Immunotherapy for ox40 expressing cancer

Abstract

Disclosed herein are methods of using OX40 as a biomarker for predicting clinical sensitivity and therapeutic response of subjects having cancer to treatment with immunotherapeutic agents, and methods of using OX40 as a biomarker for selecting patient population for cancer treatment with immunotherapeutic agents. Disclosed herein are also methods of treating OX40-expressing cancers using immunotherapeutic agents. Further provided herein are kit for predicting the responsiveness of a subject having cancer to treatment with immunotherapeutic agents.

Description

effective amount of an immunotherapeutic agent to a subject. Provided herein are methods of treating OX40-expressing MDS by administering a therapeutically effective amount of an immunotherapeutic agent to a subject. Provided herein are methods of treating OX40-expressing CMML by administering a therapeutically effective amount of an immunotherapeutic agent to a subject. Provided herein are methods of treating OX40-expressing T-ALL by administering a therapeutically effective amount of an immunotherapeutic agent to a subject. Provided herein are methods of treating OX40-expressing NK leukemia by administering a therapeutically effective amount of an immunotherapeutic agent to a subject. Provided herein are methods of treating OX40-expressing DLBCL by administering a therapeutically effective amount of an immunotherapeutic agent to a subject. Provided herein are methods of treating OX40-expressing CTCL by administering a therapeutically effective amount of an immunotherapeutic agent to a subject. Provided herein are methods of treating OX40-expressing PTCL by administering a therapeutically effective amount of an immunotherapeutic agent to a subject. Provided herein are methods of treating OX40-expressing T cell lymphoblastic lymphoma by administering a therapeutically effective amount of an immunotherapeutic agent to a subject. Provided herein are methods of treating OX40-expressing ATLL by administering a therapeutically effective amount of an immunotherapeutic agent to a subject. Provided herein are methods of treating OX40-expressing T-cell lymphoma by administering a therapeutically effective amount of an immunotherapeutic agent to a subject. Provided herein are methods of treating OX40-expressing NK / T cell lymphoma by administering a therapeutically effective amount of an immunotherapeutic agent to a subject. The immunotherapeutic agent can be, e.g, HX009, HX011, HX518, HX543, HX523, HX523-1, HX534, HX534-2, HX111, HX011-MMAF, HXOll-Dxd, HX011-SN-38, HXOll-Exatecan, HXOll-Eriblin, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, HX534-2-MMAE, or HX044. In some embodiments, the immunotherapeutic agent is an ADC having HX011 conjugated to a cytotoxic agent (e.g, HX111, HX011-MMAF, HXOll-Dxd, HX011-SN-38, HXOll-Exatecan, or HXOll-Eriblin). In some embodiments, the immunotherapeutic agent is an ADC having an anti-OX40 antibody conjugated to MMAE (e.g, HX111, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, or HX534-2-MMAE). In some embodiments, the immunotherapeutic agent is an ADC having an anti-OX40 antibody conjugated to a cytotoxic agent, wherein the anti-OX40 antibody is HX011, HX518, HX543, HX523, HX523-1, HX534, or HX534-2; and the cytotoxic agent is MMAE, MMAF, Dxd, SN-38, Exatecan, or Eribulin.

[0099] In some embodiments, cancers that can be treated with the methods provided herein are solid tumors. In some embodiments, provided herein are methods for treating 0X40 expressing solid tumor by administering a therapeutically effective amount of an immunotherapeutic agent to a subject in need thereof. In some embodiments, methods provided herein comprise assessing 0X40 expression in a sample from a subject having a solid tumor and administering a therapeutically effective amount of an immunotherapeutic agent to the subject if the subject has 0X40 expressing cancer. In some embodiments, provided herein are methods of predicting responsiveness of a subject having solid tumor to an immunotherapeutic agent, comprising measuring 0X40 expression in a sample from the subject, wherein the subject is likely responsive to the treatment if the solid tumor is an 0X40 expressing cancer. In some embodiments, provided herein are methods of selecting a subject having solid tumor for treatment with immunotherapeutic agent comprising measuring the 0X40 expression in a sample from the subject and selecting the subject for the immunotherapeutic agent treatment if the subject has an 0X40 expressing cancer.

[00100] In some embodiments, the solid tumor can be carcinomas, sarcoma, melanoma (e.g, cutaneous or intraocular malignant melanoma), endometrial cancer, glioma, glioblastoma, brain and spinal cord tumors, germ cell tumors, neuroendocrine tumors, carcinoid tumors, gastric cancer, esophageal cancer, gastroesophageal junction adenocarcinoma, liver cancer, lung cancer (e.g, small cell lung cancer, or non-small cell lung cancer), head and neck cancer, skin cancer, nasopharyngeal cancer, kidney cancer, colorectal cancer, breast cancer, pancreatic cancer, testicular cancer, cervical cancer, ovarian cancer, uterine cancer, prostate cancer (for example, hormone refractory prostate adenocarcinoma), bladder cancer, colon cancer, endocrine cancer, basal cell cancer, squamous cell cancer, dermatofibrosarcoma protuberans, mesothelioma, Merkel cell carcinoma, bone cancer, intestinal cancer, renal cancer (for example, clear cell carcinoma), throat cancer, rectal cancer, cancer of the anal region, brain cancer, stomach cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the small intestine, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, cancer of ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, malignant epithelioid mesothelioma, gallbladder cancer, epidermoid cancer, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, synovial sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, Ewing’s tumor, leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm’s tumor, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodenroglioma, schwannoma, meningioma, neuroblastoma, or retinoblastoma.

[00101] In some embodiments, provided herein are methods for treating 0X40 expressing solid tumor by administering a therapeutically effective amount of an immunotherapeutic agent to a subject in need thereof. In some embodiments, provided herein are methods for treating 0X40-expressing head and neck cancer, OX40-expressing esophageal cancer, OX40-expressing cervical cancer, OX40-expressing sarcoma, OX40-expressing small cell lung cancer, OX40-expressing nonsmall cell lung cancer, OX40-expressing gastric cancer, OX40-expressing liver cancer, 0X40-expressing melanoma, OX40-expressing colorectal cancer, OX40-expressing squamous cell carcinoma, OX40-expressing endometrial cancer, OX40-expressing breast cancer, OX40-expressing malignant epithelioid mesothelioma, OX40-expressing gallbladder cancer, OX40-expressing pancreatic cancer, OX40-expressing glioblastoma, OX40-expressing ovarian cancer, or 0X40-expressing gastroesophageal junction adenocarcinoma by administering a therapeutically effective amount of an immunotherapeutic agent to a subject in need thereof. The immunotherapeutic agent can be, e.g, HX009, HX011, HX518, HX543, HX523, HX523-1, HX534, HX534-2, HX111, HX011-MMAF, HX011-Dxd, HX01 l-SN-38, HX011-Exatecan, HX011-Eriblin, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, HX534-2-MMAE, or HX044. In some embodiments, the immunotherapeutic agent is an ADC having HX011 conjugated to a cytotoxic agent (e.g, HX111, HX011-MMAF, HXOll-Dxd, HX011-SN-38, HX011-Exatecan, or HX011-Eriblin). In some embodiments, the immunotherapeutic agent is an ADC having an anti-0X40 antibody conjugated to MMAE (e.g, HX111, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, or HX534-2-MMAE). In some embodiments, the immunotherapeutic agent is an ADC having an anti-OX40 antibody conjugated to a cytotoxic agent, wherein the anti-OX40 antibody is HX011, HX518, HX543, HX523, HX523-1, HX534, or HX534-2; and the cytotoxic agent is MMAE, MMAF, Dxd, SN-38, Exatecan, or Eribulin.

[00102] Provided herein are methods of treating OX40-expressing head and neck cancer by administering a therapeutically effective amount of an immunotherapeutic agent to a subject. Provided herein are methods of treating OX40-expressing esophageal cancer by administering a therapeutically effective amount of an immunotherapeutic agent to a subject. Provided herein are methods of treating OX40-expressing cervical cancer by administering a therapeutically effective amount of an immunotherapeutic agent to a subject. Provided herein are methods of treating 0X40-expressing sarcoma by administering a therapeutically effective amount of an immunotherapeutic agent to a subject. In some embodiments, sarcoma can be spindle cell sarcoma, small round cell sarcoma, Ewing's sarcoma, chondrosarcoma, synovial sarcoma, osteosarcoma, high-grade sarcoma, high-grade undifferentiated sarcoma, rhabdomyosarcoma, pleomorphic sarcoma, cystosarcoma, sarcomatoid carcinoma, undifferentiated pleomorphic sarcoma, or leiomyosarcoma. In some embodiments, sarcoma is spindle cell sarcoma. In some embodiments, sarcoma is small round cell sarcoma. In some embodiments, sarcoma is Ewing's sarcoma. In some embodiments, sarcoma is chondrosarcoma. In some embodiments, sarcoma is synovial sarcoma. In some embodiments, sarcoma is osteosarcoma. In some embodiments, sarcoma is high-grade sarcoma. In some embodiments, sarcoma is high-grade undifferentiated sarcoma. In some embodiments, sarcoma is rhabdomyosarcoma. In some embodiments, sarcoma is pleomorphic sarcoma. In some embodiments, sarcoma is cystosarcoma. In some embodiments, sarcoma is sarcomatoid carcinoma. In some embodiments, sarcoma is undifferentiated pleomorphic sarcoma. In some embodiments, sarcoma is leiomyosarcoma.

[00103] In some embodiments, the cancer to be treated by methods disclosed herein has a high degree of microsatellite instability. In some embodiments, the cancer is a metastatic cancer, refractory cancer, or recurrent cancer. In some embodiments, the cancer is at an advanced stage.

[00104] In some embodiments, the cancer to be treated by methods disclosed herein is EBV positive, HPV positive, HBV positive, HIV positive, or HTLV-1 positive. In some embodiments, the cancer is EBV positive. In some embodiments, the cancer is HPV positive. In some embodiments, the cancer is HBV positive. In some embodiments, the cancer is HIV positive. In some embodiments, the cancer is HTLV-1 positive.

[00105] In some embodiments, the cancer is EBV negative, or HPV negative, or HBV negative, or HIV negative, or HTLV-1 negative. In some embodiments, the cancer is EBV negative. In some embodiments, the cancer is HPV negative. In some embodiments, the cancer is HBV negative. In some embodiments, the cancer is HIV negative. In some embodiments, the cancer is HTLV-1 negative.

[00106] In some embodiments, provided herein are methods of treating a subject having an 0X40-expressing hematological cancer by administering a therapeutically effective amount of an anti-0X40 antibody (e.g, HX011, HX518, HX543, HX523, HX523-1, HX534, HX534-2) to a subject. In some embodiments, provided herein are method of predicting responsiveness of a subject having a hematological cancer to an anti-OX40 antibody (e.g., HX011, HX518, HX543, HX523, HX523-1, HX534, HX534-2), comprising measuring 0X40 expression in a sample from the subject, wherein the subject is likely responsive to the anti-OX40 antibody (e.g., HX011, HX518, HX543, HX523, HX523-1, HX534, HX534-2) if the cancer is an 0X40 expressing cancer. In some embodiments, provided herein are methods of selecting a subject having hematological cancer for treatment with an anti-OX40 antibody (e.g, HX011, HX518, HX543, HX523, HX523-1, HX534, HX534-2), comprising measuring the 0X40 expression in a sample from the subject and selecting the subject for the treatment if the subject has an 0X40 expressing cancer. In some embodiments, the 0X40-expressing hematological cancer is OX40-expressing lymphoma or OX40-expressing leukemia. In some embodiments, the OX40-expressing hematological cancer is OX40-expressing AML, 0X40-expressing CML, OX40-expressing MDS, OX40-expressing CMML, OX40-expressing T-ALL, OX40-expressing NK leukemia, OX40-expressing DLBCL, OX40-expressing CTCL, 0X40-expressing PTCL, OX40-expressing T cell lymphoblastic lymphoma, OX40-expressing ATLL, OX40-expressing angioimmunoblastic T-cell lymphoma, or OX40-expressing NK / T cell lymphoma. In some embodiments, the OX40-expressing hematological cancer is responsive to the treatment of the anti-OX40 antibody (e.g, HX011, HX518, HX543, HX523, HX523-1, HX534, HX534-2) provided herein. In some embodiments, the anti-OX40 antibody is HX011.

[00107] In some embodiments, provided herein are methods of treating a subject having an 0X40-expressing lymphoma by administering a therapeutically effective amount of an anti-OX40 antibody (e.g, HX011, HX518, HX543, HX523, HX523-1, HX534, HX534-2) to a subject. In some embodiments, provided herein are method of predicting responsiveness of a subject having a lymphoma to an anti-OX40 antibody (e.g, HX011, HX518, HX543, HX523, HX523-1, HX534, HX534-2), comprising measuring 0X40 expression in a sample from the subject, wherein the subject is likely responsive to the anti-OX40 antibody (e.g., HX011, HX518, HX543, HX523, HX523-1, HX534, HX534-2) if the lymphoma is an 0X40 expressing lymphoma. In some embodiments, provided herein are methods of selecting a subject having a lymphoma for treatment with an anti-0X40 antibody (e.g, HX011, HX518, HX543, HX523, HX523-1, HX534, HX534-2), comprising measuring the 0X40 expression in a sample from the subject and selecting the subject for the treatment if the subject has an 0X40 expressing lymphoma. In some embodiments, the anti-OX40 antibody is HX011.

[00108] In some embodiments, provided herein are methods of treating a subject having an 0X40-expressing T cell lymphoma by administering a therapeutically effective amount of an anti-OX40 antibody (e.g, HX011, HX518, HX543, HX523, HX523-1, HX534, HX534-2) to a subject. In some embodiments, provided herein are method of predicting responsiveness of a subject having a T cell lymphoma to an anti-OX40 antibody (e.g, HX011, HX518, HX543, HX523, HX523-1, HX534, HX534-2), comprising measuring 0X40 expression in a sample from the subject, wherein the subject is likely responsive to the anti-OX40 antibody (e.g., HX011, HX518, HX543, HX523, HX523-1, HX534, HX534-2) if the T cell lymphoma is an 0X40 expressing T cell lymphoma. In some embodiments, provided herein are methods of selecting a subject having a T cell lymphoma for treatment with an anti-OX40 antibody (e.g., HX011, HX518, HX543, HX523, HX523-1, HX534, HX534-2), comprising measuring the 0X40 expression in a sample from the subject and selecting the subject for the treatment if the subject has an 0X40 expressing T cell lymphoma. In some embodiments, the anti-OX40 antibody is HX011.

[00109] In some embodiments, provided herein are methods of treating a subject having an 0X40-expressing B cell lymphoma by administering a therapeutically effective amount of an anti-OX40 antibody (e.g, HX011, HX518, HX543, HX523, HX523-1, HX534, HX534-2) to a subject. In some embodiments, provided herein are method of predicting responsiveness of a subject having a B cell lymphoma to an anti-OX40 antibody (e.g, HX011, HX518, HX543, HX523, HX523-1, HX534, HX534-2), comprising measuring 0X40 expression in a sample from the subject, wherein the subject is likely responsive to the anti-OX40 antibody (e.g., HX011, HX518, HX543, HX523, HX523-1, HX534, HX534-2) if the B cell lymphoma is an 0X40 expressing B cell lymphoma. In some embodiments, provided herein are methods of selecting a subject having a B cell lymphoma for treatment with an anti-OX40 antibody (e.g, HX011, HX518, HX543, HX523, HX523-1, HX534, HX534-2), comprising measuring the 0X40 expression in a sample from the subject and selecting the subject for the treatment if the subject has an 0X40 expressing B cell lymphoma. In some embodiments, the anti-OX40 antibody is HX011.

[00110] In some embodiments, provided herein are methods of treating a subject having an 0X40-expressing DLBCL by administering a therapeutically effective amount of an anti-OX40 antibody (e.g, HX011, HX518, HX543, HX523, HX523-1, HX534, HX534-2) to a subject. In some embodiments, provided herein are method of predicting responsiveness of a subject having DLBCL to an anti-OX40 antibody (e.g, HX011, HX518, HX543, HX523, HX523-1, HX534, HX534-2), comprising measuring 0X40 expression in a sample from the subject, wherein the subject is likely responsive to the anti-OX40 antibody (e.g, HX011, HX518, HX543, HX523, HX523-1, HX534, HX534-2) if the DLBCL is an 0X40 expressing DLBCL. In some embodiments, provided herein are methods of selecting a subject having DLBCL for treatment with an anti-OX40 antibody (e.g., HX011, HX518, HX543, HX523, HX523-1, HX534, HX534-2), comprising measuring the 0X40 expression in a sample from the subject and selecting the subject for the treatment if the subject has an 0X40 expressing DLBCL. In some embodiments, the anti-OX40 antibody is HX011.

[00111] In some embodiments, provided herein are methods of treating a subject having an 0X40-expressing leukemia by administering a therapeutically effective amount of an anti-OX40 antibody (e.g, HX011, HX518, HX543, HX523, HX523-1, HX534, HX534-2) to a subject. In some embodiments, provided herein are method of predicting responsiveness of a subject having a leukemia to an anti-OX40 antibody (e.g, HX011, HX518, HX543, HX523, HX523-1, HX534, HX534-2), comprising measuring 0X40 expression in a sample from the subject, wherein the subject is likely responsive to the anti-OX40 antibody (e.g., HX011, HX518, HX543, HX523, HX523-1, HX534, HX534-2) if the leukemia is an 0X40 expressing leukemia. In some embodiments, provided herein are methods of selecting a subject having a leukemia for treatment with an anti-OX40 antibody (e.g, HX011, HX518, HX543, HX523, HX523-1, HX534, HX534-2), comprising measuring the 0X40 expression in a sample from the subject and selecting the subject for the treatment if the subject has an 0X40 expressing leukemia. In some embodiments, the anti-OX40 antibody is HX011.

[00112] In some embodiments, provided herein are methods of treating a subject having an 0X40-expressing T cell leukemia by administering a therapeutically effective amount of an anti-OX40 antibody (e.g, HX011, HX518, HX543, HX523, HX523-1, HX534, HX534-2) to a subject. In some embodiments, provided herein are method of predicting responsiveness of a subject having a T cell leukemia to an anti-OX40 antibody (e.g, HX011, HX518, HX543, HX523, HX523-1, HX534, HX534-2), comprising measuring 0X40 expression in a sample from the subject, wherein the subject is likely responsive to the anti-OX40 antibody (e.g., HX011, HX518, HX543, HX523, HX523-1, HX534, HX534-2) if the T cell leukemia is an 0X40 expressing T cell leukemia. In some embodiments, provided herein are methods of selecting a subject having a T cell leukemia for treatment with an anti-OX40 antibody (e.g, HX011, HX518, HX543, HX523, HX523-1, HX534, HX534-2), comprising measuring the 0X40 expression in a sample from the subject and selecting the subject for the treatment if the subject has an 0X40 expressing T cell leukemia. In some embodiments, the anti-OX40 antibody is HX011.

[00113] In some embodiments, provided herein are methods of treating a subject having an 0X40-expressing ATLL by administering a therapeutically effective amount of an anti-OX40 antibody (e.g., HX011, HX518, HX543, HX523, HX523-1, HX534, HX534-2) to a subject. In some embodiments, provided herein are method of predicting responsiveness of a subject having a ATLL to an anti-0X40 antibody (e.g, HX011, HX518, HX543, HX523, HX523-1, HX534, HX534-2), comprising measuring 0X40 expression in a sample from the subject, wherein the subject is likely responsive to the anti-OX40 antibody (e.g, HX011, HX518, HX543, HX523, HX523-1, HX534, HX534-2) if the ATLL is an 0X40 expressing ATLL. In some embodiments, provided herein are methods of selecting a subject having a ATLL for treatment with an anti-OX40 antibody (e.g., HX011, HX518, HX543, HX523, HX523-1, HX534, HX534-2), comprising measuring the 0X40 expression in a sample from the subject and selecting the subject for the treatment if the subject has an 0X40 expressing ATLL. In some embodiments, the anti-OX40 antibody is HX011.

[00114] In some embodiments, provided herein are methods of treating a subject having an 0X40-expressing solid tumor by administering a therapeutically effective amount of an anti-OX40 antibody (e.g, HX011, HX518, HX543, HX523, HX523-1, HX534, HX534-2) to a subject. In some embodiments, provided herein are method of predicting responsiveness of a subject having a solid tumor to an anti-OX40 antibody (e.g, HX011, HX518, HX543, HX523, HX523-1, HX534, HX534-2), comprising measuring 0X40 expression in a sample from the subject, wherein the subject is likely responsive to the anti-OX40 antibody (e.g, HX011, HX518, HX543, HX523, HX523-1, HX534, HX534-2) if the cancer is an 0X40 expressing cancer. In some embodiments, provided herein are methods of selecting a subject having a solid tumor for treatment with an anti-OX40 antibody (e.g, HX011, HX518, HX543, HX523, HX523-1, HX534, HX534-2), comprising measuring the 0X40 expression in a sample from the subject and selecting the subject for the treatment if the subject has an 0X40 expressing cancer. In some embodiments, the 0X40-expressing solid tumor is OX40-expressing head and neck cancer, OX40-expressing esophageal cancer, OX40-expressing cervical cancer, OX40-expressing sarcoma, OX40-expressing small cell lung cancer, OX40-expressing non-small cell lung cancer, OX40-expressing gastric cancer, 0X40-expressing liver cancer, OX40-expressing melanoma, OX40-expressing colorectal cancer, 0X40-expressing squamous cell carcinoma, OX40-expressing endometrial cancer, OX40-expressing breast cancer, OX40-expressing malignant epithelioid mesothelioma, OX40-expressing gallbladder cancer, OX40-expressing pancreatic cancer, OX40-expressing glioblastoma, OX40-expressing ovarian cancer, or OX40-expressing gastroesophageal junction adenocarcinoma. In some embodiments, the OX40-expressing solid tumor is responsive to the treatment of anti-OX40 antibody (e.g, HX011, HX518, HX543, HX523, HX523-1, HX534, HX534-2) provided herein. In some embodiments, the anti-OX40 antibody is HX011.

[00115] In some embodiments, provided herein are methods of treating a subject having an 0X40-expressing head and neck cancer by administering a therapeutically effective amount of an anti-0X40 antibody (e.g, HX011, HX518, HX543, HX523, HX523-1, HX534, HX534-2) to a subject. In some embodiments, provided herein are method of predicting responsiveness of a subject having a head and neck cancer to an anti-OX40 antibody (e.g, HX011, HX518, HX543, HX523, HX523-1, HX534, HX534-2), comprising measuring 0X40 expression in a sample from the subject, wherein the subject is likely responsive to the anti-OX40 antibody (e.g., HX011, HX518, HX543, HX523, HX523-1, HX534, HX534-2) if the head and neck cancer is an 0X40 expressing head and neck cancer. In some embodiments, provided herein are methods of selecting a subject having a head and neck cancer for treatment with an anti-OX40 antibody (e.g, HX011, HX518, HX543, HX523, HX523-1, HX534, HX534-2), comprising measuring the 0X40 expression in a sample from the subject and selecting the subject for the treatment if the subject has an 0X40 expressing head and neck cancer. In some embodiments, the anti-OX40 antibody is HX011.

[00116] In some embodiments, provided herein are methods of treating a subject having an 0X40-expressing sarcoma by administering a therapeutically effective amount of an anti-OX40 antibody (e.g, HX011, HX518, HX543, HX523, HX523-1, HX534, HX534-2) to a subject. In some embodiments, provided herein are method of predicting responsiveness of a subject having a sarcoma to an anti-OX40 antibody (e.g, HX011, HX518, HX543, HX523, HX523-1, HX534, HX534-2), comprising measuring 0X40 expression in a sample from the subject, wherein the subject is likely responsive to the anti-OX40 antibody (e.g, HX011, HX518, HX543, HX523, HX523-1, HX534, HX534-2) if the sarcoma is an 0X40 expressing sarcoma. In some embodiments, provided herein are methods of selecting a subject having a sarcoma for treatment with an anti-OX40 antibody (e.g, HX011, HX518, HX543, HX523, HX523-1, HX534, HX534-2), comprising measuring the 0X40 expression in a sample from the subject and selecting the subject for the treatment if the subject has an 0X40 expressing sarcoma. In some embodiments, the anti-OX40 antibody is HX011. In some embodiments, sarcoma can be spindle cell sarcoma, small round cell sarcoma, Ewing's sarcoma, chondrosarcoma, synovial sarcoma, osteosarcoma, high-grade sarcoma, high-grade undifferentiated sarcoma, rhabdomyosarcoma, pleomorphic sarcoma, cystosarcoma, sarcomatoid carcinoma, undifferentiated pleomorphic sarcoma, or leiomyosarcoma. In some embodiments, sarcoma is spindle cell sarcoma. In some embodiments, sarcoma is small round cell sarcoma. In some embodiments, sarcoma is Ewing's sarcoma. In some embodiments, sarcoma is chondrosarcoma. In some embodiments, sarcoma is synovial sarcoma. In some embodiments, sarcoma is osteosarcoma. In some embodiments, sarcoma is high-grade sarcoma. In some embodiments, sarcoma is high-grade undifferentiated sarcoma. In some embodiments, sarcoma is rhabdomyosarcoma. In some embodiments, sarcoma is pleomorphic sarcoma. In some embodiments, sarcoma is cystosarcoma. In some embodiments, sarcoma is sarcomatoid carcinoma. In some embodiments, sarcoma is undifferentiated pleomorphic sarcoma. In some embodiments, sarcoma is leiomyosarcoma.

