A method of treating tumors using a combination of IL-7 protein and immune checkpoint inhibitors.

Abstract

To provide a method of treating a tumor.SOLUTION: A method of treating a tumor of a human subject who needs treatment of the tumor includes administering an effective amount of interleukin-7(IL-7) protein in combination with an effective amount of Programmed Death-1(PD-1) pathway inhibitor to the subject, where a tumor volume in the subject after the administration is reduced compared to a reference tumor volume after administration of either the PD-1 pathway inhibitor alone or IL-7 protein alone.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Cross-reference of related applications This PCT application claims the benefit of priority under U.S. Provisional Application No. 62 / 768,355 filed November 16, 2018, No. 62 / 826,734 filed March 29, 2019, and No. 62 / 896,484 filed September 5, 2019, each of which is incorporated herein by reference as a whole.

[0002] Reference to electronically submitted sequence listings via EFS-WEB The contents of the sequence listing (name: 4241_002PC03_SequenceListing_ST25.txt, size: 78,087 bytes, creation date: November 14, 2019), submitted electronically as an ASCII text file with this application, are incorporated herein by reference in their entirety. [Background technology]

[0003] Human cancers possess numerous genetic and epigenetic alterations, generating nascent antigens that can be recognized by the immune system. (Sjoblom et al., Science) 314:268-74 (2006). The adaptive immune system, composed of T lymphocytes and B lymphocytes, possesses potent anti-cancer potential, broad ability to respond to diverse tumor antigens, and excellent specificity. Furthermore, the immune system exhibits remarkable flexibility and memory. If all these characteristics of the adaptive immune system can be utilized, immunotherapy will become unique among all forms of cancer treatment.

[0004] Immunotherapy for cancer has become well-established in recent years and is now one of the most successful treatment options available to many cancer patients. Scott, AM, et al., Cancer Immun 12:14 (2012). Antibodies can not only target antigens involved in the proliferation and survival of cancer cells, but can also activate or antagonize immunological pathways that are important for immune surveillance of cancer. Furthermore, intensive efforts have led to the successful development of several immune checkpoint pathway inhibitors, some of which have been approved by the U.S. Food and Drug Administration, such as the anti-CTLA-4 antibody ipilimumab (YERVOY®), the anti-PD-1 antibodies nivolumab (OPDIVO®) and pembrolizumab (KEYTRUDA®), and the anti-PD-L1 antibodies atezolizumab (TECENTRIQ®), durvalumab (IMFINZI®), and avelumab (BAVENCIO®). Despite these advances, patients with certain malignancies (e.g., metastatic or refractory solid tumors) still have a very poor prognosis. Only a small fraction of these patients actually experience long-term remission of cancer; many either do not respond to antibodies or, even if they initially respond, eventually develop resistance. Sharma, P., et al., Cell 168(4):707-723 (2017). Furthermore, many cancer treatments using available standard therapies (e.g., chemotherapy and radiotherapy) are known to cause lymphopenia, and many cancer patients are lymphopenic. Grossman, SA, et al., J Natl Compr Canc Netw 13(10):1225-31(2015). Checkpoint inhibitors such as anti-PD-1 antibodies have been shown to have limited efficacy in such cancer patients. Yarchoan, M., et al., J Clin Oncol 35:e14512(2017). Therefore, there is still a need for new treatment options with an acceptable safety profile and high efficacy in cancer patients, including those with lymphopenia. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Sjoblom et al.,Science 314:268-74(2006) [Non-Patent Document 2] Scott, AM, et al., Cancer Immun 12:14(2012) [Non-Patent Document 3] Sharma,P.,et al.,Cell 168(4):707-723(2017) [Non-Patent Document 4] Grossman, SA, et al., J Natl Compr Canc Netw 13(10):1225-31(2015) [Non-Patent Document 5] Yarchoan, M., et al., J Clin Oncol 35:e14512(2017) [Overview of the project]

[0006] This specification provides a method for treating a tumor in a human subject requiring treatment, comprising administering an effective amount of interleukin-7 (IL-7) protein in combination with an effective amount of a Programmed Death-1 (PD-1) pathway inhibitor to the subject, wherein the tumor volume in the subject after administration is reduced compared to a reference tumor volume after administration of either the PD-1 pathway inhibitor alone or the IL-7 protein alone. In some embodiments, the tumor volume is reduced after administration by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%.

[0007] In some embodiments, the methods of the present disclosure increase the number of tumor-infiltrating lymphocytes (TILs) in a tumor after administration compared to the number of TILs in a tumor after administration of either a PD-1 pathway inhibitor alone or IL-7 protein alone. In certain embodiments, TILs are CD4 + In another embodiment, TIL is CD8 + These are TILs. In some embodiments, the number of TILs increases after administration by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 125%, at least about 150%, at least about 200%, at least about 250%, or at least about 300%.

[0008] In some embodiments, human subjects exhibit lymphopenia prior to administration (i.e., as described herein).

[0009] Furthermore, this specification provides a method for treating a tumor in a subject requiring treatment, comprising administering to the subject an effective amount of interleukin-7 (IL-7) protein in combination with an effective amount of a programmed death-1 (PD-1) pathway inhibitor, wherein the subject exhibits lymphopenia.

[0010] In some embodiments, human subjects exhibiting lymphopenia have T lymphopenia, B lymphopenia, and / or NK lymphopenia. In some embodiments, lymphopenia is caused by or associated with a tumor. In certain embodiments, lymphopenia is caused by or associated with past therapy for a tumor. In further embodiments, lymphopenia is caused by infection, chronic right ventricular failure, Hodgkin's disease and lymphoid cancers, leukemia, thoracic duct leakage or rupture, side effects of prescription drugs including anticancer agents (e.g., chemotherapy), antiviral agents, and glucocorticoids, malnutrition resulting from a low-protein diet, radiotherapy, uremia, autoimmune disorders, immunodeficiency syndromes, high stress levels, trauma, thymectomy, or a combination thereof. In certain embodiments, lymphopenia is idiopathic. In certain embodiments, lymphopenia includes idiopathic CD4-positive T lymphopenia (ICL), acute radiation syndrome (ARS), or a combination thereof.

[0011] In some aspects, lymphopenia is characterized by a circulating total lymphocyte count that is at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% lower than the circulating total lymphocyte count of a corresponding subject not exhibiting lymphopenia. In certain aspects, lymphopenia is characterized by a circulating total lymphocyte count of less than about 1,500 lymphocytes per μL, less than about 1,000 lymphocytes per μL, less than about 800 lymphocytes per μL, less than about 500 lymphocytes per μL, or less than about 200 lymphocytes per μL.

[0012] In some embodiments, the number of tumor-infiltrating lymphocytes (TILs) in tumors exhibiting lymphopenia increases after administration compared to the number of TILs in tumors after administration of either a PD-1 pathway inhibitor alone or IL-7 protein alone. In certain embodiments, the number of TILs increases after administration by at least approximately 10%, at least approximately 20%, at least approximately 30%, at least approximately 40%, at least approximately 50%, at least approximately 60%, at least approximately 70%, at least approximately 80%, at least approximately 90%, at least approximately 100%, at least approximately 125%, at least approximately 150%, at least approximately 200%, at least approximately 250%, or at least approximately 300%. In some embodiments, TILs are CD4 + These are T cells. In some embodiments, TILs are CD8 + These are T cells.

[0013] In some aspects, the IL-7 protein is not wild-type IL-7.

[0014] In some embodiments, the IL-7 protein contains an oligopeptide consisting of 1 to 10 amino acid residues. In certain embodiments, the oligopeptide is selected from the group consisting of methionine, glycine, methionine-methionine, glycine-glycine, methionine-glycine, glycine-methionine-methionine, methionine-methionine-glycine, methionine-glycine-methionine, glycine-methionine-methionine, methionine-glycine-glycine, glycine-glycine-methionine, and glycine-glycine-glycine. In some embodiments, the oligopeptide is methionine-glycine-methionine.

[0015] In some embodiments, the IL-7 protein contains a half-life extension portion. In certain embodiments, the half-life extension portion includes Fc, albumin, albumin-binding polypeptide, Pro / Ala / Ser (PAS), the C-terminal peptide of the β-subunit of human chorionic gonadotropin (CTP), polyethylene glycol (PEG), long-chain unstructured hydrophilic amino acid sequences (XTEN), hydroxyethyl starch (HES), albumin-binding small molecules, or a combination thereof.

[0016] In some embodiments, the half-life extension portion is Fc. In certain embodiments, Fc is a hybrid Fc comprising a hinge region, a CH2 domain, and a CH3 domain, wherein the hinge region comprises a human IgD hinge region, the CD2 domain comprises a portion of the human IgD CH2 domain and a portion of the human IgG4 CH2 domain, and the CH3 domain comprises a portion of the human IgG4 CH3 domain.

[0017] In some embodiments, the IL-7 protein contains an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with SEQ ID NOs. 1-6 and 15-25.

[0018] In some embodiments, the PD-1 pathway inhibitors that can be used in the methods of the present invention include anti-PD-1 antibodies or anti-PD-L1 antibodies. In certain embodiments, the anti-PD-1 antibodies include nivolumab, pembrolizumab, MEDI0608, AMP-224, PDR001, BGB-A317, or any combination thereof. In some embodiments, the anti-PD-L1 antibodies include BMS-936559, MPDL3280A, MEDI4736, MSB0010718C, or any combination thereof.

[0019] In some embodiments, the IL-7 protein and the PD-1 pathway inhibitor are administered simultaneously. In other embodiments, the IL-7 protein and the PD-1 pathway inhibitor are administered sequentially. In certain embodiments, the IL-7 protein is administered to the subject before the administration of the PD-1 pathway inhibitor.

[0020] In some aspects, tumors are derived from cancers including breast cancer, head and neck cancer, uterine cancer, brain cancer, skin cancer, kidney cancer, lung cancer, colorectal cancer, prostate cancer, liver cancer, bladder cancer, renal cancer, pancreatic cancer, thyroid cancer, esophageal cancer, eye cancer, stomach cancer (gastric cancer), gastrointestinal cancer, ovarian cancer, carcinoma, sarcoma, leukemia, lymphoma, myeloma, or combinations thereof. In certain aspects, breast cancer is triple-negative breast cancer (TNBC). In certain aspects, brain cancer is gliablastoma. In some aspects, skin cancer is basal cell carcinoma (BCC), cutaneous squamous cell carcinoma (cSCC), melanoma, Merkel cell carcinoma (MCC), or combinations thereof. In further aspects, head and neck cancer is head and neck squamous cell carcinoma. In some aspects, lung cancer is small cell lung cancer (SCLC). In certain aspects, esophageal cancer is esophagogastric junction cancer. In some aspects, kidney cancer is renal cell carcinoma. In some aspects, liver cancer is hepatocellular carcinoma.

[0021] In some embodiments, the IL-7 protein is administered to the target as a supplement, intramuscular, subcutaneous, eye drops, intravenous, intraperitoneal, intradermal, intraorbital, intracerebral, intracranial, intraspinal, intracardiac, intrathecal, intracisional, intracavitary, intrasacral, intracapsular, or intratumoral.

[0022] In some embodiments, PD-1 pathway inhibitors are administered to the subject as supplemental, intramuscular, subcutaneous, intravenous, or intraperitoneal doses.

[0023] Furthermore, this specification provides a method for treating tumors in a human subject requiring such treatment, comprising administering an effective amount of interleukin-7 (IL-7) protein in combination with an effective amount of a CTLA-4 pathway inhibitor to the human subject. In certain embodiments, tumor volume is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% after administration. In some embodiments, the human subject exhibits lymphopenia prior to administration.

[0024] In some embodiments, the CTLA-4 pathway inhibitor comprises an anti-CTLA-4 antibody. In certain embodiments, the anti-CTLA-4 antibody comprises ipilimumab, tremelimumab (tisilimmab; CP-675,206), AGEN-1884, or a combination thereof.

[0025] In some embodiments, the IL-7 protein and the CTLA-4 pathway inhibitor are administered simultaneously. In other embodiments, the IL-7 protein and the CTLA-4 pathway inhibitor are administered sequentially. In certain embodiments, the IL-7 protein is administered to the subject before the administration of the CTLA-4 pathway inhibitor.

[0026] In some aspects, tumors originate from cancers including breast cancer, head and neck cancer, uterine cancer, brain cancer, skin cancer, kidney cancer, lung cancer, colorectal cancer, prostate cancer, liver cancer, bladder cancer, renal cancer, pancreatic cancer, thyroid cancer, esophageal cancer, eye cancer, stomach cancer (gastric cancer), gastrointestinal cancer, ovarian cancer, carcinoma, sarcoma, leukemia, lymphoma, myeloma, or combination thereof.

[0027] In some embodiments, the IL-7 protein of this disclosure is administered in doses of more than approximately 600 μg / kg, more than approximately 700 μg / kg, more than approximately 800 μg / kg, more than approximately 900 μg / kg, more than approximately 1,000 μg / kg, more than approximately 1,100 μg / kg, more than approximately 1,200 μg / kg, more than approximately 1,300 μg / kg, more than approximately 1,400 μg / kg, more than approximately 1,500 μg / kg, more than approximately 1,600 μg / kg, more than approximately 1,700 μg / kg, more than approximately 1,800 μg / kg, more than approximately 1,900 μg / kg, or more than approximately 2,000 μg / kg.

[0028] In some embodiments, the IL-7 protein is present in concentrations of approximately 610 μg / kg to 1,200 μg / kg, approximately 650 μg / kg to 1,200 μg / kg, approximately 700 μg / kg to 1,200 μg / kg, approximately 750 μg / kg to 1,200 μg / kg, approximately 800 μg / kg to 1,200 μg / kg, approximately 850 μg / kg to 1,200 μg / kg, approximately 900 μg / kg to 1,200 μg / kg, approximately 950 μg / kg to 1,200 μg / kg, approximately 1,000 μg / kg to 1,200 μg / kg, approximately 1,050 μg / kg to 1,200 μg / kg, approximately 1,100 μg / kg to 1,200 μg / kg, and approximately 1,200 μg / kg. μg / kg ~ approx. 2,000 μg / kg, approx. 1,300 μg / kg ~ approx. 2,000 μg / kg, approx. 1,500 μg / kg ~ approx. 2,000 μg / kg, approx. 1, 700μg / kg ~ approx. 2,000μg / kg, approx. 610μg / kg ~ approx. 1,000μg / kg, approx. 650μg / kg ~ approx. 1,000μg / kg, approx. 700μ It is administered in doses of approximately g / kg to 1,000 μg / kg, approximately 750 μg / kg to 1,000 μg / kg, approximately 800 μg / kg to 1,000 μg / kg, approximately 850 μg / kg to 1,000 μg / kg, approximately 900 μg / kg to 1,000 μg / kg, or approximately 950 μg / kg to 1,000 μg / kg.

[0029] In some embodiments, the IL-7 protein is administered in doses of approximately 700 μg / kg to 900 μg / kg, approximately 750 μg / kg to 950 μg / kg, approximately 700 μg / kg to 850 μg / kg, approximately 750 μg / kg to 850 μg / kg, approximately 700 μg / kg to 800 μg / kg, approximately 800 μg / kg to 900 μg / kg, approximately 750 μg / kg to 850 μg / kg, or approximately 850 μg / kg to 950 μg / kg.

[0030] In some embodiments, the IL-7 protein is present in concentrations of approximately 650 μg / kg, 680 μg / kg, 700 μg / kg, 720 μg / kg, 740 μg / kg, 750 μg / kg, 760 μg / kg, 780 μg / kg, 800 μg / kg, 820 μg / kg, 840 μg / kg, 850 μg / kg, 860 μg / kg, 880 μg / kg, 900 μg / kg, 920 μg / kg, 940 μg / kg, and 950 μg. / kg, approximately 960μg / kg, approximately 980μg / kg, approximately 1,000μg / kg, approximately 1,020μg / kg, approximately 1,040μg / kg, approximately 1,060μg / kg, approximately 1,080μg / kg, approximately 1,100μg / kg, approximately 1,2 00μg / kg, approximately 1,220μg / kg, approximately 1,240μg / kg, approximately 1,260μg / kg, approximately 1,280μg / kg, approximately 1,300μg / kg, approximately 1,320μg / kg, approximately 1,340μg / kg, approximately 1,360μg / kg, approximately 1,380μg / kg, approximately 1,400μg / kg, approximately 1,420μg / kg, approximately 1,440μg / kg, approximately 1,460μg / kg, approximately 1,480μg / kg, approximately 1,500μg / kg, approximately 1,520μg / kg, Approximately 1,540μg / kg, approximately 1,560μg / kg, approximately 1,580μg / kg, approximately 1,600μg / kg, approximately 1,620μg / kg, approximately 1,640μg / kg, approximately 1,660μg / kg, approximately 1,680μg / kg, approximately 1,7 It is administered in doses of 00 μg / kg, approximately 1,720 μg / kg, approximately 1,740 μg / kg, approximately 1,760 μg / kg, approximately 1,780 μg / kg, approximately 1,800 μg / kg, approximately 1,820 μg / kg, approximately 1,840 μg / kg, approximately 1,860 μg / kg, approximately 1,880 μg / kg, approximately 1,900 μg / kg, approximately 1,920 μg / kg, approximately 1,940 μg / kg, approximately 1,960 μg / kg, approximately 1,980 μg / kg, or approximately 2,000 μg / kg.

[0031] In some cases, the IL-7 protein is administered at a frequency of once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, once every eleven weeks, or once every twelve weeks.

[0032] In some embodiments, the IL-7 protein is administered supplementally. In some embodiments, the IL-7 protein is administered intravenously.

[0033] In some embodiments, the IL-7 protein, PD-1 pathway inhibitors, and / or CTLA-4 pathway inhibitors are incorporated into compositions comprising bulking agents, stabilizers, surfactants, buffers, or combinations thereof.

[0034] In some embodiments, the PD-1 pathway inhibitor is nivolumab, and the composition comprises (a) mannitol (e.g., about 30 mg), (b) pentetate (e.g., about 0.008 mg), (c) polysorbate 80 (e.g., about 0.2 mg), (d) sodium chloride (e.g., about 2.92 mg), and (e) anhydrous sodium citrate (e.g., about 5.88 mg). In certain embodiments, the PD-1 pathway inhibitor is administered to the subject at a constant dose of about 240 mg every two weeks or about 480 mg every four weeks. In some embodiments, the PD-1 pathway inhibitor is administered to the subject at a weight-based dose of about 3 mg / kg every two weeks.

[0035] In some embodiments, the PD-1 pathway inhibitor is pembrolizumab, and the composition comprises (a) L-histidine (e.g., about 1.55 mg), (b) polysorbate 80 (e.g., about 0.2 mg), and (c) sucrose (e.g., about 70 mg). In certain embodiments, the PD-1 pathway inhibitor is administered to the subject at a constant dose of about 200 mg every three weeks. In further embodiments, the PD-1 pathway inhibitor is administered to the subject at a weight-based dose of about 2 mg / kg every three weeks.

[0036] In some embodiments, the PD-1 pathway inhibitor is atezolizumab, and the composition comprises (a) glacial acetate (e.g., about 16.5 mg), (b) L-histidine (e.g., about 62 mg), (c) sucrose (e.g., about 821.6 mg), and (d) polysorbate 20 (e.g., about 8 mg). In certain embodiments, the PD-1 pathway inhibitor is administered to the subject at a constant dose of about 1200 mg every three weeks.

[0037] In some embodiments, the PD-1 pathway inhibitor is durvalumab, and the composition comprises (a) L-histidine (e.g., about 2 mg), (b) L-histidine hydrochloride monohydrate (e.g., about 2.7 mg), (c) α,α-trehalose dihydrate (e.g., about 104 mg), and (d) polysorbate 80 (e.g., about 0.2 mg). In certain embodiments, the PD-1 pathway inhibitor is administered to the subject at a weight-based dose of about 10 mg / kg every two weeks.

[0038] In some embodiments, the PD-1 pathway inhibitor is avelumab, and the composition comprises (a) D-mannitol (e.g., about 51 mg), (b) glacial acetic acid (e.g., about 0.6 mg), (c) polysorbate 20 (e.g., about 0.5 mg), and (d) sodium hydroxide (e.g., about 0.3 mg). In some embodiments, the PD-1 pathway inhibitor is administered to the subject at a constant dose of about 800 mg every two weeks.

[0039] In some embodiments, the CTLA-4 pathway inhibitor is ipilimumab, and the composition comprises (a) diethylenetriaminepentaacetic acid (DTPA) (e.g., about 0.04 mg), (b) mannitol (e.g., about 10 mg), (c) polysorbate 80 (plant-derived) (e.g., about 0.1 mg), (d) sodium chloride (e.g., about 5.85 mg), and (e) tris hydrochloride (e.g., about 3.15 mg). In certain embodiments, the CTLA-4 pathway inhibitor is administered to subjects at a weight-based dose of about 3 mg / kg every three weeks. In further embodiments, the CTLA-4 pathway inhibitor is administered to subjects at four doses of about 10 mg / kg every three weeks, followed by a weight-based dose of 10 mg / kg every 12 weeks.

