at least a 0.3-fold increase, Increased by at least 0.4 times, increased by at least 0.5 times, increased by at least 0.6 times, increased by at least 0.7 times, increased by at least 0.8 times, increased by at least 0.9 times, increased by at least 1.0 times, increased by at least 1.5 times, increased by at least 2.0 times, increased by at least 2.5 times, Increased by at least 3.0-fold, increased by at least 3.5-fold, increased by at least 4.0-fold, increased by at least 4.5-fold, or increased by at least 5.0-fold (e.g., compared to the number of HLA-DR ⁺ lymphocytes (e.g., HLA-DR ⁺ NK cells) in the individual before administration compared to the largest multiple increase in absolute quantity).

In some embodiments, the individual was previously diagnosed or identified as having a MICA-positive cancer (eg, any of the exemplary MICA-positive cancers described herein). In some embodiments, the individual was previously diagnosed or identified as having a MICA positive/HLA-E negative cancer. Some embodiments of these methods may further comprise identifying or diagnosing that the individual has a MICA positive cancer. Some embodiments of these methods may further comprise identifying or diagnosing the individual as having a MICA positive/HLA-E negative cancer.

Non-limiting examples of MICA-positive cancers include: adrenocortical carcinoma, cholangiocarcinoma, pancreatic cancer, renal cancer, thyroid cancer, mesothelioma, cutaneous melanoma, colorectal cancer, squamous cell carcinoma of the cervix, and endocervical glandular carcinoma. cancer. In some embodiments, MICA-positive cancers include, but are not limited to, adrenocortical cancer, 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 neoplasms diffuse large b-cell lymphoma, renal carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, renal chromophobe, liver cancer, lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, interstitial Tumors, ovarian cancer, pancreatic cancer, rectal cancer, skin cancer, stomach cancer, testicular cancer, thymoma, thyroid cancer, uterine cancer, uterine endometrial cancer, uterine carcinosarcoma, and uveal melanoma.

In some embodiments, the individual was previously diagnosed or identified as having an MICB-positive cancer (eg, any of the exemplary MICB-positive cancers described herein). In some embodiments, the individual was previously diagnosed or identified as having an MICB positive/HLA-E negative cancer. Some embodiments of these methods may further comprise identifying or diagnosing the individual as having an MICB positive cancer. Some embodiments of these methods may further comprise identifying or diagnosing that the individual has an MICB positive/HLA-E negative cancer.

Non-limiting examples of MICB-positive cancers include: acute myelogenous leukemia, lymphoid neoplasm diffuse large B-cell lymphoma, testicular germ cell tumor, gastric adenocarcinoma, ovarian serous cystadenocarcinoma, esophageal cancer, and lung cancer. In some embodiments, MICB-positive cancers include, but are not limited to: adrenocortical cancer, 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 neoplasms diffuse large b-cell lymphoma, renal carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, renal chromophobe, liver cancer, lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, interstitial Tumors, ovarian cancer, pancreatic cancer, rectal cancer, skin cancer, stomach cancer, testicular cancer, thymoma, thyroid cancer, uterine cancer, uterine endometrial cancer, uterine carcinosarcoma, and uveal melanoma.

In some embodiments, the individual was previously diagnosed or identified as having a MICA/MICB positive cancer (eg, any of the exemplary MICA/MICB positive cancers described herein). In some embodiments, the individual was previously diagnosed or identified as having a MICA/MICB positive/HLA-E negative cancer. Some embodiments of these methods may further comprise identifying or diagnosing the individual as having a MICA/MICB positive cancer. Some embodiments of these methods may further comprise identifying or diagnosing that the individual has a MICA/MICB positive/HLA-E negative cancer.

Non-limiting examples of MICA/MICB positive cancers include: adrenocortical carcinoma, acute myeloid leukemia, pancreatic cancer, cholangiocarcinoma, kidney cancer, squamous cell carcinoma of the cervix, endocervical carcinoma, colorectal cancer, mesothelioma, Cutaneous epidermal melanoma, and lung cancer. In some embodiments, MICA/MICB positive cancers include, but are not limited to, adrenocortical cancer, anal cancer, bile duct cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer , acute myelogenous leukemia, lymphoid neoplasms diffuse large b-cell lymphoma, renal carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, renal chromophobe, liver cancer, lung cancer, lung adenocarcinoma, lung squamous cell carcinoma , mesothelioma, ovarian cancer, pancreatic cancer, rectal cancer, skin cancer, gastric cancer, testicular cancer, thymoma, thyroid cancer, uterine cancer, uterine endometrial cancer, uterine carcinosarcoma, and uveal melanoma.

Enucleated erythroid cells can be administered to an individual as described herein using any dose and with any frequency. Method of increasing the ratio of NKG2D concentration to NKG2A concentration on the surface of lymphocyte cells

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 an individual previously identified or diagnosed as having a MICA-positive, MICB-positive, or MICA/MICB-positive cancer, comprising adding The subject is administered a therapeutically effective amount of a pharmaceutical composition comprising a population of enucleated erythroid cells comprising on their extracellular surface a first exogenous polypeptide comprising (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof. 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 an individual previously identified or diagnosed as having a MICA-positive, MICB-positive, or MICA/MICB-positive cancer, comprising administering to an individual a pharmaceutical composition comprising a population of enucleated erythroid cells comprising on their extracellular surface a first exogenous fusion polypeptide comprising ( i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor α or a functional fragment thereof; and a second exogenous polypeptide on the extracellular surface thereof, the second exogenous polypeptide comprising 4- 1BBL or a functional fragment thereof.

Some embodiments of these methods result in an increase of at least 1%, an increase of at least 5%, an increase of at least 10%, an increase of at least 15%, an increase of at least 20% in the ratio of NKG2D concentration to NKG2A concentration on the cell surface of lymphocytes (e.g., NK cells) in the individual , at least 30% increase, at least 40% increase, at least 50% increase, at least 60% increase, at least 70% increase, at least 80% increase, at least 90% increase, at least 100% increase, at least 110% increase, at least 120% increase , increase by at least 130%, increase by at least 140%, increase by at least 150%, increase by at least 160%, increase by at least 170%, increase by at least 180%, increase by at least 190%, increase by at least 200%, increase by at least 250%, increase by at least 300% , increase by at least 350%, increase by at least 400%, increase by at least 450%, increase by at least 500%, increase by at least 550%, increase by at least 600%, increase by at least 650%, increase by at least 700%, increase by at least 750%, increase by at least 800% , increased by at least 850%, increased by at least 900%, increased by at least 950%, or increased by at least 1000% (e.g., 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 an individual prior to administration) . Some embodiments of these methods result in an about 0.01-fold to about 10-fold increase (or any exemplary subrange of this range described herein) in the ratio of NKG2D concentration to NKG2A concentration on the cell surface of lymphocytes (e.g., NK cells) in an individual (e.g. , compared with the ratio of NKG2D concentration to NKG2A concentration on the cell surface of lymphocytes (eg, NK cells) in the individual before administration).

Some embodiments of the methods described herein result in a maximum fold change increase of at least 0.1-fold, an increase of at least 0.2-fold 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 individual's blood , increased by at least 0.3 times, increased by at least 0.4 times, increased by at least 0.5 times, increased by at least 0.6 times, increased by at least 0.7 times, increased by at least 0.8 times, increased by at least 0.9 times, increased by at least 1.0 times, increased by at least 1.5 times, increased by at least 2.0 times , increase by at least 2.5 times, increase by at least 3.0 times, increase by at least 3.5 times, increase by at least 4.0 times, increase by at least 4.5 times, increase by at least 5.0 times, increase by at least 5.5 times, increase by at least 6.0 times, increase by at least 6.5 times, increase by at least 7.0 times , an increase of at least 7.5-fold, an increase of at least 8.0-fold, an increase of at least 8.5-fold, an increase of at least 9.0-fold, an increase of at least 9.5-fold, or an increase of at least 10-fold (or any exemplary subrange of this range described herein) (e.g., with administration Ratio of NKG2D-positive lymphocytes (eg, NKG2D-positive NK cells) to NKG2A-positive lymphocytes (eg, NKG2A-positive NK cells) in the blood of the former individual). Some embodiments of the methods described herein result in a maximum fold change of about 0.01-fold to about a 10-fold increase 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 individual's blood (eg, compared to the ratio of NKG2D-positive lymphocytes (eg, NKG2D-positive NK cells) to NKG2A-positive lymphocytes (eg, NKG2A-positive NK cells) in the individual's blood prior to administration).

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

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

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

Some embodiments of these methods may further comprise identifying or diagnosing that the individual has a MICA-positive cancer, an MICB-positive cancer, or a MICA/MICB-positive cancer. Some embodiments of these methods may further comprise identifying or diagnosing that the individual has a MICA positive/HLA-E negative cancer, an MICB positive/HLA-E negative cancer, or a MICA/MICB positive/HLA-E negative cancer.

For example, in some embodiments, one or more additional therapeutic agents include blocking, reducing and/or inhibiting PD-1 and PD-L1 or PD-L2, and/or blocking, reducing and/or inhibiting PD-1 and PD-L1 PD-L1 or PD-L2 binding agents (non-limiting examples include Nivolumab (ONO-4538/BMS-936558, MDX1106, OPDIVO, BRISTOL MYERS SQUIBB), pembrolizumab (KEYTRUDA, MERCK), Piddi One or more of Zizumab (CT-011, CURE TECH), MK-3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), and MPDL328OA (ROCHE)). In some embodiments, one or more additional therapeutic agents include blocking, reducing and/or inhibiting the activity of CTLA-4 and/or the interaction of CTLA-4 with one or more of its receptors (e.g., CD80, CD86, AP2M1, SHP- 2 and PPP2R5A) binding agents. For example, in some embodiments, the agent that inhibits CTLA-4 activity is an antibody, such as ipilimumab (MDX-010, MDX-101, Yervoy, BRISTOL MYERS SQUIBB) and/or tremelimumab (PFIZER). Additionally, in some embodiments, the one or more additional therapeutic agents include blocking antibodies that target immune checkpoint molecules such as, for example, BTLA, HVEM, TIM3, GALS, LAG3, VISTA, KIR, 2B4, CD160 (also known as BY55), CGEN-15049, CHK1 and CHK2 kinases, A2aR, CEACAM (eg, CEACAM-1, CEACAM-3 and/or CEACAM-5), GITR, GITRL, Galectin-9, CD244, CD160, TIGIT, SIRPα, ICOS, CD172a, TMIGD2 and various B-7 family ligands (including but not limited to B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7- H4, B7-H5, B7-H6 and B7-H7). In some embodiments, 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 example Includes 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 therapeutic agents include one or more molecules (e.g., small molecules) that block, reduce and/or inhibit the activity of CTLA-4 and/or the interaction of CTLA-4 with one or more of its regulated binding to CD80, CD86, AP2M1, SHP-2, and PPP2R5A.

In some embodiments, the individual was previously diagnosed or identified as having a MICA-positive cancer (eg, any of the exemplary MICA-positive cancers described herein). In some embodiments, the individual was previously diagnosed or identified as having a MICA positive/HLA-E negative cancer. Some embodiments of these methods may further comprise identifying or diagnosing that the individual has a MICA positive cancer. Some embodiments of these methods may further comprise identifying or diagnosing the individual as having a MICA positive/HLA-E negative cancer.

Non-limiting examples of MICA-positive cancers include: adrenocortical carcinoma, cholangiocarcinoma, pancreatic cancer, renal cancer, thyroid cancer, mesothelioma, cutaneous melanoma, colorectal cancer, squamous cell carcinoma of the cervix, and endocervical glandular carcinoma. cancer. In some embodiments, MICA-positive cancers include, but are not limited to: adrenocortical cancer, 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 neoplasms diffuse large b-cell lymphoma, renal carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, renal chromophobe, liver cancer, lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, interstitial Tumors, ovarian cancer, pancreatic cancer, rectal cancer, skin cancer, stomach cancer, testicular cancer, thymoma, thyroid cancer, uterine cancer, uterine endometrial cancer, uterine carcinosarcoma, and uveal melanoma.

In some embodiments, the individual was previously diagnosed or identified as having an MICB-positive cancer (eg, any of the exemplary MICB-positive cancers described herein). In some embodiments, the individual was previously diagnosed or identified as having an MICB positive/HLA-E negative cancer. Some embodiments of these methods may further comprise identifying or diagnosing the individual as having an MICB positive cancer. Some embodiments of these methods may further comprise identifying or diagnosing that the individual has an MICB positive/HLA-E negative cancer.

Non-limiting examples of MICB-positive cancers include: acute myelogenous leukemia, lymphoid neoplasm diffuse large B-cell lymphoma, testicular germ cell tumor, gastric adenocarcinoma, ovarian serous cystadenocarcinoma, esophageal cancer, and lung cancer. In some embodiments, MICB-positive cancers include, but are not limited to: adrenocortical cancer, 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 neoplasms diffuse large b-cell lymphoma, renal carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, renal chromophobe, liver cancer, lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, interstitial Tumors, ovarian cancer, pancreatic cancer, rectal cancer, skin cancer, stomach cancer, testicular cancer, thymoma, thyroid cancer, uterine cancer, uterine endometrial cancer, uterine carcinosarcoma, and uveal melanoma.

In some embodiments, the individual was previously diagnosed or identified as having a MICA/MICB positive cancer (eg, any of the exemplary MICA/MICB positive cancers described herein). In some embodiments, the individual was previously diagnosed or identified as having a MICA/MICB positive/HLA-E negative cancer. Some embodiments of these methods may further comprise identifying or diagnosing the individual as having a MICA/MICB positive cancer. Some embodiments of these methods may further comprise identifying or diagnosing that the individual has a MICA/MICB positive/HLA-E negative cancer.

Non-limiting examples of MICA/MICB positive cancers include: adrenocortical carcinoma, acute myeloid leukemia, pancreatic cancer, cholangiocarcinoma, kidney cancer, squamous cell carcinoma of the cervix, endocervical carcinoma, colorectal cancer, mesothelioma, Cutaneous epidermal melanoma, and lung cancer. In some embodiments, MICA/MICB positive cancers include, but are not limited to: adrenocortical cancer, anal cancer, bile duct cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer , acute myelogenous leukemia, lymphoid neoplasm diffuse large b-cell lymphoma, renal carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, renal chromophobe, liver cancer, lung cancer, lung adenocarcinoma, lung squamous cell carcinoma , mesothelioma, ovarian cancer, pancreatic cancer, rectal cancer, skin cancer, gastric cancer, testicular cancer, thymoma, thyroid cancer, uterine cancer, uterine endometrial cancer, uterine carcinosarcoma, and uveal melanoma. Methods of treating individuals with MICA positive cancer

Also provided herein are methods of treating an individual previously identified or diagnosed as having a MICA-positive cancer, such as any of the exemplary MICA-positive cancers described herein, comprising administering to the individual a therapeutically effective amount of an enucleated erythroid cell population, such as those described herein. any of the exemplary enucleated erythroid cells described above) comprising on their extracellular surface a first exogenous fusion polypeptide comprising (i) IL-15 or a functional fragment thereof , and (ii) IL-15 receptor alpha or a functional fragment thereof.

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

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

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

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

For example, in some embodiments, one or more additional therapeutic agents include blocking, reducing and/or inhibiting PD-1 and PD-L1 or PD-L2, and/or blocking, reducing and/or inhibiting PD-1 and PD-L1 PD-L1 or PD-L2 binding agents (non-limiting examples include Nivolumab (ONO-4538/BMS-936558, MDX1106, OPDIVO, BRISTOL MYERS SQUIBB), pembrolizumab (KEYTRUDA, MERCK), Piddi One or more of Zizumab (CT-011, CURE TECH), MK-3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), and MPDL328OA (ROCHE)). In some embodiments, one or more additional therapeutic agents include blocking, reducing and/or inhibiting the activity of CTLA-4 and/or the interaction of CTLA-4 with one or more of its receptors (e.g., CD80, CD86, AP2M1, SHP- 2 and PPP2R5A) binding agents. For example, in some embodiments, the agent that inhibits CTLA-4 activity is an antibody, such as ipilimumab (MDX-010, MDX-101, Yervoy, BRISTOL MYERS SQUIBB) and/or tremelimumab (PFIZER). Additionally, in some embodiments, the one or more additional therapeutic agents include blocking antibodies that target immune checkpoint molecules such as, for example, BTLA, HVEM, TIM3, GALS, LAG3, VISTA, KIR, 2B4, CD160 (also known as BY55), CGEN-15049, CHK1 and CHK2 kinases, A2aR, CEACAM (eg, CEACAM-1, CEACAM-3 and/or CEACAM-5), GITR, GITRL, Galectin-9, CD244, CD160, TIGIT, SIRPα, ICOS, CD172a, TMIGD2 and various B-7 family ligands (including but not limited to B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7- H4, B7-H5, B7-H6 and B7-H7). In some embodiments, 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 example Includes 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 therapeutic agents include one or more molecules (e.g., small molecules) that block, reduce and/or inhibit the activity of CTLA-4 and/or the interaction of CTLA-4 with one or more of its regulated binding to CD80, CD86, AP2M1, SHP-2, and PPP2R5A.

Some embodiments of these methods may further comprise identifying or diagnosing that the individual has a MICA positive cancer. Some embodiments of these methods may further comprise identifying or diagnosing the individual as having a MICA positive/HLA-E negative cancer.

Non-limiting examples of MICA-positive cancers include: adrenocortical carcinoma, cholangiocarcinoma, pancreatic cancer, renal cancer, thyroid cancer, mesothelioma, cutaneous melanoma, colorectal cancer, squamous cell carcinoma of the cervix, and endocervical glandular carcinoma. cancer. In some embodiments, MICA-positive cancers include, but are not limited to: adrenocortical cancer, 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 neoplasms diffuse large b-cell lymphoma, renal carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, renal chromophobe, liver cancer, lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, interstitial Tumors, ovarian cancer, pancreatic cancer, rectal cancer, skin cancer, stomach cancer, testicular cancer, thymoma, thyroid cancer, uterine cancer, uterine endometrial cancer, uterine carcinosarcoma, and uveal melanoma.

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

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

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

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

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

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

For example, in some embodiments, one or more additional therapeutic agents include blocking, reducing and/or inhibiting PD-1 and PD-L1 or PD-L2, and/or blocking, reducing and/or inhibiting PD-1 and PD-L1 PD-L1 or PD-L2 binding agents (non-limiting examples include Nivolumab (ONO-4538/BMS-936558, MDX1106, OPDIVO, BRISTOL MYERS SQUIBB), pembrolizumab (KEYTRUDA, MERCK), Piddi One or more of Zizumab (CT-011, CURE TECH), MK-3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), and MPDL328OA (ROCHE)). In some embodiments, one or more additional therapeutic agents include blocking, reducing and/or inhibiting the activity of CTLA-4 and/or the interaction of CTLA-4 with one or more of its receptors (e.g., CD80, CD86, AP2M1, SHP- 2 and PPP2R5A) binding agents. For example, in some embodiments, the agent that inhibits CTLA-4 activity is an antibody, such as ipilimumab (MDX-010, MDX-101, Yervoy, BRISTOL MYERS SQUIBB) and/or tremelimumab (PFIZER). Additionally, in some embodiments, the one or more additional therapeutic agents include blocking antibodies that target immune checkpoint molecules such as, for example, BTLA, HVEM, TIM3, GALS, LAG3, VISTA, KIR, 2B4, CD160 (also known as BY55), CGEN-15049, CHK1 and CHK2 kinases, A2aR, CEACAM (eg, CEACAM-1, CEACAM-3 and/or CEACAM-5), GITR, GITRL, Galectin-9, CD244, CD160, TIGIT, SIRPα, ICOS, CD172a, TMIGD2 and various B-7 family ligands (including but not limited to B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7- H4, B7-H5, B7-H6 and B7-H7). In some embodiments, 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 example Includes 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 therapeutic agents include one or more molecules (e.g., small molecules) that block, reduce and/or inhibit the activity of CTLA-4 and/or the interaction of CTLA-4 with one or more of its regulated binding to CD80, CD86, AP2M1, SHP-2, and PPP2R5A.