[00117] In some embodiments, provided herein are methods of treating a subject having an 0X40-expressing spindle cell sarcoma by administering a therapeutically effective amount of an anti-OX40 antibody (e.g, HX011, HX518, HX543, HX523, HX523-1, HX534, HX534-2) to a subject. In some embodiments, provided herein are method of predicting responsiveness of a subject having a spindle cell sarcoma to an anti-OX40 antibody (e.g, HX011, HX518, HX543, HX523, HX523-1, HX534, HX534-2), comprising measuring 0X40 expression in a sample from the subject, wherein the subject is likely responsive to the anti-OX40 antibody (e.g., HX011, HX518, HX543, HX523, HX523-1, HX534, HX534-2) if the spindle cell sarcoma is an 0X40 expressing spindle cell sarcoma. In some embodiments, provided herein are methods of selecting a subject having a spindle cell sarcoma for treatment with an anti-OX40 antibody (e.g, HX011, HX518, HX543, HX523, HX523-1, HX534, HX534-2), comprising measuring the 0X40 expression in a sample from the subject and selecting the subject for the treatment if the subject has an 0X40 expressing spindle cell sarcoma. In some embodiments, the anti-OX40 antibody is HX011.

[00118] In some embodiments, provided herein are methods of treating a subject having an 0X40-expressing hematological cancer by administering a therapeutically effective amount of an anti-0X40 ADC (e.g, HX111, HX011-MMAF, HXOll-Dxd, HX011-SN-38, HX011-Exatecan, HX011-Eriblin, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, HX534-2-MMAE) to a subject. In some embodiments, provided herein are method of predicting responsiveness of a subject having a hematological cancer to an anti-OX40 ADC (e.g., HX111, HX011-MMAF, HX011-Dxd, HX01 l-SN-38, HX011-Exatecan, HX011-Eriblin, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, HX534-2-MMAE), comprising measuring 0X40 expression in a sample from the subject, wherein the subject is likely responsive to the anti-OX40 ADC (e.g, HX111, HX011-MMAF, HXOll-Dxd, HX01 l-SN-38, HX011-Exatecan, HX011-Eriblin, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, HX534-2-MMAE) if the cancer is an 0X40 expressing cancer. In some embodiments, provided herein are methods of selecting a subject having hematological cancer for treatment with an anti-OX40 ADC (e.g., HX111, HX011-MMAF, HX011-Dxd, HX01 l-SN-38, HX011-Exatecan, HX011-Eriblin, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, HX534-2-MMAE), comprising measuring the 0X40 expression in a sample from the subject and selecting the subject for the treatment if the subject has an 0X40 expressing cancer. In some embodiments, the OX40-expressing hematological cancer is 0X40-expressing lymphoma or OX40-expressing leukemia. In some embodiments, the OX40-expressing hematological cancer is OX40-expressing AML, OX40-expressing CML, OX40-expressing MDS, OX40-expressing CMML, OX40-expressing T-ALL, OX40-expressing NK leukemia, 0X40-expressing DLBCL, OX40-expressing CTCL, OX40-expressing PTCL, OX40-expressing T cell lymphoblastic lymphoma, OX40-expressing ATLL, OX40-expressing angioimmunoblastic T-cell lymphoma, or OX40-expressing NK / T cell lymphoma. In some embodiments, the OX40-expressing hematological cancer is responsive to the treatment of the anti-OX40 ADC (e. g.. HX111, HXO11-MMAF, HXOll-Dxd, HX011-SN-38, HXOll-Exatecan, HXOll-Eriblin, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, HX534-2-MMAE) provided herein. In some embodiments, the anti-OX40 ADC is an ADC having HX011 conjugated to a cytotoxic agent (e.g, HX111, HX011-MMAF, HXOll-Dxd, HX011-SN-38, HXOll-Exatecan, or HXOll-Eriblin). In some embodiments, the anti-OX40 ADC is an ADC having an anti-OX40 antibody conjugated to MMAE (e.g, HX111, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, or HX534-2-MMAE). In some embodiments, the anti-OX40 ADC is an ADC having an anti-OX40 antibody conjugated to a cytotoxic agent, wherein the anti-OX40 antibody is HX011, HX518, HX543, HX523, HX523-1, HX534, or HX534-2; and the cytotoxic agent is MMAE, MMAF, Dxd, SN-38, Exatecan, or Eribulin. In some embodiments, the anti-OX40 ADC is HX111.

[00119] In some embodiments, provided herein are methods of treating a subject having an 0X40-expressing lymphoma by administering a therapeutically effective amount of an anti-OX40 ADC (e.g, HX111, HX011-MMAF, HXOll-Dxd, HX011-SN-38, HXOll-Exatecan, HXOll-Eriblin, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, HX534-2-MMAE) to a subject. In some embodiments, provided herein are method of predicting responsiveness of a subject having a lymphoma to an anti-OX40 ADC (e.g., HX111, HX011-MMAF, HX011-Dxd, HX01 l-SN-38, HXO 11-Exatecan, HX011-Eriblin, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, HX534-2-MMAE), comprising measuring 0X40 expression in a sample from the subject, wherein the subject is likely responsive to the anti-0X40 ADC (e.g, HX111, HX011-MMAF, HXOll-Dxd, HX011-SN-38, HXOll-Exatecan, HXOll-Eriblin, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, HX534-2-MMAE) if the lymphoma is an 0X40 expressing lymphoma. In some embodiments, provided herein are methods of selecting a subject having a lymphoma for treatment with an anti-0X40 ADC (e.g, HX111, HX011-MMAF, HXOll-Dxd, HX011-SN-38, HXOll-Exatecan, HXOll-Eriblin, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, HX534-2-MMAE), comprising measuring the 0X40 expression in a sample from the subject and selecting the subject for the treatment if the subject has an 0X40 expressing lymphoma. In some embodiments, the anti-OX40 ADC is an ADC having HX011 conjugated to a cytotoxic agent (e.g., HX111, HX011-MMAF, HX011-Dxd, HX01 l-SN-38, HX011-Exatecan, or HX011-Eriblin). In some embodiments, the anti-OX40 ADC is an ADC having an anti-OX40 antibody conjugated to MMAE (e.g, HX111, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, or HX534-2-MMAE). In some embodiments, the anti-OX40 ADC is an ADC having an anti-OX40 antibody conjugated to a cytotoxic agent, wherein the anti-OX40 antibody is HX011, HX518, HX543, HX523, HX523-1, HX534, or HX534-2; and the cytotoxic agent is MMAE, MMAF, Dxd, SN-38, Exatecan, or Eribulin. In some embodiments, the anti-OX40 ADC is HX111.

[00120] In some embodiments, provided herein are methods of treating a subject having an 0X40-expressing T cell lymphoma by administering a therapeutically effective amount of an anti-OX40 ADC (e.g, HX111, HX011-MMAF, HX011-Dxd, HX01 l-SN-38, HX011-Exatecan, HX011-Eriblin, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, HX534-2-MMAE) to a subject. In some embodiments, provided herein are method of predicting responsiveness of a subject having aT cell lymphoma to an anti-OX40 ADC (e.g, HX111, HX011-MMAF, HXOll-Dxd, HX011-SN-38, HX011-Exatecan, HX011-Eriblin, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, HX534-2-MMAE), comprising measuring 0X40 expression in a sample from the subject, wherein the subject is likely responsive to the anti-OX40 ADC (e.g, HX111, HX011-MMAF, HXOll-Dxd, HX011-SN-38, HX011-Exatecan, HX011-Eriblin, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, HX534-2-MMAE) if the T cell lymphoma is an 0X40 expressing T cell lymphoma. In some embodiments, provided herein are methods of selecting a subject having a T cell lymphoma for treatment with an anti-OX40 ADC (e.g., HX111, HX011-MMAF, HX011-Dxd, HX01 l-SN-38, HX011-Exatecan, HX011-Eriblin, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, HX534-2-MMAE), comprising measuring the 0X40 expression in a sample from the subject and selecting the subject for the treatment if the subject has an 0X40 expressing T cell lymphoma. In some embodiments, the anti-OX40 ADC is an ADC having HX011 conjugated to a cytotoxic agent (e.g, HX111, HX011-MMAF, HXOll-Dxd, HX01 l-SN-38, HX011-Exatecan, or HX011-Eriblin). In some embodiments, the anti-OX40 ADC is an ADC having an anti-0X40 antibody conjugated to MMAE (e.g, HX111, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, or HX534-2-MMAE). In some embodiments, the anti-0X40 ADC is an ADC having an anti-OX40 antibody conjugated to a cytotoxic agent, wherein the anti-OX40 antibody is HX011, HX518, HX543, HX523, HX523-1, HX534, or HX534-2; and the cytotoxic agent is MMAE, MMAF, Dxd, SN-38, Exatecan, or Eribulin. In some embodiments, the anti-OX40 ADC is HX111.

[00121] In some embodiments, provided herein are methods of treating a subject having an 0X40-expressing B cell lymphoma by administering a therapeutically effective amount of an anti-OX40 ADC (e.g, HX111, HX011-MMAF, HXOll-Dxd, HX011-SN-38, HXOll-Exatecan, HXOll-Eriblin, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, HX534-2-MMAE) to a subject. In some embodiments, provided herein are method of predicting responsiveness of a subject having a B cell lymphoma to an anti-OX40 ADC (e.g., HX111, HX011-MMAF, HXOll-Dxd, HX011-SN-38, HXOll-Exatecan, HXOll-Eriblin, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, HX534-2-MMAE), comprising measuring 0X40 expression in a sample from the subject, wherein the subject is likely responsive to the anti-OX40 ADC (e.g, HX111, HX011-MMAF, HXOll-Dxd, HX011-SN-38, HXOll-Exatecan, HXOll-Eriblin, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, HX534-2-MMAE) if the B cell lymphoma is an 0X40 expressing B cell lymphoma. In some embodiments, provided herein are methods of selecting a subject having a B cell lymphoma for treatment with an anti-OX40 ADC (e.g., HX111, HX011-MMAF, HX011-Dxd, HX01 l-SN-38, HXOll-Exatecan, HXOll-Eriblin, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, HX534-2-MMAE), comprising measuring the 0X40 expression in a sample from the subject and selecting the subject for the treatment if the subject has an 0X40 expressing B cell lymphoma. In some embodiments, the anti-OX40 ADC is an ADC having HX011 conjugated to a cytotoxic agent (e.g, HX111, HX011-MMAF, HXOll-Dxd, HX01 l-SN-38, HXOll-Exatecan, or HX011-Eriblin). In some embodiments, the anti-OX40 ADC is an ADC having an anti-0X40 antibody conjugated to MMAE (e.g, HX111, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, or HX534-2-MMAE). In some embodiments, the anti-0X40 ADC is an ADC having an anti-OX40 antibody conjugated to a cytotoxic agent, wherein the anti-OX40 antibody is HX011, HX518, HX543, HX523, HX523-1, HX534, or HX534-2; and the cytotoxic agent is MMAE, MMAF, Dxd, SN-38, Exatecan, or Eribulin. In some embodiments, the anti-OX40 ADC is HX111.

[00122] In some embodiments, provided herein are methods of treating a subject having an 0X40-expressing DLBCL by administering a therapeutically effective amount of an anti-OX40 ADC (e.g., HX111, HX011-MMAF, HXOll-Dxd, HX011-SN-38, HXOll-Exatecan, HXOll-Eriblin, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, HX534-2-MMAE) to a subject. In some embodiments, provided herein are method of predicting responsiveness of a subject having a DLBCL to an anti-OX40 ADC (e.g, HX111, HX011-MMAF, HX011-Dxd, HX011-SN-38, HXOll-Exatecan, HXOll-Eriblin, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, HX534-2-MMAE), comprising measuring 0X40 expression in a sample from the subject, wherein the subject is likely responsive to the anti-OX40 ADC (e.g., HX111, HX011-MMAF, HX011-Dxd, HX01 l-SN-38, HX011-Exatecan, HX011-Eriblin, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, HX534-2-MMAE) if the DLBCL is an 0X40 expressing DLBCL. In some embodiments, provided herein are methods of selecting a subject having a DLBCL for treatment with an anti-OX40 ADC (e.g.. HX111, HX011-MMAF, HXOll-Dxd, HX011-SN-38, HX011-Exatecan, HX011-Eriblin, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, HX534-2-MMAE), comprising measuring the 0X40 expression in a sample from the subject and selecting the subject for the treatment if the subject has an 0X40 expressing DLBCL. In some embodiments, the anti-OX40 ADC is an ADC having HX011 conjugated to a cytotoxic agent (e.g., HX111, HX011-MMAF, HXOll-Dxd, HX01 l-SN-38, HX011-Exatecan, or HX011-Eriblin). In some embodiments, the anti-OX40 ADC is an ADC having an anti-OX40 antibody conjugated to MMAE (e.g., HX111, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, or HX534-2-MMAE). In some embodiments, the anti-OX40 ADC is an ADC having an anti-OX40 antibody conjugated to a cytotoxic agent, wherein the anti-OX40 antibody is HX011, HX518, HX543, HX523, HX523-1, HX534, or HX534-2; and the cytotoxic agent is MMAE, MMAF, Dxd, SN-38, Exatecan, or Eribulin. In some embodiments, the anti-OX40 ADC is HX111.

[00123] In some embodiments, provided herein are methods of treating a subject having an 0X40-expressing leukemia by administering a therapeutically effective amount of an anti-OX40 ADC (e.g., HX111, HX011-MMAF, HXOll-Dxd, HX011-SN-38, HX011-Exatecan, HX011-Eriblin, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, HX534-2-MMAE) to a subject. In some embodiments, provided herein are method of predicting responsiveness of a subject having a leukemia to an anti-OX40 ADC (e.g., HX111, HX011-MMAF, HX011-Dxd, HX011-SN-38, HX011-Exatecan, HX011-Eriblin, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, HX534-2-MMAE), comprising measuring 0X40 expression in a sample from the subject, wherein the subject is likely responsive to the anti-OX40 ADC (e.g., HX111, HX011-MMAF, HX011-Dxd, HX01 l-SN-38, HX011-Exatecan, HX011-Eriblin, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, HX534-2-MMAE) if the leukemia is an 0X40 expressing leukemia. In some embodiments, provided herein are methods of selecting a subject having a leukemia for treatment with an anti-OX40 ADC (e.g., HX111, HX011-MMAF, HXOll-Dxd, HX011-SN-38, HX011-Exatecan, HX011-Eriblin, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, HX534-2-MMAE), comprising measuring the 0X40 expression in a sample from the subject and selecting the subject for the treatment if the subject has an 0X40 expressing leukemia. In some embodiments, the anti-OX40 ADC is an ADC having HX011 conjugated to a cytotoxic agent (e.g.. HX111, HX011-MMAF, HX011-Dxd, HX01 l-SN-38, HX011-Exatecan, or HX011-Eriblin). In some embodiments, the anti-0X40 ADC is an ADC having an anti-OX40 antibody conjugated to MMAE (e.g., HX111, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, or HX534-2-MMAE). In some embodiments, the anti-OX40 ADC is an ADC having an anti-OX40 antibody conjugated to a cytotoxic agent, wherein the anti-OX40 antibody is HX011, HX518, HX543, HX523, HX523-1, HX534, or HX534-2; and the cytotoxic agent is MMAE, MMAF, Dxd, SN-38, Exatecan, or Eribulin. In some embodiments, the anti-OX40 ADC is HX111.

[00124] In some embodiments, provided herein are methods of treating a subject having an 0X40-expressing T cell leukemia by administering a therapeutically effective amount of an anti-OX40 ADC (e.g, HX111, HX011-MMAF, HX011-Dxd, HX01 l-SN-38, HX011-Exatecan, HX011-Eriblin, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, HX534-2-MMAE) to a subject. In some embodiments, provided herein are method of predicting responsiveness of a subject having a T cell leukemia to an anti-OX40 ADC (e.g., HX111, HX011-MMAF, HXOll-Dxd, HX011-SN-38, HX011-Exatecan, HX011-Eriblin, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, HX534-2-MMAE), comprising measuring 0X40 expression in a sample from the subject, wherein the subject is likely responsive to the anti-OX40 ADC (e.g, HX111, HX011-MMAF, HXOll-Dxd, HX011-SN-38, HX011-Exatecan, HX011-Eriblin, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, HX534-2-MMAE) if the T cell leukemia is an 0X40 expressing T cell leukemia. In some embodiments, provided herein are methods of selecting a subject having a T cell leukemia for treatment with an anti-OX40 ADC (e.g., HX111, HX011-MMAF, HX011-Dxd, HX01 l-SN-38, HX011-Exatecan, HX011-Eriblin, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, HX534-2-MMAE), comprising measuring the 0X40 expression in a sample from the subject and selecting the subject for the treatment if the subject has an 0X40 expressing T cell leukemia. In some embodiments, the anti-OX40 ADC is an ADC having HX011 conjugated to a cytotoxic agent (e.g, HX111, HX011-MMAF, HXOll-Dxd, HX01 l-SN-38, HX011-Exatecan, or HX011-Eriblin). In some embodiments, the anti-OX40 ADC is an ADC having an anti-0X40 antibody conjugated to MMAE (e.g, HX111, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, or HX534-2-MMAE). In some embodiments, the anti-0X40 ADC is an ADC having an anti-OX40 antibody conjugated to a cytotoxic agent, wherein the anti-OX40 antibody is HX011, HX518, HX543, HX523, HX523-1, HX534, or HX534-2; and the cytotoxic agent is MMAE, MMAF, Dxd, SN-38, Exatecan, or Eribulin. In some embodiments, the anti-OX40 ADC is HX111.

[00125] In some embodiments, provided herein are methods of treating a subject having an 0X40-expressing ATLL by administering a therapeutically effective amount of an anti-OX40 ADC (e.g, HX111, HX011-MMAF, HXOll-Dxd, HX011-SN-38, HXO11-Exatecan, HXOll-Eriblin, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, HX534-2-MMAE) to a subject. In some embodiments, provided herein are method of predicting responsiveness of a subject having a ATLL to an anti-OX40 ADC (e.g., HX111, HX011-MMAF, HX011-Dxd, HX011-SN-38, HXO 11-Exatecan, HXOll-Eriblin, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, HX534-2-MMAE), comprising measuring 0X40 expression in a sample from the subject, wherein the subject is likely responsive to the anti-OX40 ADC (e.g., HX111, HX011-MMAF, HX011-Dxd, HX01 l-SN-38, HX011-Exatecan, HX011-Eriblin, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, HX534-2-MMAE) if the ATLL is an 0X40 expressing ATLL. In some embodiments, provided herein are methods of selecting a subject having a ATLL for treatment with an anti-OX40 ADC (e.g, HX111, HX011-MMAF, HXOll-Dxd, HX011-SN-38, HXO 11-Exatecan, HXOll-Eriblin, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, HX534-2-MMAE), comprising measuring the 0X40 expression in a sample from the subject and selecting the subject for the treatment if the subject has an 0X40 expressing ATLL. In some embodiments, the anti-OX40 ADC is an ADC having HX011 conjugated to a cytotoxic agent (e.g., HX111, HX011-MMAF, HXOll-Dxd, HX01 l-SN-38, HX011-Exatecan, or HX011-Eriblin). In some embodiments, the anti-OX40 ADC is an ADC having an anti-OX40 antibody conjugated to MMAE (e.g., HX111, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, or HX534-2-MMAE). In some embodiments, the anti-OX40 ADC is an ADC having an anti-OX40 antibody conjugated to a cytotoxic agent, wherein the anti-OX40 antibody is HX011, HX518, HX543, HX523, HX523-1, HX534, or HX534-2; and the cytotoxic agent is MMAE, MMAF, Dxd, SN-38, Exatecan, or Eribulin. In some embodiments, the anti-OX40 ADC is HX111.

[00126] In some embodiments, provided herein are methods of treating a subject having an 0X40-expressing solid tumor by administering a therapeutically effective amount of an anti-OX40 ADC (e.g, HX111, HX011-MMAF, HXOll-Dxd, HX01 l-SN-38, HXO 11-Exatecan, HXOll-Eriblin, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, HX534-2-MMAE) to a subject. In some embodiments, provided herein are method of predicting responsiveness of a subject having a solid tumor to an anti-OX40 ADC (e.g., HX111, HX011-MMAF, HXOll-Dxd, HX011-SN-38, HXOll-Exatecan, HXOll-Eriblin, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, HX534-2-MMAE), comprising measuring 0X40 expression in a sample from the subject, wherein the subject is likely responsive to the anti-OX40 ADC (e.g, HX111, HX011-MMAF, HXOll-Dxd, HX011-SN-38, HXOll-Exatecan, HXOll-Eriblin, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, HX534-2-MMAE) if the cancer is an 0X40 expressing cancer. In some embodiments, provided herein are methods of selecting a subject having a solid tumor for treatment with an anti-0X40 ADC (e.g, HX111, HX011-MMAF, HXOll-Dxd, HX011-SN-38, HXOll-Exatecan, HXOll-Eriblin, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, HX534-2-MMAE), comprising measuring the 0X40 expression in a sample from the subject and selecting the subject for the treatment if the subject has an 0X40 expressing cancer. In some embodiments, the OX40-expressing solid tumor is OX40-expressing head and neck cancer, 0X40-expressing esophageal cancer, OX40-expressing cervical cancer, OX40-expressing sarcoma, 0X40-expressing small cell lung cancer, OX40-expressing non-small cell lung cancer, OX40-expressing gastric cancer, OX40-expressing liver cancer, OX40-expressing melanoma, OX40-expressing colorectal cancer, OX40-expressing squamous cell carcinoma, OX40-expressing endometrial cancer, OX40-expressing breast cancer, OX40-expressing malignant epithelioid mesothelioma, 0X40-expressing gallbladder cancer, OX40-expressing pancreatic cancer, OX40-expressing glioblastoma, OX40-expressing ovarian cancer, or OX40-expressing gastroesophageal junction adenocarcinoma. In some embodiments, the OX40-expressing solid tumor is responsive to the treatment of anti-OX40 ADC (e.g, HX111, HX011-MMAF, HXOll-Dxd, HX011-SN-38, HXOll-Exatecan, HXOll-Eriblin, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, HX534-2-MMAE) provided herein. In some embodiments, the anti-OX40 ADC is an ADC having HX011 conjugated to a cytotoxic agent (e.g, HX111, HX011-MMAF, HXOll-Dxd, HX011-SN-38, HXOll-Exatecan, or HX011-Eriblin). In some embodiments, the anti-OX40 ADC is an ADC having an anti-0X40 antibody conjugated to MMAE (e.g, HX111, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, or HX534-2-MMAE). In some embodiments, the anti-0X40 ADC is an ADC having an anti-OX40 antibody conjugated to a cytotoxic agent, wherein the anti-OX40 antibody is HX011, HX518, HX543, HX523, HX523-1, HX534, or HX534-2; and the cytotoxic agent is MMAE, MMAF, Dxd, SN-38, Exatecan, or Eribulin. In some embodiments, the anti-OX40 ADC is HX111.