[0040] In some embodiments, the IL-7 protein disclosed herein is incorporated into a composition comprising (a) sodium citrate (e.g., about 20 mM), (b) sucrose (e.g., about 5%), (c) sorbitol (e.g., about 1.5%), and (d) Tween80 (e.g., about 0.05%). In certain embodiments, for example, the following are provided: (Item 1) A method for treating a tumor in a human subject requiring treatment of the tumor, comprising administering to the subject an effective amount of interleukin-7 (IL-7) protein in combination with an effective amount of a Programmed Death-1 (PD-1) pathway inhibitor, wherein the tumor volume in the subject after the administration is reduced compared to a reference tumor volume after administration of either the PD-1 pathway inhibitor alone or the IL-7 protein alone. (Item 2) The method according to item 1, wherein the tumor volume is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% after the administration. (Item 3) The method according to item 1 or 2, wherein the number of tumor-infiltrating lymphocytes (TILs) in the tumor increases after the administration compared to the number of TILs in the tumor after administration of either the PD-1 pathway inhibitor alone or the IL-7 protein alone. (Item 4) The aforementioned TIL is CD4 + The method described in item 3, which is TIL. (Item 5) The aforementioned TIL is CD8 + The method described in item 3, which is TIL. (Item 6) The method according to any one of items 3 to 5, wherein the TIL count increases by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 125%, at least about 150%, at least about 200%, at least about 250%, or at least about 300% after the administration. (Item 7) The method according to any one of items 1 to 6, wherein the human subject exhibits lymphopenia before the administration. (Item 8) A method for treating a tumor in a subject requiring treatment of the tumor, comprising administering to the subject an effective amount of interleukin-7 (IL-7) protein in combination with an effective amount of a programmed death-1 (PD-1) pathway inhibitor, wherein the subject exhibits lymphopenia. (Item 9) The method according to item 7 or 8, wherein the human subject exhibiting lymphopenia has T lymphopenia, B lymphopenia, and / or NK lymphopenia. (Item 10) The method according to any one of items 7 to 9, wherein the lymphopenia is caused by or related to the tumor. (Item 11) The method according to any one of items 7 to 10, wherein the lymphopenia is caused by or related to prior treatment for the tumor. (Item 12) The method according to any one of items 7 to 11, wherein the lymphopenia is caused by infection, chronic right ventricular failure, Hodgkin's disease and cancers of the lymphatic system, leukemia, leakage or rupture of the thoracic duct, side effects of prescription drugs including anticancer agents (e.g., chemotherapy), antiviral agents, and glucocorticoids, malnutrition resulting from a low-protein diet, radiotherapy, uremia, autoimmune disorders, immunodeficiency syndromes, high stress levels, trauma, thymectomy, or a combination thereof. (Item 13) The method according to any one of items 7 to 12, wherein the lymphopenia is idiopathic. (Item 14) The method according to any one of items 7 to 13, wherein the lymphopenia includes idiopathic CD4-positive T lymphopenia (ICL), acute radiation syndrome (ARS), or a combination thereof. (Item 15) The method according to any one of items 7 to 14, wherein the lymphopenia is characterized by a total circulating lymphocyte count that is at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% lower than the total circulating lymphocyte count of a corresponding subject that does not exhibit lymphopenia. (Item 16) The method according to any one of items 7 to 15, wherein the lymphopenia is characterized by a total circulating lymphocyte count of less than approximately 1,500 lymphocytes per μL, less than approximately 1,000 lymphocytes per μL, less than approximately 800 lymphocytes per μL, less than approximately 500 lymphocytes per μL, or less than approximately 200 lymphocytes per μL. (Item 17) The method according to any one of items 8 to 16, wherein the number of tumor-infiltrating lymphocytes (TILs) in the tumor increases after the administration compared to the number of TILs in the tumor after administration of either the PD-1 pathway inhibitor alone or the IL-7 protein alone. (Item 18) The method according to item 17, wherein the TIL count increases by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 125%, at least about 150%, at least about 200%, at least about 250%, or at least about 300% after the administration. (Item 19) The aforementioned TIL is CD4 + The method described in item 17 or 18, which is TIL. (Item 20) The aforementioned TIL is CD8 + The method described in item 17 or 18, which is TIL. (Item 21) The method according to any one of items 1 to 20, wherein the IL-7 protein is not wild-type IL-7. (Item 22) The method according to any one of items 1 to 21, wherein the IL-7 protein comprises an oligopeptide consisting of 1 to 10 amino acid residues. (Item 23) The method according to item 22, wherein the oligopeptide is selected from the group consisting of methionine, glycine, methionine-methionine, glycine-glycine, methionine-glycine, glycine-methionine-methionine, methionine-methionine-glycine, methionine-glycine-methionine, glycine-methionine-methionine, methionine-glycine-glycine, glycine-methionine-glycine, glycine-glycine-methionine, and glycine-glycine-glycine. (Item 24) The method according to item 23, wherein the oligopeptide is methionine-glycine-methionine. (Item 25) The method according to any one of items 1 to 24, wherein the IL-7 protein includes a half-life extension portion. (Item 26) The method according to item 25, wherein the half-life extension portion comprises Fc, albumin, albumin-binding polypeptide, Pro / Ala / Ser (PAS), the C-terminal peptide of the β-subunit of human chorionic gonadotropin (CTP), polyethylene glycol (PEG), long-chain unstructured hydrophilic amino acid sequence (XTEN), hydroxyethyl starch (HES), albumin-binding small molecules, or a combination thereof. (Item 27) The method according to item 26, wherein the half-life extension portion is Fc. (Item 28) The Fc is a hybrid Fc comprising a hinge region, a CH2 domain, and a CH3 domain. The aforementioned hinge region includes a human IgD hinge region, The CH2 domain includes a portion of the human IgD CH2 domain and a portion of the human IgG4 CH2 domain. The method according to item 27, wherein the CH3 domain includes a portion of the human IgG4 CH3 domain. (Item 29) The method according to any one of items 1 to 28, wherein the IL-7 protein comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with SEQ ID NOs. 1 to 6 and 15 to 25. (Item 30) The method according to any one of items 1 to 29, wherein the PD-1 pathway inhibitor comprises an anti-PD-1 antibody or an anti-PD-L1 antibody. (Item 31) The method according to item 30, wherein the anti-PD-1 antibody comprises nivolumab, pembrolizumab, MEDI0608, AMP-224, PDR001, BGB-A317, or any combination thereof. (Item 32) The method according to item 31, wherein the anti-PD-L1 antibody comprises BMS-936559, MPDL3280A, MEDI4736, MSB0010718C, or any combination thereof. (Item 33) The method according to any one of items 1 to 32, wherein the IL-7 protein and the PD-1 pathway inhibitor are administered simultaneously. (Item 34) The method according to any one of items 1 to 32, wherein the IL-7 protein and the PD-1 pathway inhibitor are administered sequentially. (Item 35) The method according to item 34, wherein the IL-7 protein is administered to the subject before the administration of the PD-1 pathway inhibitor. (Item 36) The method according to any one of items 1 to 35, wherein the tumor is derived from cancer including breast cancer, head and neck cancer, uterine cancer, brain cancer, skin cancer, kidney cancer, lung cancer, colorectal cancer, prostate cancer, liver cancer, bladder cancer, renal cancer, pancreatic cancer, thyroid cancer, esophageal cancer, eye cancer, stomach cancer (gastric cancer), gastrointestinal cancer, ovarian cancer, carcinoma, sarcoma, leukemia, lymphoma, myeloma, or a combination thereof. (Item 37) The method according to item 36, wherein the breast cancer is triple-negative breast cancer (TNBC). (Item 38) The method according to item 36, wherein the brain cancer is a gliablastoma. (Item 39) The method according to item 36, wherein the skin cancer is basal cell carcinoma (BCC), cutaneous squamous cell carcinoma (cSCC), melanoma, Merkel cell carcinoma (MCC), or a combination thereof. (Item 40) The method according to item 36, wherein the head and neck cancer is head and neck squamous cell carcinoma. (Item 41) The method according to item 36, wherein the lung cancer is small cell lung cancer (SCLC). (Item 42) The method according to item 36, wherein the esophageal cancer is esophagogastric junction cancer. (Item 43) The method according to item 36, wherein the aforementioned kidney cancer is renal cell carcinoma. (Item 44) The method according to item 36, wherein the liver cancer is hepatocellular carcinoma. (Item 45) The method according to any one of items 1 to 44, wherein the IL-7 protein is administered to the subject as a supplemental, intramuscular, subcutaneous, ophthalmic, intravenous, intraperitoneal, intradermal, intraorbital, intracerebral, intracranial, intraspinal, intracardiac, intrathecal, intracisional, intracavitary, intrasacral, intracapsular, or intratumoral. (Item 46) The method according to any one of items 1 to 45, wherein the PD-1 pathway inhibitor is administered to the subject as a supplemental, intramuscular, subcutaneous, intravenous, or intraperitoneal agent. (Item 47) A method for treating a tumor in a human subject requiring treatment of the tumor, comprising administering to the human subject an effective amount of interleukin-7 (IL-7) protein in combination with an effective amount of a CTLA-4 pathway inhibitor. (Item 48) The method according to item 47, wherein the tumor volume is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% after the administration. (Item 49) The method according to item 47 or 48, wherein the human subject exhibits lymphopenia before the administration. (Item 50) The method according to any one of items 47 to 49, wherein the CTLA-4 pathway inhibitor comprises an anti-CTLA-4 antibody. (Item 51) The method according to item 50, wherein the anti-CTLA-4 antibody comprises ipilimumab, tremelimumab (tisilimmab; CP-675,206), AGEN-1884, or a combination thereof. (Item 52) The method according to any one of items 47 to 51, wherein the IL-7 protein and the CTLA-4 pathway inhibitor are administered simultaneously. (Item 53) The method according to any one of items 47 to 51, wherein the IL-7 protein and the CTLA-4 pathway inhibitor are administered sequentially. (Item 54) The method according to item 53, wherein the IL-7 protein is administered to the subject before the administration of the CTLA-4 pathway inhibitor. (Item 55) The method according to any one of items 47 to 54, wherein the tumor is derived from cancer including breast cancer, head and neck cancer, uterine cancer, brain cancer, skin cancer, kidney cancer, lung cancer, colorectal cancer, prostate cancer, liver cancer, bladder cancer, renal cancer, pancreatic cancer, thyroid cancer, esophageal cancer, eye cancer, stomach cancer (gastric cancer), gastrointestinal cancer, ovarian cancer, carcinoma, sarcoma, leukemia, lymphoma, myeloma, or a combination thereof. (Item 56) The method according to any one of items 1 to 55, wherein the IL-7 protein is administered in a dose of more than approximately 600 μg / kg, more than approximately 700 μg / kg, more than approximately 800 μg / kg, more than approximately 900 μg / kg, more than approximately 1,000 μg / kg, more than approximately 1,100 μg / kg, more than approximately 1,200 μg / kg, more than approximately 1,300 μg / kg, more than approximately 1,400 μg / kg, more than approximately 1,500 μg / kg, more than approximately 1,600 μg / kg, more than approximately 1,700 μg / kg, more than approximately 1,800 μg / kg, more than approximately 1,900 μg / kg, or more than approximately 2,000 μg / kg. (Item 57) The IL-7 protein is present in amounts of approximately 610 μg / kg to 1,200 μg / kg, approximately 650 μg / kg to 1,200 μg / kg, approximately 700 μg / kg to 1,200 μg / kg, approximately 750 μg / kg to 1,200 μg / kg, approximately 800 μg / kg to 1,200 μg / kg, and approximately 850 μg / kg to 1,200 μg / kg, approximately 900μg / kg to approximately 1,200μg / kg, approximately 950μg / kg to approximately 1,200μg / kg, approximately 1,000μg / kg to approximately 1,200μg / k g, approximately 1,050μg / kg to approximately 1,200μg / kg, approximately 1,100μg / kg to approximately 1,200μg / kg, approximately 1,200μg / kg to approximately 2,000μg / The method according to any one of items 1 to 56, administered in doses of approximately 1,300 μg / kg to 2,000 μg / kg, approximately 1,500 μg / kg to 2,000 μg / kg, approximately 1,700 μg / kg to 2,000 μg / kg, approximately 610 μg / kg to 1,000 μg / kg, approximately 650 μg / kg to 1,000 μg / kg, approximately 700 μg / kg to 1,000 μg / kg, approximately 750 μg / kg to 1,000 μg / kg, approximately 800 μg / kg to 1,000 μg / kg, approximately 850 μg / kg to 1,000 μg / kg, approximately 900 μg / kg to 1,000 μg / kg, or approximately 950 μg / kg to 1,000 μg / kg. (Item 58) The method according to any one of items 1 to 57, wherein the IL-7 protein is administered in doses of approximately 700 μg / kg to approximately 900 μg / kg, approximately 750 μg / kg to approximately 950 μg / kg, approximately 700 μg / kg to approximately 850 μg / kg, approximately 750 μg / kg to approximately 850 μg / kg, approximately 700 μg / kg to approximately 800 μg / kg, approximately 800 μg / kg to approximately 900 μg / kg, approximately 750 μg / kg to approximately 850 μg / kg, or approximately 850 μg / kg to approximately 950 μg / kg. (Item 59) The aforementioned IL-7 protein is present in approximately 650 μg / kg, approximately 680 μg / kg, approximately 700 μg / kg, approximately 720 μg / kg, approximately 740 μg / kg, approximately 750 μg / kg, approximately 760 μg / kg, approximately 780 μg / kg, approximately 800 μg / kg, approximately 820 μg / kg, approximately 840 μg / kg, approximately 850 μg / kg, approximately 860 μg / kg, approximately 880 μg / kg, approximately 900 μg / kg, approximately 920 μg / kg, approximately 940 μg / kg, approximately 950 μg / kg, approximately 960 μg / kg, approximately 980 μg / kg, approximately 1,000μg / kg, approximately 1,020μg / kg, approximately 1,040μg / kg, approximately 1,060μg / kg, approximately 1,080μg / kg, approximately 1,100μg / kg, approximately 1,120μg / kg, approximately 1,140μg / kg, approximately 1,160μg / kg, Approximately 1,180μg / kg, approximately 1,200μg / kg, approximately 1,220μg / kg, approximately 1,240μg / kg, approximately 1,260μg / kg, approximately 1,280μg / kg, approximately 1,300μg / kg, approximately 1,320μg / kg, approximately 1,340μg / kg , about 1,360μg / kg, about 1,380μg / kg, about 1,400μg / kg, about 1,420μg / kg, about 1,440μg / kg, about 1,460μg / kg, about 1,480μg / kg, about 1,500μg / kg, about 1,520μg / k g, approximately 1,540μg / kg, approximately 1,560μg / kg, approximately 1,580μg / kg, approximately 1,600μg / kg, approximately 1,620μg / kg, approximately 1,640μg / kg, approximately 1,660μg / kg, approximately 1,680μg / kg, approximately 1,700μg / The method according to any one of items 1 to 58, administered in doses of approximately 1,720 μg / kg, approximately 1,740 μg / kg, approximately 1,760 μg / kg, approximately 1,780 μg / kg, approximately 1,800 μg / kg, approximately 1,820 μg / kg, approximately 1,840 μg / kg, approximately 1,860 μg / kg, approximately 1,880 μg / kg, approximately 1,900 μg / kg, approximately 1,920 μg / kg, approximately 1,940 μg / kg, approximately 1,960 μg / kg, approximately 1,980 μg / kg, or approximately 2,000 μg / kg. (Item 60) The method according to any one of items 1 to 59, wherein the IL-7 protein is administered at a dosage frequency of once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, once every eleven weeks, or once every twelve weeks. (Item 61) The method according to any one of items 1 to 60, wherein the IL-7 protein is administered supplementally. (Item 62) The method according to any one of items 1 to 60, wherein the IL-7 protein is administered intravenously. (Item 63) The method according to any one of items 1 to 62, wherein the IL-7 protein, the PD-1 pathway inhibitor, and / or the CTLA-4 pathway inhibitor are incorporated into a composition comprising a bulking agent, a stabilizer, a surfactant, a buffer, or a combination thereof. (Item 64) The method according to item 63, wherein the PD-1 pathway inhibitor is nivolumab, and the composition comprises (a) mannitol (e.g., about 30 mg), (b) pentetic acid (e.g., about 0.008 mg), (c) polysorbate 80 (e.g., about 0.2 mg), (d) sodium chloride (e.g., about 2.92 mg), and (e) anhydrous sodium citrate (e.g., about 5.88 mg). (Item 65) The method according to item 64, wherein the PD-1 pathway inhibitor is administered to the subject at a constant dose of approximately 240 mg every two weeks or approximately 480 mg every four weeks. (Item 66) The method according to item 64, wherein the PD-1 pathway inhibitor is administered to the subject at a weight-based dose of approximately 3 mg / kg every two weeks. (Item 67) The method according to item 63, wherein the PD-1 pathway inhibitor is pembrolizumab, and the composition comprises (a) L-histidine (e.g., about 1.55 mg), (b) polysorbate 80 (e.g., about 0.2 mg), and (c) sucrose (e.g., about 70 mg). (Item 68) The method according to item 67, wherein the PD-1 pathway inhibitor is administered to the subject at a constant dose of approximately 200 mg every three weeks. (Item 69) The method according to item 67, wherein the PD-1 pathway inhibitor is administered to the subject at a weight-based dose of approximately 2 mg / kg every three weeks. (Item 70) The method according to item 63, wherein the PD-1 pathway inhibitor is atezolizumab, and the composition comprises (a) glacial acetic acid (e.g., about 16.5 mg), (b) L-histidine (e.g., about 62 mg), (c) sucrose (e.g., about 821.6 mg), and (d) polysorbate 20 (e.g., about 8 mg). (Item 71) The method according to item 70, wherein the PD-1 pathway inhibitor is administered to the subject at a constant dose of approximately 1200 mg every three weeks. (Item 72) The method according to item 63, wherein the PD-1 pathway inhibitor is durvalumab, and the composition comprises (a) L-histidine (e.g., about 2 mg), (b) L-histidine hydrochloride monohydrate (e.g., about 2.7 mg), (c) α,α-trehalose dihydrate (e.g., about 104 mg), and (d) polysorbate 80 (e.g., about 0.2 mg). (Item 73) The method according to item 72, wherein the PD-1 pathway inhibitor is administered to the subject at a weight-based dose of approximately 10 mg / kg every two weeks. (Item 74) The method according to item 63, wherein the PD-1 pathway inhibitor is avelumab, and the composition comprises (a) D-mannitol (e.g., about 51 mg), (b) glacial acetic acid (e.g., about 0.6 mg), (c) polysorbate 20 (e.g., about 0.5 mg), and (d) sodium hydroxide (e.g., about 0.3 mg). (Item 75) The method according to item 74, wherein the PD-1 pathway inhibitor is administered to the subject at a constant dose of approximately 800 mg every two weeks. (Item 76) The method according to item 63, wherein the CTLA-4 pathway inhibitor is ipilimumab, and the composition comprises (a) diethylenetriaminepentaacetic acid (DTPA) (e.g., about 0.04 mg), (b) mannitol (e.g., about 10 mg), (c) polysorbate 80 (plant-derived) (e.g., about 0.1 mg), (d) sodium chloride (e.g., about 5.85 mg), and (e) tris hydrochloride (e.g., about 3.15 mg). (Item 77) The method according to item 76, wherein the CTLA-4 pathway inhibitor is administered to the subject at a weight-based dose of approximately 3 mg / kg every three weeks. (Item 78) The method according to item 76, wherein the CTLA-4 pathway inhibitor is administered to the subject at four doses of approximately 10 mg / kg every three weeks, followed by a weight-based dose of 10 mg / kg every 12 weeks. (Item 79) The method according to any one of items 63 to 78, wherein the IL-7 protein is incorporated into a composition comprising (a) sodium citrate (e.g., about 20 mM), (b) sucrose (e.g., about 5%), (c) sorbitol (e.g., about 1.5%), and (d) Tween 80 (e.g., about 0.05%). [Brief explanation of the drawing]