Some embodiments of these methods may further comprise identifying or diagnosing the individual as having an MICB positive cancer. Some embodiments of these methods may further comprise identifying or diagnosing that the individual has an MICB positive/HLA-E negative cancer.

Non-limiting examples of MICB-positive cancers include: acute myelogenous leukemia, lymphoid neoplasm diffuse large B-cell lymphoma, testicular germ cell tumor, gastric adenocarcinoma, ovarian serous cystadenocarcinoma, esophageal cancer, and lung cancer. In some embodiments, MICB-positive cancers include, but are not limited to: adrenocortical cancer, cholangiocarcinoma, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, acute myelogenous leukemia , Lymphatic neoplasm Diffuse large B-cell lymphoma, Kidney cancer, Renal clear cell carcinoma, 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 cancer, uterine carcinosarcoma, and uveal melanoma.

Enucleated erythroid cells can be administered to an individual as described herein using any dose and with any frequency. Methods of treating individuals with MICA/MICB positive cancer

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

Also provided herein are methods of treating an individual previously identified or diagnosed as having a MICA/MICB-positive cancer, such as any of the exemplary MICA/MICB-positive cancers described herein, comprising administering to the individual a therapeutically effective amount of enucleated erythroid cells ( Such as any of the exemplary enucleated erythroid cells described herein), which include on their extracellular surface a first exogenous fusion polypeptide comprising (i) IL-15 or Its functional fragment, and (ii) IL-15 receptor α or its functional fragment; and a second exogenous polypeptide, the second exogenous polypeptide includes 4-1BBL polypeptide or its functional fragment.

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

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

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

For example, in some embodiments, one or more additional therapeutic agents include blocking, reducing and/or inhibiting PD-1 and PD-L1 or PD-L2, and/or blocking, reducing and/or inhibiting PD-1 and PD-L1 PD-L1 or PD-L2 binding agents (non-limiting examples include Nivolumab (ONO-4538/BMS-936558, MDX1106, OPDIVO, BRISTOL MYERS SQUIBB), pembrolizumab (KEYTRUDA, MERCK), Piddi One or more of Zizumab (CT-011, CURE TECH), MK-3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), and MPDL328OA (ROCHE)). In some embodiments, one or more additional therapeutic agents include blocking, reducing and/or inhibiting the activity of CTLA-4 and/or the interaction of CTLA-4 with one or more of its receptors (e.g., CD80, CD86, AP2M1, SHP- 2 and PPP2R5A) binding agents. For example, in some embodiments, the agent that inhibits CTLA-4 activity is an antibody, such as ipilimumab (MDX-010, MDX-101, Yervoy, BRISTOL MYERS SQUIBB) and/or tremelimumab (PFIZER). Additionally, in some embodiments, the one or more additional therapeutic agents include blocking antibodies that target immune checkpoint molecules such as, for example, BTLA, HVEM, TIM3, GALS, LAG3, VISTA, KIR, 2B4, CD160 (also known as BY55), CGEN-15049, CHK1 and CHK2 kinases, A2aR, CEACAM (eg, CEACAM-1, CEACAM-3 and/or CEACAM-5), GITR, GITRL, Galectin-9, CD244, CD160, TIGIT, SIRPα, ICOS, CD172a, TMIGD2 and various B-7 family ligands (including but not limited to B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7- H4, B7-H5, B7-H6 and B7-H7). In some embodiments, 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 example Includes 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 therapeutic agents include one or more molecules (e.g., small molecules) that block, reduce and/or inhibit the activity of CTLA-4 and/or the interaction of CTLA-4 with one or more of its regulated binding to CD80, CD86, AP2M1, SHP-2, and PPP2R5A.

Some embodiments of these methods may further comprise identifying or diagnosing the individual as having a MICA/MICB positive cancer. Some embodiments of these methods may further comprise identifying or diagnosing that the individual has a MICA/MICB positive/HLA-E negative cancer.

Non-limiting examples of MICA/MICB positive cancers include: adrenocortical carcinoma, acute myeloid leukemia, pancreatic cancer, cholangiocarcinoma, kidney cancer, squamous cell carcinoma of the cervix, endocervical carcinoma, colorectal cancer, mesothelioma, Cutaneous epidermal melanoma, and lung cancer. In some embodiments, MICA/MICB positive cancers include, but are not limited to: adrenocortical cancer, anal cancer, bile duct cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, esophagus cancer, eye cancer, head and neck cancer , acute myelogenous leukemia, lymphoid neoplasms diffuse large b-cell lymphoma, renal carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, renal chromophobe, liver cancer, lung cancer, lung adenocarcinoma, lung squamous cell carcinoma , mesothelioma, ovarian cancer, pancreatic cancer, rectal cancer, skin cancer, gastric cancer, testicular cancer, thymoma, thyroid cancer, uterine cancer, uterine endometrial cancer, uterine carcinosarcoma, and uveal melanoma.

Enucleated erythroid cells can be administered to an individual as described herein using any dosage and with any frequency. Method of reducing the number and / or proliferation of MICA- positive cancer cells in an individual previously identified or diagnosed with MICA -positive cancer

Also provided herein is the reduction of MICA-positive cancer cells (such as described herein or in the art) in an individual previously identified or diagnosed as having a MICA-positive cancer (such as any exemplary MICA-positive cancer described herein or known in the art). methods for the number and/or proliferation of any exemplary MICA-positive cancer cells known in the art, comprising administering to a subject a therapeutically effective amount of enucleated erythroid cells (such as any of the exemplary enucleated erythroid cells described herein), which The enucleated erythroid cell comprises on its extracellular surface a first exogenous fusion polypeptide comprising (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or Its functional fragment.

Also provided herein is the reduction of MICA-positive cancer cells (such as any of the exemplary MICA-positive cancers described herein or known in the art) in an individual previously identified or diagnosed as having a MICA-positive cancer (such as any of the exemplary MICA-positive cancers described herein). MICA-positive cancer cells) number and/or proliferation, comprising administering to an individual a therapeutically effective amount of enucleated erythroid cells (such as any of the exemplary enucleated erythroid cells described herein), the enucleated erythroid cells in their cells The outer surface includes a first exogenous fusion polypeptide comprising (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof; and a second Exogenous polypeptides, the second exogenous polypeptides include 4-1BBL polypeptides or functional fragments thereof.

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

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

In some embodiments, the MICA positive cancer cells are MICA positive/HLA-E negative cancer cells.

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

For example, in some embodiments, one or more additional therapeutic agents include blocking, reducing and/or inhibiting PD-1 and PD-L1 or PD-L2, and/or blocking, reducing and/or inhibiting PD-1 and PD-L1 PD-L1 or PD-L2 binding agents (non-limiting examples include Nivolumab (ONO-4538/BMS-936558, MDX1106, OPDIVO, BRISTOL MYERS SQUIBB), pembrolizumab (KEYTRUDA, MERCK), Piddi One or more of Zizumab (CT-011, CURE TECH), MK-3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), and MPDL328OA (ROCHE)). In some embodiments, one or more additional therapeutic agents include blocking, reducing and/or inhibiting the activity of CTLA-4 and/or the interaction of CTLA-4 with one or more of its receptors (e.g., CD80, CD86, AP2M1, SHP- 2 and PPP2R5A) binding agents. For example, in some embodiments, the agent that inhibits CTLA-4 activity is an antibody, such as ipilimumab (MDX-010, MDX-101, Yervoy, BRISTOL MYERS SQUIBB) and/or tremelimumab (PFIZER). Additionally, in some embodiments, the one or more additional therapeutic agents include blocking antibodies that target immune checkpoint molecules such as, for example, BTLA, HVEM, TIM3, GALS, LAG3, VISTA, KIR, 2B4, CD160 (also known as BY55), CGEN-15049, CHK1 and CHK2 kinases, A2aR, CEACAM (eg, CEACAM-1, CEACAM-3 and/or CEACAM-5), GITR, GITRL, Galectin-9, CD244, CD160, TIGIT, SIRPα, ICOS, CD172a, TMIGD2 and various B-7 family ligands (including but not limited to B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7- H4, B7-H5, B7-H6 and B7-H7). In some embodiments, 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 example Includes 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 therapeutic agents include one or more molecules (e.g., small molecules) that block, reduce and/or inhibit the activity of CTLA-4 and/or the interaction of CTLA-4 with one or more of its regulated binding to CD80, CD86, AP2M1, SHP-2, and PPP2R5A.

Some embodiments of these methods may further comprise identifying or diagnosing that the individual has a MICA positive cancer. Some embodiments of these methods may further comprise identifying or diagnosing the individual as having a MICA positive/HLA-E negative cancer.

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

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

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

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

Some embodiments of these methods may further comprise identifying or diagnosing that the individual has a MICA positive cancer. Some embodiments of these methods may further comprise identifying or diagnosing the individual as having a MICA positive/HLA-E negative cancer.

Non-limiting examples of MICA-positive cancer cells include: adrenocortical cancer cells, cholangiocarcinoma cells, pancreatic cancer cells, kidney cancer cells, thyroid cancer cells, mesothelioma cancer cells, skin epidermal melanoma cancer cells, colorectal cancer cells, Cervical squamous cell carcinoma, and endocervical adenocarcinoma. In some embodiments, MICA-positive cancers include, but are not limited to: adrenocortical cancer, 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 neoplasms diffuse large b-cell lymphoma, renal carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, renal chromophobe, liver cancer, lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, interstitial Tumors, ovarian cancer, pancreatic cancer, rectal cancer, skin cancer, stomach cancer, testicular cancer, thymoma, thyroid cancer, uterine cancer, uterine endometrial cancer, uterine carcinosarcoma, and uveal melanoma.

Enucleated erythroid cells can be administered to an individual as described herein using any dosage and with any frequency. Method of reducing the number and / or proliferation of MICB -positive cancer cells in an individual previously identified or diagnosed with MICB -positive cancer

Also provided herein is the reduction of MICB-positive cancer cells (such as described herein or in the art) in an individual previously identified or diagnosed as having an MICB-positive cancer (such as any exemplary MICB-positive cancer described herein or known in the art). methods for the number and/or proliferation of any exemplary MICB-positive cancer cells known in the art, comprising administering to a subject a therapeutically effective amount of enucleated erythroid cells (such as any of the exemplary enucleated erythroid cells described herein), which The enucleated erythroid cell comprises on its extracellular surface a first exogenous fusion polypeptide comprising (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or Its functional fragment.

Also provided herein is the reduction of MICB-positive cancer cells (such as any of the exemplary MICB-positive cancers described herein or known in the art) in an individual previously identified or diagnosed as having a MICA-positive cancer (such as any of the exemplary MICB-positive cancers described herein). MICB-positive cancer cells) in number and/or proliferation methods comprising administering to a subject a therapeutically effective amount of enucleated erythroid cells (such as any of the exemplary enucleated erythroid cells described herein), the enucleated erythroid cells in their cells The outer surface includes a first exogenous fusion polypeptide comprising (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof; and a second Exogenous polypeptides, the second exogenous polypeptides include 4-1BBL polypeptides or functional fragments thereof.

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

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

In some embodiments, the MICB positive cancer cells are MICB positive/HLA-E negative cancer cells.

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

For example, in some embodiments, one or more additional therapeutic agents include blocking, reducing and/or inhibiting PD-1 and PD-L1 or PD-L2, and/or blocking, reducing and/or inhibiting PD-1 and PD-L1 PD-L1 or PD-L2 binding agents (non-limiting examples include Nivolumab (ONO-4538/BMS-936558, MDX1106, OPDIVO, BRISTOL MYERS SQUIBB), pembrolizumab (KEYTRUDA, MERCK), Piddi One or more of Zizumab (CT-011, CURE TECH), MK-3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), and MPDL328OA (ROCHE)). In some embodiments, one or more additional therapeutic agents include blocking, reducing and/or inhibiting the activity of CTLA-4 and/or the interaction of CTLA-4 with one or more of its receptors (e.g., CD80, CD86, AP2M1, SHP- 2 and PPP2R5A) binding agents. For example, in some embodiments, the agent that inhibits CTLA-4 activity is an antibody, such as ipilimumab (MDX-010, MDX-101, Yervoy, BRISTOL MYERS SQUIBB) and/or tremelimumab (PFIZER). Additionally, in some embodiments, the one or more additional therapeutic agents include blocking antibodies that target immune checkpoint molecules such as, for example, BTLA, HVEM, TIM3, GALS, LAG3, VISTA, KIR, 2B4, CD160 (also known as BY55), CGEN-15049, CHK1 and CHK2 kinases, A2aR, CEACAM (eg, CEACAM-1, CEACAM-3 and/or CEACAM-5), GITR, GITRL, Galectin-9, CD244, CD160, TIGIT, SIRPα, ICOS, CD172a, TMIGD2 and various B-7 family ligands (including but not limited to B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7- H4, B7-H5, B7-H6 and B7-H7). In some embodiments, 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 example Includes 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 therapeutic agents include one or more molecules (e.g., small molecules) that block, reduce and/or inhibit the activity of CTLA-4 and/or the interaction of CTLA-4 with one or more of its regulated binding to CD80, CD86, AP2M1, SHP-2, and PPP2R5A.

Some embodiments of these methods may further comprise identifying or diagnosing the individual as having an MICB positive cancer. Some embodiments of these methods may further comprise identifying or diagnosing that the individual has an MICB positive/HLA-E negative cancer.

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

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

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

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

Some embodiments of these methods may further comprise identifying or diagnosing the individual as having an MICB positive cancer. Some embodiments of these methods may further comprise identifying or diagnosing that the individual has an MICB positive/HLA-E negative cancer.

Non-limiting examples of MICB-positive cancer cells include: acute myeloid leukemia cancer cells, lymphoid neoplasms diffuse large B-cell lymphoma cancer cells, testicular germ cell tumor cancer cells, gastric adenocarcinoma cells, ovarian serous cystadenocarcinoma cells, esophagus cancer cells, and lung cancer cells. In some embodiments, MICB-positive cancers include, but are not limited to: adrenocortical cancer, 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 neoplasms diffuse large b-cell lymphoma, renal carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, renal chromophobe, liver cancer, lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, interstitial Tumors, ovarian cancer, pancreatic cancer, rectal cancer, skin cancer, stomach cancer, testicular cancer, thymoma, thyroid cancer, uterine cancer, uterine endometrial cancer, uterine carcinosarcoma, and uveal melanoma.

Enucleated erythroid cells can be administered to an individual as described herein using any dose and with any frequency. Method of reducing the number and / or proliferation of MICA/MICB positive cancer cells in an individual previously identified or diagnosed with MICA/MICB positive cancer

Also provided herein is the reduction of MICA/MICB-positive cancer cells (e.g., MICA/MICB-positive cancers, e.g., A method for the number and/or proliferation of any exemplary MICA/MICB positive cancer cells described herein or known in the art, comprising administering to an individual a therapeutically effective amount of enucleated erythroid cells (such as any of the exemplary MICA/MICB positive cells described herein) enucleated erythroid cells) comprising on their extracellular surface a first exogenous fusion polypeptide comprising (i) IL-15 or a functional fragment thereof, and (ii ) IL-15 receptor alpha or a functional fragment thereof.

Also provided herein is the reduction of MICA/MICB positive cancer cells (such as described herein or known in the art) in an individual previously identified or diagnosed as having a MICA/MICB positive cancer (such as any of the exemplary MICA/MICB positive cancers described herein). Known methods for the number and/or proliferation of any exemplary MICA/MICB positive cancer cells) comprising administering to a subject a therapeutically effective amount of enucleated erythroid cells (such as any of the exemplary enucleated erythroid cells described herein), the Such enucleated erythroid cells comprise on their extracellular surface a first exogenous fusion polypeptide comprising (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof; and a second exogenous polypeptide comprising a 4-1BBL polypeptide or a functional fragment thereof.

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

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

In some embodiments, the MICA/MICB positive cancer cells are MICA/MICB positive/HLA-E negative cancer cells.

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

For example, in some embodiments, one or more additional therapeutic agents include blocking, reducing and/or inhibiting PD-1 and PD-L1 or PD-L2, and/or blocking, reducing and/or inhibiting PD-1 and PD-L1 PD-L1 or PD-L2 binding agents (non-limiting examples include Nivolumab (ONO-4538/BMS-936558, MDX1106, OPDIVO, BRISTOL MYERS SQUIBB), pembrolizumab (KEYTRUDA, MERCK), Piddi One or more of Zizumab (CT-011, CURE TECH), MK-3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), and MPDL328OA (ROCHE)). In some embodiments, one or more additional therapeutic agents include blocking, reducing and/or inhibiting the activity of CTLA-4 and/or the interaction of CTLA-4 with one or more of its receptors (e.g., CD80, CD86, AP2M1, SHP- 2 and PPP2R5A) binding agents. For example, in some embodiments, the agent that inhibits CTLA-4 activity is an antibody, such as ipilimumab (MDX-010, MDX-101, Yervoy, BRISTOL MYERS SQUIBB) and/or tremelimumab (PFIZER). Additionally, in some embodiments, the one or more additional therapeutic agents include blocking antibodies that target immune checkpoint molecules such as, for example, BTLA, HVEM, TIM3, GALS, LAG3, VISTA, KIR, 2B4, CD160 (also known as BY55), CGEN-15049, CHK1 and CHK2 kinases, A2aR, CEACAM (eg, CEACAM-1, CEACAM-3 and/or CEACAM-5), GITR, GITRL, Galectin-9, CD244, CD160, TIGIT, SIRPα, ICOS, CD172a, TMIGD2 and various B-7 family ligands (including but not limited to B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7- H4, B7-H5, B7-H6 and B7-H7). In some embodiments, 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 example Includes 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 therapeutic agents include one or more molecules (e.g., small molecules) that block, reduce and/or inhibit the activity of CTLA-4 and/or the interaction of CTLA-4 with one or more of its regulated binding to CD80, CD86, AP2M1, SHP-2, and PPP2R5A.

Some embodiments of these methods may further comprise identifying or diagnosing the individual as having a MICA/MICB positive cancer. Some embodiments of these methods may further comprise identifying or diagnosing that the individual has a MICA/MICB positive/HLA-E negative cancer.

In some embodiments of these methods, the method results in a reduction (e.g., at least 5% reduction, at least 10% reduction, at least 15% reduction, at least 20% reduction, at least 25% reduction, at least 25% reduction, At least 30% reduction, at least 35% reduction, at least 40% reduction, at least 45% reduction, at least 50% reduction, at least 55% reduction, at least 60% reduction, at least 65% reduction, at least 70% reduction, at least 75% reduction, A reduction of at least 80%, a reduction of at least 85%, a reduction of at least 90%, or a reduction of at least 95%, or a reduction of about 5% to a reduction of about 99% (or any exemplary subrange of such a range described herein)) (e.g., with the cast compared with the number of MICA/MICB positive cancer cells in previous individuals).