[00127] In some embodiments, provided herein are methods of treating a subject having an 0X40-expressing head and neck cancer by administering a therapeutically effective amount of an anti-0X40 ADC (e.g, HX111, HX011-MMAF, HXOll-Dxd, HX011-SN-38, HX011-Exatecan, HX011-Eriblin, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, HX534-2-MMAE) to a subject. In some embodiments, provided herein are method of predicting responsiveness of a subject having a head and neck cancer to an anti-OX40 ADC (e.g., HX111, HX011-MMAF, HX011-Dxd, HX01 l-SN-38, HX011-Exatecan, HX011-Eriblin, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, HX534-2-MMAE), comprising measuring 0X40 expression in a sample from the subject, wherein the subject is likely responsive to the anti-OX40 ADC (e.g, HX111, HX011-MMAF, HXOll-Dxd, HX01 l-SN-38, HX011-Exatecan, HX011-Eriblin, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, HX534-2-MMAE) if the head and neck cancer is an 0X40 expressing head and neck cancer. In some embodiments, provided herein are methods of selecting a subject having a head and neck cancer for treatment with an anti-OX40 ADC (e.g.. HX111, HX011-MMAF, HX011-Dxd, HX01 l-SN-38, HX011-Exatecan, HX011-Eriblin, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, HX534-2-MMAE), comprising measuring the 0X40 expression in a sample from the subject and selecting the subject for the treatment if the subject has an 0X40 expressing head and neck cancer. In some embodiments, the anti-OX40 ADC is an ADC having HX011 conjugated to a cytotoxic agent (e.g, HX111, HX011-MMAF, HX011-Dxd, HX011-SN-38, HX011-Exatecan, or HX011-Eriblin). In some embodiments, the anti-OX40 ADC is an ADC having an anti-OX40 antibody conjugated to MMAE (e.g, HX111, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, or HX534-2-MMAE). In some embodiments, the anti-OX40 ADC is an ADC having an anti-OX40 antibody conjugated to a cytotoxic agent, wherein the anti-OX40 antibody is HX011, HX518, HX543, HX523, HX523-1, HX534, or HX534-2; and the cytotoxic agent is MMAE, MMAF, Dxd, SN-38, Exatecan, or Eribulin. In some embodiments, the anti-OX40 ADC is HX111.

[00128] In some embodiments, provided herein are methods of treating a subject having an 0X40-expressing sarcoma by administering a therapeutically effective amount of an anti-OX40 ADC (e.g, HX111, HX011-MMAF, HXOll-Dxd, HX011-SN-38, HX011-Exatecan, HX011-Eriblin, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, HX534-2-MMAE) to a subject. In some embodiments, provided herein are method of predicting responsiveness of a subject having a sarcoma to an anti-OX40 ADC (e.g., HX111, HX011-MMAF, HX011-Dxd, HX011-SN-38, HX011-Exatecan, HX011-Eriblin, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, HX534-2-MMAE), comprising measuring 0X40 expression in a sample from the subject, wherein the subject is likely responsive to the anti-OX40 ADC (e.g., HX111, HX011-MMAF, HX011-Dxd, HX01 l-SN-38, HX011-Exatecan, HX011-Eriblin, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, HX534-2-MMAE) if the sarcoma is an 0X40 expressing sarcoma. In some embodiments, provided herein are methods of selecting a subject having a sarcoma for treatment with an anti-OX40 ADC (e.g, HX111, HX011-MMAF, HXOll-Dxd, HX011-SN-38, HX011-Exatecan, HX011-Eriblin, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, HX534-2-MMAE), comprising measuring the 0X40 expression in a sample from the subject and selecting the subject for the treatment if the subject has an 0X40 expressing sarcoma. In some embodiments, the anti-OX40 ADC is an ADC having HX011 conjugated to a cytotoxic agent (e.g.. HX111, HX011-MMAF, HX011-Dxd, HX01 l-SN-38, HX011-Exatecan, or HX011-Eriblin). In some embodiments, the anti-0X40 ADC is an ADC having an anti-OX40 antibody conjugated to MMAE (e.g., HX111, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, or HX534-2-MMAE). In some embodiments, the anti-OX40 ADC is an ADC having an anti-OX40 antibody conjugated to a cytotoxic agent, wherein the anti-OX40 antibody is HX011, HX518, HX543, HX523, HX523-1, HX534, or HX534-2; and the cytotoxic agent is MMAE, MMAF, Dxd, SN-38, Exatecan, or Eribulin. In some embodiments, the anti-OX40 ADC is HX111. In some embodiments, sarcoma can be spindle cell sarcoma, small round cell sarcoma, Ewing's sarcoma, chondrosarcoma, synovial sarcoma, osteosarcoma, high-grade sarcoma, high-grade undifferentiated sarcoma, rhabdomyosarcoma, pleomorphic sarcoma, cystosarcoma, sarcomatoid carcinoma, undifferentiated pleomorphic sarcoma, or leiomyosarcoma. In some embodiments, sarcoma is spindle cell sarcoma. In some embodiments, sarcoma is small round cell sarcoma. In some embodiments, sarcoma is Ewing's sarcoma. In some embodiments, sarcoma is chondrosarcoma. In some embodiments, sarcoma is synovial sarcoma. In some embodiments, sarcoma is osteosarcoma. In some embodiments, sarcoma is high-grade sarcoma. In some embodiments, sarcoma is high-grade undifferentiated sarcoma. In some embodiments, sarcoma is rhabdomyosarcoma. In some embodiments, sarcoma is pleomorphic sarcoma. In some embodiments, sarcoma is cystosarcoma. In some embodiments, sarcoma is sarcomatoid carcinoma. In some embodiments, sarcoma is undifferentiated pleomorphic sarcoma. In some embodiments, sarcoma is leiomyosarcoma.

[00129] In some embodiments, provided herein are methods of treating a subject having an 0X40-expressing spindle cell sarcoma by administering a therapeutically effective amount of an anti-OX40 ADC (e.g, HX111, HX011-MMAF, HXOll-Dxd, HX01 l-SN-38, HX011-Exatecan, HX011-Eriblin, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, HX534-2-MMAE) to a subject. In some embodiments, provided herein are method of predicting responsiveness of a subject having a spindle cell sarcoma to an anti-OX40 ADC (e.g., HX111, HX011-MMAF, HX011-Dxd, HX01 l-SN-38, HX011-Exatecan, HX011-Eriblin, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, HX534-2-MMAE), comprising measuring 0X40 expression in a sample from the subject, wherein the subject is likely responsive to the anti-OX40 ADC (e.g, HX111, HX011-MMAF, HX011-Dxd, HX01 l-SN-38, HX011-Exatecan, HX011-Eriblin, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, HX534-2-MMAE) if the spindle cell sarcoma is an 0X40 expressing spindle cell sarcoma. In some embodiments, provided herein are methods of selecting a subject having a spindle cell sarcoma for treatment with an anti-OX40 ADC (e.g., HX111, HX011-MMAF, HX011-Dxd, HX01 l-SN-38, HX011-Exatecan, HX011-Eriblin, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, HX534-2-MMAE), comprising measuring the 0X40 expression in a sample from the subject and selecting the subject for the treatment if the subject has an 0X40 expressing spindle cell sarcoma. In some embodiments, the anti-OX40 ADC is an ADC having HX011 conjugated to a cytotoxic agent (e.g., HX111, HX011-MMAF, HX011-Dxd, HX01 l-SN-38, HX011-Exatecan, or HX011-Eriblin). In some embodiments, the anti-OX40 ADC is an ADC having an anti-OX40 antibody conjugated to MMAE (e.g., HX111, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, or HX534-2-MMAE). In some embodiments, the anti-OX40 ADC is an ADC having an anti-OX40 antibody conjugated to a cytotoxic agent, wherein the anti-OX40 antibody is HX011, HX518, HX543, HX523, HX523-1, HX534, or HX534-2; and the cytotoxic agent is MMAE, MMAF, Dxd, SN-38, Exatecan, or Eribulin. In some embodiments, the anti-OX40 ADC is HX111.

[00130] In some embodiments, provided herein are methods of treating a subject having an 0X40-expressing hematological cancer by administering a therapeutically effective amount of a fusion protein targeting CD47 and PD1 (e.g., HX009) to a subject. In some embodiments, provided herein are method of predicting responsiveness of a subject having a hematological cancer to a fusion protein targeting CD47 and PD1 (e.g., HX009), comprising measuring 0X40 expression in a sample from the subject, wherein the subject is likely responsive to the fusion protein targeting CD47 and PD1 (e.g., HX009) if the cancer is an 0X40 expressing cancer. In some embodiments, provided herein are methods of selecting a subject having hematological cancer for treatment with a fusion protein targeting CD47 and PD1 (e.g., HX009), comprising measuring the 0X40 expression in a sample from the subject and selecting the subject for the treatment if the subject has an 0X40 expressing cancer. In some embodiments, the OX40-expressing hematological cancer is 0X40-expressing lymphoma or OX40-expressing leukemia. In some embodiments, the OX40-expressing hematological cancer is OX40-expressing AML, OX40-expressing CML, OX40-expressing MDS, OX40-expressing CMML, OX40-expressing T-ALL, OX40-expressing NK leukemia, 0X40-expressing DLBCL, OX40-expressing CTCL, OX40-expressing PTCL, OX40-expressing T cell lymphoblastic lymphoma, OX40-expressing ATLL, OX40-expressing angioimmunoblastic T-cell lymphoma, or OX40-expressing NK / T cell lymphoma. In some embodiments, the OX40-expressing hematological cancer is responsive to the treatment of the fusion protein targeting CD47 and PD1 (e.g., HX009) provided herein.

[00131] In some embodiments, provided herein are methods of treating a subject having an 0X40-expressing lymphoma by administering a therapeutically effective amount of a fusion protein targeting CD47 and PD1 (e.g, HX009) to a subject. In some embodiments, provided herein are method of predicting responsiveness of a subject having a lymphoma to a fusion protein targeting CD47 and PD1 (e.g, HX009), comprising measuring 0X40 expression in a sample from the subject, wherein the subject is likely responsive to the fusion protein targeting CD47 and PD1 (e.g, HX009) if the lymphoma is an 0X40 expressing lymphoma. In some embodiments, provided herein are methods of selecting a subject having lymphoma for treatment with a fusion protein targeting CD47 and PD1 (e.g, HX009), comprising measuring the 0X40 expression in a sample from the subject and selecting the subject for the treatment if the subject has an 0X40 expressing lymphoma.

[00132] In some embodiments, provided herein are methods of treating a subject having an 0X40-expressing B cell lymphoma by administering a therapeutically effective amount of a fusion protein targeting CD47 and PD1 (e.g, HX009) to a subject. In some embodiments, provided herein are method of predicting responsiveness of a subject having a B cell lymphoma to a fusion protein targeting CD47 and PD1 (e.g, HX009), comprising measuring 0X40 expression in a sample from the subject, wherein the subject is likely responsive to the fusion protein targeting CD47 and PD1 (e.g., HX009) if the B cell lymphoma is an 0X40 expressing B cell lymphoma. In some embodiments, provided herein are methods of selecting a subject having B cell lymphoma for treatment with a fusion protein targeting CD47 and PD1 (e.g., HX009), comprising measuring the 0X40 expression in a sample from the subject and selecting the subject for the treatment if the subject has an 0X40 expressing B cell lymphoma.

[00133] In some embodiments, provided herein are methods of treating a subject having an 0X40-expressing DLBCL by administering a therapeutically effective amount of a fusion protein targeting CD47 and PD1 (e.g, HX009) to a subject. In some embodiments, provided herein are method of predicting responsiveness of a subject having a DLBCL to a fusion protein targeting CD47 and PD1 (e.g, HX009), comprising measuring 0X40 expression in a sample from the subject, wherein the subject is likely responsive to the fusion protein targeting CD47 and PD1 (e.g., HX009) if the DLBCL is an 0X40 expressing DLBCL. In some embodiments, provided herein are methods of selecting a subject having DLBCL for treatment with a fusion protein targeting CD47 and PD1 (e.g, HX009), comprising measuring the 0X40 expression in a sample from the subject and selecting the subject for the treatment if the subject has an 0X40 expressing DLBCL.

[00134] In some embodiments, provided herein are methods of treating a subject having an 0X40-expressing solid tumor by administering a therapeutically effective amount of a fusion protein targeting CD47 and PD1 (e.g, HX009) to a subject. In some embodiments, provided herein are method of predicting responsiveness of a subject having a solid tumor to a fusion protein targeting CD47 and PD1 (e.g, HX009), comprising measuring 0X40 expression in a sample from the subject, wherein the subject is likely responsive to the fusion protein targeting CD47 and PD1 (e.g, HX009) if the cancer is an 0X40 expressing cancer. In some embodiments, provided herein are methods of selecting a subject having a solid tumor for treatment with a fusion protein targeting CD47 and PD1 (e.g, HX009), comprising measuring the 0X40 expression in a sample from the subject and selecting the subject for the treatment if the subject has an 0X40 expressing cancer. In some embodiments, the OX40-expressing solid tumor is OX40-expressing head and neck cancer, 0X40-expressing esophageal cancer, OX40-expressing cervical cancer, OX40-expressing sarcoma, 0X40-expressing small cell lung cancer, OX40-expressing non-small cell lung cancer, OX40-expressing gastric cancer, OX40-expressing liver cancer, OX40-expressing melanoma, OX40-expressing colorectal cancer, OX40-expressing squamous cell carcinoma, OX40-expressing endometrial cancer, OX40-expressing breast cancer, OX40-expressing malignant epithelioid mesothelioma, 0X40-expressing gallbladder cancer, OX40-expressing pancreatic cancer, OX40-expressing glioblastoma, OX40-expressing ovarian cancer, or OX40-expressing gastroesophageal junction adenocarcinoma. In some embodiments, the OX40-expressing solid tumor is responsive to the treatment of fusion protein targeting CD47 and PD1 (e.g, HX009) provided herein.

[00135] In some embodiments, provided herein are methods of treating a subject having an 0X40-expressing hematological cancer by administering a therapeutically effective amount of a fusion protein targeting CD47 and CTLA4 (e.g, HX044) to a subject. In some embodiments, provided herein are method of predicting responsiveness of a subject having a hematological cancer to a fusion protein targeting CD47 and CTLA4 (e.g, HX044), comprising measuring 0X40 expression in a sample from the subject, wherein the subject is likely responsive to the fusion protein targeting CD47 and CTLA4 (e.g., HX044) if the cancer is an 0X40 expressing cancer. In some embodiments, provided herein are methods of selecting a subject having hematological cancer for treatment with a fusion protein targeting CD47 and CTLA4 (e.g., HX044), comprising measuring the 0X40 expression in a sample from the subject and selecting the subject for the treatment if the subject has an 0X40 expressing cancer. In some embodiments, the OX40-expressing hematological cancer is OX40-expressing lymphoma or OX40-expressing leukemia. In some embodiments, the 0X40-expressing hematological cancer is OX40-expressing AML, OX40-expressing CML, 0X40-expressing MDS, OX40-expressing CMML, OX40-expressing T-ALL, OX40-expressing NK leukemia, OX40-expressing DLBCL, OX40-expressing CTCL, OX40-expressing PTCL, 0X40-expressing T cell lymphoblastic lymphoma, OX40-expressing ATLL, OX40-expressing angioimmunoblastic T-cell lymphoma, or OX40-expressing NK / T cell lymphoma. In some embodiments, the OX40-expressing hematological cancer is responsive to the treatment of the fusion protein targeting CD47 and CTLA4 (e.g, HX044) provided herein.

[00136] In some embodiments, provided herein are methods of treating a subject having an 0X40-expressing solid tumor by administering a therapeutically effective amount of a fusion protein targeting CD47 and CTLA4 (e.g., HX044) to a subject. In some embodiments, provided herein are method of predicting responsiveness of a subject having a solid tumor to a fusion protein targeting CD47 and CTLA4 (e.g., HX044), comprising measuring 0X40 expression in a sample from the subject, wherein the subject is likely responsive to the fusion protein targeting CD47 and CTLA4 (e.g, HX044) if the cancer is an 0X40 expressing cancer. In some embodiments, provided herein are methods of selecting a subject having a solid tumor for treatment with a fusion protein targeting CD47 and CTLA4 (e.g., HX044), comprising measuring the 0X40 expression in a sample from the subject and selecting the subject for the treatment if the subject has an 0X40 expressing cancer. In some embodiments, the OX40-expressing solid tumor is OX40-expressing head and neck cancer, OX40-expressing esophageal cancer, OX40-expressing cervical cancer, OX40-expressing sarcoma, OX40-expressing small cell lung cancer, OX40-expressing non-small cell lung cancer, 0X40-expressing gastric cancer, OX40-expressing liver cancer, OX40-expressing melanoma, 0X40-expressing colorectal cancer, OX40-expressing squamous cell carcinoma, OX40-expressing endometrial cancer, OX40-expressing breast cancer, OX40-expressing malignant epithelioid mesothelioma, OX40-expressing gallbladder cancer, OX40-expressing pancreatic cancer, 0X40-expressing glioblastoma, OX40-expressing ovarian cancer, or OX40-expressing gastroesophageal junction adenocarcinoma. In some embodiments, the OX40-expressing solid tumor is responsive to the treatment of fusion protein targeting CD47 and CTLA4 (e.g., HX044) provided herein.

[00137] In some embodiments, provided herein are methods of treating a subject having an 0X40-expressing melanoma by administering a therapeutically effective amount of a fusion protein targeting CD47 and CTLA4 (e.g., HX044) to a subject. In some embodiments, provided herein are method of predicting responsiveness of a subject having a melanoma to a fusion protein targeting CD47 and CTLA4 (e.g., HX044), comprising measuring 0X40 expression in a sample from the subject, wherein the subject is likely responsive to the fusion protein targeting CD47 and CTLA4 (e.g., HX044) if the melanoma is an 0X40 expressing melanoma. In some embodiments, provided herein are methods of selecting a subject having a melanoma for treatment with a fusion protein targeting CD47 and CTLA4 (e.g., HX044), comprising measuring the 0X40 expression in a sample from the subject and selecting the subject for the treatment if the subject has an 0X40 expressing melanoma.

[00138] In some embodiments, provided herein are methods of treating a subject having an 0X40-expressing colon cancer by administering a therapeutically effective amount of a fusion protein targeting CD47 and CTLA4 (e.g., HX044) to a subject. In some embodiments, provided herein are method of predicting responsiveness of a subject having a colon cancer to a fusion protein targeting CD47 and CTLA4 (e.g., HX044), comprising measuring 0X40 expression in a sample from the subject, wherein the subject is likely responsive to the fusion protein targeting CD47 and CTLA4 (e.g., HX044) if the colon cancer is an 0X40 expressing colon cancer. In some embodiments, provided herein are methods of selecting a subject having a colon cancer for treatment with a fusion protein targeting CD47 and CTLA4 (e.g, HX044), comprising measuring the 0X40 expression in a sample from the subject and selecting the subject for the treatment if the subject has an 0X40 expressing colon cancer. 6.2.2 0X40 expression profiling

[00139] Provided herein are methods for increasing the responsiveness to cancer immunotherapy by selectively treating patients having 0X40 expressing cancer. Provided herein are also methods for cancer patient population selection for immunotherapy based on 0X40 expression. Provided herein are also methods of predicting responsiveness of a subject having cancer to immunotherapy, wherein a subject is predicted to be likely response if the subject has 0X40 expressing cancer. In some embodiments, provided here are methods for treating an 0X40 expressing cancer in a subject, comprising administering a therapeutically effective amount of an immunotherapeutic agent to the subject. Accordingly, methods provided herein comprise measuring 0X40 expression in a sample from a subject having cancer. In some embodiments, methods provided herein measure mRNA expression. In some embodiments, methods provided herein measure protein expression.

[00140] Methods provided herein comprise measuring the expression level of 0X40 in a subject. In some embodiments, the methods provided herein comprise measuring the protein level. Many methods of measuring protein expression are known in the art, including qualitative, semi-quantitative and quantitative methods. It is generally known to the skilled artisan which methods are suitable for qualitative and / or for quantitative detection of a biomarker.

[00141] In some embodiments, the protein level is measured by immunohistochemistry (IHC), immunocytochemistry (ICC), an enzyme-linked immunosorbent assay (ELISA), immunoblotting assay (e.g., Western blot), flow cytometry (FACS), a fluorescent immunosorbent assay (FIA), a chemiluminescence immunoassay (CIA), a radioimmunoassay (RIA), a solid phase radioimmunoassay (SPROA), or a dot / line-immunoblot assay. Further suitable methods to detect biomarkers include measuring a physical or chemical property specific for the peptide or polypeptide such as its precise molecular mass or NMR spectrum. Said methods comprise, e.g, biosensors, optical devices coupled to immunoassays, biochips, analytical devices such as mass-spectrometers, NMR-analyzers, or chromatography devices. Further, methods include microplate ELISA-based methods, fully-automated or robotic immunoassays (available for example on Elecsys™ analyzers), CBA (an enzymatic Cobalt Binding Assay, available for example on Roche-Hitachi™ analyzers), and latex agglutination assays (available for example on Roche-Hitachi™ analyzers).

[00142] In some embodiments, the protein level is measured by IHC. IHC staining of tissue sections has been shown to be a reliable method of assessing or detecting presence of proteins in a sample. Immunohistochemistry techniques utilize an antibody to probe and visualize cellular antigens in situ, generally by chromogenic or fluorescent methods. Thus, antibodies or antisera, preferably polyclonal antisera, and most preferably monoclonal antibodies specific for each marker are used to detect expression. As discussed in greater detail below, the antibodies can be detected by direct labeling of the antibodies themselves, for example, with radioactive labels, fluorescent labels, hapten labels such as, biotin, or an enzyme such as horse radish peroxidase or alkaline phosphatase. Alternatively, unlabeled primary antibody is used in conjunction with a labeled secondary antibody, comprising antisera, polyclonal antisera or a monoclonal antibody specific for the primary antibody. IHC protocols and kits are well known in the art and are commercially available. Automated systems for slide preparation and IHC processing are available commercially. The Ventana® BenchMark XT system is an example of such an automated system. Standard immunological and immunoassay procedures can be found in Basic and Clinical Immunology (Stites & Terr eds., 7th ed. 1991). Moreover, the immunoassays can be performed in any of several configurations, which are reviewed extensively in Enzyme Immunoassay (Maggio, ed., 1980). For a review of the general immunoassays, see also Methods in Cell Biology: Antibodies in Cell Biology, volume 37 (Asai, ed. 1993); Basic and Clinical Immunology (Stites & Ten, eds., 7th ed. 1991).

[00143] Commonly used assays to detect protein level of a biomarker include noncompetitive assays, e.g, sandwich assays, and competitive assays. A wide range of immunoassay techniques using such an assay format are available, see, e.g, U.S. Pat. Nos. 4,016,043, 4,424,279, and 4,018,653, which are hereby incorporated by reference in their entireties. Sandwich immunoassays are broadly used in the detection of an analyte of interest. In such assay the analyte is “sandwiched” in between a first antibody and a second antibody. Typically, a sandwich assay requires that capture and detection antibody bind to different, non-overlapping epitopes on an analyte of interest. By appropriate means such sandwich complex is measured and the analyte thereby quantified. In a typical sandwich-type assay, a first antibody bound to the solid phase or capable of binding thereto and a detectably-labeled second antibody each bind to the analyte at different and non-overlapping epitopes. The first analytespecific binding agent (e.g., an antibody) is either covalently or passively bound to a solid surface. The solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene. The solid supports can be in the form of tubes, beads, discs of microplates, or any other surface suitable for conducting an immunoassay. The binding processes are well-known in the art and generally consist of cross-linking covalently binding or physically adsorbing, the polymer-antibody complex is washed in preparation for the test sample. An aliquot of the sample to be tested is then added to the solid phase complex and incubated for a period of time sufficient (e.g., 2-40 minutes or overnight if more convenient) and under suitable conditions (e.g., from room temperature to 40°C such as between 25° C and 37° C inclusive) to allow for binding between the first or capture antibody and the corresponding antigen. Following the incubation period, the solid phase, comprising the first or capture antibody and bound thereto the antigen can be washed, and incubated with a secondary or labeled antibody binding to another epitope on the antigen. The second antibody is linked to a reporter molecule which is used to indicate the binding of the second antibody to the complex of first antibody and the antigen of interest.

[00144] Variations of the sandwich assay technique exist. For example, in a simultaneous assay, both sample and labeled antibody are added simultaneously to the bound antibody. These techniques are well known to those skilled in the art, including any minor variations as will be readily apparent. A versatile alternative sandwich assay uses a solid phase coated with the first partner of a binding pair, e.g., paramagnetic streptavidin-coated microparticles. Such microparticles are mixed and incubated with an analyte-specific binding agent bound to the second partner of the binding pair (e.g., a biotinylated antibody), a sample suspected of comprising or comprising the analyte, wherein said second partner of the binding pair is bound to said analyte-specific binding agent, and a second analyte-specific binding agent which is detectably labeled. As obvious to the skilled person these components are incubated under appropriate conditions and for a period of time sufficient for binding the labeled antibody via the analyte, the analyte-specific binding agent (bound to) the second partner of the binding pair and the first partner of the binding pair to the solid phase microparticles. As appropriate such assay can include one or more washing step(s).