[0041] [Figure 1] Figures A, B, and C show the effects of IL-7 protein and anti-PD-1 antibody administration on tumor volume in a mouse adenocarcinoma model. Figure A shows the schedule of tumor dissemination and treatment administration. Figures B and C provide comparisons of tumor volume (mm3) for each treatment group in two different studies, respectively. Treatment groups included (1) IL-7 combined buffer + isotype control antibody (circle), (2) IL-7 combined buffer + anti-PD-1 antibody (triangle), (3) IL-7 protein + isotype control antibody (inverted triangle), and (4) IL-7 protein + anti-PD-1 antibody (diamond). Data are shown as mean ± SEM. All comparisons were performed using two-way ANOVA and Bonferroni post-hoc test. "*" and "***" indicate statistically significant differences compared to control animals (p<0.05 and p<0.0001, respectively). [Figure 2] Figures A, B, and C show the effects of IL-7 protein and anti-PD-1 antibody administration on tumor-infiltrating lymphocyte (TIL) counts in animals in each treatment group. Figure A shows the tumor dissemination and treatment schedule. Figure B presents a comparison of CD4+ TIL counts in each treatment group. Figure C presents a comparison of CD8+ TIL counts in each treatment group. Treatment groups included (1) IL-7 combined buffer + isotype control antibody, (2) IL-7 combined buffer + anti-PD-1 antibody, (3) IL-7 protein + isotype control antibody, and (4) IL-7 protein + anti-PD-1 antibody. In both B and C, CD4+ TIL counts and CD8+ TIL counts are shown as a percentage of the total CD45+ cells in the tumor. Data are shown for individual animals and as mean ± SEM. All comparisons were performed using one-way ANOVA and Tukey's multiple comparison test. "*", "**", and "***" indicate statistically significant differences compared to the control animals (p<0.05, p<0.01, and p<0.0001, respectively). [Figure 3A] Figures A, B, and C show the effects of tripartite combination therapy with cyclophosphamide (CPA), IL-7 protein, and a PD-1 pathway inhibitor on tumor volume and survival in animals in each treatment group. Figure 3A shows the schedule of tumor dissemination and treatment administration. Figure 3B presents a comparison of tumor volume (mm3) in each treatment group at various time points after CPA treatment. Figure 3C presents survival data. The treatment groups included (1) PBS + IL-7 combined buffer + isotype control antibody, (2) CPA + IL-7 combined buffer + isotype control antibody, (3) CPA + IL-7 protein + isotype control antibody, (4) CPA + IL-7 protein + anti-PD-1 antibody, and (5) CPA + IL-7 protein + anti-PD-L1 antibody. In Figure 3B, data are shown as mean ± SEM. Comparisons of each treatment group were performed using two-way ANOVA and Bonferroni post-hoc test. "*" and "***" indicate statistically significant differences compared to the control animals (p<0.05 and p<0.001, respectively). [Figure 3B]Figures A, B, and C show the effects of tripartite combination therapy with cyclophosphamide (CPA), IL-7 protein, and a PD-1 pathway inhibitor on tumor volume and survival in animals in each treatment group. Figure 3A shows the schedule of tumor dissemination and treatment administration. Figure 3B presents a comparison of tumor volume (mm3) in each treatment group at various time points after CPA treatment. Figure 3C presents survival data. The treatment groups included (1) PBS + IL-7 combined buffer + isotype control antibody, (2) CPA + IL-7 combined buffer + isotype control antibody, (3) CPA + IL-7 protein + isotype control antibody, (4) CPA + IL-7 protein + anti-PD-1 antibody, and (5) CPA + IL-7 protein + anti-PD-L1 antibody. In Figure 3B, data are shown as mean ± SEM. Comparisons of each treatment group were performed using two-way ANOVA and Bonferroni post-hoc test. "*" and "***" indicate statistically significant differences compared to the control animals (p<0.05 and p<0.001, respectively). [Figure 3C] Figures A, B, and C show the effects of tripartite combination therapy with cyclophosphamide (CPA), IL-7 protein, and a PD-1 pathway inhibitor on tumor volume and survival in animals in each treatment group. Figure 3A shows the schedule of tumor dissemination and treatment administration. Figure 3B presents a comparison of tumor volume (mm3) in each treatment group at various time points after CPA treatment. Figure 3C presents survival data. The treatment groups included (1) PBS + IL-7 combined buffer + isotype control antibody, (2) CPA + IL-7 combined buffer + isotype control antibody, (3) CPA + IL-7 protein + isotype control antibody, (4) CPA + IL-7 protein + anti-PD-1 antibody, and (5) CPA + IL-7 protein + anti-PD-L1 antibody. In Figure 3B, data are shown as mean ± SEM. Comparisons of each treatment group were performed using two-way ANOVA and Bonferroni post-hoc test. "*" and "***" indicate statistically significant differences compared to the control animals (p<0.05 and p<0.001, respectively). [Figure 4]Figures A and B show the effects of IL-7 protein and anti-PD-1 antibody administration on tumor volume in thymectomized animals. Figure A is the study design. Figure B presents a comparison of tumor volume (mm3) in each treatment group. The treatment groups included (1) IL-7 combined buffer + isotype control antibody (circle), (2) IL-7 protein + isotype control antibody (square), (3) IL-7 combined buffer + anti-PD-1 antibody (triangle), and (4) IL-7 protein + anti-PD-1 antibody (inverted triangle). Arrows indicate the effects of IL-7 protein (gray arrow) and anti-PD-1 antibody (black arrow). Data are shown as mean ± SEM. Comparisons of each treatment group were performed using two-way ANOVA and Bonferroni post-hoc test. "***" indicates a statistically significant difference (p<0.001) compared to the control animals. [Figure 5] Figures A, B, and C show the effects of IL-7 protein and anti-PD-1 antibody administration on tumor-infiltrating lymphocyte (TIL) counts in thymectomized animals. Figure A is the study design. Figures B and C present comparisons of CD4+ TIL counts and CD8+ TIL counts, respectively. Treatment groups included (1) IL-7 combined buffer + isotype control antibody ("control"), (2) IL-7 combined buffer + anti-PD-1 antibody ("α-PD1"), (3) IL-7 protein + isotype control antibody ("IL-7"), and (4) IL-7 protein + anti-PD-1 antibody ("combination"). In both B and C, CD4+ TIL counts and CD8+ TIL counts are shown as a percentage of the total CD45+ cells in the tumor. Data are shown for individual animals and as mean ± SEM. All comparisons were performed using one-way ANOVA and Tukey's multiple comparison test. "*", "**", and "***" indicate statistically significant differences compared to the control animals (p<0.05, p<0.01, and p<0.0001, respectively). [Figure 6A]This study demonstrates the effects of IL-7 protein on cytokine-induced T cell proliferation and activation in normal C57BL / 6 mice. It also shows the dynamics of CD8+ T cell subsets in the blood after treatment with IL-7 protein. The CD8+ T cell subsets shown include (i) all CD8+ T cells (left graph), (ii) CD8+CD44- cells (center graph), and (iii) CD8+CD44+ cells (right graph). The top row shows the number of CD8+ T cell subsets as a percentage of total white blood cells. The bottom row shows the percentage of Ki67+ (i.e., actively proliferating) CD8+ T cell subsets. Control animals were given only buffer (white circles). Data are shown as mean ± SD. [Figure 6B] This study demonstrates the effects of IL-7 protein on cytokine-induced T cell proliferation and activation in normal C57BL / 6 mice. The expression profiles of various activation markers in CD8+ spleen T cells 5 days after IL-7 protein administration (blue lines) are shown. The black lines correspond to isotype controls. The activation markers shown (from left to right) include T-bet, Eomes, PD-1, granzyme B (GzmB), CXCR3, IFN-γ, TNF-α, and IL-2. [Figure 7] Figures A, B, and C show the effects of IL-7 protein administration on the activation and proliferation of mouse naive T cells (top row) and central memory CD8+ T cells (bottom row). Figure A shows the CD44 and CD62L expression profiles of naive T cells and central memory T cells in the spleen 5 days after IL-7 administration. Figures B and C present proliferation data (based on CTV staining and Ki67 expression, respectively). In figures B and C, blue represents T cells from animals that received IL-7 protein, while orange represents T cells from control animals (i.e., those that received only buffer). [Figure 8A]This study demonstrates the dose-dependent antitumor effect of IL-7 protein administration in syngeneic tumor models. It presents a comparison of tumor volume (mm3) in animals treated with various concentrations of IL-7 protein: (i) 0 mg / kg (i.e., buffer only) (black), (ii) 1.25 mg / kg (orange), (iii) 2.5 mg / kg (green), (iv) 5 mg / kg (blue), and (v) 10 mg / kg (red). Two-way ANOVA with Bonferroni post-hoc test (Figure 8A), or one-way ANOVA with Dunnett post-hoc test (Figure 8C), yielded *p<0.05, **p<0.01, and ***p<0.001 compared to the buffer group. Data are presented as mean ± SD. [Figure 8B] This study demonstrates the dose-dependent antitumor effect of IL-7 protein administration in a syngeneic tumor model. The proportion of immune cell compartments within CD45+ cells in PBMCs 7 days after IL-7 protein administration is shown. Each immune cell compartment in the illustration includes (i) CD8+ T cells (blue), (ii) CD4+ T cells (orange), (iii) Foxp3+CD4+ regulatory T cells (purple), (iv) B220+ B cells (gray), and (v) other immune cells not belonging to any of the above four categories (white). Each column represents the respective concentration of IL-7 protein. [Figure 8C] This figure shows the dose-dependent antitumor effect of IL-7 protein administration in a syngeneic tumor model. It also shows the absolute number of different immune cell populations in PBMCs 7 days post-treatment. The immune cell populations shown include (i) CD8+ T cells (first graph), (ii) CD4+ T cells (second graph), (iii) Foxp3+ regulatory T cells (third graph), and (iv) B220+ B cells (fourth graph). The x-axis represents the concentration of IL-7 protein administered to each treatment group. Two-way ANOVA with Bonferroni post-hoc test (Figure 8A) or one-way ANOVA with Dunnett post-hoc test (Figure 8C) yielded *p<0.05, **p<0.01, and ***p<0.001 compared to the buffer group. Data are presented as mean ± SD. [Figure 9A]This study demonstrates that the IL-7 protein may derive antitumor activity by inducing a CD8+ T cell inflammatory tumor microenvironment. The graphs show the percentages (upper graph) and numbers (lower graph) of various tumor-infiltrating leukocytes (TILs) observed in mouse tumors 5 days after IL-7 protein administration (purple) or buffer only (orange). Each TIL in the graph includes (i) monocytic myeloid-derived suppressor cells (M-MDSCs), (ii) polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs), (iii) tumor-associated macrophages (TAMs), (iv) tumor-associated dendritic cells (TADCs), (v) CD8+ T cells, (vi) CD4+ helper T cells (CD4Th cells), (v) CD4+ regulatory T cells (Treg cells), (vi) NK cells, and (vii) B cells. In each of Figures 9A to 9H, independent t-tests for the buffer group yielded the following results: *p<0.05, **p<0.01, and ***p<0.001. Data are presented as mean ± standard deviation. [Figure 9B] This study demonstrates that the IL-7 protein may derive antitumor activity by inducing a CD8+ T cell inflammatory tumor microenvironment. We present a comparison of the ratio of CD8+ TILs to Foxp3+ regulatory T cells (left graph) or MDSCs (right graph) at 5 days post-treatment in animals treated with either buffer alone (orange) or IL-7 protein (purple). Independent t-tests for each of Figures 9A-9H yielded *p<0.05, **p<0.01, and ***p<0.001 relative to the buffer group. Data are presented as mean ± sd. [Figure 9C] This study demonstrates that the IL-7 protein may derive antitumor activity by inducing an inflammatory tumor microenvironment of CD8+ T cells. We present a comparison of the proportion of Ki67+ cells (left graph) or granzyme B+ cells (right graph) in CD8+ TILs 5 days post-treatment in animals treated with buffer alone (orange) or IL-7 protein (purple). Independent t-tests for each of Figures 9A-9H yielded *p<0.05, **p<0.01, and ***p<0.001 relative to the buffer group. Data are presented as mean ± sd. [Figure 9D]This study demonstrates that the IL-7 protein may derive antitumor activity by inducing an inflammatory tumor microenvironment of CD8+ T cells. The percentages of IFN-γ and / or TNF-α-producing CD8+ TILs at 5 days post-treatment in animals treated with buffer alone (orange) or IL-7 protein (purple) are shown. Independent t-tests for each of Figures 9A-9H yielded *p<0.05, **p<0.01, and ***p<0.001 relative to the buffer group. Data are presented as mean ± sd. [Figure 9E] This study demonstrates that the IL-7 protein may derive antitumor activity by inducing an inflammatory tumor microenvironment of CD8+ T cells. The percentage of PD-1+ cells in CD8+ TILs in animals treated with buffer alone or with IL-7 protein is shown. Independent t-tests for each of Figures 9A-9H yielded *p<0.05, **p<0.01, and ***p<0.001 relative to the buffer group. Data are presented as mean ± sd. [Figure 9F] This study demonstrates that the IL-7 protein may derive antitumor activity by inducing a CD8+ T cell inflammatory tumor microenvironment. The proportion of LAG-3+TIM-3+ cells in CD8+PD-1+TILs is shown. Independent t-tests for each of Figures 9A-9H yielded *p<0.05, **p<0.01, and ***p<0.001 relative to the buffer group. Data are presented as mean ± sd. [Figure 9G] This study demonstrates that the IL-7 protein may derive antitumor activity by inducing a CD8+ T cell inflammatory tumor microenvironment. Geometric mean fluorescence intensity (gMFI) levels of various immune checkpoint receptors are shown in PD-1+LAG-3+TIM-3+CD8+TIL cells in animals treated with buffer alone or IL-7 protein. Independent t-tests for each of Figures 9A-9H yielded *p<0.05, **p<0.01, and ***p<0.001 relative to the buffer group. Data are presented as mean ± sd. [Figure 9H]This study demonstrates that the IL-7 protein may derive antitumor activity by inducing a CD8+ T cell inflammatory tumor microenvironment. Relative expression of various chemokines (CCL2, CCL5, CXCL1, CXCL9, CXCL10, and CXCL11), measured by RT-qPCR, is shown in tumor lysates of animals treated with either buffer (white) or IL-7 protein (green). In each of Figures 9A-9H, independent t-tests for the buffer group yielded *p<0.05, **p<0.01, and ***p<0.001. Data are presented as mean ± sd. [Figure 10] This study demonstrates the antitumor effects of IL-7 protein in combination with cyclophosphamide (CPA) and / or immune checkpoint inhibitors. The graph on the left shows tumor volume (mm3) at various time points after treatment. The graph on the right shows survival data. The top row shows results for animals treated with (i) buffer only, (ii) CPA and anti-PD-1 antibody, or (iii) CPA, anti-PD-1 antibody, and IL-7 protein. The middle row shows results for animals treated with (i) buffer only, (ii) CPA and anti-PD-L1 antibody, or (iii) CPA, anti-PD-L1 antibody, and IL-7 protein. The bottom row shows results for animals treated with (i) buffer only, (ii) CPA and anti-CTLA-4 antibody, or (iii) CPA, anti-CTLA-4 antibody, and IL-7 protein. Log-rank (Mantel-Cox) tests for corresponding color groups in the graph descriptions yielded **p<0.01 and ***p<0.001. Data are presented as mean ± standard deviation. [Figure 11]A presents a comparison of CD8+ T cell counts in the spleen, peripheral blood, and lymph nodes of thymectomized animals and Siamese controls. B shows the number of each CD8+ T cell population in the spleen of tumor mice treated with PBS or IL-7 protein at various weekly time points after administration. The CD8+ T cell populations shown include (i) total CD8+ T cells (first graph), (ii) naive (CD44-CD62L+) CD8+ T cells (second graph), (iii) effector memory (CD44+CD62L-) CD8+ T cells (third graph), and (iv) central memory (CD44+CD62L+) CD8+ T cells (fourth graph). Independent t-tests between the groups shown yielded *p<0.05, **p<0.01, and ***p<0.001. [Figure 12] This is a schematic diagram of the study design for the Phase 1b clinical trial described in Example 11, which evaluates the safety and efficacy of IL-7 protein in patients with advanced solid tumors. [Figure 13] A table summarizing the adverse effects observed in patients with advanced solid tumors in the Phase 1b clinical trial described in Example 11 is presented. "TEAE" refers to any adverse event that occurred under treatment. "ADR" refers to adverse drug reaction. [Figure 14] A, B, and C present the pharmacokinetic analysis results from the Phase 1b clinical trial described in Example 11. A presents a comparison of serum IL-7 concentrations in patients with advanced solid tumors treated with various doses of IL-7 protein. As described in Example 11, the doses included (i) 60 μg / kg ("1"), (ii) 120 μg / kg ("2"), (iii) 240 μg / kg ("3"), (iv) 480 μg / kg ("4"), (v) 720 μg / kg ("5"), (vi) 960 μg / kg ("6"), and (vii) 1,200 μg / kg ("7"). B and C present a comparison of Cmax and AUC in patients with advanced solid tumors for each dose group. Data are shown as mean ± SEM for each dose level. [Figure 15A]The pharmacodynamic analysis results from the Phase 1b clinical trial described in Example 11 are presented. Figures 15A to 15D show a comparison of absolute lymphocyte count (ALC), CD3+ T cell count, CD4+ T cell count, and CD8+ T cell count in patients with advanced solid tumors before IL-7 protein administration (i.e., time point "0") and 3 weeks after the first dose, respectively. Figures 15E and 15F show a comparison of ALC in non-lymphopenic and lymphopenic patients, respectively. In each figure, patients were classified into a low-dose group (60 and 120 μg / kg) ("circle"), a medium-dose group (240 and 480 μg / kg) ("square"), and a high-dose group (720 and 1,200 μg / kg) ("triangle"). "*" means p<0.05 for the baseline (week 0) group according to the Wilcoxon matched pair signed-rank test, "**" means p<0.01, and "***" means p<0.001. [Figure 15B] The pharmacodynamic analysis results from the Phase 1b clinical trial described in Example 11 are presented. Figures 15A to 15D show a comparison of absolute lymphocyte count (ALC), CD3+ T cell count, CD4+ T cell count, and CD8+ T cell count in patients with advanced solid tumors before IL-7 protein administration (i.e., time point "0") and 3 weeks after the first dose, respectively. Figures 15E and 15F show a comparison of ALC in non-lymphopenic and lymphopenic patients, respectively. In each figure, patients were classified into a low-dose group (60 and 120 μg / kg) ("circle"), a medium-dose group (240 and 480 μg / kg) ("square"), and a high-dose group (720 and 1,200 μg / kg) ("triangle"). "*" means p<0.05 for the baseline (week 0) group according to the Wilcoxon matched pair signed-rank test, "**" means p<0.01, and "***" means p<0.001. [Figure 15C]The pharmacodynamic analysis results from the Phase 1b clinical trial described in Example 11 are presented. Figures 15A to 15D show a comparison of absolute lymphocyte count (ALC), CD3+ T cell count, CD4+ T cell count, and CD8+ T cell count in patients with advanced solid tumors before IL-7 protein administration (i.e., time point "0") and 3 weeks after the first dose, respectively. Figures 15E and 15F show a comparison of ALC in non-lymphopenic and lymphopenic patients, respectively. In each figure, patients were classified into a low-dose group (60 and 120 μg / kg) ("circle"), a medium-dose group (240 and 480 μg / kg) ("square"), and a high-dose group (720 and 1,200 μg / kg) ("triangle"). "*" means p<0.05 for the baseline (week 0) group according to the Wilcoxon matched pair signed-rank test, "**" means p<0.01, and "***" means p<0.001. [Figure 15D] The pharmacodynamic analysis results from the Phase 1b clinical trial described in Example 11 are presented. Figures 15A to 15D show a comparison of absolute lymphocyte count (ALC), CD3+ T cell count, CD4+ T cell count, and CD8+ T cell count in patients with advanced solid tumors before IL-7 protein administration (i.e., time point "0") and 3 weeks after the first dose, respectively. Figures 15E and 15F show a comparison of ALC in non-lymphopenic and lymphopenic patients, respectively. In each figure, patients were classified into a low-dose group (60 and 120 μg / kg) ("circle"), a medium-dose group (240 and 480 μg / kg) ("square"), and a high-dose group (720 and 1,200 μg / kg) ("triangle"). "*" means p<0.05 for the baseline (week 0) group according to the Wilcoxon matched pair signed-rank test, "**" means p<0.01, and "***" means p<0.001. [Figure 15E]The pharmacodynamic analysis results from the Phase 1b clinical trial described in Example 11 are presented. Figures 15A to 15D show a comparison of absolute lymphocyte count (ALC), CD3+ T cell count, CD4+ T cell count, and CD8+ T cell count in patients with advanced solid tumors before IL-7 protein administration (i.e., time point "0") and 3 weeks after the first dose, respectively. Figures 15E and 15F show a comparison of ALC in non-lymphopenic and lymphopenic patients, respectively. In each figure, patients were classified into a low-dose group (60 and 120 μg / kg) ("circle"), a medium-dose group (240 and 480 μg / kg) ("square"), and a high-dose group (720 and 1,200 μg / kg) ("triangle"). "*" means p<0.05 for the baseline (week 0) group according to the Wilcoxon matched pair signed-rank test, "**" means p<0.01, and "***" means p<0.001. [Figure 15F] The pharmacodynamic analysis results from the Phase 1b clinical trial described in Example 11 are presented. Figures 15A to 15D show a comparison of absolute lymphocyte count (ALC), CD3+ T cell count, CD4+ T cell count, and CD8+ T cell count in patients with advanced solid tumors before IL-7 protein administration (i.e., time point "0") and 3 weeks after the first dose, respectively. Figures 15E and 15F show a comparison of ALC in non-lymphopenic and lymphopenic patients, respectively. In each figure, patients were classified into a low-dose group (60 and 120 μg / kg) ("circle"), a medium-dose group (240 and 480 μg / kg) ("square"), and a high-dose group (720 and 1,200 μg / kg) ("triangle"). "*" means p<0.05 for the baseline (week 0) group according to the Wilcoxon matched pair signed-rank test, "**" means p<0.01, and "***" means p<0.001. [Figure 16A]This section presents a comparison of the effects of IL-7 protein administration on different CD4+ and CD8+ T cell subsets in patients from the Phase 1b clinical trial described in Example 11. Figures 16A and 16C show a comparison of Ki67+CD4+ T cells and Ki67+CD8+ T cells in patients before IL-7 protein administration (i.e., time point "0") and one week after administration (i.e., time point "1"), respectively. Figures 16B and 16D show a comparison of CD127+CD4+ T cells and CD127+CD8+ T cells in patients before IL-7 protein administration (i.e., time point "0") and one week after administration (i.e., time point "1"), respectively. Figure 16E shows a comparison of the CD4+ T cell / Treg ratio (left graph) and CD8+ T cell / Treg ratio (right graph) in patients before IL-7 protein administration (i.e., time point "0") and one week after administration (i.e., time point "1"). Figure 16F shows a comparison of subsets of naive (left column), effector memory (EM) (center column), and central memory (CM) (right column) for both CD4+ T cells (top row) and CD8+ T cells (bottom row) in patients before IL-7 protein administration (i.e., time point "0") and 3 weeks after administration (i.e., time point "3"). Figures 16G and 16H show a comparison of CCR5+CD4+ T cells and CCR5+CD8+ T cells in patients before IL-7 protein administration (i.e., time point "0") and 1 week after administration (i.e., time point "1", respectively). In each figure, patients were classified into a low-dose group (60 and 120 μg / kg) (circle; two columns on the left), a medium-dose group (240 and 480 μg / kg) (square; two columns in the center), and a high-dose group (720 and 1,200 μg / kg) (triangle; two columns on the right). "*" indicates p<0.05 by Wilcoxon matched-pair signed-rank test compared to the baseline (week 0) group, "**" indicates p<0.01, and "***" indicates p<0.001. [Figure 16B]This section presents a comparison of the effects of IL-7 protein administration on different CD4+ and CD8+ T cell subsets in patients from the Phase 1b clinical trial described in Example 11. Figures 16A and 16C show a comparison of Ki67+CD4+ T cells and Ki67+CD8+ T cells in patients before IL-7 protein administration (i.e., time point "0") and one week after administration (i.e., time point "1"), respectively. Figures 16B and 16D show a comparison of CD127+CD4+ T cells and CD127+CD8+ T cells in patients before IL-7 protein administration (i.e., time point "0") and one week after administration (i.e., time point "1"), respectively. Figure 16E shows a comparison of the CD4+ T cell / Treg ratio (left graph) and CD8+ T cell / Treg ratio (right graph) in patients before IL-7 protein administration (i.e., time point "0") and one week after administration (i.e., time point "1"). Figure 16F shows a comparison of subsets of naive (left column), effector memory (EM) (center column), and central memory (CM) (right column) for both CD4+ T cells (top row) and CD8+ T cells (bottom row) in patients before IL-7 protein administration (i.e., time point "0") and 3 weeks after administration (i.e., time point "3"). Figures 16G and 16H show a comparison of CCR5+CD4+ T cells and CCR5+CD8+ T cells in patients before IL-7 protein administration (i.e., time point "0") and 1 week after administration (i.e., time point "1", respectively). In each figure, patients were classified into a low-dose group (60 and 120 μg / kg) (circle; two columns on the left), a medium-dose group (240 and 480 μg / kg) (square; two columns in the center), and a high-dose group (720 and 1,200 μg / kg) (triangle; two columns on the right). "*" indicates p<0.05 by Wilcoxon matched-pair signed-rank test compared to the baseline (week 0) group, "**" indicates p<0.01, and "***" indicates p<0.001. [Figure 16C]This section presents a comparison of the effects of IL-7 protein administration on different CD4+ and CD8+ T cell subsets in patients from the Phase 1b clinical trial described in Example 11. Figures 16A and 16C show a comparison of Ki67+CD4+ T cells and Ki67+CD8+ T cells in patients before IL-7 protein administration (i.e., time point "0") and one week after administration (i.e., time point "1"), respectively. Figures 16B and 16D show a comparison of CD127+CD4+ T cells and CD127+CD8+ T cells in patients before IL-7 protein administration (i.e., time point "0") and one week after administration (i.e., time point "1"), respectively. Figure 16E shows a comparison of the CD4+ T cell / Treg ratio (left graph) and CD8+ T cell / Treg ratio (right graph) in patients before IL-7 protein administration (i.e., time point "0") and one week after administration (i.e., time point "1"). Figure 16F shows a comparison of subsets of naive (left column), effector memory (EM) (center column), and central memory (CM) (right column) for both CD4+ T cells (top row) and CD8+ T cells (bottom row) in patients before IL-7 protein administration (i.e., time point "0") and 3 weeks after administration (i.e., time point "3"). Figures 16G and 16H show a comparison of CCR5+CD4+ T cells and CCR5+CD8+ T cells in patients before IL-7 protein administration (i.e., time point "0") and 1 week after administration (i.e., time point "1", respectively). In each figure, patients were classified into a low-dose group (60 and 120 μg / kg) (circle; two columns on the left), a medium-dose group (240 and 480 μg / kg) (square; two columns in the center), and a high-dose group (720 and 1,200 μg / kg) (triangle; two columns on the right). "*" indicates p<0.05 by Wilcoxon matched-pair signed-rank test compared to the baseline (week 0) group, "**" indicates p<0.01, and "***" indicates p<0.001. [Figure 16D]This section presents a comparison of the effects of IL-7 protein administration on different CD4+ and CD8+ T cell subsets in patients from the Phase 1b clinical trial described in Example 11. Figures 16A and 16C show a comparison of Ki67+CD4+ T cells and Ki67+CD8+ T cells in patients before IL-7 protein administration (i.e., time point "0") and one week after administration (i.e., time point "1"), respectively. Figures 16B and 16D show a comparison of CD127+CD4+ T cells and CD127+CD8+ T cells in patients before IL-7 protein administration (i.e., time point "0") and one week after administration (i.e., time point "1"), respectively. Figure 16E shows a comparison of the CD4+ T cell / Treg ratio (left graph) and CD8+ T cell / Treg ratio (right graph) in patients before IL-7 protein administration (i.e., time point "0") and one week after administration (i.e., time point "1"). Figure 16F shows a comparison of subsets of naive (left column), effector memory (EM) (center column), and central memory (CM) (right column) for both CD4+ T cells (top row) and CD8+ T cells (bottom row) in patients before IL-7 protein administration (i.e., time point "0") and 3 weeks after administration (i.e., time point "3"). Figures 16G and 16H show a comparison of CCR5+CD4+ T cells and CCR5+CD8+ T cells in patients before IL-7 protein administration (i.e., time point "0") and 1 week after administration (i.e., time point "1", respectively). In each figure, patients were classified into a low-dose group (60 and 120 μg / kg) (circle; two columns on the left), a medium-dose group (240 and 480 μg / kg) (square; two columns in the center), and a high-dose group (720 and 1,200 μg / kg) (triangle; two columns on the right). "*" indicates p<0.05 by Wilcoxon matched-pair signed-rank test compared to the baseline (week 0) group, "**" indicates p<0.01, and "***" indicates p<0.001. [Figure 16E]This section presents a comparison of the effects of IL-7 protein administration on different CD4+ and CD8+ T cell subsets in patients from the Phase 1b clinical trial described in Example 11. Figures 16A and 16C show a comparison of Ki67+CD4+ T cells and Ki67+CD8+ T cells in patients before IL-7 protein administration (i.e., time point "0") and one week after administration (i.e., time point "1"), respectively. Figures 16B and 16D show a comparison of CD127+CD4+ T cells and CD127+CD8+ T cells in patients before IL-7 protein administration (i.e., time point "0") and one week after administration (i.e., time point "1"), respectively. Figure 16E shows a comparison of the CD4+ T cell / Treg ratio (left graph) and CD8+ T cell / Treg ratio (right graph) in patients before IL-7 protein administration (i.e., time point "0") and one week after administration (i.e., time point "1"). Figure 16F shows a comparison of subsets of naive (left column), effector memory (EM) (center column), and central memory (CM) (right column) for both CD4+ T cells (top row) and CD8+ T cells (bottom row) in patients before IL-7 protein administration (i.e., time point "0") and 3 weeks after administration (i.e., time point "3"). Figures 16G and 16H show a comparison of CCR5+CD4+ T cells and CCR5+CD8+ T cells in patients before IL-7 protein administration (i.e., time point "0") and 1 week after administration (i.e., time point "1", respectively). In each figure, patients were classified into a low-dose group (60 and 120 μg / kg) (circle; two columns on the left), a medium-dose group (240 and 480 μg / kg) (square; two columns in the center), and a high-dose group (720 and 1,200 μg / kg) (triangle; two columns on the right). "*" indicates p<0.05 by Wilcoxon matched-pair signed-rank test compared to the baseline (week 0) group, "**" indicates p<0.01, and "***" indicates p<0.001. [Figure 16F]This section presents a comparison of the effects of IL-7 protein administration on different CD4+ and CD8+ T cell subsets in patients from the Phase 1b clinical trial described in Example 11. Figures 16A and 16C show a comparison of Ki67+CD4+ T cells and Ki67+CD8+ T cells in patients before IL-7 protein administration (i.e., time point "0") and one week after administration (i.e., time point "1"), respectively. Figures 16B and 16D show a comparison of CD127+CD4+ T cells and CD127+CD8+ T cells in patients before IL-7 protein administration (i.e., time point "0") and one week after administration (i.e., time point "1"), respectively. Figure 16E shows a comparison of the CD4+ T cell / Treg ratio (left graph) and CD8+ T cell / Treg ratio (right graph) in patients before IL-7 protein administration (i.e., time point "0") and one week after administration (i.e., time point "1"). Figure 16F shows a comparison of subsets of naive (left column), effector memory (EM) (center column), and central memory (CM) (right column) for both CD4+ T cells (top row) and CD8+ T cells (bottom row) in patients before IL-7 protein administration (i.e., time point "0") and 3 weeks after administration (i.e., time point "3"). Figures 16G and 16H show a comparison of CCR5+CD4+ T cells and CCR5+CD8+ T cells in patients before IL-7 protein administration (i.e., time point "0") and 1 week after administration (i.e., time point "1", respectively). In each figure, patients were classified into a low-dose group (60 and 120 μg / kg) (circle; two columns on the left), a medium-dose group (240 and 480 μg / kg) (square; two columns in the center), and a high-dose group (720 and 1,200 μg / kg) (triangle; two columns on the right). "*" indicates p<0.05 by Wilcoxon matched-pair signed-rank test compared to the baseline (week 0) group, "**" indicates p<0.01, and "***" indicates p<0.001. [Figure 16G]This section presents a comparison of the effects of IL-7 protein administration on different CD4+ and CD8+ T cell subsets in patients from the Phase 1b clinical trial described in Example 11. Figures 16A and 16C show a comparison of Ki67+CD4+ T cells and Ki67+CD8+ T cells in patients before IL-7 protein administration (i.e., time point "0") and one week after administration (i.e., time point "1"), respectively. Figures 16B and 16D show a comparison of CD127+CD4+ T cells and CD127+CD8+ T cells in patients before IL-7 protein administration (i.e., time point "0") and one week after administration (i.e., time point "1"), respectively. Figure 16E shows a comparison of the CD4+ T cell / Treg ratio (left graph) and CD8+ T cell / Treg ratio (right graph) in patients before IL-7 protein administration (i.e., time point "0") and one week after administration (i.e., time point "1"). Figure 16F shows a comparison of subsets of naive (left column), effector memory (EM) (center column), and central memory (CM) (right column) for both CD4+ T cells (top row) and CD8+ T cells (bottom row) in patients before IL-7 protein administration (i.e., time point "0") and 3 weeks after administration (i.e., time point "3"). Figures 16G and 16H show a comparison of CCR5+CD4+ T cells and CCR5+CD8+ T cells in patients before IL-7 protein administration (i.e., time point "0") and 1 week after administration (i.e., time point "1", respectively). In each figure, patients were classified into a low-dose group (60 and 120 μg / kg) (circle; two columns on the left), a medium-dose group (240 and 480 μg / kg) (square; two columns in the center), and a high-dose group (720 and 1,200 μg / kg) (triangle; two columns on the right). "*" indicates p<0.05 by Wilcoxon matched-pair signed-rank test compared to the baseline (week 0) group, "**" indicates p<0.01, and "***" indicates p<0.001. [Figure 16H]This section presents a comparison of the effects of IL-7 protein administration on different CD4+ and CD8+ T cell subsets in patients from the Phase 1b clinical trial described in Example 11. Figures 16A and 16C show a comparison of Ki67+CD4+ T cells and Ki67+CD8+ T cells in patients before IL-7 protein administration (i.e., time point "0") and one week after administration (i.e., time point "1"), respectively. Figures 16B and 16D show a comparison of CD127+CD4+ T cells and CD127+CD8+ T cells in patients before IL-7 protein administration (i.e., time point "0") and one week after administration (i.e., time point "1"), respectively. Figure 16E shows a comparison of the CD4+ T cell / Treg ratio (left graph) and CD8+ T cell / Treg ratio (right graph) in patients before IL-7 protein administration (i.e., time point "0") and one week after administration (i.e., time point "1"). Figure 16F shows a comparison of subsets of naive (left column), effector memory (EM) (center column), and central memory (CM) (right column) for both CD4+ T cells (top row) and CD8+ T cells (bottom row) in patients before IL-7 protein administration (i.e., time point "0") and 3 weeks after administration (i.e., time point "3"). Figures 16G and 16H show a comparison of CCR5+CD4+ T cells and CCR5+CD8+ T cells in patients before IL-7 protein administration (i.e., time point "0") and 1 week after administration (i.e., time point "1", respectively). In each figure, patients were classified into a low-dose group (60 and 120 μg / kg) (circle; two columns on the left), a medium-dose group (240 and 480 μg / kg) (square; two columns in the center), and a high-dose group (720 and 1,200 μg / kg) (triangle; two columns on the right). "*" indicates p<0.05 by Wilcoxon matched-pair signed-rank test compared to the baseline (week 0) group, "**" indicates p<0.01, and "***" indicates p<0.001. [Figure 17]Figures A and B show a comparison of the effects of IL-7 protein administration on NK cells and B cells before administration (i.e., time point "0") and at 3 weeks after administration (i.e., time point "3") in patients from the Phase 1b clinical trial described in Example 11, respectively. In both figures, patients were classified into a low-dose group (60 and 120 μg / kg) (circle; two columns on the left), a medium-dose group (240 and 480 μg / kg) (square; two columns in the center), and a high-dose group (720 and 1,200 μg / kg) (triangle; two columns on the right). "*" indicates p<0.05 by Wilcoxon matched-pair signed-rank test compared to the baseline (week 0) group. [Figure 18] A table summarizing the adverse effects observed in gliablastoma patients in the Phase 1b clinical trial described in Example 12 is presented. "TEAE" refers to any adverse event that occurred under treatment. "ADR" refers to adverse drug reaction. [Figure 19A] The pharmacodynamic analysis results of the Phase 1b clinical trial described in Example 12 are presented. Figures 19A to 19D show a comparison of absolute lymphocyte count (ALC), CD3+ T cell count, CD4+ T cell count, and CD8+ T cell count in gliablastoma cancer patients before IL-7 protein administration (i.e., time point "0") and 3 weeks after the first dose, respectively. Figures 19E and 19F show a comparison of ALC in non-lymphopenic and lymphopenic patients, respectively. In each figure, patients were classified into a low-dose group (60 μg / kg) (circle), a medium-dose group (360 and 600 μg / kg) (square), and a high-dose group (840 and 1,440 μg / kg) (triangle). "*" means p<0.05 by Wilcoxon matched-pair signed-rank test compared to the baseline (week 0) group, and "**" means p<0.01. [Figure 19B]The pharmacodynamic analysis results of the Phase 1b clinical trial described in Example 12 are presented. Figures 19A to 19D show a comparison of absolute lymphocyte count (ALC), CD3+ T cell count, CD4+ T cell count, and CD8+ T cell count in gliablastoma cancer patients before IL-7 protein administration (i.e., time point "0") and 3 weeks after the first dose, respectively. Figures 19E and 19F show a comparison of ALC in non-lymphopenic and lymphopenic patients, respectively. In each figure, patients were classified into a low-dose group (60 μg / kg) (circle), a medium-dose group (360 and 600 μg / kg) (square), and a high-dose group (840 and 1,440 μg / kg) (triangle). "*" means p<0.05 by Wilcoxon matched-pair signed-rank test compared to the baseline (week 0) group, and "**" means p<0.01. [Figure 19C] The pharmacodynamic analysis results of the Phase 1b clinical trial described in Example 12 are presented. Figures 19A to 19D show a comparison of absolute lymphocyte count (ALC), CD3+ T cell count, CD4+ T cell count, and CD8+ T cell count in gliablastoma cancer patients before IL-7 protein administration (i.e., time point "0") and 3 weeks after the first dose, respectively. Figures 19E and 19F show a comparison of ALC in non-lymphopenic and lymphopenic patients, respectively. In each figure, patients were classified into a low-dose group (60 μg / kg) (circle), a medium-dose group (360 and 600 μg / kg) (square), and a high-dose group (840 and 1,440 μg / kg) (triangle). "*" means p<0.05 by Wilcoxon matched-pair signed-rank test compared to the baseline (week 0) group, and "**" means p<0.01. [Figure 19D]The pharmacodynamic analysis results of the Phase 1b clinical trial described in Example 12 are presented. Figures 19A to 19D show a comparison of absolute lymphocyte count (ALC), CD3+ T cell count, CD4+ T cell count, and CD8+ T cell count in gliablastoma cancer patients before IL-7 protein administration (i.e., time point "0") and 3 weeks after the first dose, respectively. Figures 19E and 19F show a comparison of ALC in non-lymphopenic and lymphopenic patients, respectively. In each figure, patients were classified into a low-dose group (60 μg / kg) (circle), a medium-dose group (360 and 600 μg / kg) (square), and a high-dose group (840 and 1,440 μg / kg) (triangle). "*" means p<0.05 by Wilcoxon matched-pair signed-rank test compared to the baseline (week 0) group, and "**" means p<0.01. [Figure 19E] The pharmacodynamic analysis results of the Phase 1b clinical trial described in Example 12 are presented. Figures 19A to 19D show a comparison of absolute lymphocyte count (ALC), CD3+ T cell count, CD4+ T cell count, and CD8+ T cell count in gliablastoma cancer patients before IL-7 protein administration (i.e., time point "0") and 3 weeks after the first dose, respectively. Figures 19E and 19F show a comparison of ALC in non-lymphopenic and lymphopenic patients, respectively. In each figure, patients were classified into a low-dose group (60 μg / kg) (circle), a medium-dose group (360 and 600 μg / kg) (square), and a high-dose group (840 and 1,440 μg / kg) (triangle). "*" means p<0.05 by Wilcoxon matched-pair signed-rank test compared to the baseline (week 0) group, and "**" means p<0.01. [Figure 19F]The pharmacodynamic analysis results of the Phase 1b clinical trial described in Example 12 are presented. Figures 19A to 19D show a comparison of absolute lymphocyte count (ALC), CD3+ T cell count, CD4+ T cell count, and CD8+ T cell count in gliablastoma cancer patients before IL-7 protein administration (i.e., time point "0") and 3 weeks after the first dose, respectively. Figures 19E and 19F show a comparison of ALC in non-lymphopenic and lymphopenic patients, respectively. In each figure, patients were classified into a low-dose group (60 μg / kg) (circle), a medium-dose group (360 and 600 μg / kg) (square), and a high-dose group (840 and 1,440 μg / kg) (triangle). "*" means p<0.05 by Wilcoxon matched-pair signed-rank test compared to the baseline (week 0) group, and "**" means p<0.01. [Figure 20A] Figures A, B, and C show the effects of IL-7 protein administration on AUC, Ki67+CD8+ T cell frequency, and Ki67+CD4+ T cell frequency, respectively, in gliablastoma patients receiving temozolomide (TMZ). Each figure indicates the day on which TMZ or IL-7 protein was administered. [Figure 20B] Figures A, B, and C show the effects of IL-7 protein administration on AUC, Ki67+CD8+ T cell frequency, and Ki67+CD4+ T cell frequency, respectively, in gliablastoma patients receiving temozolomide (TMZ). Each figure indicates the day on which TMZ or IL-7 protein was administered. [Figure 20C] Figures A, B, and C show the effects of IL-7 protein administration on AUC, Ki67+CD8+ T cell frequency, and Ki67+CD4+ T cell frequency, respectively, in gliablastoma patients receiving temozolomide (TMZ). Each figure indicates the day on which TMZ or IL-7 protein was administered. [Figure 21A]Figures A, B, and C show the effects of IL-7 protein administration on AUC, Ki67+CD8+ T cell incidence, and Ki67+CD4+ T cell incidence, respectively, in gliablastoma patients receiving Avastin / irinotecan (A / I). Each figure indicates the day on which A / I or IL-7 protein was administered. [Figure 21B] Figures A, B, and C show the effects of IL-7 protein administration on AUC, Ki67+CD8+ T cell incidence, and Ki67+CD4+ T cell incidence, respectively, in gliablastoma patients receiving Avastin / irinotecan (A / I). Each figure indicates the day on which A / I or IL-7 protein was administered. [Figure 21C] Figures A, B, and C show the effects of IL-7 protein administration on AUC, Ki67+CD8+ T cell incidence, and Ki67+CD4+ T cell incidence, respectively, in gliablastoma patients receiving Avastin / irinotecan (A / I). Each figure indicates the day on which A / I or IL-7 protein was administered. [Modes for carrying out the invention]