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

In some embodiments of these methods, the method results in a reduction (e.g., at least 5% reduction, at least 10% reduction, at least 15% reduction, at least 20% reduction, at least 25% reduction, at least 25% reduction, At least 30% reduction, at least 35% reduction, at least 40% reduction, at least 45% reduction, at least 50% reduction, at least 55% reduction, at least 60% reduction, at least 65% reduction, at least 70% reduction, at least 75% reduction, A reduction of at least 80%, a reduction of at least 85%, a reduction of at least 90%, or a reduction of at least 95%, or a reduction of about 5% to a reduction of about 99% (or any exemplary subrange of such a range described herein)) (e.g., with the cast compared with the proliferation of MICA/MICB-positive cancer cells in former individuals).

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

Some embodiments of these methods may further comprise identifying or diagnosing the individual as having a MICA/MICB positive cancer. Some embodiments of these methods may further comprise identifying or diagnosing that the individual has a MICA/MICB positive/HLA-E negative cancer.

Non-limiting examples of MICA/MICB positive cancer cells include: adrenocortical cancer cells, acute myeloid leukemia cancer cells, pancreatic cancer cells, cholangiocarcinoma cells, kidney cancer cells, cervical squamous cell cancer cells, endocervical cancer cells , colorectal cancer cells, mesothelioma cancer cells, skin epidermal melanoma cancer cells, and lung cancer cells. In some embodiments, MICA/MICB positive cancers include, but are not limited to: adrenocortical cancer, anal cancer, bile duct cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, esophagus cancer, eye cancer, head and neck cancer , acute myelogenous leukemia, lymphoid neoplasms diffuse large b-cell lymphoma, renal carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, renal chromophobe, liver cancer, lung cancer, lung adenocarcinoma, lung squamous cell carcinoma , mesothelioma, ovarian cancer, pancreatic cancer, rectal cancer, skin cancer, gastric cancer, testicular cancer, thymoma, thyroid cancer, uterine cancer, uterine endometrial cancer, uterine carcinosarcoma, and uveal melanoma.

Enucleated erythroid cells can be administered to an individual as described herein using any dose and with any frequency. Method of inducing killing of MICA- positive cancer cells in an individual previously identified or diagnosed with MICA -positive cancer

Also provided herein is the induction of killing of MICA-positive cancer cells (such as any of the exemplary MICA-positive cancer cells described herein) in an individual previously identified or diagnosed as having a MICA-positive cancer (such as any of the exemplary MICA-positive cancers described herein). A method comprising administering to an individual a therapeutically effective amount of an enucleated erythroid cell (such as any of the exemplary enucleated erythroid cells described herein) comprising a first exogenous fusion polypeptide on its extracellular surface , the first exogenous fusion polypeptide includes (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof.

Also provided herein is the induction of killing of MICA-positive cancer cells (such as any of the exemplary MICA-positive cancer cells described herein) in an individual previously identified or diagnosed as having a MICA-positive cancer (such as any of the exemplary MICA-positive cancers described herein). A method comprising administering to an individual a therapeutically effective amount of an enucleated erythroid cell (such as any of the exemplary enucleated erythroid cells described herein) comprising a first exogenous fusion polypeptide on its extracellular surface , the first exogenous fusion polypeptide includes (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof; and a second exogenous polypeptide, the second exogenous Polypeptides include 4-1BBL polypeptides or functional fragments thereof.

In some embodiments, inducing killing of MICA-positive cancer cells also results in inducing killing of non-MICA-positive cancer cells in the individual (eg, non-MICA-positive cancer cells in a tumor that also includes MICA-positive cancer cells).

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

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

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

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

For example, in some embodiments, one or more additional therapeutic agents include blocking, reducing and/or inhibiting PD-1 and PD-L1 or PD-L2, and/or blocking, reducing and/or inhibiting PD-1 and PD-L1 PD-L1 or PD-L2 binding agents (non-limiting examples include Nivolumab (ONO-4538/BMS-936558, MDX1106, OPDIVO, BRISTOL MYERS SQUIBB), pembrolizumab (KEYTRUDA, MERCK), Piddi One or more of Zizumab (CT-011, CURE TECH), MK-3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), and MPDL328OA (ROCHE)). In some embodiments, one or more additional therapeutic agents include blocking, reducing and/or inhibiting the activity of CTLA-4 and/or the interaction of CTLA-4 with one or more of its receptors (e.g., CD80, CD86, AP2M1, SHP- 2 and PPP2R5A) binding agents. For example, in some embodiments, the agent that inhibits CTLA-4 activity is an antibody, such as ipilimumab (MDX-010, MDX-101, Yervoy, BRISTOL MYERS SQUIBB) and/or tremelimumab (PFIZER). Additionally, in some embodiments, the one or more additional therapeutic agents include blocking antibodies that target immune checkpoint molecules such as, for example, BTLA, HVEM, TIM3, GALS, LAG3, VISTA, KIR, 2B4, CD160 (also known as BY55), CGEN-15049, CHK1 and CHK2 kinases, A2aR, CEACAM (eg, CEACAM-1, CEACAM-3 and/or CEACAM-5), GITR, GITRL, Galectin-9, CD244, CD160, TIGIT, SIRPα, ICOS, CD172a, TMIGD2 and various B-7 family ligands (including but not limited to B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7- H4, B7-H5, B7-H6 and B7-H7). In some embodiments, 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 example Includes 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 therapeutic agents include one or more molecules (e.g., small molecules) that block, reduce and/or inhibit the activity of CTLA-4 and/or the interaction of CTLA-4 with one or more of its regulated binding to CD80, CD86, AP2M1, SHP-2, and PPP2R5A.

Some embodiments of these methods may further comprise identifying or diagnosing that the individual has a MICA positive cancer. Some embodiments of these methods may further comprise identifying or diagnosing the individual as having a MICA positive/HLA-E negative cancer.

Non-limiting examples of MICA-positive cancer cells include: adrenocortical cancer cells, cholangiocarcinoma cells, pancreatic cancer cells, kidney cancer cells, thyroid cancer cells, mesothelioma cancer cells, skin epidermal melanoma cancer cells, colorectal cancer cells, Cervical squamous cell carcinoma, and endocervical adenocarcinoma. In some embodiments, MICA-positive cancers include, but are not limited to: adrenocortical cancer, 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 neoplasms diffuse large b-cell lymphoma, renal carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, renal chromophobe, liver cancer, lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, interstitial Tumors, ovarian cancer, pancreatic cancer, rectal cancer, skin cancer, stomach cancer, testicular cancer, thymoma, thyroid cancer, uterine cancer, uterine endometrial cancer, uterine carcinosarcoma, and uveal melanoma.

Enucleated erythroid cells can be administered to an individual as described herein using any dosage and with any frequency. Method of inducing killing of MICB positive cancer cells in an individual previously identified or diagnosed with MICB positive cancer

Also provided herein is the induction of killing of MICB-positive cancer cells (e.g., any of the exemplary MICB-positive cancer cells described herein) in an individual previously identified or diagnosed as having an MICB-positive cancer (e.g., any of the exemplary MICB-positive cancers described herein). A method comprising administering to an individual a therapeutically effective amount of an enucleated erythroid cell (such as any of the exemplary enucleated erythroid cells described herein) comprising a first exogenous fusion polypeptide on its extracellular surface , the first exogenous fusion polypeptide includes (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof.

Also provided herein is the induction of killing of MICB-positive cancer cells (e.g., any of the exemplary MICB-positive cancer cells described herein) in an individual previously identified or diagnosed as having an MICB-positive cancer (e.g., any of the exemplary MICB-positive cancers described herein). A method comprising administering to an individual a therapeutically effective amount of enucleated erythroid cells (such as any of the exemplary enucleated erythroid cells described herein) comprising a first exogenous fusion polypeptide on their extracellular surface, The first exogenous fusion polypeptide includes (i) IL-15 or its functional fragment, and (ii) IL-15 receptor α or its functional fragment; and a second exogenous polypeptide, the second exogenous polypeptide Including 4-1BBL polypeptide or its functional fragments.

In some embodiments, inducing killing of MICB-positive cancer cells also results in inducing killing of non-MICB-positive cancer cells in the individual (eg, non-MICB-positive cancer cells in a tumor that also includes MICB-positive cancer cells).

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

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

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

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

For example, in some embodiments, one or more additional therapeutic agents include blocking, reducing and/or inhibiting PD-1 and PD-L1 or PD-L2, and/or blocking, reducing and/or inhibiting PD-1 and PD-L1 PD-L1 or PD-L2 binding agents (non-limiting examples include Nivolumab (ONO-4538/BMS-936558, MDX1106, OPDIVO, BRISTOL MYERS SQUIBB), pembrolizumab (KEYTRUDA, MERCK), Piddi One or more of Zizumab (CT-011, CURE TECH), MK-3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), and MPDL328OA (ROCHE)). In some embodiments, one or more additional therapeutic agents include blocking, reducing and/or inhibiting the activity of CTLA-4 and/or the interaction of CTLA-4 with one or more of its receptors (e.g., CD80, CD86, AP2M1, SHP- 2 and PPP2R5A) binding agents. For example, in some embodiments, the agent that inhibits CTLA-4 activity is an antibody, such as ipilimumab (MDX-010, MDX-101, Yervoy, BRISTOL MYERS SQUIBB) and/or tremelimumab (PFIZER). Additionally, in some embodiments, the one or more additional therapeutic agents include blocking antibodies that target immune checkpoint molecules such as, for example, BTLA, HVEM, TIM3, GALS, LAG3, VISTA, KIR, 2B4, CD160 (also known as BY55), CGEN-15049, CHK1 and CHK2 kinases, A2aR, CEACAM (eg, CEACAM-1, CEACAM-3 and/or CEACAM-5), GITR, GITRL, Galectin-9, CD244, CD160, TIGIT, SIRPα, ICOS, CD172a, TMIGD2 and various B-7 family ligands (including but not limited to B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7- H4, B7-H5, B7-H6 and B7-H7). In some embodiments, 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 example Includes 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 therapeutic agents include one or more molecules (e.g., small molecules) that block, reduce and/or inhibit the activity of CTLA-4 and/or the interaction of CTLA-4 with one or more of its regulated binding to CD80, CD86, AP2M1, SHP-2, and PPP2R5A.

Some embodiments of these methods may further comprise identifying or diagnosing the individual as having an MICB positive cancer. Some embodiments of these methods may further comprise identifying or diagnosing that the individual has an MICB positive/HLA-E negative cancer.

Non-limiting examples of MICB-positive cancer cells include: acute myeloid leukemia cancer cells, lymphoid neoplasms diffuse large B-cell lymphoma cancer cells, testicular germ cell tumor cancer cells, gastric adenocarcinoma cells, ovarian serous cystadenocarcinoma cells, esophagus cancer cells, and lung cancer cells. In some embodiments, MICB-positive cancers include, but are not limited to: adrenocortical cancer, 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 neoplasms diffuse large b-cell lymphoma, renal carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, renal chromophobe, liver cancer, lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, interstitial Tumors, ovarian cancer, pancreatic cancer, rectal cancer, skin cancer, stomach cancer, testicular cancer, thymoma, thyroid cancer, uterine cancer, uterine endometrial cancer, uterine carcinosarcoma, and uveal melanoma.

Enucleated erythroid cells can be administered to an individual as described herein using any dose and with any frequency. Method of Inducing Killing of MICA/MICB Positive Cancer Cells in Individuals Previously Identified or Diagnosed as Having MICA/MICB Positive Cancer

Also provided herein is the induction of killing of MICA/MICB positive cancer cells (such as any of the exemplary MICA/MICB positive cancers described herein) in individuals previously identified or diagnosed as having a MICA/MICB positive cancer (such as any of the exemplary MICA/MICB positive cancers described herein). MICA/MICB-positive cancer cells) comprising administering to an individual a therapeutically effective amount of enucleated erythroid cells (such as any of the exemplary enucleated erythroid cells described herein), which have enucleated erythroid cells on their extracellular surfaces A first exogenous fusion polypeptide is included, and the first exogenous fusion polypeptide includes (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof.

Also provided herein is the induction of killing of MICA/MICB positive cancer cells (such as any of the exemplary MICA/MICB positive cancers described herein) in individuals previously identified or diagnosed as having a MICA/MICB positive cancer (such as any of the exemplary MICA/MICB positive cancers described herein). MICA/MICB positive cancer cells) comprising administering to a subject a therapeutically effective amount of enucleated erythroid cells (such as any of the exemplary enucleated erythroid cells described herein) comprising on their extracellular surface A first exogenous fusion polypeptide comprising (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor α or a functional fragment thereof; and a second exogenous polypeptide , the second exogenous polypeptide includes 4-1BBL polypeptide or a functional fragment thereof.

In some embodiments, inducing killing of MICA/MICB positive cancer cells also results in inducing killing of non-MICA/MICB positive cancer cells in the individual (e.g., non-MICA/MICB positive in tumors that also include MICA/MICB positive cancer cells cancer cell).

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

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

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

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

For example, in some embodiments, one or more additional therapeutic agents include blocking, reducing and/or inhibiting PD-1 and PD-L1 or PD-L2, and/or blocking, reducing and/or inhibiting PD-1 and PD-L1 PD-L1 or PD-L2 binding agents (non-limiting examples include Nivolumab (ONO-4538/BMS-936558, MDX1106, OPDIVO, BRISTOL MYERS SQUIBB), pembrolizumab (KEYTRUDA, MERCK), Piddi One or more of Zizumab (CT-011, CURE TECH), MK-3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), and MPDL328OA (ROCHE)). In some embodiments, one or more additional therapeutic agents include blocking, reducing and/or inhibiting the activity of CTLA-4 and/or the interaction of CTLA-4 with one or more of its receptors (e.g., CD80, CD86, AP2M1, SHP- 2 and PPP2R5A) binding agents. For example, in some embodiments, the agent that inhibits CTLA-4 activity is an antibody, such as ipilimumab (MDX-010, MDX-101, Yervoy, BRISTOL MYERS SQUIBB) and/or tremelimumab (PFIZER). Additionally, in some embodiments, the one or more additional therapeutic agents include blocking antibodies that target immune checkpoint molecules such as, for example, BTLA, HVEM, TIM3, GALS, LAG3, VISTA, KIR, 2B4, CD160 (also known as BY55), CGEN-15049, CHK1 and CHK2 kinases, A2aR, CEACAM (eg, CEACAM-1, CEACAM-3 and/or CEACAM-5), GITR, GITRL, Galectin-9, CD244, CD160, TIGIT, SIRPα, ICOS, CD172a, TMIGD2 and various B-7 family ligands (including but not limited to B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7- H4, B7-H5, B7-H6 and B7-H7). In some embodiments, 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 example Includes 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 therapeutic agents include one or more molecules (e.g., small molecules) that block, reduce and/or inhibit the activity of CTLA-4 and/or the interaction of CTLA-4 with one or more of its regulated binding to CD80, CD86, AP2M1, SHP-2, and PPP2R5A.

Some embodiments of these methods may further comprise identifying or diagnosing the individual as having a MICA/MICB positive cancer. Some embodiments of these methods may further comprise identifying or diagnosing that the individual has a MICA/MICB positive/HLA-E negative cancer.

Non-limiting examples of MICA/MICB positive cancer cells include: adrenocortical cancer cells, acute myeloid leukemia cancer cells, pancreatic cancer cells, cholangiocarcinoma cells, kidney cancer cells, cervical squamous cell cancer cells, endocervical cancer cells , colorectal cancer cells, mesothelioma cancer cells, skin epidermal melanoma cancer cells, and lung cancer cells. In some embodiments, MICA/MICB positive cancers include, but are not limited to: adrenocortical cancer, anal cancer, bile duct cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, esophagus cancer, eye cancer, head and neck cancer , acute myelogenous leukemia, lymphoid neoplasms diffuse large b-cell lymphoma, renal carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, renal chromophobe, liver cancer, lung cancer, lung adenocarcinoma, lung squamous cell carcinoma , mesothelioma, ovarian cancer, pancreatic cancer, rectal cancer, skin cancer, gastric cancer, testicular cancer, thymoma, thyroid cancer, uterine cancer, uterine endometrial cancer, uterine carcinosarcoma, and uveal melanoma.

Enucleated erythroid cells can be administered to an individual as described herein using any dosage and with any frequency. Method of reducing the volume of a solid tumor in an individual previously identified or diagnosed as having a MICA- positive cancer, a MICB- positive cancer, or a MICA/MICB -positive cancer

Also provided herein are those previously identified or diagnosed as having a MICA-positive cancer (such as any of the exemplary MICA cancers described herein or known in the art), an MICB-positive cancer (such as any of the exemplary MICA cancers described herein or known in the art). Exemplary MICB cancers) or MICA/MICB positive cancers (such as any exemplary MICA/MICB cancers described herein or known in the art), methods of reducing solid tumor volume comprising administering to the individual a therapeutically effective amount of Enucleated erythroid cells (such as any of the exemplary enucleated erythroid cells described herein) comprising on their extracellular surface a first exogenous fusion polypeptide comprising (i ) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof.

Also provided herein are those previously identified or diagnosed as having a MICA-positive cancer (such as any of the exemplary MICA-positive cancers described herein), an MICB-positive cancer (such as any of the exemplary MICB cancers described herein or known in the art), or A method of reducing the volume of a solid tumor in an individual with a MICA/MICB positive cancer, such as any exemplary MICA/MICB cancer described herein or known in the art, comprising administering to the individual a therapeutically effective amount of enucleated erythroid cells (e.g., Any of the exemplary enucleated erythroid cells described herein) that include on their extracellular surface a first exogenous fusion polypeptide that includes (i) IL-15 or a functional fragment, and (ii) IL-15 receptor alpha or a functional fragment thereof; and a second exogenous polypeptide, the second exogenous polypeptide comprising 4-1BBL polypeptide or a functional fragment thereof.

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

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

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

For example, in some embodiments, one or more additional therapeutic agents include blocking, reducing and/or inhibiting PD-1 and PD-L1 or PD-L2, and/or blocking, reducing and/or inhibiting PD-1 and PD-L1 PD-L1 or PD-L2 binding agents (non-limiting examples include Nivolumab (ONO-4538/BMS-936558, MDX1106, OPDIVO, BRISTOL MYERS SQUIBB), pembrolizumab (KEYTRUDA, MERCK), Piddi One or more of Zizumab (CT-011, CURE TECH), MK-3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), and MPDL328OA (ROCHE)). In some embodiments, one or more additional therapeutic agents include blocking, reducing and/or inhibiting the activity of CTLA-4 and/or the interaction of CTLA-4 with one or more of its receptors (e.g., CD80, CD86, AP2M1, SHP- 2 and PPP2R5A) binding agents. For example, in some embodiments, the agent that inhibits CTLA-4 activity is an antibody, such as ipilimumab (MDX-010, MDX-101, Yervoy, BRISTOL MYERS SQUIBB) and/or tremelimumab (PFIZER). Additionally, in some embodiments, the one or more additional therapeutic agents include blocking antibodies that target immune checkpoint molecules such as, for example, BTLA, HVEM, TIM3, GALS, LAG3, VISTA, KIR, 2B4, CD160 (also known as BY55), CGEN-15049, CHK1 and CHK2 kinases, A2aR, CEACAM (eg, CEACAM-1, CEACAM-3 and/or CEACAM-5), GITR, GITRL, Galectin-9, CD244, CD160, TIGIT, SIRPα, ICOS, CD172a, TMIGD2 and various B-7 family ligands (including but not limited to B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7- H4, B7-H5, B7-H6 and B7-H7). In some embodiments, 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 example Includes 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 therapeutic agents include one or more molecules (e.g., small molecules) that block, reduce and/or inhibit the activity of CTLA-4 and/or the interaction of CTLA-4 with one or more of its regulated binding to CD80, CD86, AP2M1, SHP-2, and PPP2R5A.