[00145] In the case of an enzyme immunoassay, an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate. As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan. Commonly used enzymes include horseradish peroxidase, glucose oxidase, beta-galactosidase, and alkaline phosphatase, and other are discussed herein. The substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable color change. Examples of suitable enzymes include alkaline phosphatase and peroxidase. It is also possible to employ fluorogenic substrates, which yield a fluorescent product rather than the chromogenic substrates noted above. In all cases, the enzyme-labeled antibody is added to the first antibody -molecular marker complex, allowed to bind, and then the excess reagent is washed away. A solution containing the appropriate substrate is then added to the complex of antibody-antigen-antibody. The substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of biomarker which was present in the sample. Alternately, fluorescent compounds, such as fluorescein and rhodamine, can be chemically coupled to antibodies without altering their binding capacity. When activated by illumination with light of a particular wavelength, the fluorochrome-labeled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic color visually detectable with a light microscope. As in the EIA, the fluorescent labeled antibody is allowed to bind to the first antibody-molecular marker complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to the light of the appropriate wavelength, the fluorescence observed indicates the presence of the molecular marker of interest. Immunofluorescence and EIA techniques are both very well established in the art and are discussed herein. In some embodiments, an assay such as an ELISA assay can be used. ELISA assays are known in the art and commercially available.

[00146] In some embodiments, flow cytometry (FACS) can be used to detect the protein level of a biomarker. Surface proteins can be detected using antibodies against specific biomarkers. The flow cytometer detects and reports the intensity of the fluorochrome-tagged antibody, which indicates the expression level of the biomarker. Non-fluorescent cytoplasmic proteins can also be observed by staining permeabilized cells. The stain can either be a fluorescence compound able to bind to certain molecules, or a fluorochrome-tagged antibody to bind the molecule of choice.

[00147] Antibodies that specifically bind 0X40 (e.g, human 0X40) are known in the art and commercially available from many sources, for example, Invitrogen, BioLegend, LifeSpan BioSciences, Thermo Fisher Scientific, Merck, etc.

[00148] Methods provided herein comprise measuring the expression level of 0X40 in a subject. In some embodiments, the methods provided herein comprise measuring the mRNA level. Any method as described herein or otherwise known in the art to determine the mRNA level of a gene can be used. The mRNA sequence (e.g., the mRNA of 0X40, or a fragment thereof) can be used to prepare a probe that is at least partially complementary. The probe can then be used to detect the mRNA sequence in a sample, using any suitable assay, such as PCR-based methods, northern blotting, a dipstick assay, and the like.

[00149] Measuring mRNA levels traditionally involves isolating an intact RNA fraction from samples, immobilizing it, and detecting and quantifying the RNA transcripts of interest. This can be done using a transcript-specific, labeled probe. Exemplary techniques include: northern blotting, dot blotting, ribonuclease protection assays (RPAs), serial analysis of gene expression (SAGE), and differential or subtractive hybridization.

[00150] Current approaches provide greater detection efficiency than immobilized RNA techniques, and they are adaptable for increased target and sample numbers. They usually involve adding multiple probes to an RNA fraction or directly to a cell lysate. These techniques include polymerase chain reaction (PCR)-based methods such as quantitative PCR (qPCR), digital PCR (dPCR), or realtime PCR (RT-PCR), next generation sequencing (NGS), microarrays and panels, and in situ hybridization, including fluorescent in situ hybridization (FISH).

[00151] In some embodiments, the mRNA is measured using in situ RNA hybridization (e.g., FISH), qPCR, RT-PCR, microarray analysis, SAGE, MassARRAY, or NGS. In some embodiments, the mRNA level is measured using in situ RNA hybridization.

[00152] The commonly used methods known in the art for the quantification of mRNA expression in a sample include northern blotting and in situ hybridization (Parker &Bames, Methods in Molecular Biology 106:247-283 (1999)); RNAse protection assays (Hod, Biotechniques 13 :852- 854 (1992)); and polymerase chain reaction (PCR) (Weis et al.. Trends in Genetics 8:263-264 (1992)). Alternatively, antibodies can be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Representative methods for sequencing-based gene expression analysis include Serial Analysis of Gene Expression (SAGE), and gene expression analysis by massively parallel signature sequencing (MPSS).

[00153] A sensitive and flexible quantitative method is PCR. Examples of PCR methods can be found in the literature. Examples of PCR assays can be found in U. S. Patent No. 6,927,024, which is incorporated by reference herein in its entirety. Examples of RT-PCR methods can be found in U.S. Patent No. 7, 122,799, which is incorporated by reference herein in its entirety. A method of fluorescent in situ PCR is described in U.S. Patent No. 7, 186,507, which is incorporated by reference herein in its entirety.

[00154] It is noted, however, that other nucleic acid amplification protocols (i.e., other than PCR) may also be used in the nucleic acid analytical methods described herein. For example, suitable amplification methods include ligase chain reaction (see, e.g., Wu & Wallace, Genomics 4:560-569, 1988); strand displacement assay (see, e.g., Walker et al., Proc. Natl. Acad. Sci. USA 89:392-396, 1992; U. S. Pat. No. 5,455, 166); and several transcription-based amplification systems, including the methods described in U.S. Pat. Nos. 5,437,990; 5,409,818; and 5,399,491; the transcription amplification system (TAS) (Kwoh et al., Proc. Natl. Acad. Sci. USA 86: 1173- 1177, 1989); and self-sustained sequence replication (3SR) (Guatelli et al., Proc. Natl. Acad. Sci. USA 87: 1874-1878, 1990; WO 92 / 08800). Alternatively, methods that amplify the probe to detectable levels can be used, such as Q-replicase amplification (Kramer & Lizardi, Nature 339:401-402, 1989; Lomeli et al., Clin. Chem. 35: 1826-1831, 1989). A review of known amplification methods is provided, for example, by Abramson and Myers in Current Opinion in Biotechnology 4:41-47 (1993).

[00155] mRNA can be isolated from the starting tissue sample. General methods for mRNA extraction are well known in the art and are disclosed in standard textbooks of molecular biology, including Ausubel et al., Current Protocols OF Molecular Biology, John Wiley and Sons (1997). In particular, RNA isolation can be performed using purification kit, buffer set and protease from commercial manufacturers, such as Qiagen, according to the manufacturer's instructions. For example, total RNA from cells in culture can be isolated using Qiagen RNeasy mini- columns. Other commercially available RNA isolation kits include MASTERPURE® Complete DNA and RNA Purification Kit (EPICENTRE®, Madison, Wis.), and Paraffin Block RNA Isolation Kit (Ambion, Inc.). Total RNA from tissue samples can be isolated using RNA Stat-60 (Tel-Test). RNA prepared from tumor can be isolated, for example, by cesium chloride density gradient centrifugation.

[00156] In some embodiments, the first step in gene expression profiling by PCR is the reverse transcription of the RNA template into cDNA, followed by its exponential amplification in a PCR reaction. In other embodiments, a combined reverse-transcription-polymerase chain reaction (RT-PCR) reaction may be used, e.g, as described in U.S. Pat. Nos. 5,310,652; 5,322,770; 5,561 ,058; 5,641,864; and 5,693,517. The two commonly used reverse transcriptases are avian myeloblastosis virus reverse transcriptase (AMV-RT) and Moloney murine leukemia virus reverse transcriptase (MMLV-RT). The reverse transcription step is typically primed using specific primers, random hexamers, or oligo-dT primers, depending on the circumstances and the goal of expression profiling. For example, extracted RNA can be reverse- transcribed using a GENEAMP™ RNA PCR kit (Perkin Elmer), following the manufacturer’s instructions. The derived cDNA can then be used as a template in the subsequent PCR reaction.

[00157] In some embodiments, Real-Time Reverse Transcription-PCR (qRT-PCR) can be used for both the detection and quantification of RNA targets (Bustin, et al, 2005, Clin. Sci., 109:365-379). Examples of qRT-PCR-based methods can be found, for example, in U.S. Patent No. 7, 101,663, which is incorporated by reference herein in its entirety. Instruments for real time PCR, such as the Applied Biosystems 7500, are available commercially, as are the reagents, such as TaqMan Sequence Detection chemistry.

[00158] For example, TaqMan® Gene Expression Assays can be used, following the manufacturer's instructions. These kits are pre-formulated gene expression assays for rapid, reliable detection and quantification of human, mouse and rat mRNA transcripts. TaqMan® or 5’-nuclease assay, as described in U.S. Pat. Nos. 5,210,015; 5,487,972; and 5,804,375; and Holland et al., 1988, Proc. Natl. Acad. Sci. USA 88:7276-7280, can be used. TAQMAN® PCR typically utilizes the 5’-nuclease activity of Taq or Tth polymerase to hydrolyze a hybridization probe bound to its target amplicon, but any enzyme with equivalent 5’ nuclease activity can be used. Two oligonucleotide primers are used to generate an amplicon typical of a PCR reaction. A third oligonucleotide, or probe, is designed to detect nucleotide sequence located between the two PCR primers. The probe is non-extendible by Taq DNA polymerase enzyme, and is labeled with a reporter fluorescent dye and a quencher fluorescent dye. Any laser-induced emission from the reporter dye is quenched by the quenching dye when the two dyes are located close together as they are on the probe. During the amplification reaction, the Taq DNA polymerase enzyme cleaves the probe in a template-dependent manner. The resultant probe fragments disassociate in solution, and signal from the released reporter dye is free from the quenching effect of the second fluorophore. One molecule of reporter dye is liberated for each new molecule synthesized, and detection of the unquenched reporter dye provides the basis for quantitative interpretation of the data.

[00159] Any method suitable for detecting degradation product can be used in a 5’ nuclease assay. Often, the detection probe is labeled with two fluorescent dyes, one of which can quence the fluorescence of the other dye. The dyes are attached to the probe, preferably one attached to the 5’ terminus and the other is attached to an internal site, such that quenching occurs when the probe is in an unhybridized state and such that cleavage of the probe by the 5’ to 3’ exonuclease activity of the DNA polymerase occurs in between the two dyes.

[00160] Amplification results in cleavage of the probe between the dyes with a concomitant elimination of quenching and an increase in the fluorescence observable from the initially quenched dye. The accumulation of degradation product is monitored by measuring the increase in reaction fluorescence. U.S. Pat. Nos. 5,491 ,063 and 5,571 ,673, both incorporated herein by reference, describe alternative methods for detecting the degradation of probe which occurs concomitant with amplification. 5’-Nuclease assay data may be initially expressed as Ct, or the threshold cycle. As discussed above, fluorescence values are recorded during every cycle and represent the amount of product amplified to that point in the amplification reaction. The point when the fluorescent signal is first recorded as statistically significant is the threshold cycle (Ct).

[00161] To minimize errors and the effect of sample-to-sample variation, PCR is usually performed using an internal standard. The ideal internal standard is expressed at a constant level among different tissues, and is unaffected by the experimental treatment. RNAs most frequently used to normalize patterns of gene expression are mRNAs for the housekeeping genes glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and P-actin.

[00162] PCR primers and probes are designed based upon intron sequences present in the gene to be amplified. In this embodiment, the first step in the primer / probe design is the delineation of intron sequences within the genes. This can be done by publicly available software, such as the DNA BLAST software developed by Kent, W., Genome Res. 12(4):656-64 (2002), or by the BLAST software including its variations. Subsequent steps follow well established methods of PCR primer and probe design.

[00163] In order to avoid non-specific signals, it can be important to mask repetitive sequences within the introns when designing the primers and probes. This can be easily accomplished by using the Repeat Masker program available on-line through the Baylor College of Medicine, which screens DNA sequences against a library of repetitive elements and returns a query sequence in which the repetitive elements are masked. The masked intron sequences can then be used to design primer and probe sequences using any commercially or otherwise publicly available primer / probe design packages, such as Primer Express (Applied Biosystems); MGB assay-by-design (Applied Biosystems); Primer3 (Rozen and Skaletsky (2000) Primer3 on the WWW for general users and for biologist programmers. In: Krawetz S, Misener S (eds) Bioinformatics Methods and Protocols: Methods in Molecular Biology. Humana Press, Totowa, N.J., pp 365-386).

[00164] Factors considered in PCR primer design include primer length, melting temperature (Tm), and G / C content, specificity, complementary primer sequences, and 3'-end sequence. In general, optimal PCR primers are generally 17-30 bases in length, and contain about 20-80%, such as, for example, about 50-60% G+C bases. Tm's between 50 and 80° C, e.g, about 50 to 70° C. are typically preferred. For further guidelines for PCR primer and probe design see, e.g, Dieffenbach et al., “General Concepts for PCR Primer Design” in: PCR Primer, A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1995, pp. 133-155; Innis and Gelfand, “Optimization of PCRs” in: PCR Protocols, A Guide to Methods and Applications, CRC Press, London, 1994, pp. 5-11; and Plasterer, T. N. Primer select: Primer and probe design. Methods Mol. Biol. 70:520527 (1997), the entire disclosures of which are hereby expressly incorporated by reference.

[00165] An exemplary PCR program, for example, is 50°C for 2 minutes, 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds, then 60°C for 1 minute. To determine the cycle number at which the fluorescence signal associated with a particular amplicon accumulation crosses the threshold (referred to as the CT), the data can be analyzed, for example, using a 7500 Real-Time PCR System Sequence Detection software using the comparative CT relative quantification calculation method. Using this method, the output is expressed as a fold-change of expression levels. In some embodiments, the threshold level can be selected to be automatically determined by the software. In some embodiments, the threshold level is set to be above the baseline but sufficiently low to be within the exponential growth region of an amplification curve.

[00166] RNA-Seq, also called Whole Transcriptome Shotgun Sequencing (WTSS) refers to the use of high-throughput sequencing technologies to sequence cDNA in order to get information about a sample’s RNA content. Publications describing RNA-Seq include: Wang et al., Nature Reviews Genetics 10 (1): 57-63 (2009); Ryan et al. BioTechniques 45 (1):81-94 (2008); and Maher et al., Nature 458 (7234): 97-101 (2009); which are hereby incorporated in their entirety.

[00167] Differential gene expression can also be identified or confirmed using the microarray technique. In this method, polynucleotide sequences of interest (including cDNAs and oligonucleotides) are plated, or arrayed, on a microchip substrate. The arrayed sequences are then hybridized with specific DNA probes from cells or tissues of interest.

[00168] In an embodiment of the microarray technique, PCR amplified inserts of cDNA clones are applied to a substrate in a dense array. Preferably at least 10,000 nucleotide sequences are applied to the substrate. The microarray ed genes, immobilized on the microchip at 10,000 elements each, are suitable for hybridization under stringent conditions. Fluorescently labeled cDNA probes may be generated through incorporation of fluorescent nucleotides by reverse transcription of RNA extracted from tissues of interest. Labeled cDNA probes applied to the chip hybridize with specificity to each spot of DNA on the array. After stringent washing to remove non-specifically bound probes, the chip is scanned by confocal laser microscopy or by another detection method, such as a CCD camera. The quantitation of hybridization of each arrayed element allows for assessment of corresponding mRNA abundance. With dual color fluorescence, separately labeled cDNA probes generated from two sources of RNA are hybridized pairwise to the array. The relative abundance of the transcripts from the two sources corresponding to each specified gene is thus determined simultaneously. The miniaturized scale of the hybridization affords a convenient and rapid evaluation of the expression pattern for large numbers of genes. Such methods have been shown to have the sensitivity required to detect rare transcripts, which are expressed at a few copies per cell, and to reproducibly detect at least approximately two-fold differences in the expression levels (Schena et al., Proc. Natl. Acad. Sci. USA 93(2): 106-149 (1996)). Microarray analysis can be performed by commercially available equipment, following manufacturer's protocols, such as by using the Affymetrix GENCHIP™ technology, or Incyte's microarray technology. In some embodiments, mRNA levels can be measured by next generation sequencing (NGS). In some embodiments, mRNA levels can be measured by single cell whole-exome sequencing (scWES).

[00169] Serial analysis of gene expression (SAGE) is a method that allows the simultaneous and quantitative analysis of a large number of gene transcripts, without the need of providing an individual hybridization probe for each transcript. First, a short sequence tag (about 10-14 bp) is generated that contains sufficient information to uniquely identify a transcript, provided that the tag is obtained from a unique position within each transcript. Then, many transcripts are linked together to form long serial molecules, that can be sequenced, revealing the identity of the multiple tags simultaneously. The expression pattern of any population of transcripts can be quantitatively evaluated by determining the abundance of individual tags and identifying the gene corresponding to each tag. For more details see, e.g., Velculescu et al., Science 270:484- 487 (1995); and Velculescu et al., Cell 88:243-51 (1997).

[00170] The MassARRAY (Sequenom) technology is an automated, high-throughput method of gene expression analysis using mass spectrometry (MS) for detection. According to this method, following the isolation of RNA, reverse transcription and PCR amplification, the cDNAs are subjected to primer extension. The cDNA-derived primer extension products are purified, and dispensed on a chip array that is pre- loaded with the components needed for MALTI-TOF MS sample preparation. The various cDNAs present in the reaction are quantitated by analyzing the peak areas in the mass spectrum obtained.

[00171] mRNA levels can also be measured by an assay based on hybridization. A typical mRNA assay method can contain the steps of 1) obtaining surface-bound subject probes; 2) hybridization of a population of mRNAs to the surface-bound probes under conditions sufficient to provide for specific binding (3) post-hybridization washes to remove nucleic acids not bound in the hybridization; and (4) detection of the hybridized mRNAs. The reagents used in each of these steps and their conditions for use may vary depending on the particular application.

[00172] Any suitable assay platform can be used to determine the mRNA level in a sample. For example, an assay can be in the form of a dipstick, a membrane, a chip, a disk, a test strip, a filter, a microsphere, a slide, a multi-well plate, or an optical fiber. An assay system can have a solid support on which a nucleic acid corresponding to the mRNA is attached. The solid support can have, for example, a plastic, silicon, a metal, a resin, glass, a membrane, a particle, a precipitate, a gel, a polymer, a sheet, a sphere, a polysaccharide, a capillary, a film a plate, or a slide. The assay components can be prepared and packaged together as a kit for detecting an mRNA.

[00173] Hybridization can be carried out under suitable hybridization conditions, which may vary in stringency as desired. Typical conditions are sufficient to produce probe / target complexes on a solid surface between complementary binding members, i.e., between surface-bound subject probes and complementary mRNAs in a sample. In certain embodiments, stringent hybridization conditions can be employed. Hybridization is typically performed under stringent hybridization conditions. Standard hybridization techniques (e.g, under conditions sufficient to provide for specific binding of target mRNAs in the sample to the probes) are described in Kallioniemi et al, Science 258:818-821 (1992) and WO 93 / 18186. Several guides to general techniques are available, e.g., Tijssen, Hybridization with Nucleic Acid Probes, Parts I and II (Elsevier, Amsterdam 1993). For descriptions of techniques suitable for in situ hybridizations, see Gall et al. Meth. Enzymol., 21 :470-480 (1981); and Angerer et al. in Genetic Engineering: Principles and Methods (Setlow and Hollaender, Eds.) Vol 7, pgs 43-65 (Plenum Press, New York 1985). Selection of appropriate conditions, including temperature, salt concentration, polynucleotide concentration, hybridization time, stringency of washing conditions, and the like will depend on experimental design, including source of sample, identity of capture agents, degree of complementarity expected, etc., and may be determined as a matter of routine experimentation for those of ordinary skill in the art. Those of ordinary skill will readily recognize that alternative but comparable hybridization and wash conditions can be utilized to provide conditions of similar stringency.

[00174] After the mRNA hybridization procedure, the surface bound polynucleotides are typically washed to remove unbound nucleic acids. Washing may be performed using any convenient washing protocol, where the washing conditions are typically stringent, as described above. The hybridization of the target mRNAs to the probes is then detected using standard techniques.

[00175] In some embodiments, methods of measuring the expression of OX40 (protein and / or mRNA) disclosed herein comprise using a detectable label. Directly detectable labels either provide a detectable signal or they interact with a second label to modify the detectable signal provided by the first or second label, e.g, to give FRET (fluorescence resonance energy transfer). Labels such as fluorescent dyes and luminescent (including chemiluminescent and electrochemiluminescent) dyes (Briggs et al. J. Chem. Soc., Perkin-Trans. 1 (1997) 1051-1058) provide a detectable signal and are generally applicable for labeling. In some embodiments detectable labels refers to labels providing or inducible to provide a detectable signal, i.e., to a fluorescent label, to a luminescent label (e.g., a chemiluminescent label or an electrochemiluminescent label), a radioactive label or a metal- chelate based label, respectively.

[00176] Numerous labels (also referred to as dyes) are available. Some are exemplified and grouped into the following categories. As persons skilled in the art would understand, the methods provided herein are not limited to specific means of detection or measurement, and any means known in the art for labeling and measuring a protein or mRNA analyte in a sample can be applied in methods disclosed herein.

[00177] In some embodiments, the OX40 expression is measured using fluorescent dyes. Fluorescent dyes are, e.g., described by Briggs et al., J. Chem. Soc., Perkin-Trans. 1 (1997) 10511058). Fluorescent labels or fluorophores include rare earth chelates (europium chelates), fluorescein type labels including xanthene dyes, fluorescein isothiocyanate (FITC), 5-carboxyfluorescein, 6-carboxy fluorescein (FAM), 6 carboxy-2',4',7',4,7- hexachlorofluorescein (HEX), 6 carboxy 4', 5' dichloro 2', T dimethoxyfluorescein (JOE or J), fluorescein chlorotriazinyl, and napthofluorescein eosin; rhodamine type labels including N,N,N',N' tetramethyl 6 carboxyrhodamine (TAMRA or T), tetramethylrhodamine, 6 carboxy X rhodamine (ROX or R), 5 carboxyrhodamine 6G (R6G5 or G5), 6 carboxyrhodamine 6G (R6G6 or G6), and rhodamine 110 (RI00); cyanines including Cy3, Cy5 and Cy7 dyes; Alexa dyes, e.g., Al exa-fluor-555; coumarin, di ethylaminocoumarin, umbelliferone; benzimide dyes (e.g., Hoechst 33258); phenanthridine dyes (e.g, Texas Red); ethidium dyes; acridine dyes; carbazole dyes; phenoxazine dyes; porphyrin dyes; polymethine dyes, BODIPY dyes, quinoline dyes, pyrene, phycoerythrins; dansyl; lissamine; and analogs thereof. The fluorescent labels can be conjugated to an aldehyde group comprised in target molecule using the techniques disclosed herein. Fluorescent dyes and fluorescent label reagents include those which are commercially available from Invitrogen / Molecular Probes (Eugene, Oregon, USA) and Pierce Biotechnology, Inc. (Rockford, Ill.).

[00178] In some embodiments, the 0X40 expression is measured using luminescent dyes: Luminescent dyes or labels can be further subcategorized into chemiluminescent and electrochemiluminescent dyes. The different classes of chemiluminogenic labels include luminol, acridinium compounds, coelenterazine and analogues, dioxetanes, systems based on peroxyoxalic acid and their derivatives. For immunodiagnostic procedures predominantly acridinium based labels are used (a detailed overview is given in Dodeigne C. et al.. Taianta 51 (2000) 415-439). The labels of major relevance used as electrochemiluminescent labels are the Ruthenium- and the Iridium-based electrochemiluminescent complexes, respectively.

[00179] Electrochemiluminescense (ECL) can be used in analytical applications as a highly sensitive and selective method. It combines analytical advantages of chemiluminescent analysis (absence of background optical signal) with ease of reaction control by applying electrode potential. In general Ruthenium complexes, especially [Ru (Bpy)3]2+ (which releases a photon at -620 nm) regenerating with TPA (Tripropylamine) in liquid phase or liquid-solid interface are used as ECL-labels. ECL uses labels or other reactants that can be induced to luminesce when electrochemically oxidized or reduced in an appropriate chemical environment. Such electrochemiluminescense is triggered by a voltage imposed on a working electrode at a particular time and in a particular manner. The light produced by the label is measured and indicates the presence or quantity of the analyte. For a fuller description of such ECL techniques, reference is made to US Patent Nos. 5,221,605; 5,591,581; 5,597,910; 5,679,519; and to PCT published applications W090 / 05296, W092 / 14139, W090 / 05301, WO96 / 24690, WO95 / 08644, WO96 / 06946, W096 / 33411, W087 / 06706, W096 / 39534, W096 / 41175, WO96 / 40978, and W02012107419.

[00180] In some embodiments, the OX40 expression is measured using radioactive labels. Radioactive labels make use of radioisotopes (radionuclides), such as 3H, 11C, 14C, 18F, 32P, 35S, 64Cu, 68Gn, 86Y, 89Zr, 99TC, Ulin, 1231, 1241, 1251, 1311, 133Xe, 177Lu, 211At, or 131Bi.