[0042] I. Definition To facilitate understanding of this disclosure, certain terms are defined first. As used in this application, unless otherwise specified herein, each of the following terms shall have the meaning set forth below. Further definitions are provided throughout this specification.

[0043] Throughout this disclosure, the term “a” or “an” attached to an entity means one or more such entities. For example, “an antibody” is understood to mean one or more antibodies. Thus, the terms “a” (or “an”), “one or more,” and “at least one” may be used synonymously herein.

[0044] Furthermore, when used herein, “and / or” shall be interpreted as a specific disclosure of each of two particular features or components, whether or not the other is present. Accordingly, the term “and / or” as used herein in phrases such as “A and / or B” is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Similarly, the term “and / or” as used in phrases such as “A, B, and / or C” is intended to include each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0045] Wherever an aspect is described using the term "comprising" in this specification, it should be understood that other similar aspects described using the terms "consisting of" and / or "consisting essentially of" are also provided.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in the relevant field of this disclosure. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press, The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press, and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press provide many common dictionaries of the terms used herein.

[0047] Units, prefixes, and symbols are shown in the forms recognized by the International System of Units (SI). Numerical ranges include the number defining the range. Unless otherwise specified, amino acid sequences are written from left to right in the direction of amino to carboxyl. The headings provided herein are not intended to limit the various aspects of this disclosure that can be obtained by referring to the entirety of this specification. Thus, the terms defined immediately below are more fully defined by referring to the entirety of this specification.

[0048] The term "approximately" is used herein to mean roughly, approximately, about, or within a range. When the term "approximately" is used with a numerical range, it modifies that range by extending the boundary above and below the given number. Generally, the term "approximately" can modify a number to be above or below a given value by, for example, 10 percent above or below (higher or lower).

[0049] As used herein, “administer” means physically introducing a therapeutic agent or a composition containing a therapeutic agent into a target using any of the various methods and delivery systems known to those skilled in the art. Various routes of administration of therapeutic agents described herein include, for example, intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, or other parenteral routes, by injection or infusion. As used herein, the term “parenteral administration” means, but is not limited to, injections of a mode of administration other than enteral and local administration, usually by injection, including intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intrafocal, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, intratracheal, intrapulmonary, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraventricular, intravitreous, epidural, and intrasternal injections and infusions, as well as in vivo electroporation. Alternatively, the therapeutic agents described herein may be administered via non-parenteral routes such as local, dermal, or mucosal routes, for example, intranasal, oral, vaginal, rectal, sublingual, or local administration. Furthermore, administration may be carried out, for example, once, multiple times, and / or over a longer period of time.

[0050] As used herein, the term “antigen” means any naturally occurring or synthetic immunogenic substance, such as a protein, peptide, or hapten.

[0051] The terms "antibody" and "antibodies" are technical terms and may be used synonymously herein, and mean a molecule containing an antigen-binding site that specifically binds to an antigen. These terms as used herein include whole antibodies and any antigen-binding fragments thereof (i.e., "antigen-binding portions") or single chains. "Antibody" in one aspect means a glycoprotein containing at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. In another aspect, "antibody" means a single-chain antibody containing a single variable domain, e.g., a VHH domain. Each heavy chain is composed of a heavy-chain variable region (abbreviated as VH herein) and a heavy-chain constant region. In certain naturally occurring antibodies, the heavy-chain constant region is composed of three domains, CH1, CH2 and CH3. In certain naturally occurring antibodies, each light chain is composed of a light-chain variable region (abbreviated as VL herein) and a light-chain constant region. The light-chain constant region is composed of one domain CL.

[0052] The VH region and the VL region can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), and more conserved regions called framework regions (FRs) are interspersed among the CDRs. Each VH and VL contains three CDRs and four FRs, which are arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxy terminus. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of the antibody can mediate the binding of immunoglobulins to host tissues or factors, including various immune system cells (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0053] Antibodies typically bind specifically to their cognate antigens with high affinity, reflected by a dissociation constant (K -5 ~10 -11 M or less (K D ). When K D is about 10 -4If the value exceeds M, it is generally considered to exhibit nonspecific binding. As used herein, an antibody that "specifically binds" to an antigen means that it has high affinity for the antigen and substantially the same antigen, i.e., 10 -7 M or less, 10 -8 M or less, 5×10 -9 M or less, or 10 -8 M~10 -10 K below M D This refers to an antibody that binds to a given antigen but does not bind to unrelated antigens with high affinity. An antigen is "substantially identical" to a given antigen if it exhibits a high degree of sequence identity with respect to the given antigen, for example, if it exhibits at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with the sequence of the given antigen. For example, an antibody that specifically binds to PD-1 may, in certain embodiments, exhibit cross-reactivity with PD-1 antigens from specific primate species (e.g., cynomolgus monkey anti-PD-1 antibody), but it cannot cross-react with PD-1 molecules from other species or molecules other than PD-1.

[0054] Immunoglobulins may be derived from any of the commonly known isotypes, including but not limited to IgA, secretory IgA, IgG, and IgM. IgG subclasses are also well known to those skilled in the art, and include, but are not limited to, human IgG1, IgG2, IgG3, and IgG4. “Isotype” means a class or subclass of antibody (e.g., IgM or IgG1) encoded by a heavy chain constant region gene. In certain embodiments, one or more amino acids in an isotype may be mutated to alter its effector function. The term “antibody” includes, by example, both naturally occurring and non-naturally occurring antibodies (Abs), monoclonal and polyclonal antibodies, chimeric and humanized antibodies, human or non-human antibodies, fully synthetic antibodies, and single-chain antibodies. Non-human antibodies can be humanized by recombinant methods to reduce their immunogenicity in humans. Unless otherwise specified in the context, the term “antibody” includes any antigen-binding fragment or portion of any of the aforementioned immunoglobulins, including monovalent and bivalent fragments or portions, as well as single-chain antibodies.

[0055] "Isolated antibody" means an antibody that substantially does not contain other antibodies with different antigen specificities (for example, an isolated antibody that specifically binds to PD-1 substantially does not contain antibodies that specifically bind to antigens other than PD-1). However, an isolated antibody that specifically binds to PD-1 may have cross-reactivity with other antigens, such as PD-1 molecules from different species. Furthermore, isolated antibodies may substantially not contain other cellular material and / or chemical substances.

[0056] The term “monoclonal antibody” (“mAb”) refers to an unnatural preparation of an antibody molecule having a single molecular composition, i.e., an antibody molecule whose primary sequence is essentially identical and which exhibits a single binding specificity and affinity for a particular epitope. An mAb is an example of an isolated antibody. mAbs can be produced by hybridoma techniques, recombinant techniques, transgenic techniques, or other techniques known to those skilled in the art.

[0057] A “human” antibody (HuMAb) means an antibody having a variable region in which both the framework and CDR region are derived from a human germline immunoglobulin sequence. Furthermore, if the antibody includes a constant region, the constant region is also derived from a human germline immunoglobulin sequence. The human antibodies of the present invention may include amino acid residues not encoded by a human germline immunoglobulin sequence (e.g., mutations introduced in vitro by random mutagenesis or site-directed mutagenesis, or mutations introduced in vivo by somatic mutation). However, as used herein, the term “human antibody” is not intended to include antibodies in which a CDR sequence derived from the germline of another mammalian species, such as mouse, is grafted onto a human framework sequence. The terms “human” antibody and “fully human” antibody are used synonymously.

[0058] A "humanized antibody" refers to an antibody in which some, almost all, or all of the amino acids outside the CDR domain of a non-human antibody are replaced with corresponding amino acids derived from human immunoglobulin. In one embodiment of a humanized antibody, some, almost all, or all of the amino acids outside the CDR domain are replaced with amino acids derived from human immunoglobulin, but some, almost all, or all of the amino acids within one or more CDR regions remain unchanged. Minor additions, deletions, insertions, substitutions, or modifications of amino acids are acceptable as long as they do not inhibit the antibody's ability to bind to a particular antigen. Humanized antibodies retain similar antigen specificity to that of the original antibody.

[0059] A "chimeric antibody" refers to an antibody in which the variable region originates from one species and the constant region originates from another species, such as an antibody in which the variable region originates from a mouse antibody and the constant region originates from a human antibody.

[0060] An "anti-antigen" antibody is an antibody that specifically binds to an antigen. For example, an anti-PD-1 antibody specifically binds to PD-1, and an anti-CTLA-4 antibody specifically binds to CTLA-4.

[0061] The "antigen-binding portion" (also called the "antigen-binding fragment") of an antibody refers to one or more fragments of the antibody that possess the ability to specifically bind to the antigen to which the entire antibody binds.

[0062] As used herein, the terms “specific binding,” “selective binding,” “selectively binding,” and “specifically binding” mean that the antibody binds to the epitope of a given antigen. Typically, the antibody binds to an antigen-positive cell by (i) surface plasmon resonance (SPR) technique on a BIACORE® 2000 instrument, for example, using a given antigen as the analyte and the antibody as the ligand, or by Scatchard analysis of the antibody binding to antigen-positive cells, approximately 10 -7 Less than M, for example, approximately 10 -8 M, 10 -9 M or 10 -10 Equilibrium dissociation constant (K) less than M or even lower than M D (ii) binds to the given antigen with an affinity at least twice as high as the binding affinity to a nonspecific antigen other than the given antigen or a closely related antigen (e.g., BSA, casein).

[0063] As used herein, the term “naturally occurring” refers, when applied to a subject, to the fact that the subject can be found in nature. For example, polypeptides or polynucleotide sequences that are present in living organisms (including viruses), can be isolated from natural sources, and have not been intentionally modified by humans in the laboratory are naturally occurring.

[0064] A "polypeptide" refers to a chain containing at least two consecutively linked amino acid residues, with no upper limit on chain length. One or more amino acid residues in a protein may, but are not limited to, undergo modifications such as glycosylation, phosphorylation, or disulfide bond formation. A "protein" may contain one or more polypeptides. Unless otherwise specified, the terms "protein" and "polypeptide" may be used synonymously.

[0065] As used herein, the term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. Nucleic acid molecules may be single-stranded or double-stranded, and may be cDNA.

[0066] "Conservative amino acid substitution" means that an amino acid residue is substituted with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In certain embodiments, a predicted non-essential amino acid residue in an antibody is replaced with another amino acid residue from the same side-chain family. Methods for identifying conserved nucleotide and amino acid substitutions that do not eliminate antigen binding are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993), Kobayashi et al. Protein Eng. 12(10):879-884 (1999), and Burks et al. Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

[0067] With respect to nucleic acids, the term "substantial homology" indicates that two nucleic acids or their specified sequences are identical in at least approximately 80% of nucleotides, at least approximately 90%–95%, or at least approximately 98%–99.5% of nucleotides, with appropriate nucleotide insertions or deletions, when optimally aligned and compared. Alternatively, substantial homology exists when segments hybridize to the chain complement under selective hybridization conditions.

[0068] With respect to polypeptides, the term "substantial homology" indicates that two polypeptides or their specified sequences are identical in at least about 80% of amino acids, at least about 90% to 95%, or at least about 98% to 99.5% of amino acids, with appropriate amino acid insertions or deletions, when optimally aligned and compared.

[0069] The percentage of identity between two sequences is a function of the number of identical positions common to the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap (i.e., homology % = number of identical positions / total number of positions × 100). Sequence comparison and determination of the percentage of identity between two sequences can be performed using mathematical algorithms such as those described in the non-restrictive examples below.

[0070] The percentage of identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at worldwideweb.gcg.com) with the NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70, or 80, and length weights of 1, 2, 3, 4, 5, or 6. The percentage of identity between two nucleotide or amino acid sequences can also be determined using the E. Meyers and W. Miller algorithm (CABIOS, 4:11-17 (1989)) incorporated into the ALIGN program (version 2.0) with the PAM120 residue weighting table, gap length penalty 12, and gap penalty 4. Furthermore, the percentage of identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J.Mol.Biol.(48):444-453(1970)), which is incorporated into the GAP program of the GCG software package (available at worldwideweb.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0071] The nucleic acid and protein sequences described herein can also be used, for example, as "query sequences" for searching public databases to identify related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) described in Altschul, et al. (1990) J.Mol.Biol.215:403-10. Performing a BLAST nucleotide search in the NBLAST program with a score of 100 and a word length of 12 will yield nucleotide sequences homologous to the nucleic acid molecules described herein. Performing a BLAST protein search in the XBLAST program with a score of 50 and a word length of 3 will yield amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. When using the BLAST and Gapped BLAST programs, you can use the default parameters for each program (e.g., XBLAST and NBLAST). See worldwideweb.ncbi.nlm.nih.gov for details.

[0072] Nucleic acids may be present in whole cells, in cell lysates, or in partially purified or substantially pure forms. A nucleic acid is considered "isolated" or "substantially pure" if it has been purified from other cellular components or other contaminants, e.g., other cellular nucleic acids (e.g., other parts of chromosomes) or proteins, by standard techniques including alkali / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other techniques well known in the art. (F. Ausubel, et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New) See York (1987).

[0073] Nucleic acids, such as cDNA, can be mutated according to standard techniques to yield gene sequences. For coding sequences, these mutations can affect the amino acid sequence as desired. In particular, DNA sequences substantially homologous to or derived from natural V, D, J, constant, and switch sequences, and other such sequences described herein are envisioned (where “derived” indicates that one sequence is identical to another or modified from another).