Some embodiments of these methods may further comprise identifying or diagnosing that the individual has a MICA-positive, MICB-negative, or MICA/MICB-positive cancer. Some embodiments of these methods may further comprise identifying or diagnosing that the individual has a MICA positive/HLA-E negative cancer, an MICB negative/HLA-E negative cancer, or a MICA/MICB positive/HLA-E negative cancer.

Non-limiting examples of MICA-positive cancers include: adrenocortical carcinoma, cholangiocarcinoma, pancreatic cancer, renal cancer, thyroid cancer, mesothelioma, cutaneous melanoma, colorectal cancer, squamous cell carcinoma of the cervix, and endocervical glandular carcinoma. cancer. In some embodiments, MICA-positive cancers include, but are not limited to: adrenocortical cancer, 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 neoplasms diffuse large b-cell lymphoma, renal carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, renal chromophobe, liver cancer, lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, interstitial Tumors, ovarian cancer, pancreatic cancer, rectal cancer, skin cancer, stomach cancer, testicular cancer, thymoma, thyroid cancer, uterine cancer, uterine endometrial cancer, uterine carcinosarcoma, and uveal melanoma.

Non-limiting examples of MICB-positive cancers include: acute myelogenous leukemia, lymphoid neoplasm diffuse large B-cell lymphoma, testicular germ cell tumor, gastric adenocarcinoma, ovarian serous cystadenocarcinoma, esophageal cancer, and lung cancer. In some embodiments, MICB-positive cancers include, but are not limited to: adrenocortical cancer, 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 neoplasms diffuse large b-cell lymphoma, renal carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, renal chromophobe, liver cancer, lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, interstitial Tumors, ovarian cancer, pancreatic cancer, rectal cancer, skin cancer, stomach cancer, testicular cancer, thymoma, thyroid cancer, uterine cancer, uterine endometrial cancer, uterine carcinosarcoma, and uveal melanoma.

Non-limiting examples of MICA/MICB positive cancers include: adrenocortical carcinoma, acute myeloid leukemia, pancreatic cancer, cholangiocarcinoma, kidney cancer, squamous cell carcinoma of the cervix, endocervical carcinoma, colorectal cancer, mesothelioma, Cutaneous epidermal melanoma, and lung cancer. In some embodiments, MICA/MICB positive cancers include, but are not limited to: adrenocortical cancer, anal cancer, bile duct cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, esophagus cancer, eye cancer, head and neck cancer , acute myelogenous leukemia, lymphoid neoplasms diffuse large b-cell lymphoma, renal carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, renal chromophobe, liver cancer, lung cancer, lung adenocarcinoma, lung squamous cell carcinoma , mesothelioma, ovarian cancer, pancreatic cancer, rectal cancer, skin cancer, gastric cancer, testicular cancer, thymoma, thyroid cancer, uterine cancer, uterine endometrial cancer, uterine carcinosarcoma, and uveal melanoma.

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

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

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

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

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

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

The engineered erythroid cells may be administered to the individual as frequently as several times a day, or may 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 Administration is frequent, such as every few months or even a year or less. The frequency of dosage will be apparent to those of ordinary skill and will depend on many factors such as, but not limited to, the type and severity of the disease being treated, the type and age of the individual, and the like.

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

In some embodiments, the MICB-positive cancer is a cancer selected from the group consisting of acute myelogenous leukemia, lymphoid neoplasm diffuse large B-cell lymphoma, testicular germ cell tumor, gastric adenocarcinoma, ovarian serous cystadenocarcinoma, Esophageal cancer, and lung cancer.

In some embodiments, the MICA/MICB positive cancer is a cancer selected from the group consisting of adrenocortical carcinoma, acute myelogenous leukemia, pancreatic cancer, cholangiocarcinoma, kidney cancer, squamous cell carcinoma of the cervix, endocervix carcinoma, colorectal cancer, mesothelioma, cutaneous melanoma, and lung cancer. set

Also provided herein are kits comprising any of the compositions provided herein. For example, provided herein is a kit comprising a pharmaceutical composition comprising enucleated erythroid cells (such as any of the exemplary enucleated erythroid cells described herein) comprising a first exogenous A fusion polypeptide comprising (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof; and instructions for performing any of the methods described herein . In some embodiments, the enucleated erythroid cells comprise at least 1,000 copies, at least 10,000 copies, or at least 15,000 copies of the first exogenous fusion polypeptide. In some embodiments, the enucleated erythroid cells comprise at least 1,000 replicas, at least 10,000 replicas, at least 15,000 replicas, at least 20,000 replicas, at least 25,000 replicas, at least 30,000 replicas, at least 40,000 replicas, at least 50,000 replicas , 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 comprising a pharmaceutical composition comprising an enucleated erythroid cell, such as any of the exemplary enucleated erythroid cells described herein, comprising on its extracellular surface The first exogenous fusion polypeptide, the first exogenous fusion polypeptide comprises (i) IL-15 or its functional fragment, and (ii) IL-15 receptor alpha or functional fragment; and comprises 4-1BBL polypeptide or its the second exogenous polypeptide of the functional fragment, wherein the exogenous polypeptide is present on the surface of the enucleated erythroid blood cell; and instructions for performing any of the methods described herein.

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

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

Some embodiments of the kits described herein include 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 volumes such as: 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 4 00 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 IL-15 and IL-15/IL-15RA Fusion Constructs Genetically Engineered to Express a First Fusion Polypeptide

Various DNA constructs encoding fusion polypeptides were prepared for expression in erythroid cells, as shown in Table 5 below. Table 5. IL-15 and IL-15/IL-15RA fusion constructs and polypeptides. SEQ ID NO refers to the amino acid sequence. Construct / peptide illustrate SEQ ID NO : V3 IL-15 GPA Signal Peptide (SEQ ID NO: 35) - Mature Human IL-15 (SEQ ID NO: 24) - Linker - HA - Linker (SEQ ID NO: 65) - GPA (SEQ ID NO: 44) 46 V4 IL-15 + IL-15RA (extracellular) GPA signal peptide (SEQ ID NO: 35) - mature human IL-15 (SEQ ID NO: 24) - flexible linker (SEQ ID NO: 37) - mature human extracellular IL-15RA (SEQ ID NO: 30) - Linker-HA- Linker (SEQ ID NO: 65) - GPA (SEQ ID NO: 44) 48 V5 IL-15 + IL-15RA (sushi domain + 13 aa) GPA signal peptide (SEQ ID NO: 35) - mature human IL-15 (SEQ ID NO: 24) - elastic linker (SEQ ID NO: 37) - mature human IL-15RA (sushi domain+13 aa) ( SEQ ID NO:33)-Linker-HA-Linker (SEQ ID NO:65)-GPA (SEQ ID NO:44) 50 V3.1 GPA signal peptide (SEQ ID NO: 35) - mature human IL-15 (SEQ ID NO: 24) - linker (SEQ ID NO: 43) - GPA (SEQ ID NO: 44) 52 V4.1 GPA signal peptide (SEQ ID NO: 35) - mature human IL-15 (SEQ ID NO: 24) - flexible linker (SEQ ID NO: 37) - mature human extracellular IL-15RA (SEQ ID NO: 30) - Linker (SEQ ID NO: 43) - GPA (SEQ ID NO: 44) 54 IL-15/IL-15RA fusion polypeptide Mature human IL-15 (SEQ ID NO: 24) - elastic linker (SEQ ID NO: 37) - mature human extracellular IL-15RA (SEQ ID NO: 30) 39 IL-15/IL-15RA (sushi domain + 13 aa) fusion polypeptide Mature human IL-15 (SEQ ID NO: 24) - elastic linker (SEQ ID NO: 37) - mature human IL-15RA (sushi domain + 13 aa) (SEQ ID NO: 33) 41

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

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

Peripheral mobilized human CD34 ⁺ cells derived from normal human donors were used. The expansion/differentiation procedure consisted of 3 stages. In the first stage, thawed CD34 ⁺ erythroid precursor cells were cultured in Iscove's MDM (IMDM) medium including recombinant human insulin, human transferrin, recombinant human stem cell factor, and recombinant human interleukin-3. In the second stage, erythroid cells were cultured in IMDM medium supplemented with recombinant human insulin, human transferrin, human recombinant stem cell factor, human recombinant erythropoietin, and L-glutamine. In the third stage, erythroid cells were cultured in IMDM medium supplemented with human transferrin, recombinant human insulin, human recombinant erythropoietin, and heparin. Cultures were maintained at 37 °C in a 5% _CO2 incubator. Transduction of erythroid precursor cells

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

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

DNA constructs were prepared for expression in erythroid cells as shown in Table 6 below: Table 6.4-1BBL constructs. SEQ ID NO refers to the amino acid sequence. construct illustrate SEQ ID NO: 4-1BBL GPA signal peptide (SEQ ID NO: 35) - human extracellular 4-1BBL (SEQ ID NO: 56) - 4-1BBL linker (SEQ ID NO: 60) - GPA (SEQ ID NO: 44) 58 Generating lentiviral vectors

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

Peripheral mobilized human CD34 ⁺ cells derived from normal human donors were purchased frozen from AllCells Inc. The expansion/differentiation procedure consisted of 3 stages. In the first stage, thawed CD34 ⁺ erythroid precursor cells were cultured in Iscove's MDM medium including 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. Cultures were maintained at 37 °C in a 5% _CO2 incubator. Transduction of erythroid precursor cells

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

Binding of PE-labeled anti-4-1BBL antibody (eg, purified anti-human 4-1BB ligand (CD137L) antibody, BioLegend) was used to verify the expression of 4-1BBL in engineered erythroid cells. Antibody binding was measured by flow cytometry to measure PE fluorescence. Circles were set based on stained, non-transduced cells. Example 3. Generation of erythroid- producing lentiviral vectors genetically engineered to express first and second exogenous polypeptides

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

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

Peripheral mobilized human CD34 ⁺ cells derived from normal human donors were purchased frozen from AllCells Inc. The expansion/differentiation procedure consisted of 3 stages. In the first stage, thawed CD34 ⁺ erythroid precursor cells were cultured in Iscove's MDM medium including 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. Cultures were maintained at 37 °C in a 5% _CO2 incubator. Transduction of erythroid precursor cells

Erythroid precursor cells were transduced during step 1 of the culture procedure described above. Combine erythroid cells in culture medium with lentiviral supernatant and polybrene. Infection was achieved by spin-inoculation, spinning the plate at 2000 rpm for 90 minutes at room temperature. After spin seeding, cells were incubated overnight at 37°C. antibody binding

Binding of PE-labeled anti-IL-15-RA antibodies (eg anti-IL-15RA antibody (JM7A4) (ab91270), AbCam) was used to verify expression of IL-15/IL-15-RA in engineered erythroid cells. Binding of PE-labeled anti-4-1BBL antibody (eg, purified anti-human 4-1BB ligand (CD137L) antibody, BioLegend) was used to verify the expression of 4-1BBL in engineered erythroid cells. Antibody binding was measured by flow cytometry to measure PE fluorescence. Circles were set based on stained, non-transduced cells. Example 4. Administration of Enucleated Erythroid Cells Genetically Engineered to Express IL-15/IL-15RA and 4-1BBL Increases the Ratio of NKG2D- Positive Lymphocytes to NKG2A- Positive Lymphocytes in Vivo

To assess the effect of administration of 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 individuals, flow cytometry was used in the administration of The contents of NKG2D-positive lymphocytes and NKG2A-positive lymphocytes were measured in whole blood samples obtained from individuals at different time points before and after administration.

Whole blood samples were analyzed by flow cytometry using the following antibodies: APC-H7 mouse anti-human CD45 strain 2D1 (BD Pharmingen), PE-Cy7 mouse anti-human CD16 strain B73.1 (BD Biosciences), PE small Mouse anti-human CD56 strain NCAM16.2 (BD Biosciences), APC mouse anti-human NKG2D strain 1D11 (BD Biosciences), BV421 mouse anti-human NKG2A strain 131411 (BD Biosciences), treated with 7-aminoactinomycin Survival was analyzed by D (7-AAD) (BD Biosciences). Lymphocytes were defined in terms of scatter/singlet/CD45 ⁺ (lymphocytes)/survival/CD16 ⁺ or CD56 ⁺ . NKG2D positive lymphocytes and NKG2A positive lymphocytes were identified as positive based on empirically established fringing by fluorescence minus a control sample, fluorescence compensation, and quantification. The ratio of NKG2D positive/NKG2A positive was defined as the percentage of NKG2D positive lymphocytes divided by the percentage of NKG2A positive lymphocytes. The maximum fold change is the maximum result of dividing the NKG2D positive/NKG2D positive ratio by the baseline (pre-administration) value at all available time points after administration of human enucleated erythroid cells including IL-15/IL-15RA and 4-1BBL.

As shown in Figure 1, administration of human enucleated erythroid cells genetically engineered to express IL-15/IL-15RA and 4-1BBL resulted in an increased ratio of NKG2D-positive lymphocytes to NKG2A-positive lymphocytes in the blood of human subjects. Example 5. Administration of enucleated erythroid cells genetically engineered to express IL-15/IL-15RA and 4-1BBL increases NKG2D- positive lymphocytes, NK cells and CD8+ memory T cells

Cell surface markers in fresh whole blood were analyzed by flow cytometry using greater than or equal to 1 x ¹⁰¹⁰ human enucleated erythroid cells genetically engineered to express IL-15/IL-15RA and 4-1BBL Assessments were performed at multiple time points before and during treatment of patients.

Whole blood was collected from patients at pre-treatment baseline and 4 hours, 1 day, 2 days, 7 days, and 14 days post-treatment for the first dosing cycle, and 2 days, 7 days, and 14 days post-treatment following subsequent dosing cycles . Whole blood was stained for analysis by flow cytometry using standard techniques with fluorophore-conjugated antibodies. Multiple antibody panels were used, each with separate tubes and flow cytometric analysis. All panels include markers for identification of lymphocyte, NK cell and T cell populations as well as markers for activation status. The data were obtained using the following panels: Group 1 - CD3, CD8, CD16/CD56, CD45, CD45RA, CCR7, HLA-DR and live/dead staining; Group 2 - CD3, CD4, Foxp3, CD25, CD127, Ki67, Live/dead staining; Group 3—CD45, CD16, CD56, NKG2D, NKp30, and live/dead staining. Data were then analyzed as population frequency for the percentage of established parent gates (eg, %NKG2D ⁺ of CD45 ⁺ CD56 ⁺ live lymphocytes). In the case of Group 1, quantitative cell population number data per volume of blood were also analyzed against the included quantitative count control. Data are reported as the maximum fold change (% cells or parent population per μL of whole blood) observed for each parameter of interest relative to baseline.