[00181] In some embodiments, the OX40 expression is measured using metal-chelate. Metal-chelate complexes suitable as labels for imaging purposes are well-known in the art (US 2010 / 0111861; US 5,342,606; US 5,428,155; US 5,316,757; US 5,480,990; US 5,462,725; US 5,428,139; US 5,385,893; US 5,739,294; US 5,750,660; US 5,834,461; Hnatowich et al, J. Immunol. Methods 65 (1983) 147- 157; Meares et al.. Anal. Biochem. 142 (1984) 68-78; Mirzadeheta / ., Bioconjugate Chem. 1 (1990) 59-65; Meares et al., J. Cancer (1990), Suppl. 10:21-26; Izard et al., Bioconjugate Chem. 3 (1992) 346-350; Nikulaeta / ., Nucl. Med. Biol. 22 (1995) 387-90; Camera et al., Nucl. Med. Biol. 20 (1993) 955-62; Kukis et al., J. Nucl. Med. 39 (1998) 2105-2110; Verel et al., J. Nucl. Med. 44 (2003) 16631670; Camera et al., J. Nucl. Med. 21 (1994) 640-646; Ruegge! al., Cancer Res. 50 (1990) 42214226; Verel et al., J. Nucl. Med. 44 (2003) 1663-1670; Lee et al., Cancer Res. 61 (2001) 4474- 4482; Mitchell, et al., J. Nucl. Med. 44 (2003) 1105-1112; Kobayashi et al., Bioconjugate Chem. 10 (1999) 103-111; Miederer et al., J. Nucl. Med. 45 (2004) 129-137; DeNardo et al., Clinical Cancer Research 4 (1998) 2483-90; Blend et al., Cancer Biotherapy & Radiopharmaceuticals 18 (2003) 355-363; Nikula et a / J. Nucl. Med. 40 (1999) 166-76; Kobayashi etal., J. Nucl. Med. 39 (1998) 82936; Mardirossian et al., Nucl. Med. Biol. 20 (1993) 65-74; Roselli et al., Cancer Biotherapy & Radiopharmaceuticals, 14 (1999) 209-20).

[00182] Provided herein are methods of predicting responsiveness of a subject to an immunotherapeutic agent based on the expression level (mRNA level and / or protein level) of OX40. Provided herein are also methods of selecting cancer patients for immunotherapy based on the expression level (mRNA level and / or protein level) of OX40. In some embodiments, a subject having cancer is determined to be likely responsive to immunotherapy if the expression level of OX40 in a sample from the subject is higher than a reference level. In some embodiments, a subject having cancer is selected for immunotherapy if the expression level of OX40 in a sample from the subject is higher than a reference level. In some embodiments, a subject having cancer is determined to be likely responsive to immunotherapy if the expression level of OX40 in a sample from the subject is detectable. In some embodiments, a subject having cancer is selected for immunotherapy if the expression level of OX40 in a sample from the subject is detectable.

[00183] A reference level, or alternatively, a control level or a threshold level, is the level to which the expression level of the biomarker in a sample of interest is compared. A reference level thereby provides a standard allowing for the evaluation of the information obtained from the sample of interest. A reference expression level of a gene can be a cut-off value determined by a person of ordinary skill in the art through statistical analysis of the expression levels of the gene in various sample cell populations. For example, by analyzing the expression levels of a gene in sample cell populations having at least 50%, at least 60%, at least 70%, at least 80%, at least 90% cells known to express that gene, a person of ordinary skill in the art can determine a cut-off value as the reference expression level of the gene, which can be used to indicate the percentages of cells expressing the gene in a cell population with unknown constitution. In some embodiments, the reference expression level of the gene can be the expression level at the threshold of a particular quartile or tertile, as determined by a person of ordinary skill in the art analyzing the expression levels of a gene in sample cell populations, such as sample cell populations from a group of patients having, or diagnosed as having, the same type of cancer.

[00184] For example, the reference expression level of the gene can be the expression level between the lowest quartile (first quartile) and the second lowest quartile (second quartile), or between the second lowest quartile (second quartile) and the third lowest quartile (third quartile), or between the third lowest quartile (third quartile; or second highest quartile) and the highest quartile (fourth quartile), as determined by a person of ordinary skill in the art analyzing the expression levels of a gene in sample cell populations. For example, the reference expression level of the gene can be the expression level between the lowest tertile (first tertile) and the second lowest tertile (second tertile), or between the second lowest tertile (second tertile) and the highest tertile (third tertile), as determined by a person of ordinary skill in the art analyzing the expression levels of a gene in sample cell populations.

[00185] A reference level can be an internal or an external reference. An internal reference sample is used, i.e., the marker level(s) is(are) assessed in the test sample as well as in one or more other sample(s) taken from the same subject to determine if there are any changes in the level(s) of said marker(s) in the test sample. As understood by the skilled artisan, external reference levels can be obtained from a single individual or a reference population. Typically, samples from 100 or more well-characterized individuals from the appropriate reference population are used to establish a reference level. However, reference population can also be chosen to consist of about 20, about 30, about 50, about 200, about 500 or about 1000 individuals.

[00186] In some embodiments, the reference expression level of OX40 can be determined based on statistical analysis of data from previous clinical studies, including clinical presentation of a group of patients. Many statistical methods are well known in the art to determine the reference level (or referred to as the “cut-off value”) of OX40 expression when used to assessing the likelihood of a subject for being responsive for immunotherapy.

[00187] For illustrative purpose, IHC can be used to determine the OX40 expression level and the reference expression level of 0X40, and H-Score can be used to predict the responsiveness of a subject having cancer to treatment with immunotherapeutic agents (e.g., HX009, HX011, HX518, HX543, HX523, HX523-1, HX534, HX534-2, HX111, HX011-MMAF, HXOll-Dxd, HX011-SN-38, HXOll-Exatecan, HXOll-Eriblin, HX518-MMAE, HX543-MMAE, HX523-MMAE, HX523-1-MMAE, HX534-MMAE, HX534-2-MMAE, or HX044). In some embodiments, IHC workflow as described in example 1 of the instant application is used to determine the 0X40 expression level and the reference expression level of 0X40. In some embodiments, a H-Score greater than about 0.14 predicts that the subject having cancer is likely to respond to the treatment with immunotherapeutic agents. In some embodiments, the reference expression level of 0X40 is about 0.14 in H-Score. In some embodiments, a H-Score greater than about 0.57 predicts that the subject having cancer is likely to respond to the treatment with immunotherapeutic agents. In some embodiments, the reference expression level of 0X40 is about 0.57 in H-Score. In some embodiments, a H-Score greater than about 0.89 predicts that the subject having cancer is likely to respond to the treatment with immunotherapeutic agents. In some embodiments, the reference expression level of 0X40 is about 0.89 in H-Score. In some embodiments, a H-Score greater than about 0.94 predicts that the subject having cancer is likely to respond to the treatment with immunotherapeutic agents. In some embodiments, the reference expression level of 0X40 is about 0.94 in H-Score. In some embodiments, a H-Score greater than about 1.58 predicts that the subject having cancer is likely to respond to the treatment with immunotherapeutic agents. In some embodiments, the reference expression level of 0X40 is about 1.58 in H-Score. In some embodiments, a H-Score greater than about 2.19 predicts that the subject having cancer is likely to respond to the treatment with immunotherapeutic agents. In some embodiments, the reference expression level of 0X40 is about 2.19 in H-Score. In some embodiments, a H-Score greater than about 7.26 predicts that the subject having cancer is likely to respond to the treatment with immunotherapeutic agents. In some embodiments, the reference expression level of 0X40 is about 7.26 in H-Score. In some embodiments, a H-Score greater than about 24.58 predicts that the subject having cancer is likely to respond to the treatment with immunotherapeutic agents. In some embodiments, the reference expression level of 0X40 is about 24.58 in H-Score. In some embodiments, a H-Score greater than about 0.14 predicts that the subject having lymphoma (e.g., DLBCL) is likely to respond to the treatment with immunotherapeutic agents (e.g, HX009). In some embodiments, the reference expression level of 0X40 is about 0.14 in H-Score for a subject having lymphoma (e.g., DLBCL) to receive the treatment with immunotherapeutic agents (e.g., HX009). In some embodiments, aH-Score greater than about 0.57 predicts that the subject having lymphoma (e.g, DLBCL) is likely to respond to the treatment with immunotherapeutic agents (e.g, HX009). In some embodiments, the reference expression level of 0X40 is about 0.57 in H-Score for a subject having lymphoma (e.g, DLBCL) to receive the treatment with immunotherapeutic agents (e.g., HX009). In some embodiments, a H-Score greater than about 0.89 predicts that the subject having lymphoma (e.g, DLBCL) is likely to respond to the treatment with immunotherapeutic agents (e.g, HX009). In some embodiments, the reference expression level of 0X40 is about 0.89 in H-Score for a subject having lymphoma (e.g, DLBCL) to receive the treatment with immunotherapeutic agents (e.g., HX009). In some embodiments, a H-Score greater than about 0.94 predicts that the subject having lymphoma (e.g, DLBCL) is likely to respond to the treatment with immunotherapeutic agents (e.g, HX009). In some embodiments, the reference expression level of 0X40 is about 0.94 in H-Score for a subject having lymphoma (e.g, DLBCL) to receive the treatment with immunotherapeutic agents (e.g, HX009). In some embodiments, a H-Score greater than about 1.58 predicts that the subject having lymphoma (e.g, DLBCL) is likely to respond to the treatment with immunotherapeutic agents (e.g, HX009). In some embodiments, the reference expression level of 0X40 is about 1.58 in H-Score for a subject having lymphoma (e.g, DLBCL) to receive the treatment with immunotherapeutic agents (e.g, HX009). In some embodiments, a H-Score greater than about 2.19 predicts that the subject having lymphoma (e.g, DLBCL) is likely to respond to the treatment with immunotherapeutic agents (e.g., HX009). In some embodiments, the reference expression level of 0X40 is about 2.19 in H-Score for a subject having lymphoma (e.g., DLBCL) to receive the treatment with immunotherapeutic agents (e.g, HX009). In some embodiments, a H-Score greater than about 7.26 predicts that the subject having lymphoma (e.g, DLBCL) is likely to respond to the treatment with immunotherapeutic agents (e.g, HX009). In some embodiments, the reference expression level of 0X40 is about 7.26 in H-Score for a subject having lymphoma (e.g, DLBCL) to receive the treatment with immunotherapeutic agents (e.g, HX009). In some embodiments, aH-Score greater than about 24.58 predicts that the subject having lymphoma (e.g, DLBCL) is likely to respond to the treatment with immunotherapeutic agents (e.g, HX009). In some embodiments, the reference expression level of 0X40 is about 24.58 in H-Score for a subject having lymphoma (e.g, DLBCL) to receive the treatment with immunotherapeutic agents (e.g., HX009). The H-Score values (e.g., 0.14, 0.57, 0.89, 0.94, 1.58, 2.19, 7.26, and 24.58) disclosed herein are determined with IHC workflow described in example 1 of the instant application.

[00188] One method includes analyzing gene expression profiles for biomarkers that distinguish responsive population from unresponsive population to determine the reference expression level for 0X40. For example, comparison of 0X40 expression levels between subjects responsive to a particular immunotherapy can be performed using the Mann-Whitney U-test, Chi-square test, or Fisher's Exact test. Analysis of descriptive statistics and comparisons can be performed using SigmaStat Software (Systat Software, Inc).

[00189] In some embodiments, a classification and regression tree (CART) analysis can be adopted to determine the reference level. In some embodiments, Receiver Operator Characteristic (ROC) analysis can be utilized to determine the reference expression level, or test the overall predictive value of individual genes and / or multigene classifiers (Soreide, J Clin Pathol 10.1136 (2008)). The reference level can be determined from the ROC curve of the training set to ensure both high sensitivity and high specificity. The Top Scoring Pair (TSP) algorithm first introduced by Geman et al. (2004) can also be used. A review of the methods and statistical tools useful for determining a reference level can be found in James Westgard, Ph.D., Basic Methods Validation, 3d edition (2008), which is hereby incorporated by reference in its entirety. Specific references are made to Chapter 9 ("How is reportable range of a method determined") and Chapter 15 ("How is a reference interval verified").

[00190] Clinically reportable range (CRR) is the range of analyte values that a method can measure, allowing for specimen dilution, concentration, or other pretreatment used to extend the direct analytical measurement range. As provided in the Basic Methods Validation by Dr. Westgard, the experiment to be performed is often called a “linearity experiment,” though there technically is no requirement that a method provide a linear response unless two-point calibration is being used. This range can also be referred to as the “linear range,” “analytical range,” or “working range” for a method. The reportable range is assessed by inspection of the linearity graph. That inspection can involve manually drawing the best straight line through the linear portion of the points, drawing a point-to-point line through all the points then comparing with the best straight line, or fitting a regression line through the points in the linear range. There are more complicated statistical calculations that are recommended in some guidelines, such as Clinical Laboratory Standards Institute (CLSI)’s EP-6 protocol for evaluating the linearity of analytical methods. But it is commonly accepted that the reportable range can be adequately determined from a “visual” assessment, i.e., by manually drawing the best straight line that fits the lowest points in the series. The Clinical Laboratory Standards Institute (CLSI) recommends a minimum of at least 4- preferably 5-different levels of concentrations. More than 5 can be used, particularly if the upper limit of reportable range needs to be maximized, but 5 levels are convenient and almost always sufficient.

[00191] A reference level is typically established by assaying specimens that are obtained from individuals that meet carefully defined criteria (reference sample group). Protocols such as those of the International Federation of Clinical Chemistry (IFCC) Expert Panel on Theory of Reference Values and the CLSI delineate comprehensive systematic processes that use carefully selected reference sample groups to establish reference intervals. These protocols typically need a minimum of 120 reference individuals for each group (or subgroup) that needs to be characterized.

[00192] The CLSI Approved Guideline C28-A2 describes different ways for a laboratory to validate the transference of established reference intervals to the individual laboratory that includes 1. Divine judgment, wherein the laboratory simply reviews the information submitted and subjectively verifies that the reference intervals are applicable to the adopting laboratory's patient population and test methods; 2. Verification with 20 samples, wherein experimental validation is performed by collecting and analyzing specimens from 20 individuals who represent the reference sample population; 3. Estimation with 60 samples, wherein an experimental validation is performed by collecting and analyzing specimens from 60 individuals who represent the reference sample population, and the actual reference interval is estimated and compared to the claimed or reported interval using a statistical formula comparing the means and standard deviations of the two populations; and 4. Calculation from comparative method, wherein one can adjust or correct the claimed or reported reference intervals on the basis of the observed methodological bias and the mathematical relationship demonstrated between the analytical methods being used.

[00193] Depending on the intended use, an appropriate control sample is chosen, and a reference value established therein. As also clear to the skilled artisan, the absolute marker values established in a control sample depend on the assay used for measuring the expression level.

[00194] In some embodiments, methods provided herein further comprise obtaining the sample from the subject. The sample used in the methods provided herein includes, for example, a tumor biopsy from the subject or a tissue biopsy from the subject.

[00195] In some embodiments, the sample used in the present methods includes a biopsy (e.g., a tumor biopsy). The biopsy can be from any organ or tissue, for example, skin, liver, lung, heart, colon, kidney, bone marrow, teeth, lymph node, hair, spleen, brain, breast, or other organs. Any biopsy technique known by those skilled in the art can be used for isolating a sample from a subject, for instance, open biopsy, close biopsy, core biopsy, incisional biopsy, excisional biopsy, or fine needle aspiration biopsy. In some embodiments, the sample is a lymph node biopsy. In some embodiments, the sample can be a frozen tissue sample. In some embodiments, the sample can be a formalin-fixed paraffin-embedded (“FFPE”) tissue sample. In some embodiments, the sample can be a deparaffinized tissue section.

[00196] In some embodiments, the sample is a body fluid sample. Non-limiting examples of body fluids include blood (e.g, peripheral whole blood, peripheral blood), blood plasma (plasma), bone marrow, amniotic fluid, aqueous humor, bile, lymph, menses, serum, urine, cerebrospinal fluid surrounding the brain and the spinal cord, synovial fluid surrounding bone joints.

[00197] In some embodiments, the sample is a blood sample. The blood sample can be a whole blood sample, a partially purified blood sample, a blood plasma sample, or a peripheral blood sample. The blood sample can be obtained using conventional techniques as described in, e.g. Innis et al, editors, PCR Protocols (Academic Press, 1990). White blood cells can be separated from blood samples using convention techniques or commercially available kits, e.g. RosetteSep kit (Stein Cell Technologies, Vancouver, Canada). Sub-populations of white blood cells, e.g. mononuclear cells, NK cells, B cells, T cells, monocytes, granulocytes or lymphocytes, can be further isolated using conventional techniques, e.g. magnetically activated cell sorting (MACS) (Miltenyi Biotec, Auburn, California) or fluorescently activated cell sorting (FACS) (Becton Dickinson, San Jose, California).

[00198] In one embodiment, the blood sample is from about 0.1 mL to about 10.0 mL, from about 0.2 mL to about 7 mL, from about 0.3 mL to about 5 mL, from about 0.4 mL to about 3.5

[00199] mL, or from about 0.5 mL to about 3 mL. In another embodiment, the blood sample is about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0 or 10.0 mL.

[00200] In one embodiment, the sample is a bone marrow sample. Procedures to obtain a bone marrow sample are well known in the art, including but not limited to bone marrow biopsy and bone marrow aspiration. Bone marrow has a fluid portion and a more solid portion. In bone marrow biopsy, a sample of the solid portion is taken. In bone marrow aspiration, a sample of the fluid portion is taken. Bone marrow biopsy and bone marrow aspiration can be done at the same time and referred to as a bone marrow exam. Bone marrow stromal cells (BMSCs), such as human primary BMSCs, are well known to support hematological tumor cell survival (including hematological cancer progression) and resistance to chemotherapy.

[00201] In certain embodiments, the sample used in the methods provided herein includes a plurality of cells. Such cells can include any type of cells, e.g, stem cells, blood cells (e.g, PBMCs), lymphocytes, NK cells, B cells, T cells, monocytes, granulocytes, immune cells, or tumor or cancer cells. Specific cell populations can be obtained using a combination of commercially available antibodies (e.g, Quest Diagnostic (San Juan Capistrano, Calif); Dako (Denmark)). In certain embodiments, the sample used in the methods provided herein includes PBMCs.

[00202] In certain embodiments, the sample used in the methods provided herein includes a plurality of cells from the diseased tissue, for example, the tumor sample from the subject. In some embodiments, the cells can be obtained from the tumor tissue, such as a tumor biopsy or a tumor explants. In certain embodiments, the number of cells used in the methods provided herein can range from a single cell to about 109 cells. In some embodiments, the number of cells used in the methods provided herein is about 1 x 104, 5 x 104, 1 x 105, 5 x 105, 1 x 106, 5 x 106, 1 x 107, 5 x 107, 1 x 108, or 5 x 108.

[00203] The number and type of cells collected from a subject can be monitored, for example, by measuring changes in morphology and cell surface markers using standard cell detection techniques such as flow cytometry, cell sorting, immunocytochemistry (e.g, staining with tissue specific or cellmarker specific antibodies) fluorescence activated cell sorting (FACS), magnetic activated cell sorting (MACS), by examination of the morphology of cells using light or confocal microscopy, and / or by measuring changes in gene expression using techniques well known in the art, such as PCR and gene expression profiling. These techniques can be used, too, to identify cells that are positive for one or more particular markers.

[00204] In certain embodiments, subsets of cells are used in the methods provided herein. Methods to sort and isolate specific populations of cells are well-known in the art and can be based on cell size, morphology, or intracellular or extracellular markers. Such methods include, but are not limited to, flow cytometry, flow sorting, FACS, bead-based separation such as magnetic cell sorting, size-based separation (e.g, a sieve, an array of obstacles, or a filter), sorting in a microfluidics device, antibody -based separation, sedimentation, affinity adsorption, affinity extraction, density gradient centrifugation, laser capture microdissection, etc.

[00205] In some embodiments, the sample is preserved after being obtained from the subject to minimize the possible loss of DNA, RNA, and / or protein during handling. In some embodiments, the sample is preserved before being processed or analyzed. The sample can be preserved using any means known in the art. In some embodiments, preserving the sample comprises placing the sample in a fixative. The fixative can be liquid. The fixative can be solid. In some embodiments, the fixative is neutral buffered formalin (NBF). NBF typically includes formaldehyde, methanol, and phosphate buffer. In some embodiments, the fixative is aldehyde-based. In some embodiments, the fixative is ethanol-based. The ethanol-based fixative can have >50% (e.g., 60%, 70%, 80%, 90% or 100%) ethanol. In some embodiments, the ethanol-based fixative can have 70% ethanol. In some embodiments, the fixative can have at least one component selected from glycerol, glacial acetic acid, a chao trope or denaturant (for example, guanidinium thiocyanate, guanidinium HC1, or guanidinium acetate), trehalose, polyethylene glycol (e.g., PEG200), ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis( -aminoethyl ether) ethylenediaminetetraacetic acid (EGTA), acrylamide, trichloroacetic acid, acetate salt (e.g., zinc acetate, copper acetate, or magnesium acetate), acetonitrile, and ethylene glycol. In some embodiments, the fixative is powder.

[00206] In some embodiments, the sample used in methods disclosed herein can be directly obtained from the source or pretreated prior to use. In some embodiments, a sample can be treated prior to use, such as preparing plasma from blood, diluting viscous fluids, and the like. Methods of treatment can involve filtration, distillation, extraction, concentration, inactivation of interfering components, the addition of reagents, and the like. In some embodiments, the cells contained in a sample are washed, mixed, allowed to settle, and embedded according to standard procedures. In some embodiments, the sample is treated by appropriate measures to release the gene products (protein and / or mRNA) from cellular constituents. As a result, the gene product(s) of 0X40 is obtained in soluble and easily accessible form. In some embodiments, the sample is diluted into an appropriate incubation buffer. Such incubation buffers are well-known to the skilled artisan. For illustrative purposes, if immunoassays are used, an appropriate buffer is selected that can serve both the purposes to liberate and / or solubilize proteins and to allow for immunological binding and thus for formation of an immunological complex for detection. 6.3 Kits

[00207] Provided herein are also kits for predicting responsiveness of a subject having cancer to an immunotherapeutic agent, comprising a means for measuring the expression level of 0X40 in a sample of the subject, and an ancillary reagent.

[00208] In certain embodiments, provided herein is a kit for detecting the mRNA level of 0X40. In certain embodiments, the kit includes one or more probes that bind specifically to the mRNAs of 0X40. In certain embodiments, the kit further includes a washing solution. In certain embodiments, the kit further includes reagents for performing a hybridization assay, mRNA isolation or purification means, detection means, as well as positive and negative controls. The kits can additionally include materials and reagents for isolating RNA from a biological sample. In certain embodiments, the kit further includes an instruction for using the kit. The kit can be tailored for in-home use, clinical use, or research use.

[00209] In certain embodiments, provided herein is a kit for detecting the protein level of 0X40. In certain embodiments, the kits include a solid surface coated with an antibody that recognizes 0X40, washing solutions, reagents for performing the assay, protein isolation or purification means, detection means, as well as positive and negative controls. In certain embodiments, the kit further includes an instruction for using the kit. The kits can additionally include materials and reagents for isolating proteins from a biological sample. The kit can be tailored for in-home use, clinical use, or research use.

[00210] In some embodiments, the kits provided herein include one or more containers and components for conducting in situ hybridization, RT-PCR, qPCR, deep sequencing, NGS, or a microarray. In certain embodiments, the kits provided herein employ means for detecting the expression of 0X40 by flow cytometry or immunofluorescence. In other embodiments, the expression of the biomarker is measured by ELISA-based methodologies or other similar methods known in the art.

[00211] In some embodiments, provided herein are kits for measuring the mRNA level of 0X40 by in situ hybridization. The kits can comprise a probe, The probe can be double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), RNA, or synthetic oligonucleotide (e.g., PNA, LNA). The probe can be detectably labeled. In some embodiments, the kits further comprise a labeling regent. The detectable label can be radioactive isotopes such as 32P, 35S, or 3H. The detectable label can also be biotin, digoxigenin, fluorescent dye (FISH), or any other label described herein or otherwise known in the art. The kits can further include a solid surface, such as a slide, hybridization buffer, wash buffer, mounting buffer, or any combination thereof. The kits can further include reagents for RNA isolation. Further, the kits can include instructions for performing in situ hybridization and methods for interpreting and analyzing the data resulting from the performance of the assay.