[0074] As used herein, the term “vector” is intended to mean a nucleic acid molecule capable of transporting another ligated nucleic acid. One type of vector is a “plasmid,” which means a circular double-stranded DNA loop to which an additional DNA segment can be ligated. Another type of vector is a viral vector, in which case an additional DNA segment can be ligated into the viral genome. Certain vectors can autonomously replicate in the host cell into which they are introduced (e.g., bacterial vectors with bacterial origins of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors), once introduced into a host cell, can be integrated into the host cell’s genome and thereby replicate together with the host genome. Furthermore, certain vectors can direct the expression of a functionally ligated gene. Such vectors are referred herein as “recombinant expression vectors” (or simply “expression vectors”). Generally, expression vectors useful in recombinant DNA technology are often in the form of plasmids. Since plasmids are the most commonly used form of vector, “plasmid” and “vector” may be used synonymously herein. However, other forms of expression vectors are also included, such as viral vectors that perform equal functions (e.g., replication-deficient retroviruses, adenoviruses, and adeno-associated viruses).

[0075] The term “recombinant host cell” (or simply “host cell”), as used herein, is intended to mean a cell containing nucleic acids not naturally present in the cell, which may be a cell into which a recombinant expression vector has been introduced. It should be understood that such terminology is intended to mean not only a specific target cell but also the offspring of such cell. Such offspring may not be identical to the parent cell in practice, due to certain modifications occurring in subsequent generations, either due to mutation or environmental influences, but they are still included within the scope of the term “host cell” as used herein.

[0076] As used herein, the term “conjugated” means the association of two or more molecules. This conjugation may be covalent or non-covalent. This conjugation may also be genetic (i.e., fusion by recombination). Such conjugations can be achieved using a wide range of techniques recognized in the art, such as chemical conjugation and recombinant protein synthesis.

[0077] "Cancer" refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division and growth lead to the formation of malignant tumors, which can invade adjacent tissues and, in some cases, metastasize to distal parts of the body via the lymphatic system or bloodstream. As used herein, "cancer" refers to primary cancer, metastatic cancer, and recurrent cancer.

[0078] Cytotoxic T lymphocyte antigen-4 (CTLA-4) refers to an immunosuppressive receptor belonging to the CD28 family. CTLA-4 is expressed exclusively on T cells in vivo and binds to two ligands, CD80 and CD86 (also known as B7-1 and B7-2, respectively). As used herein, the term "CTLA-4" includes human CTLA-4 (hCTLA-4), variants, isoforms, and species homologs of hCTLA-4, as well as analogs having at least one epitope in common with hCTLA-4. The complete hCTLA-4 sequence is available under Genbank acceptance number AAB59385.

[0079] The term "fusion protein" originally refers to a protein created by the fusion of two or more genes that code for separate proteins. Translation of this fusion gene yields a single polypeptide or a group of polypeptides that possess functional properties derived from each of the original proteins. In some embodiments, the two or more genes may include substitutions, deletions, and / or additions to their nucleotide sequences.

[0080] An "Fc receptor" or "FcR" is a receptor that binds to the Fc region of immunoglobulins. FcRs that bind to IgG antibodies include the FcγR family of receptors, which also include allele variants and alternative splicing forms of these receptors. The FcγR family consists of three activating receptors (FcγRI, FcγRIII, and FcγRIV in mice; FcγRIA, FcγRIIA, and FcγRIIIA in humans) and one inhibitory receptor (FcγRIIB). Various properties of human FcγR are known in the art. The majority of congenital effector cell types co-express one or more activating FcγRs and the inhibitory FcγRIIB, while natural killer (NK) cells in mice and humans selectively express one activating Fc receptor (FcγRIII in mice, FcγRIIIA in humans) but do not selectively express the inhibitory FcγRIIB. Human IgG1 binds to most human Fc receptors and is considered equivalent to mouse IgG2a in terms of the types of activated Fc receptors it binds to.

[0081] The term "Fc region" (fragment crystallizable region), "Fc domain," or "Fc" refers to the C-terminal region of an antibody's heavy chain that mediates the binding of immunoglobulins to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or to the first component (C1q) of the classical complement system. Therefore, the Fc region includes the constant region of the antibody, excluding the first constant-region immunoglobulin domain (e.g., CH1 or CL). In the antibody isotypes IgG, IgA, and IgD, the Fc region contains two identical protein fragments derived from the second (CH2) and third (CH3) constant domains of the two heavy chains of the antibody, while the Fc region of IgM and IgE contains three heavy-chain constant domains (CH domains 2-4) in each polypeptide chain. In the case of IgG, the Fc region includes the immunoglobulin domains CH2 and CH3, as well as the hinge between the CH1 and CH2 domains. As defined herein, the definition of the boundary of the Fc region of an immunoglobulin heavy chain can vary, but the human IgG heavy chain Fc region is defined as extending from amino acid residue D221 in IgG1, from V222 in IgG2, from L221 in IgG3, and from P224 in IgG4 to the carboxyl terminus of the heavy chain, with numbering here following the EU index, similar to Kabat. The CH2 domain of the human IgG Fc region extends from amino acid 237 to amino acid 340, and the CH3 domain is located on the C-terminal side of the CH2 domain of the Fc region. That is, it extends from amino acid 341 to amino acid 447 or 446 (if the C-terminal lysine residue is absent) or 445 (if the C-terminal glycine and lysine residues are absent) of IgG. As used herein, the Fc region may be any allotype variant or variant Fc (e.g., Fc that does not exist in nature), or a natural sequence Fc. Fc may refer to this region either on its own or in the context of protein polypeptides containing Fc, such as "binding proteins containing an Fc region" (e.g., antibodies or immunoadhesins), also known as "Fc fusion proteins."

[0082] The "natural sequence Fc region" or "natural sequence Fc" contains the same amino acid sequence as the naturally occurring Fc region. The natural sequence human Fc region contains natural sequence human IgG1 This includes Fc regions, natural human IgG2 Fc regions, natural human IgG3 Fc regions, and natural human IgG4 Fc regions, as well as their naturally occurring variants. Natural Fc regions include various allotypes of Fc (e.g., Jefferis See et al. (2009) mAbs 1:1).

[0083] Furthermore, the Fc (natural or variant) of the present invention may be in a form having natural glycans, increased or decreased glycans compared to the natural type, or it may be deglycosylated. Immunoglobulin Fc glycans can be modified by conventional methods such as chemical, enzymatic, and microbial genetic engineering. Removal of glycans from the Fc fragment sharply reduces the binding affinity of the first complement component C1 to the C1q portion, resulting in a decrease or loss of ADCC or CDC, and thus preventing the induction of unwanted immune responses in vivo. In this regard, deglycosylated or aglycosylated immunoglobulin Fc regions may be more suitable for the purposes of the present invention as drug carriers. As used herein, the term "deglycosylated" means an Fc region from which sugars have been removed from the Fc fragment by enzymes. Furthermore, the term "aglycosylated" means that the Fc fragment is produced in a nonglycosylated form by prokaryotes, preferably in E. coli.

[0084] As used herein, the term “immune response” refers to a biological response in vertebrates to foreign agents, meaning a response that protects the organism from these agents and the diseases they cause. An immune response is mediated by the action of immune system cells (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and soluble macromolecules (including antibodies, cytokines, and complement) produced by any of these cells or the liver, resulting in the selective targeting, binding, damage, destruction, and / or elimination of invading pathogens, pathogen-infected cells or tissues, cancer cells or other abnormal cells, or, in the case of autoimmune or pathological inflammation, normal human cells or tissues in the body of a vertebrate. Examples of immune responses include T cells, e.g., CD4 + Or CD8 + This includes activation or inhibition of effector T cells or Th cells, such as T cells, or inhibition of Treg cells.

[0085] "Immunomodulatory substances" or "immunomodulatory substances" means substances that may be involved in the modulation, control, or modification of the immune response, such as components of signaling pathways. "Modification," "control," or "modification" of the immune response means any change in the activity of immune system cells or such cells (e.g., effector T cells). Such modulation may include stimulation or suppression of the immune system, which may manifest as an increase or decrease in the number of various cell types, an increase or decrease in the activity of these cells, or any other change that may occur within the immune system. Both suppressive and stimulative immunomodulators have been identified, some of which may enhance function in the tumor microenvironment. In a preferred embodiment, immunomodulators are located on the surface of T cells. "Immunomodulatory targets" or "immunomodulatory targets" are immunomodulators that are targeted for binding by a substance, substance, part, compound, or molecule, and whose activity is altered by this binding. Examples of immunomodulatory targets include cell surface receptors ("immunomodulatory receptors") and receptor ligands ("immunomodulatory ligands").

[0086] The term “immunotherapy” means treating a diseased subject or a subject at risk of developing or relapsing a disease by means of inducing, enhancing, suppressing, or otherwise modifying the immune response. “Treatment” or “therapy” of a subject means any intervention or process or administration of an active agent performed on the subject with the aim of reversing, reducing, restoring, inhibiting, slowing, or preventing the onset, progression, manifestation, severity, or relapse of disease-related symptoms, complications, conditions, or biochemical signs.

[0087] "Immune stimulating therapy" refers to a therapy that aims to increase (induce or enhance) the immune response, for example, to treat cancer.

[0088] "Enhancing the endogenous immune response" means increasing the effectiveness or potency of the current immune response in a subject. Such increases in effectiveness and potency can be achieved, for example, by overcoming mechanisms that suppress the endogenous host immune response or by stimulating mechanisms that enhance the endogenous host immune response.

[0089] The term "effector T cell" (Teff) refers to a T cell that possesses cytolytic activity (e.g., CD4). + and CD8 + In addition to T cells, this also includes helper T (Th) cells that secrete cytokines and activate and induce other immune cells, but does not include regulatory T cells (Treg cells). The combination of IL-7 protein and immune checkpoint inhibitors (e.g., anti-PD-1 antibodies) targets Teff cells, e.g., CD4 cells, in the target tumor or blood. + and CD8 + It activates T cells and / or increases their incidence.

[0090] As used herein, the term “regulatory T cells” (Treg) means a population of T cells that have the ability to modulate the immune response by reducing or suppressing the induction and proliferation of effector T cells. In some embodiments, Tregs can suppress the immune response by secreting anti-inflammatory cytokines such as IL-10, TGF-β, and IL-35, which can interfere with the activation and differentiation of naive T cells into effector T cells. In some embodiments, Tregs can also produce cytolytic molecules such as granzyme B, which can induce apoptosis of effector T cells. In some embodiments, regulatory T cells are endogenous regulatory T cells (nTregs) (i.e., they arise in the thymus). In some embodiments, regulatory T cells are inducible regulatory T cells (i.e., naive T cells that differentiate into Tregs in peripheral tissues when exposed to specific stimuli). Methods for identifying Tregs are known in the art. For example, Tregs express specific phenotypic markers (e.g., CD25, Foxp3, or CD39) that can be measured using flow cytometry. See, for example, International Publication No. WO2017 / 062035A1, Gu J., et al., Cell Mol Immunol 14(6):521-528 (2017). In some embodiments, Tregs express CD45RA - CD39 + These are T cells.

[0091] As used herein, the terms “tumor-infiltrating lymphocytes” or “TILs” mean lymphocytes (e.g., effector T cells) that have migrated into a tumor from a peripheral source (e.g., from the blood). In some embodiments, tumor-infiltrating lymphocytes are CD4+ TILs. In other embodiments, tumor-infiltrating lymphocytes are CD8+ TILs.

[0092] The increased ability to stimulate the immune response or immune system may be due to improved agonist activity of T cell costimulatory receptors and / or improved antagonist activity of inhibitory receptors. This increased ability to stimulate the immune response or immune system may be reflected in the increased EC50 or maximum activity level in assays measuring immune responses, such as cytokine or chemokine release, cytolytic activity (determined directly or indirectly by detecting CD107a or granzymes in target cells), and proliferation changes. The ability to stimulate the immune response or immune system activity may increase by at least 10%, 30%, 50%, 75%, 2x, 3x, 5x, or more.

[0093] As used herein, the terms “interleukin-7” or “IL-7” mean IL-7 polypeptides and their derivatives and analogs that have substantial amino acid sequence identity and substantially equivalent biological activity to wild-type mature mammalian IL-7 in, for example, a standard bioassay or assay of IL-7 receptor binding affinity. For example, IL-7 means i) a natural IL-7 polypeptide or a naturally occurring allele variant of an IL-7 polypeptide, ii) a biologically active fragment of an IL-7 polypeptide, iii) a biologically active polypeptide analog of an IL-7 polypeptide, or iv) the amino acid sequence of a recombinant or non-recombinant polypeptide having the amino acid sequence of a biologically active variant of an IL-7 polypeptide. The IL-7 polypeptides of the present invention can be obtained from any species, e.g., humans, cattle, or sheep. The IL-7 nucleic acid and amino acid sequence are well known in the art. For example, the human IL-7 amino acid sequence is Genbank acceptance number P13232 (SEQ ID NO: 1), the mouse IL-7 amino acid sequence is Genbank acceptance number P10168 (SEQ ID NO: 3), the rat IL-7 amino acid sequence is Genbank acceptance number P56478 (SEQ ID NO: 2), the monkey IL-7 amino acid sequence is Genbank acceptance number NP_001279008 (SEQ ID NO: 4), the bovine IL-7 amino acid sequence is Genbank acceptance number P26895 (SEQ ID NO: 5), and the sheep IL-7 amino acid sequence is Genbank acceptance number Q28540 (SEQ ID NO: 6). In some embodiments, the IL-7 polypeptides of this disclosure are variants of the IL-7 protein.

[0094] A “variant” of the IL-7 protein is defined as an amino acid sequence in which one or more amino acids are altered. Variants may have “conservative” changes, such as the substitution of leucine with isoleucine, where the structural or chemical properties of the substituted amino acid are similar. Relatively rare, variants may also have “non-conservative” changes, such as the substitution of glycine with tryptophan. Similar minor changes may include the deletion or insertion of amino acids, or both. Guidance in determining the types and number of amino acid residues that can be substituted, inserted, or deleted without loss of biological activity can be found using computer programs well known in the art, such as molecular modeling or alignment generation software. Variant IL-7 proteins included in the present invention include IL-7 proteins that retain IL-7 activity. Similarly, IL-7 polypeptides containing additions, substitutions, or deletions are also included in the present invention, insofar as the protein retains substantially equivalent biological IL-7 activity. For example, cleavage forms of IL-7 that retain biological activity equivalent to that of the full-length IL-7 protein are included in the present invention. The activity of the IL-7 protein can be measured using an in vitro cell proliferation assay as described in Example 6 below. The activity of the IL-7 variant of the present invention maintains at least 10%, 20%, 40%, 60%, more preferably 80%, 90%, 95%, and even more preferably 99% of the biological activity of wild-type IL-7.

[0095] Variant IL-7 proteins also include polypeptides that have at least approximately 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, or more sequence identity with wild-type IL-7. To determine the percentage of identity between two amino acid sequences or two nucleic acids, the sequences are aligned for optimal comparison purposes (for example, a gap may be introduced into the first amino acid sequence or nucleic acid sequence for optimal alignment with the second amino acid or nucleic acid sequence). The percentage of identity between two sequences is a function of the number of identical positions common to the sequences (i.e., homology % = number of identical positions / total number of positions × 100). The determination of the percentage of homology between two sequences can be performed using mathematical algorithms. A preferred non-restrictive example of a mathematical algorithm used to compare two sequences is the algorithm described in Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as described in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such algorithms are incorporated into the NBLAST and XBLAST programs in Altschul, et al., (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed using the NBLAST program with a score of 100 and a word length of 12. BLAST protein searches can be performed using the XBLAST program with a score of 50 and a word length of 3. To obtain gapped alignments for comparative purposes, Gapped BLAST can be used, as described in Altschul et al., (1997) Nucleic Acids Research 25(17):3389-3402. When using the BLAST and Gapped BLAST programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used.

[0096] As used herein, the term "Programmed Death-1 (PD-1)" means an immunosuppressive receptor belonging to the CD28 family. PD-1 is in It is primarily expressed in T cells that have been previously activated in vivo and binds to two ligands, PD-L1 and PD-L2. As used herein, the term "PD-1" includes human PD-1 (hPD-1), variants, isoforms, and species homologs of hPD-1, as well as analogs having at least one epitope in common with hPD-1. The complete hPD-1 sequence is available under Genbank acceptance number U64863.

[0097] As used herein, the term “Programmed Death Ligand-1 (PD-L1)” refers to one of the two cell surface glycoprotein ligands of PD-1 that, upon binding to PD-1, downregulate T cell activation and cytokine secretion (the other being PD-L2). As used herein, the term “PD-L1” includes human PD-L1 (hPD-L1), variants, isoforms, and species homologs of hPD-L1, as well as analogs having at least one epitope in common with hPD-L1. The complete hPD-L1 sequence is available under Genbank acceptance number Q9NZQ7.

[0098] The term "subject" includes all humans or non-human animals. The term "non-human animals" includes, but is not limited to, non-human primates, sheep, dogs, and vertebrates such as mice, rats, and guinea pigs. In some embodiments, the subject is human. The terms "subject" and "patient" are used synonymously herein.

[0099] The term “therapeutic effective dose” or “therapeutic effective dose” means the amount of a drug that produces a desired biological, therapeutic, and / or preventive outcome. The outcome may be a reduction, recovery, mitigation, attenuation, delay, and / or mitigation of one or more signs, symptoms, or causes of a disease, or any other desired change in the biological system. With respect to solid tumors, an effective dose includes an amount sufficient to shrink the tumor and / or reduce the rate of tumor growth (e.g., inhibit tumor growth), or to prevent or delay other undesirable cell proliferation. In some embodiments, an effective dose is sufficient to delay tumor development. In some embodiments, an effective dose is sufficient to prevent or delay tumor recurrence. An effective dose may be administered in one or more doses. An effective dose of a drug or composition can (i) reduce the number of cancer cells, (ii) reduce tumor size, (iii) inhibit, delay, to some extent slow, and possibly halt the invasion of cancer cells into peripheral organs, (iv) inhibit tumor metastasis (i.e., to some extent slow, and possibly halt), (v) inhibit tumor growth, (vi) prevent or delay tumor development and / or recurrence, and / or (vii) to some extent alleviate one or more of the symptoms associated with cancer. In some embodiments, the “therapeutic effective dose” is a combination of the amount of IL-7 protein and the amount of an immune checkpoint inhibitor (e.g., PD-1 pathway inhibitor, e.g., anti-PD-1 antibody) that has been clinically proven to result in a significant reduction in cancer, such as advanced solid tumors, or a slowing (regression) of cancer progression. The ability of a therapeutic agent to promote disease regression can be evaluated using a variety of methods known to skilled physicians, for example, in human subjects during clinical trials, in animal model systems to predict efficacy in humans, or by assaying the activity of the drug in in vitro assays.

[0100] The term “medication frequency” refers to the number of times a therapeutic agent (e.g., IL-7 protein or immune checkpoint inhibitor) is administered to a subject during a given period. Medication frequency can be expressed as the number of doses per unit of time, for example, once a day, once a week, or once every two weeks. As used herein, “medication frequency” applies when a subject receives multiple (or repeated) doses of the therapeutic agent.

[0101] As used herein, the term “standard treatment” means a treatment that is recognized by healthcare professionals as appropriate for a particular type of disease and is widely used by healthcare professionals. This term may be used synonymously with the following terms: “best practice,” “standard medical care,” and “standard therapy.”

[0102] As used herein, the term “drug” means any bioactive agent (e.g., IL-7 protein or immune checkpoint inhibitor) intended to be administered to humans or non-human mammals to prevent or treat disease or other undesirable conditions. Drugs include hormones, growth factors, proteins, peptides, and other compounds. In some embodiments, the drugs disclosed herein are anticancer agents.

[0103] For example, an "anti-cancer drug" promotes the regression of the target cancer or prevents further tumor growth. In certain embodiments, a therapeutically effective dose of the drug promotes cancer regression to the point of eliminating the cancer. "Promoting cancer regression" means that, by administering an effective dose of the drug alone or in combination with an anti-neoplastic agent, it results in a reduction of tumor growth or size, tumor necrosis, a decrease in the severity of at least one disease symptom, an increase in the frequency and duration of asymptomatic periods of the disease, or prevention of functional impairment or disability caused by the disease. Furthermore, the terms "effective" and "effective" in relation to treatment include both pharmacological efficacy and physiological safety. Pharmacological efficacy means the ability of a drug to promote cancer regression in a patient. Physiological safety means the level of toxicity or other harmful physiological effects (adverse effects) at the cellular, organ, and / or biological level resulting from the administration of the drug.

[0104] As an example of tumor treatment, a therapeutically effective dose of an anticancer drug may inhibit cell growth or tumor growth by at least about 10%, at least about 20%, at least about 40%, at least about 60%, or at least about 80% compared to an untreated subject, or in certain embodiments, compared to a patient treated with standard therapy. In other embodiments of the invention, tumor regression may be observed and persist over a period of at least about 20 days, at least about 40 days, or at least about 60 days. Regardless of these final measures of therapeutic efficacy, the evaluation of immunotherapies must also take into account “immune-related” response patterns.

[0105] As used herein, the term “immune checkpoint inhibitor” means a molecule that completely or partially reduces, inhibits, interferes with, or modulates one or more checkpoint proteins. Checkpoint proteins regulate the activation or function of T cells. Numerous checkpoint proteins are known, including CTLA-4 and its ligands CD80 and CD86, and PD-1 and its ligands PD-L1 and PD-L2. Pardoll, DM, Nat Rev Cancer 12(4):252-64 (2012). These proteins are responsible for co-stimulatory or inhibitory interactions in the T cell response. Immune checkpoint proteins regulate and maintain self-tolerance, as well as the duration and extent of the physiological immune response. Immune checkpoint inhibitors contain or are derived from antibodies.

[0106] As used herein, the term "reference" means a corresponding subject (e.g., a cancer subject) that did not receive the combination of IL-7 protein and an immune checkpoint inhibitor, for example, a subject that received IL-7 protein alone or an immune checkpoint inhibitor alone. In some embodiments, the reference subject is one that did not receive either IL-7 protein or an immune checkpoint inhibitor. The term "reference" may also mean the same cancer subject but before the combination administration of IL-7 protein and an immune checkpoint inhibitor. In certain embodiments, the term "reference" means the average of the population of subjects (e.g., cancer subjects).

[0107] As used herein, the terms "ug" and "uM" are synonymous with "μg" and "μM," respectively.

[0108] The various embodiments described herein are described in more detail in the following subsections.

[0109] II. Method of Disclosure This disclosure relates to a method for treating a tumor (or cancer) in a subject requiring such treatment, comprising administering to the subject an effective amount of interleukin-7 (IL-7) protein in combination with an effective amount of an immune checkpoint inhibitor. Non-limiting examples of immune checkpoint inhibitors that can be used in the method of the present invention include anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, and combinations thereof.

[0110] In some embodiments, a combination of the IL-7 protein and an immune checkpoint inhibitor can increase the absolute number of lymphocytes in a subject when administered to that subject. In certain embodiments, the absolute number of lymphocytes increases by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% or more compared to a reference (e.g., the value of the corresponding subject after administration of the IL-7 protein alone or an immune checkpoint inhibitor alone).

[0111] In some embodiments, when the combinations disclosed herein (i.e., combinations of IL-7 protein and immune checkpoint inhibitors) are administered to a target, the proliferation of the target T cells (e.g., CD8) is stimulated. + T cells may increase. In certain embodiments, increased T cell proliferation occurs peripherally (e.g., outside the tumor). In some embodiments, administration of a combination of IL-7 protein and an immune checkpoint inhibitor may increase effector T cells (e.g., cytotoxic CD8) to the tumor in the target. + This can increase the recruitment of T lymphocytes.

[0112] In certain embodiments, T cell proliferation increases by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% or more compared to a reference (e.g., the corresponding target value after administration of IL-7 protein alone or an immune checkpoint inhibitor alone). In certain embodiments, T cells that proliferate in response to IL-7 administration (e.g., CD8 + T cells express one or more of the following markers: Eomesodermin (Eomes), granzyme B, CXCR3, IFN-γ, or a combination thereof.

[0113] In certain embodiments, the recruitment of effector T cells to the tumor is increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% or more compared to a reference (e.g., the corresponding target value after administration of IL-7 protein alone or an immune checkpoint inhibitor alone).

[0114] In some embodiments, administration of a combination of IL-7 protein and an immune checkpoint inhibitor inhibits and / or reduces tumor growth in a subject. In some embodiments, tumor growth (e.g., tumor volume or weight) is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% compared to a reference (e.g., tumor volume in a corresponding subject after administration of IL-7 protein alone or an immune checkpoint inhibitor alone).