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

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

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

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

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

The data in Figure 4B show that there was no significant change in the percentage of CD8 ⁺ T cells to total CD3 ⁺ T cells. Sequence Appendix SEQ ID NO : identifier sequence 1 NKG2D (NP_031386.2) MGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCRENASPFFFCCFIAVAMGIRFIIMVTIWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSTVQWEDGSILSPNLLTICIEMQKGDCALYASCMS 2 NKG2D (nucleic acid sequence encoding NP_031386.2) ATGGGGTGGATTCGTGGTCGGAGGTCTCGACACAGCTGGGAGATGAGTGAATTTCATAATTATAACTTGGATCTGAAGAAGAGTGATTTTTCAACACGATGGCAAAAGCAAAGATGTCCAGTAGTCAAAAGCAAATGTAGAGAAAATGCATCTCCATTTTTTTTCTGCTGCTTCATCGCTGTAGCCATGGGAATCCGTTTCATTATTATGGTAACAATATGGAGTGCTGTATTCCTAAACTCATTATTCAACCAAGAAGTTCAAATTCCCTTGACCGAAAGTTACTGTGGCCCATGTCCTAAAAACTGGATATGTTACAAAAATAACTGCTACCAATTTTTTGATGAGAGTAAAAACTGGTATGAGAGCCAGGCTTCTTGTATGTCTCAAAATGCCAGCCTTCTGAAAGTATACAGCAAAGAGGACCAGGATTTACTTAAACTGGTGAAGTCATATCATTGGATGGGACTAGTACACATTCCAACAAATGGATCTTGGCAGTGGGAAGATGGCTCCATTCTCTCACCCAACCTACTAACAATAATTGAAATGCAGAAGGGAGACTGTGCACTCTATGCCTCGAGCTTTAAAGGCTATATAGAAAACTGTTCAACTCCAAATACGTACATCTGCATGCAAAGGACTGTGTAA 3 MICA (NP_000238.1) MGLGPVFLLLAGIFPFAPPGAAA EPHSLRYNLTVLSWDGSVQSGFLTEVHLDGQPFLRCDRQKCRAKPQGQWAEDVLGNKTWDRETRDLTGNGKDLRMTLAHIKDQKEGLHSLQEIRVCEIHEDNSTRSSQHFYYDGELFLSQNLETKEWTMPQSSRAQTLAMNVRNFLKEDAMKTKTHYHAMHADCLQELRRYLKSGVVLRRTVPPMVNVTRSEASEGNITVTCRASGFYPWNITLSWRQDGVSLSHDTQQWGDVLPDGNGTYQTWVATRICQGEEQRFTCYMEHSGNHSTHPVPSGKVLVLQSHWQTFHVSAVAAAAIFVIIIFYVRCCKKKTSAAEGPELVSLQVLDQHPVGTSDHRDATQLGFQPLMSDLGSTGSTEGA 4 MICA ATGGGGCTGGGCCCGGTCTTCCTGCTTCTGGCTGGCATCTTCCCTTTTGCACCTCCGGGAGCTGCTGCTGAGCCCCACAGTCTTCGTTATAACCTCACGGTGCTGTCCTGGGATGGATCTGTGCAGTCAGGGTTTCTCACTGAGGTACATCTGGATGGTCAGCCCTTCCTGCGCTGTGACAGGCAGAAATGCAGGGCAAAGCCCCAGGGACAGTGGGCAGAAGATGTCCTGGGAAATAAGACATGGGACAGAGAGACCAGAGACTTGACAGGGAACGGAAAGGACCTCAGGATGACCCTGGCTCATATCAAGGACCAGAAAGAAGGCTTGCATTCCCTCCAGGAGATTAGGGTCTGTGAGATCCATGAAGACAACAGCACCAGGAGCTCCCAGCATTTCTACTACGATGGGGAGCTCTTCCTCTCCCAAAACCTGGAGACTAAGGAATGGACAATGCCCCAGTCCTCCAGAGCTCAGACCTTGGCCATGAACGTCAGGAATTTCTTGAAGGAAGATGCCATGAAGACCAAGACACACTATCACGCTATGCATGCAGACTGCCTGCAGGAACTACGGCGATATCTAAAATCCGGCGTAGTCCTGAGGAGAACAGTGCCCCCCATGGTGAATGTCACCCGCAGCGAGGCCTCAGAGGGCAACATTACCGTGACATGCAGGGCTTCTGGCTTCTATCCCTGGAATATCACACTGAGCTGGCGTCAGGATGGGGTATCTTTGAGCCACGACACCCAGCAGTGGGGGGATGTCCTGCCTGATGGGAATGGAACCTACCAGACCTGGGTGGCCACCAGGATTTGCCAAGGAGAGGAGCAGAGGTTCACCTGCTACATGGAACACAGCGGGAATCACAGCACTCACCCTGTGCCCTCTGGGAAAGTGCTGGTGCTTCAGAGTCATTGGCAGACATTCCATGTTTCTGCTGTTGCTGCTGCTGCTATTTTTGTTATTATTATTTTCTATGTCCGTTGTTGTAAGAAGA AAACATCAGCTGCAGAGGGTCCAGAGCTCGTGAGCCTGCAGGTCCTGGATCAACACCCAGTTGGGACGAGTGACCACAGGGATGCCACACAGCTCGGATTTCAGCCTCTGATGTCAGATCTTGGGTCCACTGGCTCCACTGAGGGCGCCTAG 5 MICA (NP_001170990.1) MGLGPVFLLLAGIFPFAPPGAAA EPHSLRYNLTVLSWDGSVQSGFLAEVHLDGQPFLRYDRQKCRAKPQGQWAEDVLGNKTWDRETRDLTGNGKDLRMTLAHIKDQKEGLHSLQEIRVCEIHEDNSTRSSQHFYYDGELFLSQNLETEEWTVPQSSRAQTLAMNVRNFLKEDAMKTKTHYHAMHADCLQELRRYLESGVVLRRTVPPMVNVTRSEASEGNITVTCRASSFYPRNIILTWRQDGVSLSHDTQQWGDVLPDGNGTYQTWVATRICRGEEQRFTCYMEHSGNHSTHPVPSGKVLVLQSHWQTFHVSAVAAGCCYFCYYYFLCPLL 6 MICA (CCDS 56412.1) ATGGGGCTGGGCCCGGTCTTTCTGCTTCTGGCTGGCATCTTCCCTTTTGCACCTCCGGGAGCTGCTGCTGAGCCCCACAGTCTTCGTTATAACCTCACGGTGCTGTCCTGGGATGGATCTGTGCAGTCAGGGTTTCTTGCTGAGGTACATCTGGATGGTCAGCCCTTCCTGCGCTATGACAGGCAGAAATGCAGGGCAAAGCCCCAGGGACAGTGGGCAGAAGATGTCCTGGGAAATAAGACATGGGACAGAGAGACCAGGGACTTGACAGGGAACGGAAAGGACCTCAGGATGACCCTGGCTCATATCAAGGACCAGAAAGAAGGCTTGCATTCCCTCCAGGAGATTAGGGTCTGTGAGATCCATGAAGACAACAGCACCAGGAGCTCCCAGCATTTCTACTACGATGGGGAGCTCTTCCTCTCCCAAAACCTGGAGACTGAGGAATGGACAGTGCCCCAGTCCTCCAGAGCTCAGACCTTGGCCATGAACGTCAGGAATTTCTTGAAGGAAGATGCCATGAAGACCAAGACACACTATCACGCTATGCATGCAGACTGCCTGCAGGAACTACGGCGATATCTAGAATCCGGCGTAGTCCTGAGGAGAACAGTGCCCCCCATGGTGAATGTCACCCGCAGCGAGGCCTCAGAGGGCAACATCACCGTGACATGCAGGGCTTCCAGCTTCTATCCCCGGAATATCATACTGACCTGGCGTCAGGATGGGGTATCTTTGAGCCACGACACCCAGCAGTGGGGGGATGTCCTGCCTGATGGGAATGGAACCTACCAGACCTGGGTGGCCACCAGGATTTGCCGAGGAGAGGAGCAGAGGTTCACCTGCTACATGGAACACAGCGGGAATCACAGCACTCACCCTGTGCCCTCTGGGAAAGTGCTGGTGCTTCAGAGTCATTGGCAGACATTCCATGTTTCTGCTGTTGCTGCTGGCTGCTGCTATTTTTGTTATTATTATTTTCTATGTCCGTTGTTGTAA 7 MICA (NP_001276081.1) MTLAHIKDQKEGLHSLQEIRVCEIHEDNSTRSSQHFYYDGELFLSQNLETEEWTVPQSSRAQTLAMNVRNFLKEDAMKTKTHYHAMHADCLQELRRYLESGVVLRRTVPPMVNVTRSEASEGNITVTCRASSFYPRNIILTWRQDGVSLSHDTQQWGDVLPDGNGTYQTWVATRICRGEEQRFTCYMEHSGNHSTHPVPSGKVLVLQSHWQTFHVSAVAAGCCYFCYYYFLCPLL 8 MICA (CCDS75421.1) ATGACCCTGGCTCATATCAAGGACCAGAAAGAAGGCTTGCATTCCCTCCAGGAGATTAGGGTCTGTGAGATCCATGAAGACAACAGCACCAGGAGCTCCCAGCATTTCTACTACGATGGGGAGCTCTTCCTCTCCCAAAACCTGGAGACTGAGGAATGGACAGTGCCCCAGTCCTCCAGAGCTCAGACCTTGGCCATGAACGTCAGGAATTTCTTGAAGGAAGATGCCATGAAGACCAAGACACACTATCACGCTATGCATGCAGACTGCCTGCAGGAACTACGGCGATATCTAGAATCCGGCGTAGTCCTGAGGAGAACAGTGCCCCCCATGGTGAATGTCACCCGCAGCGAGGCCTCAGAGGGCAACATCACCGTGACATGCAGGGCTTCCAGCTTCTATCCCCGGAATATCATACTGACCTGGCGTCAGGATGGGGTATCTTTGAGCCACGACACCCAGCAGTGGGGGGATGTCCTGCCTGATGGGAATGGAACCTACCAGACCTGGGTGGCCACCAGGATTTGCCGAGGAGAGGAGCAGAGGTTCACCTGCTACATGGAACACAGCGGGAATCACAGCACTCACCCTGTGCCCTCTGGGAAAGTGCTGGTGCTTCAGAGTCATTGGCAGACATTCCATGTTTCTGCTGTTGCTGCTGGCTGCTGCTATTTTTGTTATTATTATTTTCTATGTCCGTTGTTGTAA 9 MICA (NP_001276083.1) MGQRDQGLDRERKGPQDDPGSYQGPERRNFLKEDAMKTKTHYHAMHADCLQELRRYLESGVVLRRTVPPMVNVTRSEASEGNITVTCRASSFYPRNIILTWRQDGVSLSHDTQQWGDVLPDGNGTYQTWVATRICRGEEQRFTCYMEHSGNHSTHPVPSGKVLVLQSHWQTFHVSAVAAGCPCCY 10 MICA ATGGGACAGAGAGACCAGGGACTTGACAGGGAACGGAAAGGACCTCAGGATGACCCTGGCTCATATCAAGGACCAGAAAGAAGGAATTTCTTGAAGGAAGATGCCATGAAGACCAAGACACACTATCACGCTATGCATGCAGACTGCCTGCAGGAACTACGGCGATATCTAGAATCCGGCGTAGTCCTGAGGAGAACAGTGCCCCCCATGGTGAATGTCACCCGCAGCGAGGCCTCAGAGGGCAACATCACCGTGACATGCAGGGCTTCCAGCTTCTATCCCCGGAATATCATACTGACCTGGCGTCAGGATGGGGTATCTTTGAGCCACGACACCCAGCAGTGGGGGGATGTCCTGCCTGATGGGAATGGAACCTACCAGACCTGGGTGGCCACCAGGATTTGCCGAGGAGAGGAGCAGAGGTTCACCTGCTACATGGAACACAGCGGGAATCACAGCACTCACCCTGTGCCCTCTGGGAAAGTGCTGGTGCTTCAGAGTCATTGGCAGACATTCCATGTTTCTGCTGTTGCTGCTGGCTGCTGCTATTTTTGTTATTATTATTTTCTATGTCCGTTGTTGTAA 11 MICB (NP_001276089.1) MVLSQDGSVQSGFLAEGHLDGQPFLRYDRQKRRAKPQGQWAENVLGAKTWDTETEDLTENGQDLRRTLTHIKDQKGGLHSLQEIRVCEIHEDSSTRGSRHFYYDGELFLSQNLETQESTVPQSSRAQTLAMNVTNFWKEDAMKTKTHYRAMQADCLQKLQRYLKSGVAIRRTVPPMVNVTCSEVSEGNITVTCRASSFYPRNITLTWRQDGVSLSHNTQQWGDVLPDGNGTYQTWVATRIRQGEEQRFTCYMEHSGNHGTHPVPSGKALVLQSQRTDFPYVSAAMPCFVIIIILCVPCCKKKTSAAEGPELVSLQVLDQHPVGTGDHRDAAQLGFQPLMSATGSTGSTEGT 12 MICB (CCDS75423.1) ATGGTGCTGTCCCAGGATGGATCTGTGCAGTCAGGGTTTCTCGCTGAGGGACATCTGGATGGTCAGCCCTTCCTGCGCTATGACAGGCAGAAACGCAGGGCAAAGCCCCAGGGACAGTGGGCAGAAAATGTCCTGGGAGCTAAGACCTGGGACACAGAGACCGAGGACTTGACAGAGAATGGGCAAGACCTCAGGAGGACCCTGACTCATATCAAGGACCAGAAAGGAGGCTTGCATTCCCTCCAGGAGATTAGGGTCTGTGAGATCCATGAAGACAGCAGCACCAGGGGCTCCCGGCATTTCTACTACGATGGGGAGCTCTTCCTCTCCCAAAACCTGGAGACTCAAGAATCGACAGTGCCCCAGTCCTCCAGAGCTCAGACCTTGGCTATGAACGTCACAAATTTCTGGAAGGAAGATGCCATGAAGACCAAGACACACTATCGCGCTATGCAGGCAGACTGCCTGCAGAAACTACAGCGATATCTGAAATCCGGGGTGGCCATCAGGAGAACAGTGCCCCCCATGGTGAATGTCACCTGCAGCGAGGTCTCAGAGGGCAACATCACCGTGACATGCAGGGCTTCCAGCTTCTATCCCCGGAATATCACACTGACCTGGCGTCAGGATGGGGTATCTTTGAGCCACAACACCCAGCAGTGGGGGGATGTCCTGCCTGATGGGAATGGAACCTACCAGACCTGGGTGGCCACCAGGATTCGCCAAGGAGAGGAGCAGAGGTTCACCTGCTACATGGAACACAGCGGGAATCACGGCACTCACCCTGTGCCCTCTGGGAAGGCGCTGGTGCTTCAGAGTCAACGGACAGACTTTCCATATGTTTCTGCTGCTATGCCATGTTTTGTTATTATTATTATTCTCTGTGTCCCTTGTTGCAAGAAGAAAACATCAGCGGCAGAGGGTCCAGAGCTTGTGAGCCTGCAGGTCCTGGATCAACACCCAGTTGGGACAGGAGACCACAGGGATGCAGCACAGCTGG GATTTCAGCCTCTGATGTCAGCTACTGGGTCCACTGGTTCCACTGAGGGCACCTAG 13 MICB (NP_001276090.1) MGLGRVLLFLAVAFPFAPPAAAAEPHSLRYNLMVLSQDGSVQSGFLAEGHLDGQPFLRYDRQKRRAKPQGQWAENVLGAKTWDTETEDLTENGQDLRRTLTHIKDQKGVPQSSRAQTLAMNVTNFWKEDAMKTKTHYRAMQADCLQKLQRYLKSGVAIRRTVPPMVNVTCSEVSEGNITVTCRASSFYPRNITLTWRQDGVSLSHNTQQWGDVLPDGNGTYQTWVATRIRQGEEQRFTCYMEHSGNHGTHPVPSGKALVLQSQRTDFPYVSAAMPCFVIIIILCVPCCKKKTSAAEGPELVSLQVLDQHPVGTGDHRDAAQLGFQPLMSATGSTGSTEGT 14 MICB (CCDS75422.1) ATGGGGCTGGGCCGGGTCCTGCTGTTTCTGGCCGTCGCCTTCCCTTTTGCACCCCCGGCAGCCGCCGCTGAGCCCCACAGTCTTCGTTACAACCTCATGGTGCTGTCCCAGGATGGATCTGTGCAGTCAGGGTTTCTCGCTGAGGGACATCTGGATGGTCAGCCCTTCCTGCGCTATGACAGGCAGAAACGCAGGGCAAAGCCCCAGGGACAGTGGGCAGAAAATGTCCTGGGAGCTAAGACCTGGGACACAGAGACCGAGGACTTGACAGAGAATGGGCAAGACCTCAGGAGGACCCTGACTCATATCAAGGACCAGAAAGGAGTGCCCCAGTCCTCCAGAGCTCAGACCTTGGCTATGAACGTCACAAATTTCTGGAAGGAAGATGCCATGAAGACCAAGACACACTATCGCGCTATGCAGGCAGACTGCCTGCAGAAACTACAGCGATATCTGAAATCCGGGGTGGCCATCAGGAGAACAGTGCCCCCCATGGTGAATGTCACCTGCAGCGAGGTCTCAGAGGGCAACATCACCGTGACATGCAGGGCTTCCAGCTTCTATCCCCGGAATATCACACTGACCTGGCGTCAGGATGGGGTATCTTTGAGCCACAACACCCAGCAGTGGGGGGATGTCCTGCCTGATGGGAATGGAACCTACCAGACCTGGGTGGCCACCAGGATTCGCCAAGGAGAGGAGCAGAGGTTCACCTGCTACATGGAACACAGCGGGAATCACGGCACTCACCCTGTGCCCTCTGGGAAGGCGCTGGTGCTTCAGAGTCAACGGACAGACTTTCCATATGTTTCTGCTGCTATGCCATGTTTTGTTATTATTATTATTCTCTGTGTCCCTTGTTGCAAGAAGAAAACATCAGCGGCAGAGGGTCCAGAGCTTGTGAGCCTGCAGGTCCTGGATCAACACCCAGTTGGGACAGGAGACCACAGGGATGCAGCACAGCTGGGATTTCAGCCTCTGATGTCAGCTACTGGGTCCA CTGGTTCCACTGAGGGCACCTAG 15 MICB (NP_005922.2) MGLGRVLLFLAVAFPFAPPAAA AEPHSLRYNLMVLSQDGSVQSGFLAEGHLDGQPFLRYDRQKRRAKPQGQWAENVLGAKTWDTETEDLTENGQDLRRTLTHIKDQKGGLHSLQEIRVCEIHEDSSTRGSRHFYYDGELFLSQNLETQESTVPQSSRAQTLAMNVTNFWKEDAMKTKTHYRAMQADCLQKLQRYLKSGVAIRRTVPPMVNVTCSEVSEGNITVTCRASSFYPRNITLTWRQDGVSLSHNTQQWGDVLPDGNGTYQTWVATRIRQGEEQRFTCYMEHSGNHGTHPVPSGKALVLQSQRTDFPYVSAAMPCFVIIIILCVPCCKKKTSAAEGPELVSLQVLDQHPVGTGDHRDAAQLGFQPLMSATGSTGSTEGT 16 MICB (CCDS43449.1) ATGGGGCTGGGCCGGGTCCTGCTGTTTCTGGCCGTCGCCTTCCCTTTTGCACCCCCGGCAGCCGCCGCTGAGCCCCACAGTCTTCGTTACAACCTCATGGTGCTGTCCCAGGATGGATCTGTGCAGTCAGGGTTTCTCGCTGAGGGACATCTGGATGGTCAGCCCTTCCTGCGCTATGACAGGCAGAAACGCAGGGCAAAGCCCCAGGGACAGTGGGCAGAAAATGTCCTGGGAGCTAAGACCTGGGACACAGAGACCGAGGACTTGACAGAGAATGGGCAAGACCTCAGGAGGACCCTGACTCATATCAAGGACCAGAAAGGAGGCTTGCATTCCCTCCAGGAGATTAGGGTCTGTGAGATCCATGAAGACAGCAGCACCAGGGGCTCCCGGCATTTCTACTACGATGGGGAGCTCTTCCTCTCCCAAAACCTGGAGACTCAAGAATCGACAGTGCCCCAGTCCTCCAGAGCTCAGACCTTGGCTATGAACGTCACAAATTTCTGGAAGGAAGATGCCATGAAGACCAAGACACACTATCGCGCTATGCAGGCAGACTGCCTGCAGAAACTACAGCGATATCTGAAATCCGGGGTGGCCATCAGGAGAACAGTGCCCCCCATGGTGAATGTCACCTGCAGCGAGGTCTCAGAGGGCAACATCACCGTGACATGCAGGGCTTCCAGCTTCTATCCCCGGAATATCACACTGACCTGGCGTCAGGATGGGGTATCTTTGAGCCACAACACCCAGCAGTGGGGGGATGTCCTGCCTGATGGGAATGGAACCTACCAGACCTGGGTGGCCACCAGGATTCGCCAAGGAGAGGAGCAGAGGTTCACCTGCTACATGGAACACAGCGGGAATCACGGCACTCACCCTGTGCCCTCTGGGAAGGCGCTGGTGCTTCAGAGTCAACGGACAGACTTTCCATATGTTTCTGCTGCTATGCCATGTTTTGTTATTATTATTATTCTCTGTGTCCCTTGTTGCAAGAAGA AAACATCAGCGGCAGAGGGTCCAGAGCTTGTGAGCCTGCAGGTCCTGGATCAACACCCAGTTGGGACAGGAGACCACAGGGATGCAGCACAGCTGGGATTTCAGCTCTGATGTCAGCTACTGGGTCCACTGGTTCCACTGAGGGCACCTAG 17 NKG2A (NP 002250.2) polypeptide MDNQGVIYSDLNLPPNPKRQQRKPKGNKNSILATEQEITYAELNLQKASQDFQGNDKTYHCKDLPSAPEKLIVGILGIICLILMASVVTIVVIPSTLIQRHNNSSLNTRTQKARHCGHCPEEWITYSNSCYYIGKERRTWEESLLACTSKNSSLLSIDNEEEMKFLSIISPSSWIGVFRNSSHHPWVTMNGLAFKHEIKDSDNAELNCAVLQVNRLKSAQCGSSIIYHCKHKL 18 Nucleic acid sequence encoding SEQ ID NO: 17 ATGGATAACCAAGGAGTAATCTACTCAGACCTGAATCTGCCCCCAAACCCAAAGAGGCAGCAACGAAAACCTAAAGGCAATAAAAACTCCATTTTAGCAACTGAACAGGAAATAACCTATGCGGAATTAAACCTTCAAAAAGCTTCTCAGGATTTTCAAGGGAATGACAAAACCTATCACTGCAAAGATTTACCATCAGCTCCAGAGAAGCTCATTGTTGGGATCCTGGGAATTATCTGTCTTATCTTAATGGCCTCTGTGGTAACGATAGTTGTTATTCCCTCTACATTAATACAGAGGCACAACAATTCTTCCCTGAATACAAGAACTCAGAAAGCACGTCATTGTGGCCATTGTCCTGAGGAGTGGATTACATATTCCAACAGTTGTTACTACATTGGTAAGGAAAGAAGAACTTGGGAAGAGAGTTTGCTGGCCTGTACTTCGAAGAACTCCAGTCTGCTTTCTATAGATAATGAAGAAGAAATGAAATTTCTGTCCATCATTTCACCATCCTCATGGATTGGTGTGTTTCGTAACAGCAGTCATCATCCATGGGTGACAATGAATGGTTTGGCTTTCAAACATGAGATAAAAGACTCAGATAATGCTGAACTTAACTGTGCAGTGCTACAAGTAAATCGACTTAAATCAGCCCAGTGTGGATCTTCAATAATATATCATTGTAAGCATAAGCTTTAG 19 NKG2A (NP_998823.1) polypeptide MDNQGVIYSDLNLPPNPKRQQRKPKGNKSSILATEQEITYAELNLQKASQDFQGNDKTYHCKDLPSAPEKLIVGILGIICLILMASVVTIVVIPSTLIQRHNNSSLNTRTQKARHCGHCPEEWITYSNSCYYIGKERRTWEESLLACTSKNSSLLSIDNEEEMKFLSIISPSSWIGVFRNSSHHPWVTMNGLAFKHEIKDSDNAELNCAVLQVNRLKSAQCGSSIIYHCKHKL 20 Nucleic acid sequence encoding SEQ ID NO: 19 ATGGATAACCAAGGAGTAATCTACTCAGACCTGAATCTGCCCCCAAACCCAAAGAGGCAGCAACGAAAACCTAAAGGCAATAAAAACTCCATTTTAGCAACTGAACAGGAAATAACCTATGCGGAATTAAACCTTCAAAAAGCTTCTCAGGATTTTCAAGGGAATGACAAAACCTATCACTGCAAAGATTTACCATCAGCTCCAGAGAAGCTCATTGTTGGGATCCTGGGAATTATCTGTCTTATCTTAATGGCCTCTGTGGTAACGATAGTTGTTATTCCCTCTACATTAATACAGAGGCACAACAATTCTTCCCTGAATACAAGAACTCAGAAAGCACGTCATTGTGGCCATTGTCCTGAGGAGTGGATTACATATTCCAACAGTTGTTACTACATTGGTAAGGAAAGAAGAACTTGGGAAGAGAGTTTGCTGGCCTGTACTTCGAAGAACTCCAGTCTGCTTTCTATAGATAATGAAGAAGAAATGAAATTTCTGTCCATCATTTCACCATCCTCATGGATTGGTGTGTTTCGTAACAGCAGTCATCATCCATGGGTGACAATGAATGGTTTGGCTTTCAAACATGAGATAAAAGACTCAGATAATGCTGAACTTAACTGTGCAGTGCTACAAGTAAATCGACTTAAATCAGCCCAGTGTGGATCTTCAATAATATATCATTGTAAGCATAAGCTTTAG twenty one HLA-E (NP 005507.3) polypeptide MVDGTLLLLLSEALALTQTWAGSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRFDNDAASPRMVPRAPWMEQEGSEYWDRETRSARDTAQIFRVNLRTLRGYYNQSEAGSHTLQWMHGCELGPDGRFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDASEAEHQRAYLEDTCVEWLHKYLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQQDGEGHTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPVTLRWKPASQPTIPIVGIIAGLVLLGSVVSGAVVAAVIWRKKSSGGKGGSYSKAEWSDSAQGSESHSL twenty two Nucleic acid sequence encoding SEQ ID NO: 21 ATGGTAGATGGAACCCTCCTTTTACTCCTCTCGGAGGCCCTGGCCCTTACCCAGACCTGGGCGGGCTCCCACTCCTTGAAGTATTTCCACACTTCCGTGTCCCGGCCCGGCCGCGGGGAGCCCCGCTTCATCTCTGTGGGCTACGTGGACGACACCCAGTTCGTGCGCTTCGACAACGACGCCGCGAGTCCGAGGATGGTGCCGCGGGCGCCGTGGATGGAGCAGGAGGGGTCAGAGTATTGGGACCGGGAGACACGGAGCGCCAGGGACACCGCACAGATTTTCCGAGTGAATCTGCGGACGCTGCGCGGCTACTACAATCAGAGCGAGGCCGGGTCTCACACCCTGCAGTGGATGCATGGCTGCGAGCTGGGGCCCGACGGGCGCTTCCTCCGCGGGTATGAACAGTTCGCCTACGACGGCAAGGATTATCTCACCCTGAATGAGGACCTGCGCTCCTGGACCGCGGTGGACACGGCGGCTCAGATCTCCGAGCAAAAGTCAAATGATGCCTCTGAGGCGGAGCACCAGAGAGCCTACCTGGAAGACACATGCGTGGAGTGGCTCCACAAATACCTGGAGAAGGGGAAGGAGACGCTGCTTCACCTGGAGCCCCCAAAGACACACGTGACTCACCACCCCATCTCTGACCATGAGGCCACCCTGAGGTGCTGGGCCCTGGGCTTCTACCCTGCGGAGATCACACTGACCTGGCAGCAGGATGGGGAGGGCCATACCCAGGACACGGAGCTCGTGGAGACCAGGCCTGCAGGGGATGGAACCTTCCAGAAGTGGGCAGCTGTGGTGGTGCCTTCTGGAGAGGAGCAGAGATACACGTGCCATGTGCAGCATGAGGGGCTACCCGAGCCCGTCACCCTGAGATGGAAGCCGGCTTCCCAGCCCACCATCCCCATCGTGGGCATCATTGCTGGCCTGGTTCTCCTTGGATCTGTGGTCTCTGGAGCTGTGGTTGCTGCTGTGATATGGAGGAAGAAGAGCT CAGGTGGAAAAGGAGGGAGCTACTCTAAGGCTGAGTGGAGCGACAGTGCCCAGGGGTCTGAGTCTCACAGCTTGTAA twenty three immature human IL-15 MRISKPHLRSISIQCYLCLLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS twenty four mature human IL-15 NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 25 Nucleic acid sequence encoding mature human IL-15 (encoding SEQ ID NO: 24) AACTGGGTGAACGTTATTAGTGACCTTAAAAAGATCGAAGATTTGATACAGTCAATGCACATAGACGCGACGCTTTATACAGAATCTGATGTACATCCTTCATGCAAGGTTACTGCTATGAAGTGTTTTCTTCTCGAACTCCAAGTAATAAGTCTTGAGAGCGGAGATGCGAGCATTCATGACACCGTTGAGAATCTTATTATATTGGCTAACAACTCTCTGTCCAGCAATGGTAATGTGACAGAAAGCGGGTGTAAGGAGTGCGAGGAACTCGAGGAGAAGAACATCAAAGAGTTCTTGCAGTCTTTCGTCCATATTGTCCAGATGTTCATAAATACTAGC 26 Nucleic acid sequence encoding mature human IL-15 (encoding SEQ ID NO: 