[00212] In some embodiments, kits for measuring the expression of 0X40 are provided herein, which comprise materials and reagents that are necessary for measuring the expression of 0X40. For example, a RT-PCR kit can be produced for a specific condition and contain the reagents and materials necessary for measuring the levels of 0X40 mRNA. In some embodiments, such kits can further include materials and reagents for synthesizing cDNA from RNA isolated from a sample. The kits generally include probes attached to a solid support surface. In one such embodiment, probes can be either be oligonucleotides or longer length probes including probes ranging from 150 nucleotides in length to 800 nucleotides in length. The probes can be attached to a detectable label. Included within the kits are probes specific for 0X40. In some embodiments, such kits can include primers for PCR as well as probes for Quantitative PCR. The kits can include instructions for performing the assay and methods for interpreting and analyzing the data resulting from the performance of the assay. The kits can also include hybridization reagents and / or reagents necessary for detecting a signal produced when a probe hybridizes to a target nucleic acid sequence. Generally, the materials and reagents for the kits are in one or more containers. Each component of the kit is generally in its own a suitable container.

[00213] For Quantitative PCR, the kits can include pre-selected primers specific for particular nucleic acid sequences. The Quantitative PCR kits can also include enzymes suitable for amplifying nucleic acids (e.g, polymerases such as Taq), and deoxynucleotides and buffers needed for the reaction mixture for amplification. The Quantitative PCR kits can also include probes specific for the nucleic acid sequences associated with or indicative of a condition. The probes can be labeled with a fluorophore. The probes can also be labeled with a quencher molecule. In some embodiments the Quantitative PCR kits can also include components suitable for reverse-transcribing RNA including enzymes (e.g.. reverse transcriptases such as AMV, MMLV and the like) and primers for reverse transcription along with deoxynucleotides and buffers needed for the reverse transcription reaction. Each component of the quantitative PCR kit is generally in its own suitable container. Thus, these kits generally include distinct containers suitable for each individual reagent, enzyme, primer and probe. Further, the quantitative PCR kits can include instructions for performing the assay and methods for interpreting and analyzing the data resulting from the performance of the assay.

[00214] For antibody-based kits (e.g, IHC kits, or ELISA kits), the kit can include, for example: (1) a first antibody which binds to 0X40; and, optionally, (2) a second, different antibody which binds to either 0X40, or the first antibody and is conjugated to a detectable label (e.g, a fluorescent label, radioactive isotope or enzyme). The first antibody can be attached to a solid support. In a specific embodiment, the polypeptide or protein of interest is a biomarker provided herein. The antibodybased kits can also include beads for conducting immunoprecipitation. The kits can further comprise blocking buffers, diluents, mounting medium (e.g, mounting reagents for fluorescent dye, or mounting medium for IHC with chromogen), or any combination thereof. Each component of the antibody-based kits is generally in its own suitable container. Thus, these kits generally include distinct containers suitable for each antibody. Further, the antibody-based kits can include instructions for performing the assay and methods for interpreting and analyzing the data resulting from the performance of the assay.

[00215] In certain embodiments of the methods and kits provided herein, solid phase supports are used for purifying proteins, labeling samples or carrying out the solid phase assays. Examples of solid phases suitable for carrying out the methods disclosed herein include beads, particles, colloids, single surfaces, tubes, multiwell plates, microtiter plates, slides, membranes, gels and electrodes. The solid support of the kit can be, for example, a plastic, silicon, a metal, a resin, glass, a membrane, a particle, a precipitate, a gel, a polymer, a sheet, a sphere, a polysaccharide, a capillary, a film, a plate, or a slide. When the solid phase is a particulate material (e.g., beads), it is, in one embodiment, distributed in the wells of multi-well plates to allow for parallel processing of the solid phase supports. The kits provided herein can employ, for example, a dipstick, a membrane, a chip, a disk, a test strip, a filter, a microsphere, a slide, a multiwell plate, or an optical fiber.

[00216] The kit of this disclosure can include an ancillary reagent. In some embodiments, the ancillary reagent can be a secondary antibody, a detection reagent, a detection buffer, an immobilization buffer, a dilution buffer, a washing buffer, or any combination thereof.

[00217] Secondary antibodies can be monoclonal or polyclonal antibodies. Secondary antibodies can be derived from any mammalian organism, including bovine, mice, rats, hamsters, goats, camels, chicken, rabbit, and others. Secondary antibodies can include, for example, an anti-human IgA antibody, an anti-human IgD antibody, an anti-human IgE antibody, an anti- human IgG antibody, or an anti-human IgM antibody. Secondary antibodies can be conjugated to enzymes (e.g, horseradish peroxidase (HRP), alkaline phosphatase (AP), luciferase, and the like) or dyes (e.g, colorimetric dyes, fluorescent dyes, fluorescence resonance energy transfer (FRET)-dyes, time-resolved (TR)-FRET dyes, and the like). In some embodiments, the secondary antibody is a polyclonal rabbit-anti-human IgG antibody, which is HRP-conjugated.

[00218] Any detection reagent known in the art can be included in a kit of this disclosure. In some embodiments, the detection reagent is a colorimetric detection reagent, a fluorescent detection reagent, or a chemiluminescent detection reagent. In some embodiments, the colorimetric detection reagent includes PPP (p-nitrophenyl phosphate), ABTS (2,2'-azino- bis(3-ethylbenzothiazoline-6-sulphonic acid)) or OPD (o-phenylenediamine). In some embodiments, the fluorescent detection reagent includes QuantaBluTM or QuantaRedTM (Thermo Scientific, Waltham, MA). In some embodiments, the luminescent detection reagent includes luminol or luciferin. In some embodiments, the detection reagent includes a trigger (e.g., H202) and a tracer (e.g., isoluminol-conjugate).

[00219] Any detection buffer known in the art can be included in a kit of this disclosure. In some embodiments the detection buffer is a citrate-phosphate buffer (e.g, about pH 4.2).

[00220] Any stop solution known in the art can be included in a kit of this disclosure. The stop solutions of this disclosure terminate or delay the further development of the detection reagent and corresponding assay signals. Stop solutions can include, for example, low-pH buffers (e.g., glycine-buffer, pH 2.0), chaotrophic agents (e.g, guanidinium chloride, sodium- dodecylsulfate (SDS)) or reducing agents (e.g, dithiothreitol, mecaptoethanol), or the like.

[00221] In some embodiments, the ancillary reagent is an immobilization reagent, which can be any immobilization reagent known in the art, including covalent and non-covalent immobilization reagents. Covalent immobilization reagents can include any chemical or biological reagent that can be used to covalently immobilize a peptide or a nucleic acid on a surface. Covalent immobilization reagents can include, for example, a carboxyl-to-amine reactive group (e.g., carbodiimides such as EDC or DCC), an amine reactive group (e.g, N-hydroxysuccinimide (NHS) esters, imidoesters), a sulfhydryl-reactive crosslinker (e.g., mal eimides, haloacetyls, pyridyl disulfides), a carbonyl-reactive crosslinker groups (e.g, hydrazides, alkoxyamines), a photoreactive crosslinker (e.g., aryl azides, dizirines), or a chemoselective ligation group (e.g, a Staudinger reaction pair). Non-covalent immobiliazation reagents include any chemical or biological reagent that can be used to immobilize a peptide or a nucleic acid non-covalently on a surface, such as affinity tags (e.g., biotin) or capture ragents (e.g., streptavidin or anti-tag antibodies, such as anti-His6 or anti-Myc antibodies). The kits of this disclosure can include combinations of immobilization reagents. Such combinations include, for example, EDC and NHS, which can be used, for example, to immobilize a protein of this disclosure on a surface, such as a carboxylated dextrane matrix (e.g, on a BIAcoreTM CM5 chip or a dextrane-based bead). Combinations of immobilization reagents can be stored as premixed reagent combinations or with one or more immobilization reagents of the combination being stored separately from other immobilization reagents.

[00222] A large selection of washing buffers is known in the art, such as tris(hydroxymethyl)aminom ethane (Tris)-based buffers (e.g, Tris-buffered saline, TBS) or phosphate buffers (e.g, phosphate-buffered saline, PBS). Washing buffers can include detergents, such as ionic or non-ionic detergents. In some embodiments, the washing buffer is a PBS buffer (e.g, about pH 7.4) including Tween®20 (e.g, about 0.05% Tween®20).

[00223] Any dilution buffer known in the art can be included in a kit of this disclosure. Dilution buffers can include a carrier protein (e.g, bovine serum albumin, BSA) and a detergent (e.g, Tween®20). In some embodiments, the dilution buffer is PBS (e.g, about pH 7.4) including BSA (e.g, about 1% BSA) and Tween®20 (e.g., about 0.05% Tween®20).

[00224] In some embodiments, the kit of this disclosure includes a cleaning reagent for an automated assay system. An automated assay system can include systems by any manufacturer. In some embodiments, the automated assay systems include, for example, the BIO-FLASHTM, the BEST 2000TM, the DS2TM, the ELx50 WASHER, the ELx800 WASHER, and the ELx800 READER. A cleaning reagent can include any cleaning reagent known in the art.

[00225] It is noted that any combination of the above-listed embodiments, for example, with respect to one or more reagents, such as, without limitation, nucleic acid primers, solid support and the like, are also contemplated in relation to any of the various methods and / or kits provided herein.

[00226] For illustrative purposes, in some embodiments, provided herein are kits of companion diagnostic for a cancer immunotherapy, comprising a means for detecting 0X40 expression. The kits can comprise IHC reagents, in situ hybridization reagents, ELISA reagents, or RT-PCR regents, all of which are well known in the art. The kits can further include a sampler for collecting a sample from a patient. The kits can further comprise a control sample which provides a reference level of the 0X40 expression. The kits can further comprise instructions for conducting the assay and / or for interpreting the results.

[00227] The practice of the invention employs, unless otherwise indicated, conventional techniques in molecular biology, cell biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields within the skill of the art. These techniques are described in the references cited herein and are fully explained in the literature. See, e.g., Maniatis et al. (1982) MOLECULAR Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Sambrook et al. (1989), Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press; Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al.. CURRENT PROTOCOLS IN Molecular Biology, John Wiley & Sons (1987 and annual updates); Current Protocols in Immunology, John Wiley & Sons (1987 and annual updates) Gait (ed.) (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein (ed.) (1991) Oligonucleotides and Analogues: A Practical Approach, IRL Press; Birren et al. (eds.) (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Borrebaeck (ed.) (1995); each of which is incorporated herein by reference in its entirety. 6.4 Exemplified Embodiments

[00228] Embodiment 1. A method of treating an 0X40 expressing cancer in a subject in need thereof, comprising administering a therapeutically effective amount of an immunotherapeutic agent to the subject.

[00229] Embodiment 2. The method of Embodiment 1, wherein the cancer is a hematological cancer or a solid tumor.

[00230] Embodiment 3. The method of Embodiment 2, wherein the cancer is a hematological cancer selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia (CMML), T cell acute lymphoblastic leukemia (T-ALL), natural killer cell leukemia (NK leukemia), Diffuse Large B-cell lymphoma (DLBCL), T cell lymphoblastic lymphoma, cutaneous T-Cell lymphoma (CTCL), peripheral T-cell lymphoma (PTCL), adult T-cell leukemia / lymphoma (ATLL), angioimmunoblastic T-cell lymphoma and natural killer cell / T cell lymphoma (NK / T cell lymphoma).

[00231] Embodiment 4. The method of Embodiment 2, wherein the cancer is a solid tumor selected from the group consisting of sarcoma, small cell lung cancer, non-small cell lung cancer, gastric cancer, liver cancer, melanoma, colorectal cancer, squamous cell carcinoma, endometrial cancer, breast cancer, malignant epithelioid mesothelioma, gallbladder cancer, pancreatic cancer, glioblastoma, ovarian cancer, and gastroesophageal junction adenocarcinoma.

[00232] Embodiment 5. The method of any one of Embodiments 1 to 4, wherein the cancer is at an advanced stage or metastatic.

[00233] Embodiment 6. The method of any one of Embodiments 1 to 5, wherein the cancer is EBV positive, HPV positive, HBV positive, HIV positive, orHTLV-1 positive.

[00234] Embodiment 7. The method of any one of Embodiments 1 to 6, wherein the immunotherapeutic agent targets PD1, PD-L1, OX40, CD47, CTLA-4, 4-1 BBL (CD137L), 4-IBB (CD 137), LAG-3, or TIGIT.

[00235] Embodiment 8. The method of Embodiment 7, wherein the immunotherapeutic agent targets PD-1.

[00236] Embodiment 9. The method of Embodiment 7, wherein the immunotherapeutic agent targets 0X40.

[00237] Embodiment 10. The method of Embodiment 7, wherein the immunotherapeutic agent targets CD47.

[00238] Embodiment 11. The method of Embodiment 7, wherein the immunotherapeutic agent targets CD 137.

[00239] Embodiment 12. The method of Embodiment 7, wherein the immunotherapeutic agent targets CTLA-4.

[00240] Embodiment 13. The method of Embodiment 7, wherein the immunotherapeutic agent comprises pembrolizumab, nivolumab, atezolizumab, durvalumab, cemiplimab, ipilimumab, tislelizumab, bempegaldesleukin, spartalizumab, sintilimab, toripalimab, envafolimab, tremelimumab, magrolimab, istiratumab, mavolimab, MGA012, BMS-986218, MK-1308, SHR-1210, GSK3359609, LY3415244, CA-170, Hu5F9-G4, TTI-621, AO-176, SRF231, CC-90002, IBI188, GSK3174998, BMS-986178, KHK4083, PF-04518600, or INCMGA00012, cinrebafusp alfa, RG7827, ADG106, NBRX-105, CTX-471, Genl046, MCLA-145, RG6076, MP0310, Genl042, AGEN2373, LVGN6051, ATOR-1017, STA551, ND-021, emfizatamab, DSP107, FS120, FS222, HOT-1030, ABL503, IBI319, GNC-039, EU101, CB307, ABL111, GNC-035, PRS-344, BI 765179, QL301, ATG-101, BT7480, PM1003, YH004, LBL-024, PM1032, HLX35 / BNA035, HBM7008, ABL105, BGB-B167, ADG206 or PE0116.

[00241] Embodiment 14. The method of Embodiment 7, wherein the immunotherapeutic agent comprises a bispecific antibody targeting CD47 and CTLA4 comprising a first peptide chain, a second peptide chain and a third peptide chain, having the amino acid sequences of SEQ ID NOs: 1, 2 and 3, respectively.

[00242] Embodiment 15. The method of Embodiment 7, wherein the immunotherapeutic agent comprises an anti-OX40 antibody comprising a light chain variable region (VL) and heavy chain variable region (VH) having the amino acid sequences of (1) SEQ ID NOs:4 and 5, respectively; (2) SEQ ID NOs: 38 and 39, respectively; (3) SEQ ID NOs: 38 and 42, respectively; (4) SEQ ID NOs: 44 and 45, respectively; or (5) SEQ ID NOs: 49 and 50, respectively.

[00243] Embodiment 16. The method of Embodiment 15, wherein the immunotherapeutic agent is an antibody-drug conjugate (“ADC”) having the anti-OX40 antibody conjugated to a cytotoxic agent.

[00244] Embodiment 17. The method of Embodiment 16, wherein the cytotoxic agent is dolastatin 10 or a derivative thereof (e.g, monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF)), Exatecan or a derivative thereof (e.g., Dxd), SN-38, or halichondrin B or a derivative thereof (e.g., Eribulin).

[00245] Embodiment 18. The method of Embodiment 7, wherein the immunotherapeutic agent is a fusion protein targeting CD47 and PD1 comprising a first peptide chain and a second peptide chain, having the amino acid sequences of SEQ ID NOs: 10 and 11, respectively.

[00246] Embodiment 19. The method of any one of Embodiments 1 to 18, comprising determining OX40 expression in a sample from the subject.

[00247] Embodiment 20. The method of Embodiment 19, wherein the 0X40 expression in the sample is determined to be detectable or higher than a reference level.

[00248] Embodiment 21. The method of Embodiment 19 or 20, wherein the 0X40 expression is determined at protein level.

[00249] Embodiment 22. The method of Embodiment 21, wherein the 0X40 expression is measured by immunohistochemistry (IHC), immunocytochemistry (ICC), an enzyme-linked immunosorbent assay (ELISA), immunoblotting assay (e.g, Western blot), flow cytometry (FACS), a fluorescent immunosorbent assay (FIA), a chemiluminescence immunoassay (CIA), a radioimmunoassay (RIA), a solid phase radioimmunoassay (SPROA), or a dot / line-immunoblot assay.

[00250] Embodiment 23. The method of Embodiment 22, wherein the 0X40 expression is measured by IHC.

[00251] Embodiment 24. The method of Embodiment 19 or 20, wherein the 0X40 expression is determined at mRNA level.

[00252] Embodiment 25. The method of Embodiment 24, wherein the 0X40 expression is measured by RNA-Seq, in situ RNA hybridization (e.g, fluorescence in situ hybridization, or FISH), quantitative polymerase chain reaction (qPCR), real-time polymerase chain reaction (RT-PCR), microarray analysis, serial analysis of gene expression (SAGE), MassARRAY, next generation sequencing (NGS), or single cell whole-exome sequencing (scWES).

[00253] Embodiment 26. The method of Embodiment 25, wherein the 0X40 expression is measured by RNA-Seq, or qPCR.

[00254] Embodiment 27. The method of any one of Embodiments 19 to 26, wherein the sample is a tissue biopsy or tumor biopsy.

[00255] Embodiment 28. The method of any one of Embodiments 19 to 26, wherein the sample is a blood sample, a serum sample, or a bone marrow sample.

[00256] Embodiment 29. The method of any one of Embodiments 19 to 26, wherein the sample is patient-derived xenograft (PDX) sample.

[00257] Embodiment 30. The method of any one of Embodiments 19 to 29, further comprising obtaining the sample from the subject.

[00258] Embodiment 31. A method of predicting responsiveness of a subject having cancer to an immunotherapeutic agent, comprising measuring OX40 expression in a sample from the subject, wherein the subject is likely responsive to the immunotherapeutic agent if the cancer is an 0X40 expressing cancer.

[00259] Embodiment 32. A method of selecting a subject having cancer for treatment with an immunotherapeutic agent, comprising measuring the 0X40 expression in a sample from the subject and selecting the subject for the treatment if the subject has an 0X40 expressing cancer.

[00260] Embodiment 33. The method of Embodiment 31 or 32, wherein the cancer is a hematological cancer or a solid tumor.

[00261] Embodiment 34. The method of Embodiment 33, wherein the cancer is a hematological cancer selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia (CMML), T cell acute lymphoblastic leukemia (T-ALL), natural killer cell leukemia (NK leukemia), Diffuse Large B-cell lymphoma (DLBCL), T cell lymphoblastic lymphoma, cutaneous T-Cell lymphoma (CTCL), peripheral T-cell lymphoma (PTCL), adult T-cell leukemia / lymphoma (ATLL), angioimmunoblastic T-cell lymphoma and natural killer cell / T cell lymphoma (NK / T cell lymphoma).

[00262] Embodiment 35. The method of Embodiment 33, wherein the cancer is a solid tumor selected from the group consisting of sarcoma, small cell lung cancer, non-small cell lung cancer, gastric cancer, liver cancer, melanoma, colorectal cancer, squamous cell carcinoma, endometrial cancer, breast cancer, malignant epithelioid mesothelioma, gallbladder cancer, pancreatic cancer, glioblastoma, ovarian cancer, and gastroesophageal junction adenocarcinoma.,

[00263] Embodiment 36. The method of any one of Embodiments 31 to 35, wherein the cancer is at an advanced stage or metastatic.

[00264] Embodiment 37. The method of any one of Embodiments 31 to 36, wherein the cancer is EBV positive, HPV positive, HBV positive, HIV positive, or HTLV-1 positive.

[00265] Embodiment 38. The method of any one of Embodiments 31 to 37, wherein the immunotherapeutic agent targets PD1, PD-L1, OX40, CD47, CTLA-4, 4-1 BBL (CD137L), 4-IBB (CD 137), LAG-3, or TIGIT.

[00266] Embodiment 39. The method of Embodiment 38, wherein the immunotherapeutic agent targets PD-1.

[00267] Embodiment 40. The method of Embodiment 38, wherein the immunotherapeutic agent targets OX40.

[00268] Embodiment 41. The method of Embodiment 38, wherein the immunotherapeutic agent targets CD47.

[00269] Embodiment 42. The method of Embodiment 38, wherein the immunotherapeutic agent targets CD 137.

[00270] Embodiment 43. The method of Embodiment 38, wherein the immunotherapeutic agent targets CTLA-4.

[00271] Embodiment 44. The method of Embodiment 38, wherein the immunotherapeutic agent comprises pembrolizumab, nivolumab, atezolizumab, durvalumab, cemiplimab, ipilimumab, tislelizumab, bempegaldesleukin, spartalizumab, sintilimab, toripalimab, envafolimab, tremelimumab, magrolimab, istiratumab, mavolimab, MGA012, BMS-986218, MK-1308, SHR-1210, GSK3359609, LY3415244, CA-170, Hu5F9-G4, TTI-621, AO-176, SRF231, CC-90002, IBI188, GSK3174998, BMS-986178, KHK4083, PF-04518600, INCMGA00012, cinrebafusp alfa, RG7827, ADG106, NBRX-105, CTX-471, Genl046, MCLA-145, RG6076, MP0310, Genl042, AGEN2373, LVGN6051, ATOR-1017, STA551, ND-021, emfizatamab, DSP107, FS120, FS222, HOT-1030, ABL503, IBI319, GNC-039, EU101, CB307, ABL111, GNC-035, PRS-344, BI 765179, QL301, ATG-101, BT7480, PM1003, YH004, LBL-024, PM1032, HLX35 / BNA035, HBM7008, ABL105, BGB-B167, ADG206 or PE0116.

[00272] Embodiment 45. The method of Embodiment 38, wherein the immunotherapeutic agent comprises a bispecific antibody targeting CD47 and CTLA4 comprising a first peptide chain, a second peptide chain and a third peptide chain, having the amino acid sequences of SEQ ID NOs: 1, 2 and 3, respectively.

[00273] Embodiment 46. The method of Embodiment 38, wherein the immunotherapeutic agent comprises an anti-OX40 antibody comprising a light chain variable region (VL) and heavy chain variable region (VH) having the amino acid sequences of (1) SEQ ID NOs:4 and 5, respectively; (2) SEQ ID NOs: 38 and 39, respectively; (3) SEQ ID NOs: 38 and 42, respectively; (4) SEQ ID NOs: 44 and 45, respectively; or (5) SEQ ID NOs: 49 and 50, respectively.

[00274] Embodiment 47. The method of Embodiment 46, wherein the immunotherapeutic agent is an ADC having the anti-OX40 antibody conjugated to a cytotoxic agent.

[00275] Embodiment 48. The method of Embodiment 47, wherein the cytotoxic agent is dolastatin 10 or a derivative thereof (e.g, monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF)), Exatecan or a derivative thereof (e.g., Dxd), SN-38, or halichondrin B or a derivative thereof (e.g., Eribulin).

[00276] Embodiment 49. The method of Embodiment 38, wherein the immunotherapeutic agent is a fusion protein targeting CD47 and PD1 comprising a first peptide chain and a second peptide chain, having the amino acid sequences of SEQ ID NOs: 10 and 11, respectively.

[00277] Embodiment 50. The method of any one of Embodiments 31 to 49, wherein the cancer is determined to be OX40 expressing cancer if the 0X40 expression in the sample is detectable or higher than a reference level.

[00278] Embodiment 51. The method of claim Embodiment 50, wherein the 0X40 expression is determined at protein level.

[00279] Embodiment 52. The method of Embodiment 51, wherein the 0X40 expression is measured by immunohistochemistry (IHC), immunocytochemistry (ICC), an enzyme-linked immunosorbent assay (ELISA), immunoblotting assay (e.g, Western blot), flow cytometry (FACS), a fluorescent immunosorbent assay (FIA), a chemiluminescence immunoassay (CIA), a radioimmunoassay (RIA), a solid phase radioimmunoassay (SPROA), or a dot / line-immunoblot assay.

[00280] Embodiment 53. The method of Embodiment 52, wherein the 0X40 expression is measured by IHC.

[00281] Embodiment 54. The method of Embodiment 50, wherein the 0X40 expression is determined at mRNA level.