[0115] In some embodiments, a tumor or a subject suffering from a tumor is treated by administering a combination of the IL-7 protein and an immune checkpoint inhibitor to promote and / or enhance the immune response to tumor antigens. In some embodiments, administration of the compositions of the Disclosure promotes the immune response to tumor-infiltrating lymphocytes (TILs) (e.g., CD4) in the tumor of the subject. + or CD8 + ) increases the number and / or proportion of TILs. In some embodiments, the number and / or proportion of TILs increases after administration by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 125%, at least about 150%, at least about 200%, at least about 250%, or at least about 300%, compared to a reference (e.g., the number and / or proportion of TILs in a target tumor treated with either IL-7 protein alone or an immune checkpoint inhibitor alone).

[0116] In some embodiments, administration of a combination of IL-7 protein and an immune checkpoint inhibitor reduces the number and / or proportion of regulatory T cells (Tregs) in the target tumor. In some embodiments, regulatory T cells are CD4 + These are regulatory T cells. In some embodiments, regulatory T cells are Foxp3 +In certain embodiments, the number and / or proportion of regulatory T cells in a tumor is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least 100% compared to a reference (e.g., the corresponding number and / or proportion in a subject treated with IL-7 protein alone or an immune checkpoint inhibitor alone).

[0117] In some embodiments, administration of a combination of IL-7 protein and an immune checkpoint inhibitor can induce CD8 in the target tumor. + The ratio of TIL to Treg increases. In certain embodiments, CD8 + The ratio of TILs to Tregs increases after administration by at least approximately 5%, at least approximately 10%, at least approximately 20%, at least approximately 30%, at least approximately 40%, at least approximately 50%, at least approximately 60%, at least approximately 70%, at least approximately 80%, at least approximately 90%, at least approximately 100%, at least approximately 125%, at least approximately 150%, at least approximately 200%, at least approximately 250%, or at least approximately 300%, compared to a reference (e.g., the number and / or percentage of TILs in the target tumor treated with either IL-7 protein alone or an immune checkpoint inhibitor alone).

[0118] In some embodiments, administration of a combination of IL-7 protein and an immune checkpoint inhibitor reduces the number and / or proportion of myeloid-derived suppressor cells (MDSCs) in the tumor of interest. As used herein, the term “myeloid-derived suppressor cells” (MDSCs) means a heterogeneous population of immune cells defined by their myeloid origin, immature state, and ability to potently suppress T cell responses. They are known to proliferate in certain pathological conditions, such as chronic infection and cancer. In certain embodiments, the MDSCs are monocytic MDSCs (M-MDSCs). In other embodiments, the MDSCs are polymorphonuclear MDSCs (PMN-MDSCs). In some embodiments, the number and / or proportion of MDSCs in the tumor is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least 100% compared to a reference (e.g., the value of the corresponding subject after administration of IL-7 protein alone or an immune checkpoint inhibitor alone).

[0119] In some embodiments, administration of a combination of IL-7 protein and an immune checkpoint inhibitor can induce CD8 in the target tumor. + The ratio of TIL to MDSC increases. In certain embodiments, CD8 + The ratio of TIL to MDSC increases after administration by at least approximately 5%, at least approximately 10%, at least approximately 20%, at least approximately 30%, at least approximately 40%, at least approximately 50%, at least approximately 60%, at least approximately 70%, at least approximately 80%, at least approximately 90%, at least approximately 100%, at least approximately 125%, at least approximately 150%, at least approximately 200%, at least approximately 250%, or at least approximately 300%, compared to a reference (e.g., the value of the corresponding target after administration of IL-7 protein alone or an immune checkpoint inhibitor alone).

[0120] In some embodiments, administration of a combination of IL-7 protein and an immune checkpoint inhibitor reduces the expression of an immune checkpoint inhibitor molecule (e.g., PD-1) in the target TIL. In certain embodiments, the combination of IL-7 protein and an immune checkpoint inhibitor reduces the mean fluorescence index (MFI) of the immune checkpoint inhibitor molecule (e.g., PD-1) in the TIL. In some embodiments, the immune checkpoint inhibitor molecule is PD-1. In certain embodiments, administration of a combination of IL-7 protein and an immune checkpoint inhibitor reduces the expression of CD8 + MFI of PD-1 expression in TIL is reduced by at least approximately 5%, at least approximately 10%, at least approximately 20%, at least approximately 30%, at least approximately 40%, at least approximately 50%, at least approximately 60%, at least approximately 70%, at least approximately 80%, at least approximately 90%, or at least approximately 100% compared to a reference (e.g., the corresponding number and / or percentage in subjects treated with IL-7 protein alone or an immune checkpoint inhibitor alone).

[0121] In some embodiments, administration of a combination of IL-7 protein and an immune checkpoint inhibitor increases the expression of markers associated with effector (e.g., antitumor) activity in the target TIL. Non-limiting examples of effector activity-related markers include Ki-67, granzyme B, T-bet, Eomes, CXCR3, IFN-γ, TNF-α, and IL-2. In certain embodiments, the effector activity-related markers include Ki-67 and granzyme B. In certain embodiments, the combination of IL-7 protein and an immune checkpoint inhibitor increases the mean fluorescence index (MFI) of the expression of effector activity-related markers in TIL by at least approximately 5%, at least approximately 10%, at least approximately 20%, at least approximately 30%, at least approximately 40%, at least approximately 50%, at least approximately 60%, at least approximately 70%, at least approximately 80%, at least approximately 90%, or at least approximately 100%, compared to a reference (e.g., the corresponding value in the target that did not receive the combination of IL-7 protein and an immune checkpoint inhibitor).

[0122] As described above, many cancer treatments using available standard therapies (e.g., chemotherapy and radiotherapy) are known to cause lymphopenia, and many cancer patients are lymphopenic. Therefore, the methods disclosed herein can also be used to treat cancers targeting lymphopenia.

[0123] As used herein, the term “lymphopenic subject” means a subject having lymphopenia. As used herein, the terms “lymphopenia” and “lymphopenia” are used synonymously and refer to a condition characterized by an abnormally low number of circulating immune cells (e.g., lymphocytes). In patients suffering from lymphopenia, all types of lymphocytes or subgroups of lymphocytes (e.g., CD4) +Peripheral circulation of T cells may be depleted or abnormally low. See, for example, Lymphopenia Description, The Merck Manual (18th Edition, 2006, Merck & Co.). In some embodiments, compared to normal subjects (e.g., healthy subjects), lymphopenic subjects have fewer T lymphocytes ("T lymphopenia"), B lymphocytes ("B lymphopenia"), and / or NK cells ("NK lymphopenia").

[0124] Lymphopenia can be quantitatively expressed by various cutoffs. In some embodiments, a lymphopenic subject has a circulating total lymphocyte count that is at least approximately 5%, at least approximately 10%, at least approximately 20%, at least approximately 30%, at least approximately 40%, at least approximately 50%, at least approximately 60%, at least approximately 70%, at least approximately 80%, at least approximately 90%, or at least approximately 100% lower than that of a corresponding subject without lymphopenia. In some embodiments, a subject has lymphopenia if its circulating total lymphocyte count is less than approximately 1,500 lymphocytes per μL, less than approximately 1,000 lymphocytes per μL, less than approximately 800 lymphocytes per μL, less than approximately 500 lymphocytes per μL, or less than approximately 200 lymphocytes per μL.

[0125] Lymphopenia has a wide range of possible causes. In some aspects, lymphopenia is caused by or associated with tumors. In some aspects, lymphopenia is associated with or caused by past treatments for tumors (e.g., chemotherapy or radiotherapy). In some aspects, lymphopenia is caused by or associated with infections, including viral infections (e.g., HIV infection or hepatitis infection), bacterial infections (e.g., active tuberculosis infection), and fungal infections, chronic right ventricular failure, Hodgkin's disease and lymphatic cancers, leukemia, thoracic duct leakage or rupture, side effects of prescription drugs, including anticancer drugs, antiviral drugs, and glucocorticoids, malnutrition resulting from a low-protein diet, radiotherapy, uremia, autoimmune disorders, immunodeficiency syndromes, high stress levels, and trauma.

[0126] In some aspects, lymphopenia is idiopathic (i.e., of unknown etiology). Non-exclusive examples of idiopathic lymphopenia include idiopathic CD4-positive T lymphopenia (ICL), acute radiation syndrome (ARS), or a combination thereof.

[0127] In some embodiments, administration of IL-7 protein in combination with an immune checkpoint inhibitor to lymphopenic subjects with tumors inhibits and / or reduces tumor growth in the subjects. In some embodiments, tumor growth (e.g., tumor volume or weight) is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% compared to a reference (e.g., tumor volume in the corresponding subject after administration of IL-7 protein alone or an immune checkpoint inhibitor alone).

[0128] In some embodiments, when the IL-7 protein is administered in combination with an immune checkpoint inhibitor to lymphopenic subjects with tumors, tumor-infiltrating lymphocytes (TILs) (e.g., CD4) in the target tumors are stimulated. + or CD8 + The number and / or proportion of TILs increases. In some embodiments, the number and / or proportion of TILs increases after administration by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 125%, at least about 150%, at least about 200%, at least about 250%, or at least about 300%, compared to a reference (e.g., the number and / or proportion of TILs in a target tumor treated with either IL-7 protein alone or an immune checkpoint inhibitor alone).

[0129] In some embodiments, administration of the IL-7 protein in combination with an immune checkpoint inhibitor to lymphopenic subjects with tumors reduces the number and / or proportion of regulatory T cells in the subjects' tumors. In some embodiments, regulatory T cells are Foxp3 + In certain embodiments, the number and / or proportion of regulatory T cells in a tumor is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least 100% compared to a reference (e.g., the corresponding number and / or proportion in a subject treated with IL-7 protein alone or an immune checkpoint inhibitor alone).

[0130] In some embodiments, administration of a combination of IL-7 protein and an immune checkpoint inhibitor to lymphopenic subjects leads to CD8 in the tumors of the subjects. + The ratio of TIL to Treg increases. In certain embodiments, CD8 + The ratio of TILs to Tregs increases after administration by at least approximately 5%, at least approximately 10%, at least approximately 20%, at least approximately 30%, at least approximately 40%, at least approximately 50%, at least approximately 60%, at least approximately 70%, at least approximately 80%, at least approximately 90%, at least approximately 100%, at least approximately 125%, at least approximately 150%, at least approximately 200%, at least approximately 250%, or at least approximately 300%, compared to a reference (e.g., the number and / or percentage of TILs in the target tumor treated with either IL-7 protein alone or an immune checkpoint inhibitor alone).

[0131] In some embodiments, administration of a combination of IL-7 protein and an immune checkpoint inhibitor to lymphopenic subjects reduces the expression of immune checkpoint inhibitor molecules (e.g., PD-1) in the subject's TILs. In specific embodiments, the combination of IL-7 protein and an immune checkpoint inhibitor reduces the mean fluorescence index (MFI) of immune checkpoint inhibitor molecule (e.g., PD-1) expression in TILs. In some embodiments, the immune checkpoint inhibitor molecule is PD-1. In specific embodiments, administration of a combination of IL-7 protein and an immune checkpoint inhibitor reduces CD8 + MFI of PD-1 expression in TIL is reduced by at least approximately 5%, at least approximately 10%, at least approximately 20%, at least approximately 30%, at least approximately 40%, at least approximately 50%, at least approximately 60%, at least approximately 70%, at least approximately 80%, at least approximately 90%, or at least approximately 100% compared to a reference (e.g., the corresponding number and / or percentage in subjects treated with IL-7 protein alone or an immune checkpoint inhibitor alone).

[0132] Non-limiting examples of cancers (or tumors) that can be treated by the methods disclosed herein include squamous cell carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer, squamous non-small cell lung cancer (NSCLC), non-squamous NSCLC, gastrointestinal cancer, kidney cancer (e.g., clear cell carcinoma), ovarian cancer, liver cancer (e.g., hepatocellular carcinoma), colorectal cancer, endometrial cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), prostate cancer (e.g., hormone-refractory prostate cancer), thyroid cancer, pancreatic cancer, cervical cancer, stomach cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, and head and neck cancer (and more). Carcinomas, gastric cancer, germ cell tumors, pediatric sarcomas, sinus natural killer cancers, melanoma (e.g., metastatic melanoma, malignant melanoma of the skin or eyeball), bone cancer, skin cancer, uterine cancer, anal cancer, testicular cancer, fallopian tube cancer, endometrial carcinoma, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer (e.g., gastroesophageal junction cancer), small intestine cancer, endocrine cancers, parathyroid cancer, adrenal cancer, soft tissue sarcomas, urethral cancer, penile cancer, solid tumors in childhood, ureteral cancer, renal pelvis cancer, tumor angiogenesis, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, as Environmentally induced cancers, including those induced by the best, virus-associated cancers or cancers of viral origin (e.g., human papillomavirus (tumors associated with or derived from HPV)), and hematological malignancies originating from either of the two main hematological cell lines, namely the myeloid line (producing granulocytes, erythrocytes, platelets, macrophages, and mast cells) or the lymphoid line (producing B cells, T cells, NK cells, and plasma cells), such as all types of leukemia, lymphoma, and myeloma, e.g., acute, chronic, lymphocytic and / or This includes myeloid leukemias, such as acute leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML), anaplastic AML (MO), myeloblastic leukemia (M1), myeloblastic leukemia (M2; with cell maturation), promyelocytic leukemia (M3 or M3 variant [M3V]), myelomonocytic leukemia (M4 or M4 variant with eosinophilia [M4E]), monocytic leukemia (M5), erythroleukemia (M6), megakaryoblastic leukemia (M7), solitary granulocytic sarcoma, and chloroplast;Lymphomas, e.g., Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL); B-cell hematological malignancies, e.g., B-cell lymphoma, T-cell lymphoma, lymphoplasmacytic lymphoma, monocyticoid B-cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, undifferentiated (e.g., Ki1); + ) Large cell lymphoma, adult T-cell lymphoma / leukemia, mantle cell lymphoma, angioimmunoblastic T-cell lymphoma, vascular central lymphoma, intestinal T-cell lymphoma, mediastinal primary B-cell lymphoma, progenitor T-lymphoblastic lymphoma, T-lymphoblastic lymphoma; and lymphoma / leukemia (T-Lbly / T-ALL), peripheral T-cell lymphoma, lymphoblastic lymphoma, post-transplant lymphoproliferative disorder, histiocytic lymphoma, primary body Humoral lymphoma, B-cell lymphoma, lymphoblastic lymphoma (LBL), hematopoietic neoplasms of the lymphatic system, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt lymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma, progenitor B-lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTLC) (also known as mycosis fungoides or Sézary syndrome), and Waldenström. Lymphoplasmacytic lymphoma (LPL) with macroglobulinemia; myeloma, e.g., IgG myeloma, light chain myeloma, nonsecretory myeloma, smoldering myeloma (also called asymptomatic myeloma), solitary plasmacytoma, and multiple myeloma; chronic lymphocytic leukemia (CLL), hairy cell lymphoma; hematopoietic neoplasms of the myeloid system, mesenchymal tumors including fibrosarcoma and rhabdomyosarcoma; seminomas, teratomas, fibrosarcomas, rhabdomyosarcomas Mesenchymal tumors, including monosarcoma and osteosarcoma; as well as other tumors, such as melanoma, xeroderma pigmentosum, keratosacral cell tumor, seminoma, follicular thyroid carcinoma and teratoma; hematopoietic tumors of the lymphatic system, such as T-cell tumors and B-cell tumors; T-cell disorders, such as pre-T lymphocytic leukemia (T-PLL), including, but not limited to, small cell and cerebral-like cell types; large granular lymphocytic leukemia (LGL) of the T-cell type; a / d T-NHL hepatosplenic lymphoma; peripheral / postthymic T-cell lymphoma (pleomorphic and immunoblastic subtypes); vascular central (nasal) T-cell lymphoma; head and neck cancer, renal cancer, rectal cancer, thyroid cancer; acute myeloid lymphoma; and any combination thereof.

[0133] In some embodiments, the cancers (or tumors) that can be treated include breast cancer, head and neck cancer, uterine cancer, brain cancer, skin cancer, kidney cancer, lung cancer, colorectal cancer, prostate cancer, liver cancer, bladder cancer, renal cancer, pancreatic cancer, thyroid cancer, esophageal cancer, eye cancer, stomach cancer (gastric cancer), gastrointestinal cancer, carcinoma, sarcoma, leukemia, lymphoma, myeloma, or combinations thereof. In a particular embodiment, the cancer (or tumor) that can be treated by the method of the present invention is breast cancer. In some embodiments, breast cancer is triple-negative breast cancer (TNBC). In some embodiments, the cancer (or tumor) that can be treated is brain cancer. In a particular embodiment, brain cancer is gliablastoma. In some embodiments, the cancer (or tumor) that can be treated by the method of the present invention is skin cancer. In some embodiments, skin cancer is basal cell carcinoma (BCC), cutaneous squamous cell carcinoma (cSCC), melanoma, Merkel cell carcinoma (MCC), or combinations thereof. In certain aspects, head and neck cancer is head and neck squamous cell carcinoma. In further aspects, lung cancer is small cell lung cancer (SCLC). In some aspects, esophageal cancer is esophagogastric junction cancer. In certain aspects, kidney cancer is renal cell carcinoma. In some aspects, liver cancer is hepatocellular carcinoma.

[0134] In some embodiments, the methods described herein may also be used to treat metastatic cancer, unresectable refractory cancer (e.g., cancer that was refractory to previous cancer therapies, e.g., immunotherapy, e.g., therapy with anti-PD-1 blocking antibodies), and / or recurrent cancer. In certain embodiments, previous cancer therapies include chemotherapy. In some embodiments, chemotherapy includes platinum-based therapies. In some embodiments, platinum-based therapies include platinum-based antineoplastic agents selected from the group consisting of cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, satraplatin, and any combination thereof. In certain embodiments, platinum-based therapies include cisplatin. In further embodiments, platinum-based therapies include carboplatin.

[0135] In some embodiments, subjects treated by the methods disclosed herein have previously received one, two, three, four, five, or more cancer treatments. In other embodiments, subjects are treatment-naive (i.e., have never received cancer treatment before). In some embodiments, subjects are undergoing other cancer treatments. In certain embodiments, previous cancer treatments included immunotherapy (e.g., using anti-PD-1 antibodies). In other embodiments, previous cancer treatments included chemotherapy. In some embodiments, the tumor is recurrent. In some embodiments, the tumor is metastatic. In other embodiments, the tumor is not metastatic.

[0136] In some embodiments, the methods disclosed herein effectively extend the survival of subjects in need of it (e.g., those suffering from tumors). For example, in some embodiments, the survival of a subject is extended by at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 1 year compared to a reference subject (e.g., a corresponding subject treated with IL-7 protein alone or an immune checkpoint inhibitor alone). In other embodiments, the methods disclosed herein extend the survival of a subject to a level higher than the survival of a reference subject (e.g., a corresponding subject treated with IL-7 protein alone or an immune checkpoint inhibitor alone) (about 1 month higher, about 2 months higher, about 3 months higher, about 4 months higher, about 5 months higher, about 6 months higher, about 7 months higher, about 8 months higher, about 9 months higher, about 10 months higher, about 11 months higher, or about 1 year higher).

[0137] In some embodiments, the methods of the present disclosure effectively extend progression-free survival in subjects (e.g., cancer patients). For example, the progression-free survival of a subject is extended by at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 1 year compared to a reference subject (e.g., a corresponding subject treated with IL-7 protein alone or an immune checkpoint inhibitor alone).

[0138] In some embodiments, the methods disclosed herein effectively increase the response rate of the control group. For example, the response rate of the control group increases by at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% compared to a reference group (e.g., a corresponding group treated with IL-7 protein alone or an immune checkpoint inhibitor alone).

[0139] In some embodiments, the subject treated by this method is a non-human animal such as a rat or mouse. In some embodiments, the subject treated by this method is a human.

[0140] In some embodiments, the unit dose of the IL-7 protein disclosed herein (e.g., for human use) may be in the range of 0.001 mg / kg to 10 mg / kg. In certain embodiments, the unit dose of the IL-7 protein is in the range of 0.01 mg / kg to 2 mg / kg. In some embodiments, the unit dose is in the range of 0.02 mg / kg to 1 mg / kg. The unit dose may vary depending on the disease being treated and the presence of adverse effects. The IL-7 protein can be administered by periodic bolus injections or by external reservoirs (e.g., intravenous bags), or by continuous intravenous, subcutaneous, or intraperitoneal administration from internal sources (e.g., biodegradable implants). In certain embodiments, the IL-7 protein disclosed herein is administered by intramuscular injection.

[0141] In some embodiments, the IL-7 protein disclosed herein may be administered to a subject in a weight-based dose. In certain embodiments, the IL-7 protein may be administered in weight-based doses ranging from about 20 μg / kg to about 600 μg / kg. In further embodiments, the IL-7 protein of this disclosure may be administered in weight-based doses of about 20 μg / kg, about 60 μg / kg, about 120 μg / kg, about 240 μg / kg, about 360 μg / kg, about 480 μg / kg, or about 600 μg / kg.

[0142] In some embodiments, the IL-7 protein disclosed herein may be administered to subjects at doses greater than approximately 600 μg / kg. In certain embodiments, the IL-7 protein is administered to subjects at doses greater than approximately 600 μg / kg, greater than approximately 700 μg / kg, greater than approximately 800 μg / kg, greater than approximately 900 μg / kg, greater than approximately 1,000 μg / kg, greater than approximately 1,100 μg / kg, greater than approximately 1,200 μg / kg, greater than approximately 1,300 μg / kg, greater than approximately 1,400 μg / kg, greater than approximately 1,500 μg / kg, greater than approximately 1,600 μg / kg, greater than approximately 1,700 μg / kg, greater than approximately 1,800 μg / kg, greater than approximately 1,900 μg / kg, or greater than approximately 2,000 μg / kg.

[0143] In some embodiments, the IL-7 protein of this disclosure is available in concentrations of approximately 610 μg / kg to 1,200 μg / kg, 650 μg / kg to 1,200 μg / kg, 700 μg / kg to 1,200 μg / kg, 750 μg / kg to 1,200 μg / kg, 800 μg / kg to 1,200 μg / kg, 850 μg / kg to 1,200 μg / kg, 900 μg / kg to 1,200 μg / kg, 950 μg / kg to 1,200 μg / kg, 1,000 μg / kg to 1,200 μg / kg, 1,050 μg / kg to 1,200 μg / kg, 1,100 μg / kg to 1,200 μg / kg, and approximately 1,200 μg / kg. 0 μg / kg ~ approx. 2,000 μg / kg, approx. 1,300 μg / kg ~ approx. 2,000 μg / kg, approx. 1,500 μg / kg ~ approx. 2,000 μg / kg, approx. 1 , 700 μg / kg ~ approx. 2,000 μg / kg, approx. 610 μg / kg ~ approx. 1,000 μg / kg, approx. 650 μg / kg ~ approx. 1,000 μg / kg, approx. 700 It is administered in doses of approximately μg / kg to 1,000 μg / kg, approximately 750 μg / kg to 1,000 μg / kg, approximately 800 μg / kg to 1,000 μg / kg, approximately 850 μg / kg to 1,000 μg / kg, approximately 900 μg / kg to 1,000 μg / kg, or approximately 950 μg / kg to 1,000 μg / kg.

[0144] In some embodiments, the IL-7 protein of this disclosure is administered in doses ranging from 610 μg / kg to about 1,200 μg / kg. In certain embodiments, the IL-7 protein is administered in doses ranging from 650 μg / kg to about 1,200 μg / kg. In some embodiments, the IL-7 protein is administered in doses ranging from about 700 μg / kg to about 1,200 μg / kg. In further embodiments, the IL-7 protein is administered in doses ranging from about 750 μg / kg to about 1,200 μg / kg. In certain embodiments, the IL-7 protein is administered in doses ranging from about 800 μg / kg to about 1,200 μg / kg. In some embodiments, the IL-7 protein is administered in doses ranging from about 850 μg / kg to about 1,200 μg / kg. In some embodiments, the IL-7 protein is administered in doses ranging from about 900 μg / kg to about 1,200 μg / kg. In further embodiments, the IL-7 protein is administered in doses of approximately 950 μg / kg to approximately 1,200 μg / kg. In some embodiments, the IL-7 protein disclosed herein is administered in doses of approximately 1,000 μg / kg to approximately 1,200 μg / kg. In some embodiments, the IL-7 protein is administered in doses of approximately 1,050 μg / kg to approximately 1,200 μg / kg. In some embodiments, the IL-7 protein is administered in doses of approximately 1,100 μg / kg to approximately 1,200 μg / kg. In some embodiments, the IL-7 protein is administered in doses of approximately 1,200 μg / kg to approximately 2,000 μg / kg. In further embodiments, the IL-7 protein is administered in doses of approximately 1,300 μg / kg to approximately 2,000 μg / kg. In some embodiments, the IL-7 protein is administered in doses of approximately 1,500 μg / kg to approximately 2,000 μg / kg. In some embodiments, the IL-7 protein is administered in doses of approximately 1,700 μg / kg to approximately 2,000 μg / kg. In certain embodiments, the IL-7 protein is administered in doses of approximately 610 μg / kg to approximately 1,000 μg / kg. In some embodiments, the IL-7 protein is administered in doses of approximately 650 μg / kg to approximately 1,000 μg / kg. In further embodiments, the IL-7 protein is administered in doses of approximately 700 μg / kg to approximately 1,000 μg / kg.In further embodiments, the IL-7 protein is administered at a dose of approximately 750 μg / kg to approximately 1,000 μg / kg. In certain embodiments, the IL-7 protein is administered at a dose of approximately 800 μg / kg to approximately 1,000 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of approximately 850 μg / kg to approximately 1,000 μg / kg. In some embodiments, the IL-7 protein of this disclosure is administered at a dose of approximately 900 μg / kg to approximately 1,000 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of approximately 950 μg / kg to approximately 1,000 μg / kg.