24) AACTGGGTAAACGTCATAAGCGACCTCAAAAAGATCGAGGACCTTATACAGTCTATGCACATAGATGCGACACTTTACACGGAATCAGACGTGCACCCGTCCTGCAAAGTCACAGCCATGAAGTGCTTTCTTCTCGAACTGCAGGTAATTTCTCTCGAATCAGGTGACGCATCTATCCACGACACAGTTGAAAATCTTATTATCCTGGCTAATAACTCCCTTAGCTCAAACGGCAATGTCACCGAGAGTGGATGTAAAGAATGTGAGGAACTTGAAGAGAAAAACATAAAGGAATTCTTGCAGAGTTTCGTTCATATTGTGCAAATGTTCATCAATACTAGT 27 Immature full-length human IL-15 receptor alpha MAPRRARGCRTLGLPALLLLLLLRPPATRGITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVE MEAMEALPVTWGTSSRDEDLENCSHHL 28 Mature full-length human IL-15 receptor alpha ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSLLECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTTVAISTSTVLLCGLSAVSLLALACYLKSRQTPPLASTLEDSH 29 immature extracellular human IL-15 receptor alpha MAPRRARGCRTLGLPALLLLLLRPPATRGITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTT 30 Mature extracellular human IL-15 receptor alpha ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTT 31 Nucleic acid sequence encoding SEQ ID NO: 30 (mature extracellular human IL-15 receptor α) ATCACATGCCCACCGCCCATGTCTGTTGAACACGCAGACATTTGGGTTAAAAGTTACTCACTTTACTCACGCGAGAGATATATATGCAACAGCGGCTTCAAGCGCAAAGCAGGCACTAGTAGTCTTACAGAGTGCGTGCTCAATAAAGCTACAAATGTAGCTCATTGGACTACTCCTAGTCTCAAATGCATTCGGGACCCCGCGCTTGTGCACCAGAGACCTGCGCCGCCGTCCACAGTGACGACAGCTGGTGTAACCCCCCAACCTGAATCCCTTAGTCCGTCTGGTAAAGAACCGGCGGCGTCTTCACCTTCCAGCAATAATACTGCGGCGACAACAGCCGCGATAGTTCCTGGATCCCAACTCATGCCGTCAAAGTCTCCTTCAACGGGAACGACAGAGATCTCTTCACATGAAAGTTCTCATGGAACACCGAGCCAAACTACGGCAAAGAACTGGGAACTGACTGCCTCAGCAAGCCACCAGCCGCCAGGGGTGTACCCGCAAGGGCACTCAGATACTACT 32 Human IL-15 receptor sushi domain ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR 33 Human IL-15 receptor sushi domain + 13 aa of IL-15 receptor alpha ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLETCVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPS 34 Nucleic acid sequence encoding SEQ ID NO: 33 ATCACCTGCCCGCCTCCCATGAGCGTGGAACACGCGGACATTTGGGTTAAGAGCTACAGTCTTTACAGCCGGGAGCGCTATATCTGCAACTCAGGGTTTAAGCGGAAAGCAGGGACATCAAGTTTGACAGAATGTGTGTTGAACAAGGCTACAAATGTTGCTCACTGGACCACGCCATCTTTGAAGTGTATCCGAGATCCCGCGCTTGTCCATCAGCGCCCAGCGCCT 35 GPA signal peptide MYGKIIFVLLLSEIVSISA 36 Nucleic acid sequence encoding SEQ ID NO: 35 ATGTATGGAAAAATAATCTTTGTATTACTATTGTCAGAAATTGTGAGCATATCAGCA 37 Linker GGGGSGGGGSGGGGS 38 (GGGGS)n (GGGGS)n, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 39 Mature human IL-15 + (G4S)3 linker + mature extracellular soluble human IL-15 receptor alpha NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGGSGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTT 40 Nucleic acid sequence encoding SEQ ID NO: 39 AACTGGGTGAACGTTATTAGTGACCTTAAAAAGATCGAAGATTTGATACAGTCAATGCACATAGACGCGACGCTTTATACAGAATCTGATGTACATCCTTCATGCAAGGTTACTGCTATGAAGTGTTTTCTTCTCGAACTCCAAGTAATAAGTCTTGAGAGCGGAGATGCGAGCATTCATGACACCGTTGAGAATCTTATTATATTGGCTAACAACTCTCTGTCCAGCAATGGTAATGTGACAGAAAGCGGGTGTAAGGAGTGCGAGGAACTCGAGGAGAAGAACATCAAAGAGTTCTTGCAGTCTTTCGTCCATATTGTCCAGATGTTCATAAATACTAGCGGGGGAGGTGGCTCTGGTGGAGGCGGGAGTGGCGGGGGCGGCTCAATCACATGCCCACCGCCCATGTCTGTTGAACACGCAGACATTTGGGTTAAAAGTTACTCACTTTACTCACGCGAGAGATATATATGCAACAGCGGCTTCAAGCGCAAAGCAGGCACTAGTAGTCTTACAGAGTGCGTGCTCAATAAAGCTACAAATGTAGCTCATTGGACTACTCCTAGTCTCAAATGCATTCGGGACCCCGCGCTTGTGCACCAGAGACCTGCGCCGCCGTCCACAGTGACGACAGCTGGTGTAACCCCCCAACCTGAATCCCTTAGTCCGTCTGGTAAAGAACCGGCGGCGTCTTCACCTTCCAGCAATAATACTGCGGCGACAACAGCCGCGATAGTTCCTGGATCCCAACTCATGCCGTCAAAGTCTCCTTCAACGGGAACGACAGAGATCTCTTCACATGAAAGTTCTCATGGAACACCGAGCCAAACTACGGCAAAGAACTGGGAACTGACTGCCTCAGCAAGCCACCAGCCGCCAGGGGTGTACCCGCAAGGGCACTCAGATACTACT 41 Mature human IL-15 + (G4S)3 linker + IL-15 receptor alpha sushi domain + 13 aa of IL-15 receptor alpha NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGGSGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAPPSLTECVLNKATNVAHWTTPSLK 42 Nucleic acid sequence encoding SEQ ID NO: 41 AACTGGGTAAACGTCATAAGCGACCTCAAAAAGATCGAGGACCTTATACAGTCTATGCACATAGATGCGACACTTTACACGGAATCAGACGTGCACCCGTCCTGCAAAGTCACAGCCATGAAGTGCTTTCTTCTCGAACTGCAGGTAATTTCTCTCGAATCAGGTGACGCATCTATCCACGACACAGTTGAAAATCTTATTATCCTGGCTAATAACTCCCTTAGCTCAAACGGCAATGTCACCGAGAGTGGATGTAAAGAATGTGAGGAACTTGAAGAGAAAAACATAAAGGAATTCTTGCAGAGTTTCGTTCATATTGTGCAAATGTTCATCAATACTAGTGGCGGGGGGGGAAGCGGTGGTGGAGGGAGCGGGGGTGGTGGATCCATCACCTGCCCGCCTCCCATGAGCGTGGAACACGCGGACATTTGGGTTAAGAGCTACAGTCTTTACAGCCGGGAGCGCTATATCTGCAACTCAGGGTTTAAGCGGAAAGCAGGGACATCAAGTTTGACAGAATGTGTGTTGAACAAGGCTACAAATGTTGCTCACTGGACCACGCCATCTTTGAAGTGTATCCGAGATCCCGCGCTTGTCCATCAGCGCCCAGCGCCTCCCTCC 43 Linker GGSGGSGGGGGSGGGSGGGSGGGS 44 GPA peptide LSTTEVAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRAHEVSEISVRTVYPPEEETGERVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDDTDVPLSSVEIEENPETSDQ 45 Nucleic acid sequence encoding SEQ ID NO: 44 TTAAGTACCACTGAGGTGGCAATGCACACTTCAACCTCTTCTTCAGTCACAAAGAGTTACATCTCATCACAGACAAATGATACGCACAAACGGGACACATATGCAGCCACTCCTAGAGCTCATGAAGTTTCAGAAATTTCTGTTAGAACTGTTTACCCTCCAGAAGAGGAAACCGGAGAAAGGGTACAACTTGCCCATCATTTCTCTGAACCAGAGATAACACTCATTATTTTTGGGGTGATGGCTGGTGTTATTGGAACGATCCTCTTAATTTCTTACGGTATTCGCCGACTGATAAAGAAAAGCCCATCTGATGTAAAACCTCTCCCCTCACCTGACACAGACGTGCCTTTAAGTTCTGTTGAAATAGAAAATCCAGAGACAAGTGATCAA 46 IL-15 V3 construct MYGKIIFVLLLSEIVSISANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGSGGSGGYPYDVPDYAGGGSGGGSLSTTEVAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRAHEVSEISVRTVYPPEEETGERVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDTDVPLSSVEIENPETSDQ 47 Nucleic acid sequence encoding SEQ ID NO: 46 (IL-15 V3) ATGTATGGAAAAATAATCTTTGTATTACTATTGTCAGAAATTGTGAGCATATCAGCAAACTGGGTAAATGTGATTTCAGACCTGAAAAAAATCGAAGACCTTATTCAATCCATGCACATCGACGCGACACTTTATACTGAATCAGACGTACACCCGTCCTGTAAGGTTACTGCGATGAAGTGCTTTCTGTTGGAATTGCAAGTGATCTCCCTCGAATCAGGGGATGCATCCATTCATGATACCGTCGAGAATTTGATCATTCTGGCAAATAACTCCCTCAGTAGTAACGGGAATGTGACCGAGTCTGGGTGTAAGGAGTGCGAAGAGTTGGAGGAAAAGAATATCAAAGAATTCCTTCAGTCCTTTGTTCACATCGTGCAAATGTTTATTAATACATCTGGAGGATCTGGCGGGTCTGGAGGCTACCCCTATGACGTGCCCGACTATGCCGGCGGAGGGTCTGGAGGCGGTTCCTTAAGTACCACTGAGGTGGCAATGCACACTTCAACCTCTTCTTCAGTCACAAAGAGTTACATCTCATCACAGACAAATGATACGCACAAACGGGACACATATGCAGCCACTCCTAGAGCTCATGAAGTTTCAGAAATTTCTGTTAGAACTGTTTACCCTCCAGAAGAGGAAACCGGAGAAAGGGTACAACTTGCCCATCATTTCTCTGAACCAGAGATAACACTCATTATTTTTGGGGTGATGGCTGGTGTTATTGGAACGATCCTCTTAATTTCTTACGGTATTCGCCGACTGATAAAGAAAAGCCCATCTGATGTAAAACCTCTCCCCTCACCTGACACAGACGTGCCTTTAAGTTCTGTTGAAATAGAAAATCCAGAGACAAGTGATCAA 48 IL-15/IL-15Ra V4 construct MYGKIIFVLLLSEIVSISANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGGSGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTGGSGGSGGYPYDVPDYAGGGSGGGSLSTTEVAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRAHEVSEISVRTVYPPEEETGERVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDTDVPLSSVEIENPETSDQ 49 Nucleic acid sequence encoding SEQ ID NO: 48 (IL-15-V4) ATGTATGGAAAAATAATCTTTGTATTACTATTGTCAGAAATTGTGAGCATATCAGCAAACTGGGTGAACGTTATTAGTGACCTTAAAAAGATCGAAGATTTGATACAGTCAATGCACATAGACGCGACGCTTTATACAGAATCTGATGTACATCCTTCATGCAAGGTTACTGCTATGAAGTGTTTTCTTCTCGAACTCCAAGTAATAAGTCTTGAGAGCGGAGATGCGAGCATTCATGACACCGTTGAGAATCTTATTATATTGGCTAACAACTCTCTGTCCAGCAATGGTAATGTGACAGAAAGCGGGTGTAAGGAGTGCGAGGAACTCGAGGAGAAGAACATCAAAGAGTTCTTGCAGTCTTTCGTCCATATTGTCCAGATGTTCATAAATACTAGCGGGGGAGGTGGCTCTGGTGGAGGCGGGAGTGGCGGGGGCGGCTCAATCACATGCCCACCGCCCATGTCTGTTGAACACGCAGACATTTGGGTTAAAAGTTACTCACTTTACTCACGCGAGAGATATATATGCAACAGCGGCTTCAAGCGCAAAGCAGGCACTAGTAGTCTTACAGAGTGCGTGCTCAATAAAGCTACAAATGTAGCTCATTGGACTACTCCTAGTCTCAAATGCATTCGGGACCCCGCGCTTGTGCACCAGAGACCTGCGCCGCCGTCCACAGTGACGACAGCTGGTGTAACCCCCCAACCTGAATCCCTTAGTCCGTCTGGTAAAGAACCGGCGGCGTCTTCACCTTCCAGCAATAATACTGCGGCGACAACAGCCGCGATAGTTCCTGGATCCCAACTCATGCCGTCAAAGTCTCCTTCAACGGGAACGACAGAGATCTCTTCACATGAAAGTTCTCATGGAACACCGAGCCAAACTACGGCAAAGAACTGGGAACTGACTGCCTCAGCAAGCCACCAGCCGCCAGGGGTGTACCCGCAAGGGCACTCAGATACTACTGGAGGATCTGGCGGGTCTGGAGGCTACCCCT ATGACGTGCCCGACTATGCCGGCGGAGGGTCTGGAGGCGGTTCCTTAAGTACCACTGAGGTGGCAATGCACACTTCAACCTCTTCTTCAGTCACAAAGAGTTACATCTCATCACAGACAAATGATACGCACAAACGGGACACATATGCAGCCACTCCTAGAGCTCATGAAGTTTCAGAAATTTCTGTTAGAACTGTTTACCCTCCAGAAGAGGAAACCGGAGAAAGGGTACAACTTGCCCATCATTTCTCTGAACCAGAGATAACACTCATTATTTTTGGGGTGATGGCTGGTGTTATTGGAACGATCCTCTTAATTTCTTACGGTATTCGCCGACTGATAAAGAAAAGCCCATCTGATGTAAAACCTCTCCCCTCACCTGACACAGACGTGCCTTTAAGTTCTGTTGAAATAGAAAATCCAGAGACAAGTGATCAA 50 IL-15/IL-15Ra V5 MYGKIIFVLLLSEIVSISANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGGSGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSGGSGGSGGYPYDVPDYAGGGSGGGSLSTTEVAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRAHEVSEISVRTVYPPEEETGERVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDTDVPLSSVEIENPETSDQ 51 Nucleic acid sequence encoding SEQ ID NO: 50 (IL-15 V5) ATGTATGGAAAAATAATCTTTGTATTACTATTGTCAGAAATTGTGAGCATATCAGCAAACTGGGTAAACGTCATAAGCGACCTCAAAAAGATCGAGGACCTTATACAGTCTATGCACATAGATGCGACACTTTACACGGAATCAGACGTGCACCCGTCCTGCAAAGTCACAGCCATGAAGTGCTTTCTTCTCGAACTGCAGGTAATTTCTCTCGAATCAGGTGACGCATCTATCCACGACACAGTTGAAAATCTTATTATCCTGGCTAATAACTCCCTTAGCTCAAACGGCAATGTCACCGAGAGTGGATGTAAAGAATGTGAGGAACTTGAAGAGAAAAACATAAAGGAATTCTTGCAGAGTTTCGTTCATATTGTGCAAATGTTCATCAATACTAGTGGCGGGGGGGGAAGCGGTGGTGGAGGGAGCGGGGGTGGTGGATCCATCACCTGCCCGCCTCCCATGAGCGTGGAACACGCGGACATTTGGGTTAAGAGCTACAGTCTTTACAGCCGGGAGCGCTATATCTGCAACTCAGGGTTTAAGCGGAAAGCAGGGACATCAAGTTTGACAGAATGTGTGTTGAACAAGGCTACAAATGTTGCTCACTGGACCACGCCATCTTTGAAGTGTATCCGAGATCCCGCGCTTGTCCATCAGCGCCCAGCGCCTCCCTCCGGAGGATCTGGCGGGTCTGGAGGCTACCCCTATGACGTGCCCGACTATGCCGGCGGAGGGTCTGGAGGCGGTTCCTTAAGTACCACTGAGGTGGCAATGCACACTTCAACCTCTTCTTCAGTCACAAAGAGTTACATCTCATCACAGACAAATGATACGCACAAACGGGACACATATGCAGCCACTCCTAGAGCTCATGAAGTTTCAGAAATTTCTGTTAGAACTGTTTACCCTCCAGAAGAGGAAACCGGAGAAAGGGTACAACTTGCCCATCATTTCTCTGAACCAGAGATAACACTCATTATTTTTGGGGTGATGGCTG GTGTTATTGGAACGATCCTCTTAATTTCTTACGGTATTCGCCGACTGATAAAGAAAAGCCCATCTGATGTAAAACCTCTCCCCTCACCTGACACAGACGTGCCTTTAAGTTCTGTTGAAATAGAAAATCCAGGACAAGTGATCAA 52 IL-15 V3.1 construct MYGKIIFVLLLSEIVSISANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGSGGSGGGGGSGGGSGGGSGGGSLSTTEVAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRAHEVSEISVRTVYPPEEETGERVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDTDVPLSSVEIENPETSDQ 53 Nucleic acid sequence encoding SEQ ID NO: 52 (IL-15 V3.1) ATGTATGGAAAAATAATCTTTGTATTACTATTGTCAGAAATTGTGAGCATATCAGCAAACTGGGTAAATGTGATTTCAGACCTGAAAAAAATCGAAGACCTTATTCAATCCATGCACATCGACGCGACACTTTATACTGAATCAGACGTACACCCGTCCTGTAAGGTTACTGCGATGAAGTGCTTTCTGTTGGAATTGCAAGTGATCTCCCTCGAATCAGGGGATGCATCCATTCATGATACCGTCGAGAATTTGATCATTCTGGCAAATAACTCCCTCAGTAGTAACGGGAATGTGACCGAGTCTGGGTGTAAGGAGTGCGAAGAGTTGGAGGAAAAGAATATCAAAGAATTCCTTCAGTCCTTTGTTCACATCGTGCAAATGTTTATTAATACATCTGGAGGATCTGGCGGGTCTGGAGGCGGCGGCGGCAGCGGCGGCGGCAGCGGCGGAGGGTCTGGAGGCGGTTCCTTAAGTACCACTGAGGTGGCAATGCACACTTCAACCTCTTCTTCAGTCACAAAGAGTTACATCTCATCACAGACAAATGATACGCACAAACGGGACACATATGCAGCCACTCCTAGAGCTCATGAAGTTTCAGAAATTTCTGTTAGAACTGTTTACCCTCCAGAAGAGGAAACCGGAGAAAGGGTACAACTTGCCCATCATTTCTCTGAACCAGAGATAACACTCATTATTTTTGGGGTGATGGCTGGTGTTATTGGAACGATCCTCTTAATTTCTTACGGTATTCGCCGACTGATAAAGAAAAGCCCATCTGATGTAAAACCTCTCCCCTCACCTGACACAGACGTGCCTTTAAGTTCTGTTGAAATAGAAAATCCAGAGACAAGTGATCAA 54 IL-15/IL-15Ra V4.1 construct MYGKIIFVLLLSEIVSISANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGGSGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTGGSGGSGGGGGSGGGSGGGSGGGSLSTTEVAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRAHEVSEISVRTVYPPEEETGERVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDTDVPLSSVEIENPETSDQ 55 Nucleic acid sequence encoding SEQ ID NO: 54 (IL-15-V4.1) ATGTATGGAAAAATAATCTTTGTATTACTATTGTCAGAAATTGTGAGCATATCAGCAAACTGGGTGAACGTTATTAGTGACCTTAAAAAGATCGAAGATTTGATACAGTCAATGCACATAGACGCGACGCTTTATACAGAATCTGATGTACATCCTTCATGCAAGGTTACTGCTATGAAGTGTTTTCTTCTCGAACTCCAAGTAATAAGTCTTGAGAGCGGAGATGCGAGCATTCATGACACCGTTGAGAATCTTATTATATTGGCTAACAACTCTCTGTCCAGCAATGGTAATGTGACAGAAAGCGGGTGTAAGGAGTGCGAGGAACTCGAGGAGAAGAACATCAAAGAGTTCTTGCAGTCTTTCGTCCATATTGTCCAGATGTTCATAAATACTAGCGGGGGAGGTGGCTCTGGTGGAGGCGGGAGTGGCGGGGGCGGCTCAATCACATGCCCACCGCCCATGTCTGTTGAACACGCAGACATTTGGGTTAAAAGTTACTCACTTTACTCACGCGAGAGATATATATGCAACAGCGGCTTCAAGCGCAAAGCAGGCACTAGTAGTCTTACAGAGTGCGTGCTCAATAAAGCTACAAATGTAGCTCATTGGACTACTCCTAGTCTCAAATGCATTCGGGACCCCGCGCTTGTGCACCAGAGACCTGCGCCGCCGTCCACAGTGACGACAGCTGGTGTAACCCCCCAACCTGAATCCCTTAGTCCGTCTGGTAAAGAACCGGCGGCGTCTTCACCTTCCAGCAATAATACTGCGGCGACAACAGCCGCGATAGTTCCTGGATCCCAACTCATGCCGTCAAAGTCTCCTTCAACGGGAACGACAGAGATCTCTTCACATGAAAGTTCTCATGGAACACCGAGCCAAACTACGGCAAAGAACTGGGAACTGACTGCCTCAGCAAGCCACCAGCCGCCAGGGGTGTACCCGCAAGGGCACTCAGATACTACTGGAGGATCTGGCGGGTCTGGAGGCGGCGGCG GCAGCGGCGGCGGCAGCGGCGGAGGGTCTGGAGGCGGTTCCTTAAGTACCACTGAGGTGGCAATGCACACTTCAACCTCTTCTTCAGTCACAAAGAGTTACATCTCATCACAGACAAATGATACGCACAAACGGGACACATATGCAGCCACTCCTAGAGCTCATGAAGTTTCAGAAATTTCTGTTAGAACTGTTTACCCTCCAGAAGAGGAAACCGGAGAAAGGGTACAACTTGCCCATCATTTCTCTGAACCAGAGATAACACTCATTATTTTTGGGGTGATGGCTGGTGTTATTGGAACGATCCTCTTAATTTCTTACGGTATTCGCCGACTGATAAAGAAAAGCCCATCTGATGTAAAACCTCTCCCCTCACCTGACACAGACGTGCCTTTAAGTTCTGTTGAAATAGAAAATCCAGAGACAAGTGATCAA 56 extracellular 4-1BBL polypeptide ACPWAVSGARASPPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPPEIPAGL 57 Nucleic acid sequence encoding SEQ ID NO: 56 (extracellular 4-1BBL) GCCTGCCCCTGGGCCGTGTCCGGGGCTCGCGCCTCGCCCGGCTCCGCGGCCAGCCCGAGACTCCGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA 58 4-1BBL construct MYGKIIFVLLLSEIVSISAACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSEGGSGGSGGGPEDEPGSGSGGGSGGGSLSTTEVAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRAHEVSEISVRTVYPPEEETGERVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDTDVPLSSVEIENPETSDQ 59 Nucleic acid sequence encoding SEQ ID NO: 58 (4-1BBL construct) ATGTATGGAAAAATAATCTTTGTATTACTATTGTCAGAAATTGTGAGCATATCAGCAGCCTGCCCCTGGGCCGTGTCCGGGGCTCGCGCCTCGCCCGGCTCCGCGGCCAGCCCGAGACTCCGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAAGGAGGATCTGGCGGGTCTGGAGGCGGCCCCGAGGACGAGCCCGGCAGCGGCAGCGGCGGAGGGTCTGGAGGCGGTTCCTTAAGTACCACTGAGGTGGCAATGCACACTTCAACCTCTTCTTCAGTCACAAAGAGTTACATCTCATCACAGACAAATGATACGCACAAACGGGACACATATGCAGCCACTCCTAGAGCTCATGAAGTTTCAGAAATTTCTGTTAGAACTGTTTACCCTCCAGAAGAGGAAACCGGAGAAAGGGTACAACTTGCCCATCATTTCTCTGAACCAGAGATAACACTCATTATTTTTGGGGTGATGGCTGGTG TTATTGGAACGATCCTCTTAATTTCTTACGGTATTCGCCGACTGATAAAGAAAAGCCCATCTGATGTAAAACCTCTCCCCTCACCTGACACAGACGTGCCTTTAAGTTCTGTTGAAATAGAAAATCCAGAGACAAGTGATCAA 60 4-1BBL linker GGSGGSGGGPEDEPGSGSGGGSGGGS 61 Nucleic acid sequence encoding SEQ ID NO: 60 GGAGGATCTGGCGGGTCTGGAGGCGGCCCCGAGGACGAGCCCGGCAGCGGCAGCGGCGGAGGGTCTGGAGGCGGTTCC 62 Full-length GPA Amino Acids MYGKIIFVLLLSAIVSISALSTTEVAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRAHEVSEISVRTVYPPEEETGERVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRRLIKKSPSDVKPLPSPDDVPLSSVEIEENPETSDQ 63 SMIM1 polypeptide MQPQESHVHYSRWEDGSRDGVSLGAVSSTEEASRCRRISQRLCTGKLGIAMKVLGGVALFWIIFILGYLTGYYVHKCK 64 G4S linker GGGGS 65 Linker-HA-Linker GGSGGSGGYPYDVPDYAGGGSGGGS 66 Linker GSGSGSGSGSSEEDEDEDEDGSGSGSGSGS 67 Linker GGGGSGGGGSGGGGSGGGGS 68 Linker GSGSGSGSEDGSGSGSGS 69 Linker GSGSGSGSGSGSGSGSGSGS 70 Linker GCGGSGGGGSGGGGS 71 Linker SGRGGGGSGGGGSGGGGSGGGGSSPA 72 Linker GGGGSGGGGSGGGGSGGGGSGGGG 73 Snorkel linker SGRGASSGSSGSGSQKKPRYEIRWKVVVISAILALVVLTVISLIILIMLWGSGMQSPA 74 Ig heavy chain V region 3 signal sequence MGWSCIILFLVATATGVHS 75 light chain leader MRVPAQLLGLLLLWLPGARC