[00282] Embodiment 55. The method of Embodiment 54, wherein the 0X40 expression is measured by RNA-Seq, in situ RNA hybridization (e.g, fluorescence in situ hybridization, or FISH), quantitative polymerase chain reaction (qPCR), real-time polymerase chain reaction (RT-PCR), microarray analysis, serial analysis of gene expression (SAGE), MassARRAY, next generation sequencing (NGS), or single cell whole-exome sequencing (scWES).

[00283] Embodiment 56. The method of Embodiment 55, wherein the 0X40 expression is measured by RNA-Seq or qPCR.

[00284] Embodiment 57. The method of any one of Embodiments 31 to 56, wherein the sample is a tissue biopsy or tumor biopsy.

[00285] Embodiment 58. The method of any one of Embodiments 31 to 56, wherein the sample is a blood sample, a serum sample, or a bone marrow sample.

[00286] Embodiment 59. The method of any one of Embodiments 31 to 56, wherein the sample is a PDX sample.

[00287] Embodiment 60. The method of any one of Embodiments 31 to 59, further comprising obtaining the sample from the subject.

[00288] Embodiment 61. A kit for predicting the responsiveness of a subject having cancer to treatment with an immunotherapeutic agent, comprising a means for measuring the expression of OX40 in a sample of the subject, and an ancillary reagent.

[00289] Embodiment 62. The kit of Embodiment 61, wherein the means for measuring the expression of OX40 comprises an anti-OX40 antibody.

[00290] Embodiment 63. The kit of Embodiment 61, wherein the means for measuring the expression of 0X40 comprises a nucleic acid probe for detecting the 0X40 mRNA.

[00291] Embodiment 64. The kit of any one of Embodiments 61 to 63, wherein the ancillary reagent comprises a reaction buffer, a dilution buffer, or a wash buffer, or any combination thereof.

[00292] Embodiment 65. The kit of any one of Embodiments 61 to 64, wherein said kit further comprises a solid support.

[00293] Embodiment 66. The kit of any one of Embodiments 61 to 65, wherein said kit further comprises a container for sample collection.

[00294] Embodiment 67. The kit of any one of Embodiments 61 to 66, wherein the cancer is a hematological cancer or a solid tumor.

[00295] Embodiment 68. The kit of Embodiment 67, wherein the cancer is a hematological cancer selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia (CMML), T cell acute lymphoblastic leukemia (T-ALL), natural killer cell leukemia (NK leukemia), Diffuse Large B-cell lymphoma (DLBCL), T cell lymphoblastic lymphoma, cutaneous T-Cell lymphoma (CTCL), peripheral T-cell lymphoma (PTCL), adult T-cell leukemia / lymphoma (ATLL), angioimmunoblastic T-cell lymphoma and natural killer cell / T cell lymphoma (NK / T cell lymphoma).

[00296] Embodiment 69. The kit of Embodiment 67, wherein the cancer is a solid tumor selected from the group consisting of sarcoma, small cell lung cancer, non-small cell lung cancer, gastric cancer, liver cancer, melanoma, colorectal cancer, squamous cell carcinoma, endometrial cancer, breast cancer, malignant epithelioid mesothelioma, gallbladder cancer, pancreatic cancer, glioblastoma, ovarian cancer, and gastroesophageal junction adenocarcinoma.,

[00297] Embodiment 70. The kit of any one of Embodiments 61 to 69, wherein the cancer is at an advanced stage or metastatic.

[00298] Embodiment 71. The kit of any one of Embodiments 61 to 70, wherein the cancer is EBV positive, HPV positive, HBV positive, HIV positive, orHTLV-1 positive.

[00299] Embodiment 72. The kit of any one of Embodiments 61 to 71, wherein the immunotherapeutic agent targets PD1, PD-L1, OX40, CD47, CTLA-4, 4-1 BBL (CD137L), 4-IBB (CD 137), LAG-3, or TIGIT.

[00300] Embodiment 73. The kit of Embodiment 72, wherein the immunotherapeutic agent targets PD-1.

[00301] Embodiment 74. The kit of Embodiment 72, wherein the immunotherapeutic agent targets 0X40.

[00302] Embodiment 75. The kit of Embodiment 72, wherein the immunotherapeutic agent targets CD47.

[00303] Embodiment 76. The kit of Embodiment 72, wherein the immunotherapeutic agent targets CD137.

[00304] Embodiment 77. The kit of Embodiment 72, wherein the immunotherapeutic agent targets CTLA-4.

[00305] Embodiment 78. The kit of Embodiment 72, wherein the immunotherapeutic agent comprises pembrolizumab, nivolumab, atezolizumab, durvalumab, cemiplimab, ipilimumab, tislelizumab, bempegaldesleukin, spartalizumab, sintilimab, toripalimab, envafolimab, tremelimumab, magrolimab, istiratumab, mavolimab, MGA012, BMS-986218, MK-1308, SHR-1210, GSK3359609, LY3415244, CA-170, Hu5F9-G4, TTI-621, AO-176, SRF231, CC-90002, IBI188, GSK3174998, BMS-986178, KHK4083, PF-04518600, INCMGA00012, cinrebafusp alfa, RG7827, ADG106, NBRX-105, CTX-471, Genl046, MCLA-145, RG6076, MP0310, Genl042, AGEN2373, LVGN6051, ATOR-1017, STA551, ND-021, emfizatamab, DSP107, FS120, FS222, HOT-1030, ABL503, IBI319, GNC-039, EU101, CB307, ABL111, GNC-035, PRS-344, BI 765179, QL301, ATG-101, BT7480, PM1003, YH004, LBL-024, PM1032, HLX35 / BNA035, HBM7008, ABL105, BGB-B167, ADG206 or PE0116.

[00306] Embodiment 79. The kit of Embodiment 72, wherein the immunotherapeutic agent comprises a bispecific antibody targeting CD47 and CTLA4 comprising a first peptide chain, a second peptide chain and a third peptide chain, having the amino acid sequences of SEQ ID NOs: 1, 2 and 3, respectively.

[00307] Embodiment 80. The kit of Embodiment 72, wherein the immunotherapeutic agent comprises an anti-OX40 antibody comprising a light chain variable region (VL) and heavy chain variable region (VH) having the amino acid sequences of (1) SEQ ID NOs:4 and 5, respectively; (2) SEQ ID NOs: 38 and 39, respectively; (3) SEQ ID NOs: 38 and 42, respectively; (4) SEQ ID NOs: 44 and 45, respectively; or (5) SEQ ID NOs: 49 and 50, respectively.

[00308] Embodiment 81. The kit of Embodiment 80, wherein the immunotherapeutic agent is an ADC having the anti-OX40 antibody conjugated to a cytotoxic agent.

[00309] Embodiment 82. The kit of Embodiment 81, wherein the cytotoxic agent is dolastatin 10 or a derivative thereof (e.g., monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF)), Exatecan or a derivative thereof (e.g., Dxd), SN-38, or halichondrin B or a derivative thereof (e.g., Eribulin).

[00310] Embodiment 83. The kit of Embodiment 72, wherein the immunotherapeutic agent is a fusion protein targeting CD47 and PD1 comprising a first peptide chain and a second peptide chain, having the amino acid sequences of SEQ ID NOs: 10 and 11, respectively. 6.5 Experimental

[00311] The examples provided below are for purposes of illustration only, which are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein. 6.5.1 Example 1: Validation of 0X40 IHC workflow

[00312] Ninety (90) leukemia and lymphoma PDXs of Western patient origins were comprehensively annotated, including RNAseq and histopathology data (HuBaseTM https: / / hubase.crownbio.com). The RNASeq transcriptome analysis displayed 0X40 gene overexpression (Log2 (FPKM)>3 in 8 / 90 models) in 8.9% of models. An IHC process was developed and used to further evaluate these PDXs, which also revealed high tumor 0X40 expression, with IHC scores largely correlated with the RNA data.

[00313] IHC staining of cell lines or cancer PDX models on FFPE slides was performed in the Bond RX Automated IHC / ISH Staining System (Leica Biosystems) using a commercial 0X40 antibody (CST, #61637), followed by whole slide imaging using the NanoZoomer NDP2.0-HT Digital Slide System (Hamamatsu) and quantified by HALO™ image analysis software (Indica labs). 0X40 IHC was performed according to the following protocol:

[00314] Samples Preparation

[00315] 1) Specimens were collected freshly and placed in 10% NBF (fixative volume / tissue, 10-20 folds), fixed at room temperature for 24-48 hours.

[00316] 2) The fixed tissues were trimmed to a thickness of 3-5 mm.

[00317] 3) The trimmed tissues were moved into the embedding box, and the box was snapped into deionized water for 30 minutes. Water was changed every 30 minutes, and the step was repeated twice.

[00318] 4) The fixed tissues were transferred to the HistoCore PEARL - Tissue Processor for dehydration.

[00319] Dehydration

[00320] The following automatized dehydration procedure for HistoCore PEARL - Tissue Processor was adopted: Reagent Duration Temperature °C Pressure / Vacuum 70% ethanol 20 min RT — 80% ethanol 20 min RT — 90% ethanol 20 min RT — 95% ethanol 25 min RT on 100% ethanol 25 min RT — 100% ethanol 35 min RT — 100% ethanol 35 min RT on Xylene 35 min RT — Xylene 35 min RT on Paraffin I 30 min 60 — Paraffin II 30 min 60 — Paraffin III 30 min 60 on

[00321] FFPE blocks preparation

[00322] Standard embedding process was applied to samples to form FFPE block. Tissue was embedded by paraffin on HistoCore Arcadia Embedding Center with Heated Paraffin Embedding Station and Cold Plate.

[00323] FFPE slides preparation

[00324] FFPE blocks were sectioned at 4 pm thickness by Semi-Automated Rotary Microtome, and the tissue slice was attached to the glass slide. The slides were baked at 60 °C for 60 min.

[00325] IHC staining

[00326] Anti-OX40 (rabbit IgG, 475 pg / mL, Cell Signaling, Cat. 61637) was used for staining. Antigen retrieval was performed at 100 °C in EDTA buffer, pH9.0 for 20min.

[00327] The staining was performed following the procedure below on Bond RX autostainer Step Reagent Time (minute) per cycle Temperature (°C) 1 Bond Dewax Solution 0.5 72 2 Bond Dewax Solution 0 72 3 Bond Dewax Solution 0 RT 4 Alcohol 0 RT 5 Alcohol 0 RT 6 Alcohol 0 RT 7 Bond Wash Solution 0 RT 8 Bond Wash Solution 0 RT 9 Bond Wash Solution 0 RT 10 Bond ER Solution 1 / 2 0 RT 11 Bond ER Solution 1 / 2 0 RT 12 Bond ER Solution 1 / 2 20 100 13 Bond ER Solution 1 / 2 0 RT 14 Bond Wash Solution 0 RT 15 Bond Wash Solution 0 RT 16 Bond Wash Solution 0 RT 17 Bond Wash Solution 0 RT 18 Bond Wash Solution 3 RT 19 Peroxide Block 10 RT 20 Bond Wash Solution 0 RT 21 Bond Wash Solution 0 RT 22 Bond Wash Solution 0 RT 23 Blocking Reagent 20 RT 24 Primary Antibody 60 RT 25 Bond Wash Solution 0 RT 26 Bond Wash Solution 2 RT 27 Bond Wash Solution 2 RT 28 Bond Wash Solution 2 RT 29 Secondary Antibody 20 RT 30 Bond Wash Solution 0 RT 31 Bond Wash Solution 2 RT 32 Bond Wash Solution 2 RT 33 Bond Wash Solution 2 RT 34 Deionized Water 0 RT 35 Mixed DAB Refine 0 RT 36 Mixed DAB Refine 5 RT 37 Deionized Water 0 RT 38 Deionized Water 0 RT 39 Deionized Water 0 RT 40 Hematoxylin 10 RT 41 Deionized Water 0 RT 42 Bond Wash Solution 0 RT 43 Deionized Water 0 RT

[00328] As provided below in Table 1 (cell lines) and Tables 2-4 (PDX models), 0X40 protein expression largely correlated with the mRNA expression, validating the IHC assay.

[00329] Table 1: 0X40 mRNA / protein expression in cell lines Cell line 0X40 mRNA Log2 (FPKM) OX40 Protein Viral status HuT-102 8.395 ++++ HTLV+ NAMALWA CSN / 70 7.482 NA EBV+ HuT-78 6.090 NA HTLV+ ARH-77 5.303 +++ EBV+ Jurkat E6-1 3.291 - NA Karpas 299 3.084 - NA GRANTA-519 2.971 ++ EBV+ NAMALWA 1.997 + NA JEKO-1 -0.297 - HTLV+ Note: NAMALWA CSN / 70 and HuT-78 did not have FFPE blocks.

[00330] Table 2: 0X40 mRNA / protein expression in lymphoma PDX models Model ID Cancer type 0X40 protein % Positive cells H-Score 0X40 mRNA Log2 (FPKM) Viral status LY12886 Lymphoma ++++ 99.91 258.70 N / A EBV+ LY9596 Lymphoma ++++ 97.83 251.36 8.49 EBV- LY6698 Lymphoma +++ 97.36 147.95 6.181 EBV+ LY6938 Lymphoma +++ 93.20 142.05 4.225 EBV- LY3654 Lymphoma +++ 88.82 122.03 2.868 EBV+ LY2219 Lymphoma ++ 29.34 30.24 4.434 EBV+ LY2301 Lymphoma +++ 20.73 26.14 0.826 EBV+ LY4091 Lymphoma ++ 24.03 24.58 4.916 EBV+ LY3161 Lymphoma ++ 10.14 11.33 3.437 EBV+ LY2345 Lymphoma + 7.13 7.26 2.185 EBV+ LY2037 Lymphoma ++ 6.66 6.89 4.037 EBV+ LY2324 Lymphoma ++ 5.52 5.92 1.237 EBV+ LY1487 Lymphoma + 2.35 2.61 1.858 EBV+ LY3071 Lymphoma + 1.89 2.19 -0.591 EBV+ LY3032 Lymphoma + 1.51 1.77 1.198 EBV+ LY6933 Lymphoma - 1.41 1.58 -2 EBV- LY3604 Lymphoma + 0.91 0.94 2.094 EBV- LY6483 Lymphoma - / + 0.84 0.89 0.234 EBV+ LY11632 Lymphoma + 0.76 0.79 3.076 EBV+ LY2201 Lymphoma + 0.71 0.77 2.891 EBV+ LY6696 Lymphoma -1+ 0.55 0.57 2.906 EBV+ LY2118 Lymphoma - / + 0.44 0.46 1.615 EBV+ LY12873 Lymphoma - 0.19 0.19 0.831 EBV- LY9595 Lymphoma - 0.17 0.18 0.615 EBV- LY12880 Lymphoma +1- 0.13 0.15 N / A NA LY 12962 Lymphoma - / + 0.14 0.14 0.268 EBV- LY2266 Lymphoma - 0.11 0.11 1.157 EBV+ LY9603 Lymphoma - 0.09 0.09 0.445 EBV+ LY9615 Lymphoma - 0.08 0.09 0.952 EBV- LY3463 Lymphoma - 0.08 0.09 0.068 EBV- LY12922 Lymphoma - 0.07 0.07 2.215 EBV+ LY2104 Lymphoma - 0.06 0.06 2.552 EBV+ LY0257 Lymphoma - 0.05 0.05 0.749 EBV- LY9606 Lymphoma - 0.04 0.04 -1.195 EBV- LY2318 Lymphoma - 0.03 0.03 -0.902 EBV+ LY9594 Lymphoma - 0.02 0.03 -0.931 EBV- LY2310 Lymphoma - 0.03 0.03 -2 EBV+ LY 12954 Lymphoma - 0.02 0.03 -2 EBV- LY9592 Lymphoma - 0.02 0.02 -2 EBV- LY9607 Lymphoma - 0.02 0.02 -2 EBV- LY2264 Lymphoma - 0.02 0.02 -2 EBV- LY6443 Lymphoma - 0.02 0.02 -1.242 EBV+ LY12895 Lymphoma - 0.01 0.01 -0.432 EBV- LY12935 Lymphoma - 0.01 0.01 -1.126 EBV- LY12864 Lymphoma - 0.01 0.01 -2 EBV- LY2240 Lymphoma - 0.01 0.01 0.601 EBV+ LY3148 Lymphoma - 0.01 0.01 -2 EBV- LY6931 Lymphoma - 0.01 0.01 N / A NA LY2298 Lymphoma - 0.01 0.01 -0.193 EBV- LY9604 Lymphoma - 0.01 0.01 -2 EBV- LY3786 Lymphoma - 0.01 0.01 0.636 EBV- LY6701 Lymphoma - 0.01 0.01 -2 EBV- LY2214 Lymphoma - 0.01 0.01 -2 EBV- LY12936 Lymphoma - 0.00 0.00 2.405 EBV- LY12960 Lymphoma - 0.00 0.00 N / A NA LY6934 Lymphoma - 0.00 0.00 -2 EBV- LY 12966 Lymphoma - 0.00 0.00 -2 EBV- LY12921 Lymphoma - 0.00 0.00 -1.025 EBV- LY9602 Lymphoma - 0.00 0.00 -2 EBV- LY9605 Lymphoma - 0.00 0.00 -2 EBV-

[00331] Table 3: 0X40 mRNA / protein expression in PDX models for head and neck cancer, esophageal cancer and cervical cancer Model ID Cancer type 0X40 mRNA Log2 (FPKM) 0X40 Protein Viral Status HN9285 Head and neck cancer 5.553 +++ HPV16+ HN2400 Head and neck cancer 5.334 + HPV16+ ES3876 Esophageal cancer 4.892 +++ EBV+ CV10941 Cervical cancer 5.353 ++ HPV18+

[00332] Table 4: 0X40 mRNA expression level and 0X40 protein expression level in sarcoma Model ID Cancer type OX40 mRNA Log2 (FPKM) OX40 Protein SA12961 Spindle cell sarcoma 7.508 ++++ SA13412 Small round cell sarcoma 6.657 +++ SA10199 Spindle cell sarcoma 5.71 +++ SAI 3542 Ewing's sarcoma 5.644 +++ SAI 0233 Ewing's sarcoma 5.608 ++ SA3838 Chondrosarcoma 5.471 ++ SA10153 Synovial Sarcoma 5.382 + SAI 3346 Small round cell sarcoma 4.974 +++ SAI 3543 Ewing's sarcoma 4.931 + SAI 0242 Spindle cell sarcoma 4.924 + SA4106 Spindle cell sarcoma 4.723 +1- SA4190 Spindle cell sarcoma 4.682 +++ SA4225 Osteosarcoma 4.648 + SA10162 Synovial sarcoma 4.584 +1- SA13271 Spindle cell sarcoma 4.402 - SAI 6056 Ewing's sarcoma 4.047 +1- SA4218 Ewing's sarcoma 4.005 + SA4094 Synovial sarcoma 3.907 + SA4134 synovial sarcoma 3.683 - SA4013 Osteosarcoma 3.215 - SA4098 Synovial sarcoma 2.932 + S A3 849 Osteosarcoma 2.227 - SA4008 Osteosarcoma 1.9 - S A3 841 High-grade sarcoma 1.737 - SA4028 High-grade undifferentiated sarcoma 0.944 - SA4078 Osteosarcoma 0.062 +1- SA2466 Rhabdomyosarcoma -0.701 - SA4109 Chondrosarcoma -0.872 - S A3 829 Ewing's sarcoma -1.156 +1- SA4061 Osteosarcoma -1.337 - SA3302 Pleomorphic sarcoma -1.385 - SA3296 Cystosarcoma -1.749 +1- SA0271 Spindle cell sarcoma -2 - SA0319 Spindle cell sarcoma -2 - SA0639 Rhabdomyosarcoma -2 - SA2398 Sarcoma -2 - SA3224 sarcomatoid carcinoma -2 - SA3283 Undifferentiated pleomorphic sarcoma -2 - SA3313 Osteosarcoma -2 - S A3 831 leiomyosarcoma -2 - S A3 839 High-grade undifferentiated sarcoma -2 - S A3 840 Osteosarcoma -2 - SA4033 Leiomyosarcoma -2 - SA4062 osteosarcoma -2 + / - SA4117 Spindle cell sarcoma -2 - SA4136 spindle cell sarcoma -2 - 6.5.2 Example 2: Anti-PDl-sirpa fusion effectively treated OX40-expressing lymphoma

[00333] Samples of a cohort of lymphoma (DLBCL)-PDXs were analyzed by whole transcriptome sequencing (RNAseq) and IHC for PD-L1, PD-L2, sirpa, CD47, and 0X40, etc, followed by various statistical analysis. Lymphoma PDXs were subjected to PDX trial in clinical trial style (Guo et al. (2019) BMC Cancer 19, 718; Li et al. (2017) Pharmacol Ther 173, 34-46) with the anti-PDl-sirpa fusion protein HX009. Tumor growth inhibition (TGI) was calculated by measuring tumor volume biweekly and the TGI values were statistically analyzed against the expression of these immune checkpoint proteins

[00334] Table 5: HX009 treatment results in TGI Model ID Cancer type % Positive cells H-Score 0X40 mRNA Log2 (FPKM) TGI (%) LY4091 Lymphoma 24.03 24.58 4.916 35.69 LY2345 Lymphoma 7.13 7.26 2.185 47.87 LY3071 Lymphoma 1.89 2.19 -0.591 28.28 LY6933 Lymphoma 1.41 1.58 -2 18.18 LY3604 Lymphoma 0.91 0.94 2.094 29.56 LY6483 Lymphoma 0.84 0.89 0.234 4.97 LY6696 Lymphoma 0.55 0.57 2.906 38.24 LY12962 Lymphoma 0.14 0.14 0.268 51.58 LY2266 Lymphoma 0.11 0.11 1.157 37.15 LY3463 Lymphoma 0.08 0.09 0.068 1.73 LY0257 Lymphoma 0.05 0.05 0.749 49.38 LY2318 Lymphoma 0.03 0.03 -0.902 26.19 LY2310 Lymphoma 0.03 0.03 -2 17.48 LY2264 Lymphoma 0.02 0.02 -2 4.50 LY6443 Lymphoma 0.02 0.02 -1.242 32.27 LY2298 Lymphoma 0.01 0.01 -0.193 -1.83 LY3786 Lymphoma 0.01 0.01 0.636 -0.02 LY6701 Lymphoma 0.01 0.01 -2 15.13 LY2214 Lymphoma 0.01 0.01 -2 63.25 LY6934 Lymphoma 0.00 0.00 -2 45.45 LY 12966 Lymphoma 0.00 0.00 -2 19.22 LY9602 Lymphoma 0.00 0.00 -2 31.86

[00335] As shown in Table 5, FIG. 1A and FIG. IB, analysis revealed a trend of correlation between the TGI of HX009 in a panel of DLBCL PDX and the 0X40 expression level (both protein and mRNA), indicating that 0X40 could serve as a biomarker to predict the responsiveness of a lymphoma patient to a treatment of anti-PDl-sirpa fusion protein (e.g., HX009). Correlation between the efficacy and the expression of other tested markers has not been observed. 6.5.3 Example 3: Anti-OX40 ADC effectively treated 0X40 expressing cancers

[00336] Samples of PDXs of a variety of cancers were analyzed by whole transcriptome sequencing (RNAseq) and IHC for 0X40, followed by various statistical analysis. The PDXs with 0X40 expression were subjected to PDX trial in clinical trial style (Guo et al. (2019) BMC Cancer 19, 718; Li et al. (2017) Pharmacol Ther 173, 34-46) with the anti-OX40 ADC HX111. 6-8 weeks old female SCID immunodeficient mice were inoculated with various of 0X40 expressing PDX tumor chunks in the flank region for tumor development. The mice were treated with either vehicle control or HX111 at 5 mg / kg weekly once their mean tumor size reached approximately 150 mm3. Tumor growth inhibition (TGI) was calculated by measuring tumor volume biweekly and the TGI values were statistically analyzed against the 0X40 expression.

[00337] A strong correlation between 0X40 expression and tumor inhibition by anti-OX40 ADC was observed, while In vitro data showed that HX111 didn’t have anti-tumor activities in 0X40-tumors. FIGs.3A-3E provide exemplary results of tumor inhibition by HX111 treatment in PDX models of various types of cancers with high 0X40 expression (shown in Tables 2-4 above). As shown in, HX111 showed robust activities in tumor growth inhibition in B-lymphoma PDX models LY6698 (FIG.3A; 2 of 5 CR) and LY2219 (FIG.3B; 4 of 5 CR), T-lymphoma PDX model LY9596 (FIG.3C), head and neck cancer PDX model HN9285 (FIG.3D), and sarcoma PDX model SA12961 (FIG.3E).