[0145] In some embodiments, IL-7 protein is administered in doses of approximately 700 μg / kg to 900 μg / kg, approximately 750 μg / kg to 950 μg / kg, approximately 700 μg / kg to 850 μg / kg, approximately 750 μg / kg to 850 μg / kg, approximately 700 μg / kg to 800 μg / kg, approximately 800 μg / kg to 900 μg / kg, approximately 750 μg / kg to 850 μg / kg, or approximately 850 μg / kg to 950 μg / kg. In some embodiments, IL-7 protein is administered in doses of approximately 700 μg / kg to 900 μg / kg. In certain embodiments, IL-7 protein is administered in doses of approximately 750 μg / kg to 950 μg / kg. In further embodiments, the IL-7 protein is administered at a dose of approximately 700 μg / kg to approximately 850 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of approximately 750 μg / kg to approximately 850 μg / kg. In other embodiments, the IL-7 protein is administered at a dose of approximately 700 μg / kg to approximately 800 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of approximately 800 μg / kg to approximately 900 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of approximately 750 μg / kg to approximately 850 μg / kg. In certain embodiments, the IL-7 protein is administered at a dose of approximately 850 μg / kg to approximately 950 μg / kg.

[0146] In some embodiments, the IL-7 protein is present in concentrations of approximately 650 μg / kg, 680 μg / kg, 700 μg / kg, 720 μg / kg, 740 μg / kg, 750 μg / kg, 760 μg / kg, 780 μg / kg, 800 μg / kg, 820 μg / kg, 840 μg / kg, 850 μg / kg, 860 μg / kg, 880 μg / kg, 900 μg / kg, 920 μg / kg, 940 μg / kg, 950 μg / kg, and 960 μg / kg. g, approximately 980μg / kg, approximately 1,000μg / kg, approximately 1,020μg / kg, approximately 1,040μg / kg, approximately 1,060μg / kg, approximately 1,080μg / kg, approximately 1,100μg / kg, approximately 1,120μg / kg, approximately 1,140μg / k g, approximately 1,160μg / kg, approximately 1,180μg / kg, approximately 1,200μg / kg, approximately 1,220μg / kg, approximately 1,240μg / kg, approximately 1,260μg / kg, approximately 1,280μg / kg, approximately 1,300μg / kg, approximately 1,320μg / kg, approximately 1,340μg / kg, approximately 1,360μg / kg, approximately 1,380μg / kg, approximately 1,400μg / kg, approximately 1,420μg / kg, approximately 1,440μg / kg, approximately 1,460μg / kg, approximately 1,480μg / kg, approximately 1,500 1,6 It is administered in doses of 80 μg / kg, approximately 1,700 μg / kg, approximately 1,720 μg / kg, approximately 1,740 μg / kg, approximately 1,760 μg / kg, approximately 1,780 μg / kg, approximately 1,800 μg / kg, approximately 1,820 μg / kg, approximately 1,840 μg / kg, approximately 1,860 μg / kg, approximately 1,880 μg / kg, approximately 1,900 μg / kg, approximately 1,920 μg / kg, approximately 1,940 μg / kg, approximately 1,960 μg / kg, approximately 1,980 μg / kg, or approximately 2,000 μg / kg. In some embodiments, the IL-7 protein is administered in a dose of approximately 650 μg / kg. In other embodiments, the IL-7 protein disclosed herein is administered in a dose of approximately 680 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of approximately 700 μg / kg.In some embodiments, the IL-7 protein is administered at a dose of approximately 720 μg / kg. In certain embodiments, the IL-7 protein is administered at a dose of approximately 740 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of approximately 750 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of approximately 760 μg / kg. In certain embodiments, the IL-7 protein is administered at a dose of approximately 780 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of approximately 800 μg / kg. In further embodiments, the IL-7 protein is administered at a dose of approximately 820 μg / kg. In certain embodiments, the IL-7 protein is administered at a dose of approximately 840 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of approximately 850 μg / kg. In certain embodiments, the IL-7 protein is administered at a dose of approximately 860 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of approximately 880 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of approximately 900 μg / kg. In further embodiments, the IL-7 protein is administered at a dose of approximately 920 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of approximately 940 μg / kg. In further embodiments, the IL-7 protein is administered at a dose of approximately 950 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of approximately 960 μg / kg. In certain embodiments, the IL-7 protein is administered at a dose of approximately 980 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of approximately 1,000 μg / kg. In certain embodiments, the IL-7 protein is administered at a dose of approximately 1,020 μg / kg. In further embodiments, the IL-7 protein is administered at a dose of approximately 1,040 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of approximately 1,060 μg / kg. In other embodiments, the IL-7 protein is administered at a dose of approximately 1,080 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of approximately 1,100 μg / kg. In certain embodiments, the IL-7 protein is administered at a dose of approximately 1,120 μg / kg.In further embodiments, the IL-7 protein is administered at a dose of approximately 1,140 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of approximately 1,160 μg / kg. In other embodiments, the IL-7 protein is administered at a dose of approximately 1,180 μg / kg. In certain embodiments, the IL-7 protein is administered at a dose of approximately 1,200 μg / kg. In certain embodiments, the IL-7 protein is administered at a dose of approximately 1,220 μg / kg. In further embodiments, the IL-7 protein is administered at a dose of approximately 1,240 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of approximately 1,260 μg / kg. In other embodiments, the IL-7 protein is administered at a dose of approximately 1,280 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of approximately 1,300 μg / kg. In certain embodiments, the IL-7 protein is administered at a dose of approximately 1,320 μg / kg. In further embodiments, the IL-7 protein is administered at a dose of approximately 1,340 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of approximately 1,360 μg / kg. In other embodiments, the IL-7 protein is administered at a dose of approximately 1,380 μg / kg. In further embodiments, the IL-7 protein is administered at a dose of approximately 1,400 μg / kg. In certain embodiments, the IL-7 protein is administered at a dose of approximately 1,420 μg / kg. In further embodiments, the IL-7 protein is administered at a dose of approximately 1,440 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of approximately 1,460 μg / kg. In other embodiments, the IL-7 protein is administered at a dose of approximately 1,480 μg / kg. In certain embodiments, the IL-7 protein is administered at a dose of approximately 1,500 μg / kg. In certain embodiments, the IL-7 protein is administered at a dose of approximately 1,520 μg / kg. In further embodiments, the IL-7 protein is administered at a dose of approximately 1,540 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of approximately 1,560 μg / kg. In other embodiments, the IL-7 protein is administered at a dose of approximately 1,580 μg / kg.In some embodiments, the IL-7 protein is administered at a dose of approximately 1,600 μg / kg. In certain embodiments, the IL-7 protein is administered at a dose of approximately 1,620 μg / kg. In further embodiments, the IL-7 protein is administered at a dose of approximately 1,640 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of approximately 1,660 μg / kg. In other embodiments, the IL-7 protein is administered at a dose of approximately 1,680 μg / kg. In certain embodiments, the IL-7 protein is administered at a dose of approximately 1,700 μg / kg. In certain embodiments, the IL-7 protein is administered at a dose of approximately 1,720 μg / kg. In further embodiments, the IL-7 protein is administered at a dose of approximately 1,740 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of approximately 1,760 μg / kg. In other embodiments, the IL-7 protein is administered at a dose of approximately 1,780 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of approximately 1,800 μg / kg. In certain embodiments, the IL-7 protein is administered at a dose of approximately 1,820 μg / kg. In further embodiments, the IL-7 protein is administered at a dose of approximately 1,840 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of approximately 1,860 μg / kg. In other embodiments, the IL-7 protein is administered at a dose of approximately 1,880 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of approximately 1,900 μg / kg. In certain embodiments, the IL-7 protein is administered at a dose of approximately 1,920 μg / kg. In further embodiments, the IL-7 protein is administered at a dose of approximately 1,940 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of approximately 1,960 μg / kg. In other embodiments, the IL-7 protein is administered at a dose of approximately 1,980 μg / kg. In further embodiments, the IL-7 protein is administered at a dose of approximately 2,000 μg / kg.

[0147] In some embodiments, the IL-7 protein may be administered in a constant dose. In certain embodiments, the IL-7 protein may be administered in a constant dose of approximately 0.25 mg to approximately 9 mg. In some embodiments, the IL-7 protein may be administered in a constant dose of approximately 0.25 mg, approximately 1 mg, approximately 3 mg, approximately 6 mg, or approximately 9 mg.

[0148] In some embodiments, the IL-7 protein disclosed herein is administered to a subject in multiple doses (i.e., repeated doses). In certain embodiments, the IL-7 protein is administered to a subject at least two, three, four, five, six, seven, eight, nine, or ten or more times. In other embodiments, the subject receives a single dose of the IL-7 protein (for example, before, concurrently with, or after administration of an immune checkpoint inhibitor).

[0149] In some embodiments, the IL-7 protein is administered at a frequency of approximately once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, once every eleven weeks, or once every twelve weeks. In certain embodiments, the IL-7 protein is administered at a frequency of approximately every ten days, once every twenty days, once every thirty days, once every forty days, once every fifty days, once every sixty days, once every seventy days, once every eighty days, once every ninety days, or once every hundred days. In some embodiments, the IL-7 protein is administered once every three weeks. In some embodiments, the IL-7 protein is administered once a week. In some embodiments, the IL-7 protein is administered once every two weeks. In some embodiments, the IL-7 protein is administered once every four weeks. In certain embodiments, the IL-7 protein is administered once every six weeks. In further embodiments, the IL-7 protein is administered once every eight weeks. In some embodiments, the IL-7 protein is administered once every nine weeks. In certain embodiments, the IL-7 protein is administered once every twelve weeks. In some embodiments, the IL-7 protein is administered once every ten days. In certain embodiments, the IL-7 protein is administered once every twenty days. In other embodiments, the IL-7 protein is administered once every thirty days. In some embodiments, the IL-7 protein is administered once every forty days. In certain embodiments, the IL-7 protein is administered once every fifty days. In some embodiments, the IL-7 protein is administered once every sixty days. In further embodiments, the IL-7 protein is administered once every seventy days. In some embodiments, the IL-7 protein is administered once every 80 days. In certain embodiments, the IL-7 protein is administered once every 90 days. In some embodiments, the IL-7 protein is administered once every 100 days.

[0150] In some embodiments, IL-7 protein is administered at a dose of approximately 720 μg / kg in two or more doses at intervals of approximately 1 week, 2 weeks, 3 weeks, or 4 weeks. In some embodiments, IL-7 protein is administered at a dose of approximately 840 μg / kg in two or more doses at intervals of approximately 2 weeks, 3 weeks, 4 weeks, or 5 weeks. In some embodiments, IL-7 protein is administered at a dose of approximately 960 μg / kg in two or more doses at intervals of approximately 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks. In some embodiments, IL-7 protein is administered at a dose of approximately 1200 μg / kg in two or more doses at intervals of approximately 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks. In some embodiments, the IL-7 protein is administered in doses of approximately 1440 μg / kg in two or more doses at intervals of approximately 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 2 months, 8 weeks, 10 weeks, 12 weeks, or 3 months.

[0151] In some embodiments, the IL-7 protein is administered at a dose of 60 μg / kg once a week. In some embodiments, the IL-7 protein is administered at a dose of 120 μg / kg once a week. In some embodiments, the IL-7 protein is administered at a dose of 240 μg / kg once a week. In some embodiments, the IL-7 protein is administered at a dose of 480 μg / kg once a week. In some embodiments, the IL-7 protein is administered at a dose of 720 μg / kg once a week. In some embodiments, the IL-7 protein is administered at a dose of 960 μg / kg once a week. In some embodiments, the IL-7 protein is administered at a dose of 1,200 μg / kg once a week. In some embodiments, the IL-7 protein is administered at a dose of 1,300 μg / kg once a week. In some embodiments, the IL-7 protein is administered at a dose of 1,400 μg / kg once a week. In other embodiments, the IL-7 protein is administered at a dose of 1,420 μg / kg once a week. In certain embodiments, the IL-7 protein is administered at a dose of 1,440 μg / kg once a week. In further embodiments, the IL-7 protein is administered at a dose of 1,460 μg / kg once a week. In certain embodiments, the IL-7 protein is administered at a dose of 1,480 μg / kg once a week. In some embodiments, the IL-7 protein is administered at a dose of 1,500 μg / kg once a week. In a further embodiment, the IL-7 protein is administered at a dose of 1,600 μg / kg once a week. In some embodiments, the IL-7 protein is administered at a dose of 1,700 μg / kg once a week. In some embodiments, the IL-7 protein is administered at a dose of 2,000 μg / kg once a week.

[0152] In some embodiments, the IL-7 protein is administered at a dose of 60 μg / kg every two weeks. In some embodiments, the IL-7 protein is administered at a dose of 120 μg / kg every two weeks. In some embodiments, the IL-7 protein is administered at a dose of 240 μg / kg every two weeks. In some embodiments, the IL-7 protein is administered at a dose of 480 μg / kg every two weeks. In some embodiments, the IL-7 protein is administered at a dose of 720 μg / kg every two weeks. In some embodiments, the IL-7 protein is administered at a dose of 960 μg / kg every two weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,200 μg / kg every two weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,300 μg / kg every two weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,400 μg / kg every two weeks. In other embodiments, the IL-7 protein is administered at a dose of 1,420 μg / kg every two weeks. In a specific embodiment, the IL-7 protein is administered at a dose of 1,440 μg / kg every two weeks. In a further embodiment, the IL-7 protein is administered at a dose of 1,460 μg / kg every two weeks. In a specific embodiment, the IL-7 protein is administered at a dose of 1,480 μg / kg every two weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,500 μg / kg every two weeks. In a further embodiment, the IL-7 protein is administered at a dose of 1,600 μg / kg every two weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,700 μg / kg every two weeks. In some embodiments, the IL-7 protein is administered at a dose of 2,000 μg / kg every two weeks.

[0153] In some embodiments, the IL-7 protein is administered at a dose of 60 μg / kg every three weeks. In some embodiments, the IL-7 protein is administered at a dose of 120 μg / kg every three weeks. In some embodiments, the IL-7 protein is administered at a dose of 240 μg / kg every three weeks. In some embodiments, the IL-7 protein is administered at a dose of 480 μg / kg every three weeks. In some embodiments, the IL-7 protein is administered at a dose of 720 μg / kg every three weeks. In some embodiments, the IL-7 protein is administered at a dose of 960 μg / kg every three weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,200 μg / kg every three weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,300 μg / kg every three weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,400 μg / kg every three weeks. In other embodiments, the IL-7 protein is administered at a dose of 1,420 μg / kg every three weeks. In a specific embodiment, the IL-7 protein is administered at a dose of 1,440 μg / kg every three weeks. In a further embodiment, the IL-7 protein is administered at a dose of 1,460 μg / kg every three weeks. In a specific embodiment, the IL-7 protein is administered at a dose of 1,480 μg / kg every three weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,500 μg / kg every three weeks. In further embodiments, the IL-7 protein is administered at a dose of 1,600 μg / kg every three weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,700 μg / kg every three weeks. In some embodiments, the IL-7 protein is administered at a dose of 2,000 μg / kg every three weeks.

[0154] In some embodiments, the IL-7 protein is administered at a dose of 60 μg / kg once every four weeks. In some embodiments, the IL-7 protein is administered at a dose of 120 μg / kg once every four weeks. In some embodiments, the IL-7 protein is administered at a dose of 240 μg / kg once every four weeks. In some embodiments, the IL-7 protein is administered at a dose of 480 μg / kg once every four weeks. In some embodiments, the IL-7 protein is administered at a dose of 720 μg / kg once every four weeks. In some embodiments, the IL-7 protein is administered at a dose of 960 μg / kg once every four weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,200 μg / kg once every four weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,300 μg / kg every four weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,400 μg / kg every four weeks. In other embodiments, the IL-7 protein is administered at a dose of 1,420 μg / kg every four weeks. In a specific embodiment, the IL-7 protein is administered at a dose of 1,440 μg / kg every four weeks. In a further embodiment, the IL-7 protein is administered at a dose of 1,460 μg / kg every four weeks. In a specific embodiment, the IL-7 protein is administered at a dose of 1,480 μg / kg every four weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,500 μg / kg every four weeks. In further embodiments, the IL-7 protein is administered at a dose of 1,600 μg / kg every four weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,700 μg / kg every four weeks. In some embodiments, the IL-7 protein is administered at a dose of 2,000 μg / kg every four weeks.

[0155] In some embodiments, the IL-7 protein is administered at a dose of 60 μg / kg every five weeks. In some embodiments, the IL-7 protein is administered at a dose of 120 μg / kg every five weeks. In some embodiments, the IL-7 protein is administered at a dose of 240 μg / kg every five weeks. In some embodiments, the IL-7 protein is administered at a dose of 480 μg / kg every five weeks. In some embodiments, the IL-7 protein is administered at a dose of 720 μg / kg every five weeks. In some embodiments, the IL-7 protein is administered at a dose of 960 μg / kg every five weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,200 μg / kg every five weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,300 μg / kg every five weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,400 μg / kg every five weeks. In other embodiments, the IL-7 protein is administered at a dose of 1,420 μg / kg every five weeks. In a specific embodiment, the IL-7 protein is administered at a dose of 1,440 μg / kg every five weeks. In a further embodiment, the IL-7 protein is administered at a dose of 1,460 μg / kg every five weeks. In a specific embodiment, the IL-7 protein is administered at a dose of 1,480 μg / kg every five weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,500 μg / kg every five weeks. In a further embodiment, the IL-7 protein is administered at a dose of 1,600 μg / kg at a frequency of once every 5 weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,700 μg / kg at a frequency of once every 5 weeks. In some embodiments, the IL-7 protein is administered at a dose of 2,000 μg / kg at a frequency of once every 5 weeks.

[0156] In some embodiments, the IL-7 protein is administered at a dose of 60 μg / kg once every six weeks. In some embodiments, the IL-7 protein is administered at a dose of 120 μg / kg once every six weeks. In some embodiments, the IL-7 protein is administered at a dose of 240 μg / kg once every six weeks. In some embodiments, the IL-7 protein is administered at a dose of 480 μg / kg once every six weeks. In some embodiments, the IL-7 protein is administered at a dose of 720 μg / kg once every six weeks. In some embodiments, the IL-7 protein is administered at a dose of 960 μg / kg once every six weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,200 μg / kg once every six weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,300 μg / kg, once every six weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,400 μg / kg, once every six weeks. In other embodiments, the IL-7 protein is administered at a dose of 1,420 μg / kg, once every six weeks. In a specific embodiment, the IL-7 protein is administered at a dose of 1,440 μg / kg, once every six weeks. In a further embodiment, the IL-7 protein is administered at a dose of 1,460 μg / kg, once every six weeks. In a specific embodiment, the IL-7 protein is administered at a dose of 1,480 μg / kg, once every six weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,500 μg / kg, once every six weeks. In further embodiments, the IL-7 protein is administered at a dose of 1,600 μg / kg once every six weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,700 μg / kg once every six weeks. In some embodiments, the IL-7 protein is administered at a dose of 2,000 μg / kg once every six weeks.

[0157] In some embodiments, the IL-7 protein is administered at a dose of 60 μg / kg once every 7 weeks. In some embodiments, the IL-7 protein is administered at a dose of 120 μg / kg once every 7 weeks. In some embodiments, the IL-7 protein is administered at a dose of 240 μg / kg once every 7 weeks. In some embodiments, the IL-7 protein is administered at a dose of 480 μg / kg once every 7 weeks. In some embodiments, the IL-7 protein is administered at a dose of 720 μg / kg once every 7 weeks. In some embodiments, the IL-7 protein is administered at a dose of 960 μg / kg once every 7 weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,200 μg / kg once every 7 weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,300 μg / kg, once every 7 weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,400 μg / kg, once every 7 weeks. In other embodiments, the IL-7 protein is administered at a dose of 1,420 μg / kg, once every 7 weeks. In a specific embodiment, the IL-7 protein is administered at a dose of 1,440 μg / kg, once every 7 weeks. In further embodiments, the IL-7 protein is administered at a dose of 1,460 μg / kg, once every 7 weeks. In a specific embodiment, the IL-7 protein is administered at a dose of 1,480 μg / kg, once every 7 weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,500 μg / kg, once every 7 weeks. In a further embodiment, the IL-7 protein is administered at a dose of 1,600 μg / kg once every 7 weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,700 μg / kg once every 7 weeks. In some embodiments, the IL-7 protein is administered at a dose of 2,000 μg / kg once every 7 weeks.

[0158] In some embodiments, the IL-7 protein is administered at a dose of 60 μg / kg once every 8 weeks. In some embodiments, the IL-7 protein is administered at a dose of 120 μg / kg once every 8 weeks. In some embodiments, the IL-7 protein is administered at a dose of 240 μg / kg once every 8 weeks. In some embodiments, the IL-7 protein is administered at a dose of 480 μg / kg once every 8 weeks. In some embodiments, the IL-7 protein is administered at a dose of 720 μg / kg once every 8 weeks. In some embodiments, the IL-7 protein is administered at a dose of 960 μg / kg once every 8 weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,200 μg / kg once every 8 weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,300 μg / kg, once every 8 weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,400 μg / kg, once every 8 weeks. In other embodiments, the IL-7 protein is administered at a dose of 1,420 μg / kg, once every 8 weeks. In a specific embodiment, the IL-7 protein is administered at a dose of 1,440 μg / kg, once every 8 weeks. In a further embodiment, the IL-7 protein is administered at a dose of 1,460 μg / kg, once every 8 weeks. In a specific embodiment, the IL-7 protein is administered at a dose of 1,480 μg / kg, once every 8 weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,500 μg / kg, once every 8 weeks. In further embodiments, the IL-7 protein is administered at a dose of 1,600 μg / kg once every 8 weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,700 μg / kg once every 8 weeks. In some embodiments, the IL-7 protein is administered at a dose of 2,000 μg / kg once every 8 weeks.

[0159] In some embodiments, the IL-7 protein is administered at a dose of 60 μg / kg once every 9 weeks. In some embodiments, the IL-7 protein is administered at a dose of 120 μg / kg once every 9 weeks. In some embodiments, the IL-7 protein is administered at a dose of 240 μg / kg once every 9 weeks. In some embodiments, the IL-7 protein is administered at a dose of 480 μg / kg once every 9 weeks. In some embodiments, the IL-7 protein is administered at a dose of 720 μg / kg once every 9 weeks. In some embodiments, the IL-7 protein is administered at a dose of 960 μg / kg once every 9 weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,200 μg / kg once every 9 weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,300 μg / kg, once every 9 weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,400 μg / kg, once every 9 weeks. In other embodiments, the IL-7 protein is administered at a dose of 1,420 μg / kg, once every 9 weeks. In a specific embodiment, the IL-7 protein is administered at a dose of 1,440 μg / kg, once every 9 weeks. In a further embodiment, the IL-7 protein is administered at a dose of 1,460 μg / kg, once every 9 weeks. In a specific embodiment, the IL-7 protein is administered at a dose of 1,480 μg / kg, once every 9 weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,500 μg / kg, once every 9 weeks. In a further embodiment, the IL-7 protein is administered at a dose of 1,600 μg / kg at a frequency of once every 9 weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,700 μg / kg at a frequency of once every 9 weeks. In some embodiments, the IL-7 protein is administered at a dose of 2,000 μg / kg at a frequency of once every 9 weeks.