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Figure 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 administration genetically engineered to express IL-15/IL-15RA and 4-1BBL human enucleated erythroid cells before (baseline) and after (post-treatment) human subjects. Figure 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 administration genetically engineered to express IL-15/IL-15RA and 4-1 BBL of greater than or equal to ^1x1010 human enucleated erythroid cells before (baseline) and after (post-treatment) human subjects. Post-treatment measurements were performed over multiple dosing cycles. Figure 2B is a graph showing the maximum fold change from baseline in the percentage of NKG2D expressing CD45 ⁺ CD16 ⁺ CD56 ^dim lymphocytes in whole blood samples obtained from administration of genetically engineered IL-15/IL expressing IL-15/IL Human subjects before (baseline) and after (post-treatment) greater than or equal to ^1x1010 human enucleated erythroid cells of -15RA and 4-1BBL. Post-treatment measurements were performed over multiple dosing cycles. Figure 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 administration genetically engineered to express IL-15/ IL-15RA and 4-1BBL in human individuals greater than or equal to ^1x1010 human enucleated erythroid cells before (baseline) and after (post-treatment). Post-treatment measurements were performed over multiple dosing cycles. Figure 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 administration of gene Human subjects before (baseline) and after (post-treatment) engineered to express IL-15/IL-15RA and 4-1BBL greater than or equal to ^1x1010 human enucleated erythroid cells. Post-treatment measurements were performed over multiple dosing cycles. Figure 4A is a graph showing the maximum fold change from baseline in the percentage of CD4 ⁺ T cells as a percentage of total T cells (CD3 ⁺ ) in whole blood samples obtained from administration genetically engineered to express IL-15/ IL-15RA and 4-1BBL in human individuals greater than or equal to ^1x1010 human enucleated erythroid cells before (baseline) and after (post-treatment). Post-treatment measurements were performed over multiple dosing cycles. Figure 4B is a graph showing the maximum fold change from baseline in the percentage of CD8 ⁺ T cells as a percentage of total T cells (CD3 ⁺ ) in whole blood samples obtained from administration of genes engineered to express IL-15/ IL-15RA and 4-1BBL in human individuals greater than or equal to ^1x1010 human enucleated erythroid cells before (baseline) and after (post-treatment). Post-treatment measurements were performed over multiple dosing cycles.

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Claims (121)