[00338] Furthermore, a correlation was observed between 0X40 mRNA expression and the PD-L1 mRNA expression in lymphoma PDX (FIG.2), demonstrating that 0X40 expression could predict the response to PD1 / PD-L1 blockade. 6.5.4 Example 4: Additional anti-OX40 antibodies and ADCs effectively killed 0X40-expressing cancer cells

[00339] In vitro antigen binding:

[00340] The ELISA binding assay was conducted for HX518, HX518-MMAE, HX523-1, HX523-1-MMAE, HX534, HX534-MMAE, HX543, and HX543-MMAE.

[00341] Results: As shown in FIG.4 and the table below, all tested anti-OX40 antibodies and anti-0X40 ADCs showed comparable antigen binding affinities and ECso values. Coating 0.5 pg / mL OX40-his Sampling (nM) HX011 HX518 HX518-MMAE HX523-1 HX523-1-MMAE HX534 HX534-MMAE HX543 HX543-MMAE 20 2.937 2.810 2.733 2.802 2.824 2.900 2.826 2.795 2.805 4 2.910 2.753 2.683 2.694 2.760 2.822 2.759 2.758 2.866 0.8 2.911 2.704 2.621 2.672 2.702 2.766 2.706 2.710 2.803 0.16 2.561 2.341 2.095 2.329 2.287 2.475 2.321 2.207 2.228 0.032 1.331 1.135 0.869 1.183 1.039 1.249 1.036 0.951 0.911 0.0064 0.395 0.331 0.246 0.350 0.308 0.360 0.293 0.270 0.263 0.00128 0.125 0.111 0.087 0.113 0.104 0.115 0.100 0.094 0.094 0.000256 0.064 0.059 0.055 0.059 0.058 0.061 0.056 0.056 0.056 Secondary antibody HRP-Goat Anti-Human, 10000X coloring 10 min EC50 nM 0.038 0.045 0.062 0.042 0.051 0.040 0.050 0.057 0.061

[00342] Internalization:

[00343] A sufficient volume of 4X working solution of antibody was prepared in cell culture medium, and a 4X working solution of Zenon™ pHrodo™ iFL IgG Labeling Reagent was also prepared. 25 pL 4X Zenon™ working solution was combined with 25 pL of HX011 and incubated for 5 minutes at room temperature to allow the labeling complexes to form. HuT-102 cells were plated at IxlO5 cells / well in 50 pL culture medium, and 50 pL of the labeling complex was added to each well. The cells were incubated with the labeling complex for 24 hours under standard cell culture conditions. Flow cytometry was used to analyze the cells.

[00344] Results: As shown in FIG.5, HX011, HX111, HX518, HX518-MMAE, HX523-1, HX523-1-MMAE, HX534, HX534-MMAE, HX543, and HX543-MMAE all showed significant internalization in HuT-102 cells compared to blank control, confirming that the conjugation had little impact on endocytosis kinetics of the naked antibodies.

[00345] In vitro cell killing:

[00346] HuT-102 cells were plated at IxlO3 cells / well. 100 pL antibody or ADC was added into the cells and incubated at 37°C for 6 days. 50 pl CellCounting-Lite2.0 reagent was in each well. Contents were mixed for 3 minutes on an orbital shaker to induce cell lysis. The plate was incubated at room temperature for 10 minutes to stabilize luminescent signal. Luminescence was recorded on the 2104 EnVision plate reader.

[00347] Results: As shown in FIG.6, ADCs (HX111, HX518-MMAE, HX523-1-MMAE, HX534-MMAE, and HX543-MMAE) showed significant killing activity against OX40-expressing HuT-102 cells.

[00348] HX01 l-SN-38, HX011-Exd, and HX011-Eriblin are also expected to exhibit efficient killing activity when subjected to same study. * * *

[00349] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[00350] Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims. 6.6 References

[00351] The following publications are hereby incorporated by reference in their entireties: 1. Kraehenbuehl, L., et al., Enhancing immunotherapy in cancer by targeting emerging immunomodulatory pathways. Nat Rev Clin Oncol, 2022. 19(1): p. 37-50. 2. Duhen, R., et al., Neoadjuvant anti-OX40 (MEDI6469) therapy in patients with head and neck squamous cell carcinoma activates and expands antigen-specific tumor-infiltrating T cells. Nat Commun, 2021. 12(1): p. 1047. 3. Jiang, B., et al., BGB-A445, a novel non-ligand-blocking agonistic anti-OX40 antibody, exhibits superior immune activation and antitumor effects in preclinical models. Front Med, 2023. 4. Postel-Vinay, S., et al., First-in-human phase I study of the OX40 agonist GSK3174998 with or without pembrolizumab in patients with selected advanced solid tumors (ENGAGE-1). J Immunother Cancer, 2023. 11(3). 5. Ke, H., Zhang, F., Wang, J., Xiong, L., An, X., Tu, X., Chen, C., Wang, Y., Mao, B., Guo, S., et al. (2023). HX009, a novel BsAb dual targeting PD1 x CD47, demonstrates potent antilymphoma activity in preclinical models. Sci Rep 13, 5419. 10.1038 / s41598-023-32547-y. 6. Roohullah, A., et al., (2021). First-in-human phase I dose escalation study of HX009, a novel recombinant humanized anti-CD47 / PD-l bispecific antibody, in patients with advanced malignancies. https: / / ascopubs.org / doi / abs / 10.1200 / JCO.2021.39.15_suppl.2517. 7. Lu, Y., Li, Y., Yu, J., Meng, S., Bi, C., Guan, Q., Li, L., Qiu, L., Qian, Z., Zhou, S., et al. (2023). 0X40 shapes an inflamed tumor immune microenvironment and predicts response to immunochemotherapy in diffuse large B-cell lymphoma. Clin Immunol 251, 109637. 10.1016 / j. dim. 2023.109637. 8. Guo, S., Jiang, X., Mao, B., and Li, Q.X. (2019). The design, analysis and application of mouse clinical trials in oncology drug development. BMC Cancer 19, 718. 10.1186 / s 12885-0195907-7. 9. Li, Q.X., Feuer, G., Ouyang, X., and An, X. (2017). Experimental animal modeling for immuno-oncology. Pharmacol Ther 173, 34-46. 10.1016 / j.pharmthera.2017.02.002.

[00352] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

Claims

1. A method of treating an 0X40 expressing cancer in a subject in need thereof, comprising administering a therapeutically effective amount of an immunotherapeutic agent to the subject.

2. The method of claim 1, wherein the cancer is a hematological cancer or a solid tumor.

3. The method of claim 2, wherein the cancer is a hematological cancer selected from the groupconsisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia (CMML), T cell acute lymphoblastic leukemia (T-ALL), natural killer cell leukemia (NK leukemia), Diffuse Large B-cell lymphoma (DLBCL), T cell lymphoblastic lymphoma, cutaneous T-Cell lymphoma (CTCL), peripheral T-cell lymphoma (PTCL), adult T-cell leukemia / lymphoma (ATLL), angioimmunoblastic T-cell lymphoma and natural killer cell / T cell lymphoma (NK / T cell lymphoma).

4. The method of claim 2, wherein the cancer is a solid tumor selected from the group consisting of sarcoma, small cell lung cancer, non-small cell lung cancer, gastric cancer, liver cancer, melanoma, colorectal cancer, squamous cell carcinoma, endometrial cancer, breast cancer, malignant epithelioid mesothelioma, gallbladder cancer, pancreatic cancer, glioblastoma, ovarian cancer, and gastroesophageal junction adenocarcinoma.

5. The method of any one of claims 1 to 4, wherein the cancer is at an advanced stage or metastatic.

6. The method of any one of claims 1 to 5, wherein the cancer is EBV positive, HPV positive, HBV positive, HIV positive, or HTLV-1 positive.

7. The method of any one of claims 1 to 6, wherein the immunotherapeutic agent targets PD1, PD-L1, 0X40, CD47, CTLA-4, 4-1BBL (CD137L), 4-1BB (CD137), LAG-3, or TIGIT.

8. The method of claim 7, wherein the immunotherapeutic agent targets PD-1.

9. The method of claim 7, wherein the immunotherapeutic agent targets 0X40.

10. The method of claim 7, wherein the immunotherapeutic agent targets CD47.

11. The method of claim 7, wherein the immunotherapeutic agent targets CD137.

12. The method of claim 7, wherein the immunotherapeutic agent targets CTLA-4.

13. The method of claim 7, wherein the immunotherapeutic agent comprises pembrolizumab, nivolumab, atezolizumab, durvalumab, cemiplimab, ipilimumab, tislelizumab, bempegaldesleukin, spartalizumab, sintilimab, toripalimab, envafolimab, tremelimumab, magrolimab, istiratumab, mavolimab, MGA012, BMS-986218, MK-1308, SHR-1210, GSK3359609, LY3415244, CA-170, Hu5F9-G4, TTI-621, AO-176, SRF231, CC-90002, IBI188, GSK3174998, BMS-986178, KHK4083, PF-04518600, or INCMGA00012, cinrebafusp alfa, RG7827, ADG106, NBRX-105, CTX-471, Genl046, MCLA-145, RG6076, MP0310, Genl042, AGEN2373, LVGN6051, ATOR-1017, STA551, ND-021, emfizatamab, DSP107, FS120, FS222, HOT-1030, ABL503, IBI319, GNC-039, EU101, CB307, ABL111, GNC-035, PRS-344, BI 765179, QL301, ATG-101, BT7480, PM1003, YH004, LBL-024, PM1032, HLX35 / BNA035, HBM7008, ABL105, BGB-B167, ADG206 or PE0116.

14. The method of claim 7, wherein the immunotherapeutic agent comprises a bispecific antibody targeting CD47 and CTLA4 comprising a first peptide chain, a second peptide chain and a third peptide chain, having the amino acid sequences of SEQ ID NOs: 1, 2 and 3, respectively.

15. The method of claim 7, wherein the immunotherapeutic agent comprises an anti-OX40 antibody comprising a light chain variable region (VL) and heavy chain variable region (VH) having the amino acid sequences of (1) SEQ ID N0s:4 and 5, respectively; (2) SEQ ID NOs: 38 and 39, respectively; (3) SEQ ID NOs: 38 and 42, respectively; (4) SEQ ID NOs: 44 and 45, respectively; or (5) SEQ ID NOs: 49 and 50, respectively.

16. The method of claim 15, wherein the immunotherapeutic agent is an antibody-drug conjugate (“ADC”) having the anti-OX40 antibody conjugated to a cytotoxic agent.

17. The method of claim 16, wherein the cytotoxic agent is dolastatin 10 or a derivative thereof (e.g., monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF)), Exatecan or a derivative thereof (e.g, Dxd), SN-38, or halichondrin B or a derivative thereof (e.g, Eribulin).

18. The method of claim 7, wherein the immunotherapeutic agent is a fusion protein targeting CD47 and PD1 comprising a first peptide chain and a second peptide chain, having the amino acid sequences of SEQ ID NOs: 10 and 11, respectively.

19. The method of any one of claims 1 to 18, comprising determining 0X40 expression in a sample from the subject.

20. The method of claim 19, wherein the 0X40 expression in the sample is determined to be detectable or higher than a reference level.

21. The method of claim 19 or 20, wherein the 0X40 expression is determined at protein level.

22. The method of claim 21, wherein the 0X40 expression is measured byimmunohistochemistry (IHC), immunocytochemistry (ICC), an enzyme-linked immunosorbent assay (ELISA), immunoblotting assay (e.g., Western blot), flow cytometry (FACS), a fluorescent immunosorbent assay (FIA), a chemiluminescence immunoassay (CIA), a radioimmunoassay (RIA), a solid phase radioimmunoassay (SPROA), or a dot / line-immunoblot assay.

23. The method of claim 22, wherein the 0X40 expression is measured by IHC.

24. The method of claim 19 or 20, wherein the 0X40 expression is determined at mRNA level.

25. The method of claim 24, wherein the 0X40 expression is measured by RNA-Seq, in situRNA hybridization (e.g., fluorescence in situ hybridization, or FISH), quantitative polymerase chain reaction (qPCR), real-time polymerase chain reaction (RT-PCR), microarray analysis, serial analysis of gene expression (SAGE), MassARRAY, next generation sequencing (NGS), or single cell whole-exome sequencing (scWES).

26. The method of claim 25, wherein the 0X40 expression is measured by RNA-Seq, or qPCR.

27. The method of any one of claims 19 to 26, wherein the sample is a tissue biopsy or tumorbiopsy.

28. The method of any one of claims 19 to 26, wherein the sample is a blood sample, a serum sample, or a bone marrow sample.

29. The method of any one of claims 19 to 26, wherein the sample is patient-derived xenograft (PDX) sample.

30. The method of any one of claims 19 to 29, further comprising obtaining the sample from the subject.

31. A method of predicting responsiveness of a subject having cancer to an immunotherapeuticagent, comprising measuring 0X40 expression in a sample from the subject, wherein the subject is likely responsive to the immunotherapeutic agent if the cancer is an 0X40 expressing cancer.

32. A method of selecting a subject having cancer for treatment with an immunotherapeutic agent, comprising measuring the 0X40 expression in a sample from the subject and selecting the subject for the treatment if the subject has an 0X40 expressing cancer.

33. The method of claim 31 or 32, wherein the cancer is a hematological cancer or a solid tumor.

34. The method of claim 33, wherein the cancer is a hematological cancer selected from thegroup consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia (CMML), T cell acute lymphoblastic leukemia (T-ALL), natural killer cell leukemia (NK leukemia), Diffuse Large B-cell lymphoma (DLBCL), T cell lymphoblastic lymphoma, cutaneous T-Cell lymphoma (CTCL), peripheral T-cell lymphoma (PTCL), adult T-cell leukemia / lymphoma (ATLL), angioimmunoblastic T-cell lymphoma and natural killer cell / T cell lymphoma (NK / T cell lymphoma).

35. The method of claim 33, wherein the cancer is a solid tumor selected from the group consisting of sarcoma, small cell lung cancer, non-small cell lung cancer, gastric cancer, liver cancer, melanoma, colorectal cancer, squamous cell carcinoma, endometrial cancer, breast cancer, malignant epithelioid mesothelioma, gallbladder cancer, pancreatic cancer, glioblastoma, ovarian cancer, and gastroesophageal junction adenocarcinoma.,36. The method of any one of claims 31 to 35, wherein the cancer is at an advanced stage or metastatic.

37. The method of any one of claims 31 to 36, wherein the cancer is EBV positive, HPV positive, HBV positive, HIV positive, or HTLV-1 positive.

38. The method of any one of claims 31 to 37, wherein the immunotherapeutic agent targets PD1, PD-L1, 0X40, CD47, CTLA-4, 4-1BBL (CD137L), 4-1BB (CD137), LAG-3, or TIGIT.

39. The method of claim 38, wherein the immunotherapeutic agent targets PD-1.

40. The method of claim 38, wherein the immunotherapeutic agent targets 0X40.

41. The method of claim 38, wherein the immunotherapeutic agent targets CD47.

42. The method of claim 38, wherein the immunotherapeutic agent targets CD137.

43. The method of claim 38, wherein the immunotherapeutic agent targets CTLA-4.

44. The method of claim 38, wherein the immunotherapeutic agent comprises pembrolizumab, nivolumab, atezolizumab, durvalumab, cemiplimab, ipilimumab, tislelizumab, bempegaldesleukin, spartalizumab, sintilimab, toripalimab, envafolimab, tremelimumab, magrolimab, istiratumab, mavolimab, MGA012, BMS-986218, MK-1308, SHR-1210, GSK3359609, LY3415244, CA-170, Hu5F9-G4, TTI-621, AO-176, SRF231, CC-90002, IBI188, GSK3174998, BMS-986178, KHK4083, PF-04518600, INCMGA00012, cinrebafusp alfa, RG7827, ADG106, NBRX-105, CTX-471, Genl046, MCLA-145, RG6076, MP0310, Genl042, AGEN2373, LVGN6051, ATOR-1017, STA551, ND-021, emfizatamab, DSP107, FS120, FS222, HOT-1030, ABL503, IBI319, GNC-039, EU101, CB307, ABL111, GNC-035, PRS-344, BI 765179, QL301, ATG-101, BT7480, PM1003, YH004, LBL-024, PM1032, HLX35 / BNA035, HBM7008, ABL105, BGB-B167, ADG206 or PE0116.

45. The method of claim 38, wherein the immunotherapeutic agent comprises a bispecific antibody targeting CD47 and CTLA4 comprising a first peptide chain, a second peptide chain and a third peptide chain, having the amino acid sequences of SEQ ID NOs: 1, 2 and 3, respectively.

46. The method of claim 38, wherein the immunotherapeutic agent comprises an anti-OX40 antibody comprising a light chain variable region (VL) and heavy chain variable region (VH) having the amino acid sequences of (1) SEQ ID N0s:4 and 5, respectively; (2) SEQ ID NOs: 38 and 39, respectively; (3) SEQ ID NOs: 38 and 42, respectively; (4) SEQ ID NOs: 44 and 45, respectively; or (5) SEQ ID NOs: 49 and 50, respectively.

47. The method of claim 46, wherein the immunotherapeutic agent is an ADC having the anti-0X40 antibody conjugated to a cytotoxic agent.

48. The method of claim 47, wherein the cytotoxic agent is dolastatin 10 or a derivative thereof (e.g., monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF)), Exatecan or a derivative thereof (e.g, Dxd), SN-38, or halichondrin B or a derivative thereof (e.g, Eribulin).

49. The method of claim 38, wherein the immunotherapeutic agent is a fusion protein targeting CD47 and PD1 comprising a first peptide chain and a second peptide chain, having the amino acid sequences of SEQ ID NOs: 10 and 11, respectively.

50. The method of any one of claims 31 to 49, wherein the cancer is determined to be 0X40 expressing cancer if the 0X40 expression in the sample is detectable or higher than a reference level.

51. The method of claim 50, wherein the 0X40 expression is determined at protein level.

52. The method of claim 51, wherein the 0X40 expression is measured byimmunohistochemistry (IHC), immunocytochemistry (ICC), an enzyme-linked immunosorbent assay (ELISA), immunoblotting assay (e.g., Western blot), flow cytometry (FACS), a fluorescent immunosorbent assay (FIA), a chemiluminescence immunoassay (CIA), a radioimmunoassay (RIA), a solid phase radioimmunoassay (SPROA), or a dot / line-immunoblot assay.

53. The method of claim 52, wherein the 0X40 expression is measured by IHC.

54. The method of claim 50, wherein the 0X40 expression is determined at mRNA level.

55. The method of claim 54, wherein the 0X40 expression is measured by RNA-Seq, in situRNA hybridization (e.g., fluorescence in situ hybridization, or FISH), quantitative polymerase chain reaction (qPCR), real-time polymerase chain reaction (RT-PCR), microarray analysis, serial analysis of gene expression (SAGE), MassARRAY, next generation sequencing (NGS), or single cell whole-exome sequencing (scWES).

56. The method of claim 55, wherein the 0X40 expression is measured by RNA-Seq or qPCR.

57. The method of any one of claims 31 to 56, wherein the sample is a tissue biopsy or tumorbiopsy.

58. The method of any one of claims 31 to 56, wherein the sample is a blood sample, a serum sample, or a bone marrow sample.

59. The method of any one of claims 31 to 56, wherein the sample is a PDX sample.

60. The method of any one of claims 31 to 59, further comprising obtaining the sample from thesubject.

61. A kit for predicting the responsiveness of a subject having cancer to treatment with an immunotherapeutic agent, comprising a means for measuring the expression of 0X40 in a sample of the subject, and an ancillary reagent.

62. The kit of claim 61, wherein the means for measuring the expression of 0X40 comprises an anti-OX40 antibody.

63. The kit of claim 61, wherein the means for measuring the expression of 0X40 comprises a nucleic acid probe for detecting the 0X40 mRNA.

64. The kit of any one of claims 61 to 63, wherein the ancillary reagent comprises a reaction buffer, a dilution buffer, or a wash buffer, or any combination thereof.

65. The kit of any one of claims 61 to 64, wherein said kit further comprises a solid support.

66. The kit of any one of claims 61 to 65, wherein said kit further comprises a container forsample collection.

67. The kit of any one of claims 61 to 66, wherein the cancer is a hematological cancer or a solid tumor.

68. The kit of claim 67, wherein the cancer is a hematological cancer selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia (CMML), T cell acute lymphoblastic leukemia (T-ALL), natural killer cell leukemia (NK leukemia), Diffuse Large B-cell lymphoma (DLBCL), T cell lymphoblastic lymphoma, cutaneous T-Cell lymphoma (CTCL), peripheral T-cell lymphoma (PTCL), adult T-cell leukemia / lymphoma (ATLL), angioimmunoblastic T-cell lymphoma and natural killer cell / T cell lymphoma (NK / T cell lymphoma).

69. The kit of claim 67, wherein the cancer is a solid tumor selected from the group consisting of sarcoma, small cell lung cancer, non-small cell lung cancer, gastric cancer, liver cancer, melanoma, colorectal cancer, squamous cell carcinoma, endometrial cancer, breast cancer, malignant epithelioid mesothelioma, gallbladder cancer, pancreatic cancer, glioblastoma, ovarian cancer, and gastroesophageal junction adenocarcinoma.,70. The kit of any one of claims 61 to 69, wherein the cancer is at an advanced stage or metastatic.

71. The kit of any one of claims 61 to 70, wherein the cancer is EBV positive, HPV positive, HBV positive, HIV positive, or HTLV-1 positive.

72. The kit of any one of claims 61 to 71, wherein the immunotherapeutic agent targets PD1, PD-Ll, 0X40, CD47, CTLA-4, 4-1BBL (CD137L), 4-1BB (CD137), LAG-3, or TIGIT.

73. The kit of claim 72, wherein the immunotherapeutic agent targets PD-1.

74. The kit of claim 72, wherein the immunotherapeutic agent targets 0X40.

75. The kit of claim 72, wherein the immunotherapeutic agent targets CD47.

76. The kit of claim 72, wherein the immunotherapeutic agent targets CD137.

77. The kit of claim 72, wherein the immunotherapeutic agent targets CTLA-4.

78. The kit of claim 72, wherein the immunotherapeutic agent comprises pembrolizumab, nivolumab, atezolizumab, durvalumab, cemiplimab, ipilimumab, tislelizumab, bempegaldesleukin, spartalizumab, sintilimab, toripalimab, envafolimab, tremelimumab, magrolimab, istiratumab, mavolimab, MGA012, BMS-986218, MK-1308, SHR-1210, GSK3359609, LY3415244, CA-170, Hu5F9-G4, TTI-621, AO-176, SRF231, CC-90002, IBI188, GSK3174998, BMS-986178, KHK4083, PF-04518600, INCMGA00012, cinrebafusp alfa, RG7827, ADG106, NBRX-105, CTX-471, Genl046, MCLA-145, RG6076, MP0310, Genl042, AGEN2373, LVGN6051, ATOR-1017, STA551, ND-021, emfizatamab, DSP107, FS120, FS222, HOT-1030, ABL503, IBI319, GNC-039, EU101, CB307, ABL111, GNC-035, PRS-344, BI 765179, QL301, ATG-101, BT7480, PM1003, YH004, LBL-024, PM1032, HLX35 / BNA035, HBM7008, ABL105, BGB-B167, ADG206 or PE0116.

79. The kit of claim 72, wherein the immunotherapeutic agent comprises a bispecific antibody targeting CD47 and CTLA4 comprising a first peptide chain, a second peptide chain and a third peptide chain, having the amino acid sequences of SEQ ID NOs: 1, 2 and 3, respectively.

80. The kit of claim 72, wherein the immunotherapeutic agent comprises an anti-OX40 antibody comprising a light chain variable region (VL) and heavy chain variable region (VH) having the amino acid sequences of (1) SEQ ID NOs:4 and 5, respectively; (2) SEQ ID NOs: 38 and 39, respectively; (3) SEQ ID NOs: 38 and 42, respectively; (4) SEQ ID NOs: 44 and 45, respectively; or (5) SEQ ID NOs: 49 and 50, respectively.

81. The kit of claim 80, wherein the immunotherapeutic agent is an ADC having the anti-OX40 antibody conjugated to a cytotoxic agent.

82. The kit of claim 81, wherein the cytotoxic agent is dolastatin 10 or a derivative thereof (e.g., monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF)), Exatecan or a derivative thereof (e.g, Dxd), SN-38, or halichondrin B or a derivative thereof (e.g., Eribulin).

83. The kit of claim 72, wherein the immunotherapeutic agent is a fusion protein targeting CD47 and PD1 comprising a first peptide chain and a second peptide chain, having the amino acid sequences of SEQ ID NOs:10 and 11, respectively.

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