A method of increasing the number of NKG2D-positive lymphocytes in an individual previously identified or diagnosed with a MICA-positive cancer comprising administering to the individual a therapeutically effective amount of a pharmaceutical composition comprising an enucleated erythroid population, the Such enucleated erythroid cells comprise on their extracellular surface a first exogenous fusion polypeptide comprising (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or its function fragment. The method of claim 1, wherein the method further comprises identifying or diagnosing the individual as having a MICA-positive cancer. The method according to claim 1 or 2, wherein the MICA-positive cancer is selected from the group consisting of adrenocortical carcinoma, cholangiocarcinoma, pancreatic cancer, kidney cancer, thyroid cancer, mesothelioma, skin epidermal melanoma, Colorectal cancer, squamous cell carcinoma of the cervix, and endocervical adenocarcinoma. The method of any one of claims 1 to 3, wherein the MICA-positive cancer is a MICA-positive/HLA-E-negative cancer. A method of increasing the number of NKG2D-positive lymphocytes in an individual previously identified or diagnosed with an MICB-positive cancer comprising administering to the individual a therapeutically effective amount of a pharmaceutical composition comprising an enucleated erythroid population, the Such enucleated erythroid cells comprise on their extracellular surface a first exogenous fusion polypeptide comprising (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or its function fragment. The method of claim 5, wherein the method further comprises identifying or diagnosing the individual as having MICB-positive cancer. The method according to claim 5 or 6, wherein the MICB-positive cancer is selected from the group consisting of acute myelogenous leukemia, lymphoid neoplasm diffuse large B-cell lymphoma, testicular germ cell tumor, gastric adenocarcinoma, ovarian serous cystadenocarcinoma, esophageal cancer, and lung cancer. The method of any one of claims 5 to 7, wherein the MICB positive cancer is MICB positive/HLA-E negative cancer. The method of any one of claims 1 to 8, wherein the administering results in an increase of at least 5% in the number of NKG2D positive lymphocytes in the individual compared to the number of NKG2D positive lymphocytes in the individual before the administration. The method of claim 9, wherein the administration results in an increase in the number of NKG2D positive lymphocytes in the individual by at least 10% compared to the number of NKG2D positive lymphocytes in the individual before administration. The method according to any one of claims 1 to 10, wherein the NKG2D-positive lymphocytes are NKG2D-positive NK cells. The method of any one of claims 1 to 11, wherein the method further results in an increase in the number of NKG2D positive/NKG2A negative lymphocytes in the individual. The method of claim 12, wherein the administering step results in an increase in the number of NKG2D-positive/NKG2A-negative lymphocytes in the individual by at least 5%. The method of claim 13, wherein the step of administering results in an increase in the number of NKG2D-positive/NKG2A-negative lymphocytes in the individual by at least 10%. The method of claim 14, wherein the step of administering results in an increase in the number of NKG2D positive/NKG2A negative lymphocytes in the individual by at least 15%. The method according to any one of claims 12 to 15, wherein the NKG2D-positive/NKG2A-negative lymphocytes are NKG2D-positive/NKG2A-negative NK cells. The method of any one of claims 1 to 16, wherein the method further comprises administering an NKG2A inhibitor to the individual. The method of claim 17, wherein the NKG2A inhibitor is an antagonistic antibody that specifically binds to NKG2A. A method of treating an individual previously identified or diagnosed as having a MICA-positive cancer, the method comprising administering to the individual previously identified or diagnosed as having a MICA-positive cancer a therapeutically effective amount of a pharmaceutical composition comprising an enucleated A population of erythroid cells comprising on their extracellular surface a first exogenous polypeptide comprising (i) IL-15 or a functional fragment thereof, and (ii) IL-15 Receptor alpha or functional fragments thereof. The method of claim 19, wherein the method further comprises identifying or diagnosing the individual as having a MICA-positive cancer. The method of claim 19 or 20, wherein the MICA-positive cancer is selected from the group consisting of: adrenocortical carcinoma, cholangiocarcinoma, pancreatic cancer, kidney cancer, thyroid cancer, mesothelioma, skin epidermal melanoma, Colorectal cancer, squamous cell carcinoma of the cervix, and endocervical adenocarcinoma. The method of any one of claims 19 to 21, wherein the MICA positive cancer is a MICA positive/HLA-E negative cancer. A method of treating an individual previously identified or diagnosed as having an MICB-positive cancer, the method comprising administering to the individual previously identified or diagnosed as having an MICB-positive cancer a therapeutically effective amount of a pharmaceutical composition comprising an enucleated A population of erythroid cells comprising on their extracellular surface a first exogenous polypeptide comprising (i) IL-15 or a functional fragment thereof, and (ii) IL-15 Receptor alpha or functional fragments thereof. The method of claim 23, wherein the method further comprises identifying or diagnosing the individual as having MICB-positive cancer. The method of claim 23 or 24, wherein the MICB-positive cancer is selected from the group consisting of acute myelogenous leukemia, lymphoid neoplasm diffuse large B-cell lymphoma, testicular germ cell tumor, gastric adenocarcinoma, ovarian serous cystadenocarcinoma, esophageal cancer, and lung cancer. The method of any one of claims 23 to 25, wherein the MICB positive cancer is MICB positive/HLA-E negative cancer. The method of any one of claims 19 to 26, wherein the administration results in an increase in the number of NKG2D positive lymphocytes in the individual and/or an increase in the number of NKG2D positive/NKG2A negative lymphocytes in the individual. The method according to claim 27, wherein the NKG2D-positive lymphocytes are NKG2D-positive NK cells. The method according to claim 27 or 28, wherein the NKG2D-positive/NKG2A-negative lymphocytes are NKG2D-positive/NKG2A-negative NK cells. The method of any one of claims 19 to 29, wherein the method further comprises administering an NKG2A inhibitor to the individual. The method of claim 30, wherein the NKG2A inhibitor is an antagonistic antibody that specifically binds to NKG2A. A method of reducing the number and/or proliferation of MICA-positive cancer cells in an individual previously identified or diagnosed with MICA-positive cancer, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an enucleated A population of erythrocytes comprising on their extracellular surface a first exogenous polypeptide comprising (i) IL-15 or a functional fragment thereof, and (ii) an IL-15 receptor body α or its functional fragments. The method of claim 32, wherein the administering results in a reduction in the number of MICA-positive cancer cells in the individual. The method of claim 33, wherein the administering results in at least a 5% reduction in the number of MICA-positive cancer cells in the individual compared to the number of MICA-positive cancer cells in the individual prior to administration. The method of claim 34, wherein the administering results in at least a 10% reduction in the number of MICA-positive cancer cells in the individual compared to the number of MICA-positive cancer cells in the individual prior to administration. The method of claim 32, wherein the administering results in decreased proliferation of MICA-positive cancer cells in the individual. The method of claim 36, wherein the administering results in at least a 5% reduction in the proliferation of MICA-positive cancer cells in the individual compared to the proliferation of MICA-positive cancer cells in the individual prior to administration. The method of claim 37, wherein the administering results in at least a 10% reduction in the proliferation of MICA-positive cancer cells in the individual compared to the proliferation of MICA-positive cancer cells in the individual prior to administration. The method of any one of claims 32 to 38, wherein the method further comprises identifying or diagnosing the individual as having a MICA-positive cancer. The method of any one of claims 32 to 39, wherein the MICA-positive cancer is selected from the group consisting of adrenocortical carcinoma, cholangiocarcinoma, pancreatic cancer, kidney cancer, thyroid cancer, mesothelioma, skin Epidermal melanoma, colorectal cancer, squamous cell carcinoma of the cervix, and endocervical adenocarcinoma. The method of any one of claims 32 to 40, wherein the MICA positive cancer is a MICA positive/HLA-E negative cancer. The method of claim 41, wherein the administering results in a reduction in the number of MICA-positive/HLA-E-negative cancer cells in the individual. The method of claim 42, wherein the administration results in a reduction in the number of MICA-positive/HLA-E-negative cancer cells in the individual compared to the number of MICA-positive/HLA-E-negative cancer cells in the individual prior to administration At least 5%. The method of claim 43, wherein the administration results in a reduction in the number of MICA-positive/HLA-E-negative cancer cells in the individual compared to the number of MICA-positive/HLA-E-negative cancer cells in the individual prior to administration At least 10%. The method of claim 41, wherein the administering results in decreased proliferation of MICA-positive/HLA-E-negative cancer cells in the individual. The method of claim 45, wherein the administration results in decreased proliferation of MICA-positive/HLA-E-negative cancer cells in the individual compared to proliferation of MICA-positive/HLA-E-negative cancer cells in the individual prior to administration At least 5%. The method of claim 46, wherein the administration results in decreased proliferation of MICA-positive/HLA-E-negative cancer cells in the individual compared to proliferation of MICA-positive/HLA-E-negative cancer cells in the individual prior to administration At least 10%. A method of reducing the number and/or proliferation of MICB-positive cancer cells in an individual previously identified or diagnosed with MICB-positive cancer, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an enucleated A population of erythrocytes comprising on their extracellular surface a first exogenous polypeptide comprising (i) IL-15 or a functional fragment thereof, and (ii) an IL-15 receptor body α or its functional fragments. The method of claim 48, wherein the administering results in a reduction in the number of MICB-positive cancer cells in the individual. The method of claim 49, wherein the administering results in at least a 5% reduction in the number of MICB positive cancer cells in the individual compared to the number of MICB positive cancer cells in the individual prior to administration. The method of claim 50, wherein the administering results in at least a 10% reduction in the number of MICB positive cancer cells in the individual compared to the number of MICB positive cancer cells in the individual prior to administration. The method of claim 48, wherein the administering results in decreased proliferation of MICB-positive cancer cells in the individual. The method of claim 52, wherein the administering results in at least a 5% reduction in the proliferation of MICB-positive cancer cells in the individual compared to the proliferation of MICB-positive cancer cells in the individual prior to administration. The method of claim 53, wherein the administering results in at least a 10% reduction in the proliferation of MICB-positive cancer cells in the individual compared to the proliferation of MICB-positive cancer cells in the individual prior to administration. The method of any one of claims 48 to 54, wherein the method further comprises identifying or diagnosing the individual as having MICB-positive cancer. The method of any one of claims 48 to 55, wherein the MICB-positive cancer is selected from the group consisting of acute myelogenous leukemia, lymphoid neoplasm diffuse large B-cell lymphoma, testicular germ cell tumor, gastric gland carcinoma, ovarian serous cystadenocarcinoma, esophageal carcinoma, and lung carcinoma. The method of any one of claims 48 to 56, wherein the MICB positive cancer is MICB positive/HLA-E negative cancer. The method of claim 57, wherein the administering results in a reduction in the number of MICB positive/HLA-E negative cancer cells in the individual. The method of claim 58, wherein the administration results in a reduction in the number of MICB-positive/HLA-E-negative cancer cells in the individual compared to the number of MICB-positive/HLA-E-negative cancer cells in the individual prior to administration At least 5%. The method of claim 59, wherein the administration results in a reduction in the number of MICB-positive/HLA-E-negative cancer cells in the individual compared to the number of MICB-positive/HLA-E-negative cancer cells in the individual prior to administration At least 10%. The method of claim 57, wherein the administration results in decreased proliferation of MICB-positive/HLA-E-negative cancer cells in the individual. The method of claim 61, wherein the administering results in decreased proliferation of MICB-positive/HLA-E-negative cancer cells in the individual compared to proliferation of MICB-positive/HLA-E-negative cancer cells in the individual prior to administration At least 5%. The method of claim 62, wherein the administering results in decreased proliferation of MICB-positive/HLA-E-negative cancer cells in the individual compared to proliferation of MICB-positive/HLA-E-negative cancer cells in the individual prior to administration At least 10%. The method of any one of claims 32 to 63, wherein the method further comprises administering an NKG2A inhibitor to the individual. The method of claim 64, wherein the NKG2A inhibitor is an antagonistic antibody that specifically binds to NKG2A. A method of inducing killing of MICA-positive cancer cells in an individual previously identified or diagnosed with MICA-positive cancer, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an enucleated erythroid population, the Such enucleated erythroid cells comprise on their extracellular surface a first exogenous fusion polypeptide comprising (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or its function fragment. The method according to claim 66, wherein the MICA-positive cancer cells are cancer cells selected from the group consisting of: adrenocortical cancer cells, cholangiocarcinoma cells, pancreatic cancer cells, kidney cancer cells, thyroid cancer cells, mesothelial cells tumor cells, skin epidermal melanoma cells, colorectal cancer cells, cervical squamous cell carcinoma cells, and endocervical adenocarcinoma cells. The method of claim 66 or 67, wherein the method further comprises identifying or diagnosing that the individual has a MICA-positive cancer. The method of claim 68, wherein the MICA-positive cancer is selected from the group consisting of adrenocortical carcinoma, cholangiocarcinoma, pancreatic cancer, kidney cancer, thyroid cancer, mesothelioma, cutaneous melanoma, colorectal carcinoma, squamous cell carcinoma of the cervix, and endocervical adenocarcinoma. The method of claim 68 or 69, wherein the MICA-positive cancer is a MICA-positive/HLA-E-negative cancer. The method of claim 70, wherein the MICA-positive cancer cells are MICA-positive and HLA-E-negative cancer cells. A method of inducing killing of MICB-positive cancer cells in an individual previously identified or diagnosed with MICB-positive cancer, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an enucleated erythroid population, the The enucleated erythroid cells comprise on their extracellular surface a first exogenous fusion polypeptide comprising (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or its function fragment. The method of claim 72, wherein the MICB-positive cancer cells are cancer cells selected from the group consisting of: acute myeloid leukemia cancer cells, lymphoid tumor diffuse large B-cell lymphoma cancer cells, testicular germ cell tumor Cancer cells, gastric adenocarcinoma cells, ovarian serous cystadenocarcinoma cells, esophageal cancer cells, and lung cancer cells. The method of claim 72 or 73, wherein the method further comprises identifying or diagnosing the individual as having MICB-positive cancer. The method of claim 74, wherein the MICB-positive cancer is selected from the group consisting of acute myelogenous leukemia, lymphoid neoplasm diffuse large B-cell lymphoma, testicular germ cell tumor, gastric adenocarcinoma, ovarian serous cyst Adenocarcinoma, esophageal cancer, and lung cancer. The method of claim 74 or 75, wherein the MICB positive cancer is MICB positive/HLA-E negative cancer. The method of claim 76, wherein the MICB positive cancer cells are MICB positive and HLA-E negative cancer cells. The method of any one of claims 66 to 77, wherein the killing comprises necrosis. The method of any one of claims 66 to 77, wherein the killing comprises apoptosis. The method of any one of claims 66 to 77, wherein the killing is mediated through NK cell-mediated lysis. The method of any one of claims 66 to 80, wherein the administering also results in killing of non-MICA-positive and non-MICB-positive cancer cells in the subject. The method of any one of claims 66 to 81, wherein the method further comprises administering an NKG2A inhibitor to the individual. The method of claim 82, wherein the NKG2A inhibitor is an antagonistic antibody that specifically binds to NKG2A. A method of reducing the volume of a solid tumor in an individual previously identified or diagnosed with a MICA-positive cancer, the method comprising administering a therapeutically effective amount of a population of enucleated erythroid cells comprising on their extracellular surfaces The first exogenous fusion polypeptide, the first exogenous fusion polypeptide comprises (i) IL-15 or its functional fragment, and (ii) IL-15 receptor α or its functional fragment. The method of claim 84, wherein the method further comprises identifying or diagnosing the individual as having a MICA-positive cancer. The method of claim 84 or 85, wherein the MICA-positive cancer is selected from the group consisting of adrenocortical carcinoma, cholangiocarcinoma, pancreatic cancer, kidney cancer, thyroid cancer, mesothelioma, cutaneous epidermal melanoma, Colorectal cancer, squamous cell carcinoma of the cervix, and endocervical adenocarcinoma. The method of any one of claims 84 to 86, wherein the MICA positive cancer is a MICA positive/HLA-E negative cancer. A method of reducing the volume of a solid tumor in an individual previously identified or diagnosed with an MICB-positive cancer, the method comprising administering a therapeutically effective amount of a population of enucleated erythroid cells comprising on their extracellular surfaces The first exogenous fusion polypeptide, the first exogenous fusion polypeptide comprises (i) IL-15 or its functional fragment, and (ii) IL-15 receptor α or its functional fragment. The method of claim 88, wherein the method further comprises identifying or diagnosing the individual as having MICB-positive cancer. The method of claim 88 or 89, wherein the MICB-positive cancer is selected from the group consisting of acute myelogenous leukemia, lymphoid neoplasm diffuse large B-cell lymphoma, testicular germ cell tumor, gastric adenocarcinoma, ovarian serous cystadenocarcinoma, esophageal cancer, and lung cancer. The method of any one of claims 88 to 90, wherein the MICB positive cancer is MICB positive/HLA-E negative cancer. The method of any one of claims 84 to 91, wherein the administering results in at least a 5% reduction in the volume of the solid tumor compared to the volume of the solid tumor in the individual prior to administration. The method of claim 92, wherein the administering results in a reduction in the volume of the solid tumor of at least 10% compared to the volume of the solid tumor in the individual prior to administration. The method of claim 93, wherein the administering results in at least a 15% reduction in the volume of the solid tumor compared to the volume of the solid tumor in the individual prior to administration. The method of any one of claims 84 to 94, wherein the method further comprises administering an NKG2A inhibitor to the individual. The method of claim 95, wherein the NKG2A inhibitor is an antagonistic antibody that specifically binds to NKG2A. The method of any one of claims 1-96, wherein the enucleated erythroid blood cells comprise at least 1,000 copies of the first exogenous fusion polypeptide. The method according to any one of claims 1 to 97, wherein the enucleated erythroid blood cells are produced by a method comprising: introducing a nucleic acid encoding the first exogenous fusion polypeptide into a nucleated erythroid precursor cell; and The nucleated erythroid precursor cells are cultured under conditions sufficient to express the first exogenous fusion polypeptide and enucleate the nucleated erythroid precursor cells. The method according to any one of claims 1 to 98, wherein the enucleated erythroid cells further comprise a second exogenous polypeptide comprising 4-1BBL or a functional fragment thereof, wherein the second exogenous polypeptide is present in the On the extracellular surface of enucleated erythroid cells. The method of claim 99, wherein the enucleated erythroid blood cells comprise at least 1,000 copies of the second exogenous polypeptide. The method as claimed in claim 99 or 100, wherein the enucleated erythroid blood cells are produced by a method comprising: introducing a nucleic acid encoding the first exogenous fusion polypeptide or a functional fragment thereof and a nucleic acid encoding the second exogenous polypeptide into a nucleated erythroid precursor cell; and The nucleated erythroid precursor cells are cultured under conditions sufficient to express the first exogenous fusion polypeptide and the second exogenous polypeptide and to enucleate the nucleated erythroid precursor cells. The method according to any one of claims 1 to 101, wherein the enucleated erythroid cells are not hypodialysis cells. The method of any one of claims 1 to 102, wherein the enucleated erythroid cells do not include a sortase-transfer signature. The method of claim 103, wherein the individual is human and the enucleated erythroid cells are human cells. A kit comprising: A pharmaceutical composition comprising enucleated erythrocytes comprising on their extracellular surface a first exogenous fusion polypeptide comprising (i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof; and Instructions for carrying out the method of any one of claims 1 to 135. The kit according to claim 105, wherein the enucleated erythroid blood cells comprise at least 1,000 copies of the first exogenous fusion polypeptide. The kit according to claim 105 or 106, wherein the enucleated erythroid blood cells are produced by a method comprising: introducing a nucleic acid encoding the first exogenous fusion polypeptide into a nucleated erythroid precursor cell; and The nucleated erythroid precursor cells are cultured under conditions sufficient to express the first exogenous fusion polypeptide and enucleate the nucleated erythroid precursor cells. The set according to any one of claims 105 to 107, wherein the enucleated erythrocytes further comprise a second exogenous polypeptide comprising 4-1BBL or a functional fragment thereof, wherein the second exogenous polypeptide is present in The extracellular surface of the enucleated erythroid cells. The kit of claim 108, wherein the enucleated erythroid blood cells comprise at least 1,000 copies of the second exogenous polypeptide. The kit according to claim 108 or 109, wherein the enucleated erythroid blood cells are produced by a method comprising: introducing a nucleic acid encoding the first exogenous fusion polypeptide and a nucleic acid encoding the second exogenous polypeptide into a nucleated erythroid precursor cell; and The nucleated erythroid precursor cells are cultured under conditions sufficient to express the first exogenous fusion polypeptide and the second exogenous polypeptide and to enucleate the nucleated erythroid precursor cells. The set according to any one of claims 105 to 110, wherein the enucleated erythroid cells are not hypodialyzed cells. The kit of any one of claims 105 to 111, wherein the enucleated erythroid cells do not include sortase-transferred features. The set of any one of claims 105 to 112, wherein the enucleated erythroid cells are human cells. A method of selecting a pharmaceutical composition comprising a population of enucleated erythrocytes comprising a first exogenous fusion polypeptide on their extracellular surface, the first exogenous fusion polypeptide comprising ( i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof, for use in an individual previously identified or diagnosed with a MICA positive cancer. The method of claim 114, wherein the method further comprises identifying or diagnosing the individual as having a MICA-positive cancer. The method of claim 114 or 115, wherein the MICA-positive cancer is selected from the group consisting of adrenocortical carcinoma, cholangiocarcinoma, pancreatic cancer, kidney cancer, thyroid cancer, mesothelioma, cutaneous epidermal melanoma, Colorectal cancer, squamous cell carcinoma of the cervix, and endocervical adenocarcinoma. The method of any one of claims 114 to 116, wherein the MICA positive cancer is a MICA positive/HLA-E negative cancer. A method of selecting a pharmaceutical composition comprising a population of enucleated erythrocytes comprising a first exogenous fusion polypeptide on their extracellular surface, the first exogenous fusion polypeptide comprising ( i) IL-15 or a functional fragment thereof, and (ii) IL-15 receptor alpha or a functional fragment thereof, for use in individuals previously identified or diagnosed with MICB positive cancer. The method of claim 118, wherein the method further comprises identifying or diagnosing the individual as having an MICB-positive cancer. The method of claim 118 or 119, wherein the MICB-positive cancer is selected from the group consisting of acute myelogenous leukemia, lymphoid neoplasm diffuse large B-cell lymphoma, testicular germ cell tumor, gastric adenocarcinoma, ovarian serous cystadenocarcinoma, esophageal cancer, and lung cancer. The method of any one of claims 118 to 120, wherein the MICB positive cancer is MICB positive/HLA-E negative cancer.

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