[0160] In some embodiments, the IL-7 protein is administered at a dose of 60 μg / kg once every 10 weeks. In some embodiments, the IL-7 protein is administered at a dose of 120 μg / kg once every 10 weeks. In some embodiments, the IL-7 protein is administered at a dose of 240 μg / kg once every 10 weeks. In some embodiments, the IL-7 protein is administered at a dose of 480 μg / kg once every 10 weeks. In some embodiments, the IL-7 protein is administered at a dose of 720 μg / kg once every 10 weeks. In some embodiments, the IL-7 protein is administered at a dose of 960 μg / kg once every 10 weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,200 μg / kg once every 10 weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,300 μg / kg, once every 10 weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,400 μg / kg, once every 10 weeks. In other embodiments, the IL-7 protein is administered at a dose of 1,420 μg / kg, once every 10 weeks. In a specific embodiment, the IL-7 protein is administered at a dose of 1,440 μg / kg, once every 10 weeks. In a further embodiment, the IL-7 protein is administered at a dose of 1,460 μg / kg, once every 10 weeks. In a specific embodiment, the IL-7 protein is administered at a dose of 1,480 μg / kg, once every 10 weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,500 μg / kg at a frequency of once every 10 weeks. In further embodiments, the IL-7 protein is administered at a dose of 1,600 μg / kg at a frequency of once every 10 weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,700 μg / kg at a frequency of once every 10 weeks. In some embodiments, the IL-7 protein is administered at a dose of 2,000 μg / kg at a frequency of once every 10 weeks.

[0161] In some embodiments, the IL-7 protein is administered at a dose of 60 μg / kg once every 11 weeks. In some embodiments, the IL-7 protein is administered at a dose of 120 μg / kg once every 11 weeks. In some embodiments, the IL-7 protein is administered at a dose of 240 μg / kg once every 11 weeks. In some embodiments, the IL-7 protein is administered at a dose of 480 μg / kg once every 11 weeks. In some embodiments, the IL-7 protein is administered at a dose of 720 μg / kg once every 11 weeks. In some embodiments, the IL-7 protein is administered at a dose of 960 μg / kg once every 11 weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,200 μg / kg once every 11 weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,300 μg / kg once every 11 weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,400 μg / kg once every 11 weeks. In other embodiments, the IL-7 protein is administered at a dose of 1,420 μg / kg once every 11 weeks. In a specific embodiment, the IL-7 protein is administered at a dose of 1,440 μg / kg once every 11 weeks. In a further embodiment, the IL-7 protein is administered at a dose of 1,460 μg / kg once every 11 weeks. In a specific embodiment, the IL-7 protein is administered at a dose of 1,480 μg / kg once every 11 weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,500 μg / kg once every 11 weeks. In further embodiments, the IL-7 protein is administered at a dose of 1,600 μg / kg once every 11 weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,700 μg / kg once every 11 weeks. In some embodiments, the IL-7 protein is administered at a dose of 2,000 μg / kg once every 11 weeks.

[0162] In some embodiments, the IL-7 protein is administered at a dose of 60 μg / kg once every 12 weeks. In some embodiments, the IL-7 protein is administered at a dose of 120 μg / kg once every 12 weeks. In some embodiments, the IL-7 protein is administered at a dose of 240 μg / kg once every 12 weeks. In some embodiments, the IL-7 protein is administered at a dose of 480 μg / kg once every 12 weeks. In some embodiments, the IL-7 protein is administered at a dose of 720 μg / kg once every 12 weeks. In some embodiments, the IL-7 protein is administered at a dose of 960 μg / kg once every 12 weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,200 μg / kg once every 12 weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,300 μg / kg, once every 12 weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,400 μg / kg, once every 12 weeks. In other embodiments, the IL-7 protein is administered at a dose of 1,420 μg / kg, once every 12 weeks. In a specific embodiment, the IL-7 protein is administered at a dose of 1,440 μg / kg, once every 12 weeks. In a further embodiment, the IL-7 protein is administered at a dose of 1,460 μg / kg, once every 12 weeks. In a specific embodiment, the IL-7 protein is administered at a dose of 1,480 μg / kg, once every 12 weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,500 μg / kg once every 12 weeks. In further embodiments, the IL-7 protein is administered at a dose of 1,600 μg / kg once every 12 weeks. In some embodiments, the IL-7 protein is administered at a dose of 1,700 μg / kg once every 12 weeks. In some embodiments, the IL-7 protein is administered at a dose of 2,000 μg / kg once every 12 weeks.

[0163] In some embodiments, the IL-7 protein is administered at a dose of 60 μg / kg, once every 10 days. In some embodiments, the IL-7 protein is administered at a dose of 120 μg / kg, once every 10 days. In some embodiments, the IL-7 protein is administered at a dose of 240 μg / kg, once every 10 days. In some embodiments, the IL-7 protein is administered at a dose of 480 μg / kg, once every 10 days. In some embodiments, the IL-7 protein is administered at a dose of 720 μg / kg, once every 10 days. In some embodiments, the IL-7 protein is administered at a dose of 960 μg / kg, once every 10 days. In some embodiments, the IL-7 protein is administered at a dose of 1,200 μg / kg, once every 10 days. In some embodiments, the IL-7 protein is administered at a dose of 1,300 μg / kg, once every 10 days. In some embodiments, the IL-7 protein is administered at a dose of 1,400 μg / kg, once every 10 days. In other embodiments, the IL-7 protein is administered at a dose of 1,420 μg / kg, once every 10 days. In a specific embodiment, the IL-7 protein is administered at a dose of 1,440 μg / kg, once every 10 days. In a further embodiment, the IL-7 protein is administered at a dose of 1,460 μg / kg, once every 10 days. In a specific embodiment, the IL-7 protein is administered at a dose of 1,480 μg / kg, once every 10 days. In some embodiments, the IL-7 protein is administered at a dose of 1,500 μg / kg, once every 10 days. In further embodiments, the IL-7 protein is administered at a dose of 1,600 μg / kg, once every 10 days. In some embodiments, the IL-7 protein is administered at a dose of 1,700 μg / kg, once every 10 days. In some embodiments, the IL-7 protein is administered at a dose of 2,000 μg / kg, once every 10 days.

[0164] In some embodiments, the IL-7 protein is administered at a dose of 60 μg / kg with a dosing frequency of once every 20 days. In some embodiments, the IL-7 protein is administered at a dose of 120 μg / kg with a dosing frequency of once every 20 days. In some embodiments, the IL-7 protein is administered at a dose of 240 μg / kg with a dosing frequency of once every 20 days. In some embodiments, the IL-7 protein is administered at a dose of 480 μg / kg with a dosing frequency of once every 20 days. In some embodiments, the IL-7 protein is administered at a dose of 720 μg / kg with a dosing frequency of once every 20 days. In some embodiments, the IL-7 protein is administered at a dose of 960 μg / kg with a dosing frequency of once every 20 days. In some embodiments, the IL-7 protein is administered at a dose of 1,200 μg / kg with a dosing frequency of once every 20 days. In some embodiments, the IL-7 protein is administered at a dose of 1,300 μg / kg with a dosing frequency of once every 20 days. In some embodiments, the IL-7 protein is administered at a dose of 1,400 μg / kg with a dosing frequency of once every 20 days. In other embodiments, the IL-7 protein is administered at a dose of 1,420 μg / kg with a dosing frequency of once every 20 days. In certain embodiments, the IL-7 protein is administered at a dose of 1,440 μg / kg with a dosing frequency of once every 20 days. In a further embodiment, the IL-7 protein is administered at a dose of 1,460 μg / kg with a dosing frequency of once every 20 days. In certain embodiments, the IL-7 protein is administered at a dose of 1,480 μg / kg with a dosing frequency of once every 20 days. In some embodiments, the IL-7 protein is administered at a dose of 1,500 μg / kg with a dosing frequency of once every 20 days. In a further embodiment, the IL-7 protein is administered at a dose of 1,600 μg / kg with a dosing frequency of once every 20 days. In some embodiments, the IL-7 protein is administered at a dose of 1,700 μg / kg with a dosing frequency of once every 20 days. In some embodiments, the IL-7 protein is administered at a dose of 2,000 μg / kg with a dosing frequency of once every 20 days.

[0165] In some embodiments, the IL-7 protein is administered at a dose of 60 μg / kg, once every 30 days. In some embodiments, the IL-7 protein is administered at a dose of 120 μg / kg, once every 30 days. In some embodiments, the IL-7 protein is administered at a dose of 240 μg / kg, once every 30 days. In some embodiments, the IL-7 protein is administered at a dose of 480 μg / kg, once every 30 days. In some embodiments, the IL-7 protein is administered at a dose of 720 μg / kg, once every 30 days. In some embodiments, the IL-7 protein is administered at a dose of 960 μg / kg, once every 30 days. In some embodiments, the IL-7 protein is administered at a dose of 1,200 μg / kg, once every 30 days. In some embodiments, the IL-7 protein is administered at a dose of 1,300 μg / kg, once every 30 days. In some embodiments, the IL-7 protein is administered at a dose of 1,400 μg / kg, once every 30 days. In other embodiments, the IL-7 protein is administered at a dose of 1,420 μg / kg, once every 30 days. In a specific embodiment, the IL-7 protein is administered at a dose of 1,440 μg / kg, once every 30 days. In a further embodiment, the IL-7 protein is administered at a dose of 1,460 μg / kg, once every 30 days. In a specific embodiment, the IL-7 protein is administered at a dose of 1,480 μg / kg, once every 30 days. In some embodiments, the IL-7 protein is administered at a dose of 1,500 μg / kg, once every 30 days. In further embodiments, the IL-7 protein is administered at a dose of 1,600 μg / kg, once every 30 days. In some embodiments, the IL-7 protein is administered at a dose of 1,700 μg / kg, once every 30 days. In some embodiments, the IL-7 protein is administered at a dose of 2,000 μg / kg, once every 30 days.

[0166] In some embodiments, the IL-7 protein is administered at a dose of 60 μg / kg, once every 40 days. In some embodiments, the IL-7 protein is administered at a dose of 120 μg / kg, once every 40 days. In some embodiments, the IL-7 protein is administered at a dose of 240 μg / kg, once every 40 days. In some embodiments, the IL-7 protein is administered at a dose of 480 μg / kg, once every 40 days. In some embodiments, the IL-7 protein is administered at a dose of 720 μg / kg, once every 40 days. In some embodiments, the IL-7 protein is administered at a dose of 960 μg / kg, once every 40 days. In some embodiments, the IL-7 protein is administered at a dose of 1,200 μg / kg, once every 40 days. In some embodiments, the IL-7 protein is administered at a dose of 1,300 μg / kg, once every 40 days. In some embodiments, the IL-7 protein is administered at a dose of 1,400 μg / kg, once every 40 days. In other embodiments, the IL-7 protein is administered at a dose of 1,420 μg / kg, once every 40 days. In a specific embodiment, the IL-7 protein is administered at a dose of 1,440 μg / kg, once every 40 days. In a further embodiment, the IL-7 protein is administered at a dose of 1,460 μg / kg, once every 40 days. In a specific embodiment, the IL-7 protein is administered at a dose of 1,480 μg / kg, once every 40 days. In some embodiments, the IL-7 protein is administered at a dose of 1,500 μg / kg, once every 40 days. In further embodiments, the IL-7 protein is administered at a dose of 1,600 μg / kg, once every 40 days. In some embodiments, the IL-7 protein is administered at a dose of 1,700 μg / kg, once every 40 days. In some embodiments, the IL-7 protein is administered at a dose of 2,000 μg / kg, once every 40 days.

[0167] In some embodiments, the IL-7 protein is administered at a dose of 60 μg / kg with a dosing frequency of once every 50 days. In some embodiments, the IL-7 protein is administered at a dose of 120 μg / kg with a dosing frequency of once every 50 days. In some embodiments, the IL-7 protein is administered at a dose of 240 μg / kg with a dosing frequency of once every 50 days. In some embodiments, the IL-7 protein is administered at a dose of 480 μg / kg with a dosing frequency of once every 50 days. In some embodiments, the IL-7 protein is administered at a dose of 720 μg / kg with a dosing frequency of once every 50 days. In some embodiments, the IL-7 protein is administered at a dose of 960 μg / kg with a dosing frequency of once every 50 days. In some embodiments, the IL-7 protein is administered at a dose of 1,200 μg / kg with a dosing frequency of once every 50 days. In some embodiments, the IL-7 protein is administered at a dose of 1,300 μg / kg with a dosing frequency of once every 50 days. In some embodiments, the IL-7 protein is administered at a dose of 1,400 μg / kg with a dosing frequency of once every 50 days. In other embodiments, the IL-7 protein is administered at a dose of 1,420 μg / kg with a dosing frequency of once every 50 days. In certain embodiments, the IL-7 protein is administered at a dose of 1,440 μg / kg with a dosing frequency of once every 50 days. In a further embodiment, the IL-7 protein is administered at a dose of 1,460 μg / kg with a dosing frequency of once every 50 days. In certain embodiments, the IL-7 protein is administered at a dose of 1,480 μg / kg with a dosing frequency of once every 50 days. In some embodiments, the IL-7 protein is administered at a dose of 1,500 μg / kg with a dosing frequency of once every 50 days. In a further embodiment, the IL-7 protein is administered at a dose of 1,600 μg / kg with a dosing frequency of once every 50 days. In some embodiments, the IL-7 protein is administered at a dose of 1,700 μg / kg with a dosing frequency of once every 50 days. In some embodiments, the IL-7 protein is administered at a dose of 2,000 μg / kg with a dosing frequency of once every 50 days.

[0168] In some embodiments, the IL-7 protein is administered at a dose of 60 μg / kg once every 60 days. In some embodiments, the IL-7 protein is administered at a dose of 120 μg / kg once every 60 days. In some embodiments, the IL-7 protein is administered at a dose of 240 μg / kg once every 60 days. In some embodiments, the IL-7 protein is administered at a dose of 480 μg / kg once every 60 days. In some embodiments, the IL-7 protein is administered at a dose of 720 μg / kg once every 60 days. In some embodiments, the IL-7 protein is administered at a dose of 960 μg / kg once every 60 days. In some embodiments, the IL-7 protein is administered at a dose of 1,200 μg / kg once every 60 days. In some embodiments, the IL-7 protein is administered at a dose of 1,300 μg / kg, once every 60 days. In some embodiments, the IL-7 protein is administered at a dose of 1,400 μg / kg, once every 60 days. In other embodiments, the IL-7 protein is administered at a dose of 1,420 μg / kg, once every 60 days. In a specific embodiment, the IL-7 protein is administered at a dose of 1,440 μg / kg, once every 60 days. In a further embodiment, the IL-7 protein is administered at a dose of 1,460 μg / kg, once every 60 days. In a specific embodiment, the IL-7 protein is administered at a dose of 1,480 μg / kg, once every 60 days. In some embodiments, the IL-7 protein is administered at a dose of 1,500 μg / kg, once every 60 days. In further embodiments, the IL-7 protein is administered at a dose of 1,600 μg / kg, once every 60 days. In some embodiments, the IL-7 protein is administered at a dose of 1,700 μg / kg, once every 60 days. In some embodiments, the IL-7 protein is administered at a dose of 2,000 μg / kg, once every 60 days.

[0169] In some embodiments, the IL-7 protein is administered at a dose of 60 μg / kg, once every 70 days. In some embodiments, the IL-7 protein is administered at a dose of 120 μg / kg, once every 70 days. In some embodiments, the IL-7 protein is administered at a dose of 240 μg / kg, once every 70 days. In some embodiments, the IL-7 protein is administered at a dose of 480 μg / kg, once every 70 days. In some embodiments, the IL-7 protein is administered at a dose of 720 μg / kg, once every 70 days. In some embodiments, the IL-7 protein is administered at a dose of 960 μg / kg, once every 70 days. In some embodiments, the IL-7 protein is administered at a dose of 1,200 μg / kg, once every 70 days. In some embodiments, the IL-7 protein is administered at a dose of 1,300 μg / kg, once every 70 days. In some embodiments, the IL-7 protein is administered at a dose of 1,400 μg / kg, once every 70 days. In other embodiments, the IL-7 protein is administered at a dose of 1,420 μg / kg, once every 70 days. In a specific embodiment, the IL-7 protein is administered at a dose of 1,440 μg / kg, once every 70 days. In a further embodiment, the IL-7 protein is administered at a dose of 1,460 μg / kg, once every 70 days. In a specific embodiment, the IL-7 protein is administered at a dose of 1,480 μg / kg, once every 70 days. In some embodiments, the IL-7 protein is administered at a dose of 1,500 μg / kg, once every 70 days. In further embodiments, the IL-7 protein is administered at a dose of 1,600 μg / kg, once every 70 days. In some embodiments, the IL-7 protein is administered at a dose of 1,700 μg / kg, once every 70 days. In some embodiments, the IL-7 protein is administered at a dose of 2,000 μg / kg, once every 70 days.

[0170] In some embodiments, the IL-7 protein is administered at a dose of 60 μg / kg, once every 80 days. In some embodiments, the IL-7 protein is administered at a dose of 120 μg / kg, once every 80 days. In some embodiments, the IL-7 protein is administered at a dose of 240 μg / kg, once every 80 days. In some embodiments, the IL-7 protein is administered at a dose of 480 μg / kg, once every 80 days. In some embodiments, the IL-7 protein is administered at a dose of 720 μg / kg, once every 80 days. In some embodiments, the IL-7 protein is administered at a dose of 960 μg / kg, once every 80 days. In some embodiments, the IL-7 protein is administered at a dose of 1,200 μg / kg, once every 80 days. In some embodiments, the IL-7 protein is administered at a dose of 1,300 μg / kg, once every 80 days. In some embodiments, the IL-7 protein is administered at a dose of 1,400 μg / kg, once every 80 days. In other embodiments, the IL-7 protein is administered at a dose of 1,420 μg / kg, once every 80 days. In a specific embodiment, the IL-7 protein is administered at a dose of 1,440 μg / kg, once every 80 days. In a further embodiment, the IL-7 protein is administered at a dose of 1,460 μg / kg, once every 80 days. In a specific embodiment, the IL-7 protein is administered at a dose of 1,480 μg / kg, once every 80 days. In some embodiments, the IL-7 protein is administered at a dose of 1,500 μg / kg, once every 80 days. In further embodiments, the IL-7 protein is administered at a dose of 1,600 μg / kg, once every 80 days. In some embodiments, the IL-7 protein is administered at a dose of 1,700 μg / kg, once every 80 days. In some embodiments, the IL-7 protein is administered at a dose of 2,000 μg / kg, once every 80 days.

[0171] In some embodiments, the IL-7 protein is administered at a dose of 60 μg / kg, once every 90 days. In some embodiments, the IL-7 protein is administered at a dose of 120 μg / kg, once every 90 days. In some embodiments, the IL-7 protein is administered at a dose of 240 μg / kg, once every 90 days. In some embodiments, the IL-7 protein is administered at a dose of 480 μg / kg, once every 90 days. In some embodiments, the IL-7 protein is administered at a dose of 720 μg / kg, once every 90 days. In some embodiments, the IL-7 protein is administered at a dose of 960 μg / kg, once every 90 days. In some embodiments, the IL-7 protein is administered at a dose of 1,200 μg / kg, once every 90 days. In some embodiments, the IL-7 protein is administered at a dose of 1,300 μg / kg, once every 90 days. In some embodiments, the IL-7 protein is administered at a dose of 1,400 μg / kg, once every 90 days. In other embodiments, the IL-7 protein is administered at a dose of 1,420 μg / kg, once every 90 days. In a specific embodiment, the IL-7 protein is administered at a dose of 1,440 μg / kg, once every 90 days. In a further embodiment, the IL-7 protein is administered at a dose of 1,460 μg / kg, once every 90 days. In a specific embodiment, the IL-7 protein is administered at a dose of 1,480 μg / kg, once every 90 days. In some embodiments, the IL-7 protein is administered at a dose of 1,500 μg / kg, once every 90 days. In further embodiments, the IL-7 protein is administered at a dose of 1,600 μg / kg, once every 90 days. In some embodiments, the IL-7 protein is administered at a dose of 1,700 μg / kg, once every 90 days. In some embodiments, the IL-7 protein is administered at a dose of 2,000 μg / kg, once every 90 days.

[0172] In some embodiments, the IL-7 protein is administered at a dose of 60 μg / kg, once every 100 days. In some embodiments, the IL-7 protein is administered at a dose of 120 μg / kg, once every 100 days. In some embodiments, the IL-7 protein is administered at a dose of 240 μg / kg, once every 100 days. In some embodiments, the IL-7 protein is administered at a dose of 480 μg / kg, once every 100 days. In some embodiments, the IL-7 protein is administered at a dose of 720 μg / kg, once every 100 days. In some embodiments, the IL-7 protein is administered at a dose of 960 μg / kg, once every 100 days. In some embodiments, the IL-7 protein is administered at a dose of 1,200 μg / kg, once every 100 days. In some embodiments, the IL-7 protein is administered at a dose of 1,300 μg / kg, once every 100 days. In some embodiments, the IL-7 protein is administered at a dose of 1,400 μg / kg, once every 100 days. In other embodiments, the IL-7 protein is administered at a dose of 1,420 μg / kg, once every 100 days. In a specific embodiment, the IL-7 protein is administered at a dose of 1,440 μg / kg, once every 100 days. In a further embodiment, the IL-7 protein is administered at a dose of 1,460 μg / kg, once every 100 days. In a specific embodiment, the IL-7 protein is administered at a dose of 1,480 μg / kg, once every 100 days. In some embodiments, the IL-7 protein is administered at a dose of 1,500 μg / kg, once every 100 days. In further embodiments, the IL-7 protein is administered at a dose of 1,600 μg / kg, once every 100 days. In some embodiments, the IL-7 protein is administered at a dose of 1,700 μg / kg, once every 100 days. In some embodiments, the IL-7 protein is administered at a dose of 2,000 μg / kg, once every 100 days.

[0173] In some embodiments, the methods disclosed herein (e.g., administering IL-7 protein in combination with an immune checkpoint inhibitor) can be used in combination with one or more additional anticancer agents and / or immunomodulators. Such agents may include, for example, chemotherapeutic agents, small molecule drugs, or antibodies that stimulate an immune response against a given cancer. In some embodiments, the methods described herein are used in combination with standard treatments (e.g., surgery, radiation, and chemotherapy). The methods described herein can also be used as maintenance therapy, for example, therapy aimed at preventing tumor development or recurrence.

[0174] In some embodiments, methods for treating tumors disclosed herein may involve administering the IL-7 protein in combination with one or more immuno-oncological agents so as to target multiple elements of the immune pathway. Such combinations are not limited to: therapies that enhance tumor antigen presentation (e.g., dendritic cell vaccines, GM-CSF secretory cell vaccines, CpG oligonucleotides, imiquimod); therapies that inhibit negative immune regulation, for example, by inhibiting the CTLA-4 and / or PD1 / PD-L1 / PD-L2 pathways and / or depleting or blocking Treg or other immunosuppressive cells (e.g., myeloid-derived suppressor cells); therapies that stimulate positive immune regulation, for example, by agonists that stimulate the CD-137, OX-40, and / or CD40 or GITR pathways and / or T cell effector function; therapies that systemically increase the incidence of antitumor T cells; for example, by using a CD25 antagonist (e.g., daclizumab), or ex Therapies include: depletion or inhibition of Treg cells in tumors by depletion with vivo anti-CD25 beads; therapies that affect the function of suppressor myeloid cells in tumors; therapies that enhance the immunogenicity of tumor cells (e.g., anthracyclines); adoptive transfer of T cells or NK cells, including genetically modified cells, e.g., cells modified with chimeric antigen receptors (CAR-T therapy); therapies that inhibit metabolic enzymes such as indoleamine dioxygenase (IDO), dioxygenase, arginase, or nitric oxide synthase; therapies that reverse / prevent T cell anergy or depletion; therapies that activate innate immunity and / or induce inflammation at the tumor site; administration of immunostimulatory cytokines; or blockade of immunosuppressive cytokines.

[0175] In some embodiments, immuno-oncological agents that can be used in combination with the IL-7 protein disclosed herein include immune checkpoint inhibitors (i.e., those that block signaling by a specific immune checkpoint pathway). Non-limiting examples of immune checkpoint inhibitors that can be used in the methods of the present invention include CTLA-4 antagonists (e.g., anti-CTLA-4 antibodies), PD-1 antagonists (e.g., anti-PD-1 antibodies, anti-PD-L1 antibodies), TIM-3 antagonists (e.g., anti-TIM-3 antibodies), or combinations thereof.

[0176] In some embodiments, immuno-oncological agents include immune checkpoint activators (i.e., they promote signaling by specific immune checkpoint pathways). In certain embodiments, immune checkpoint activators include OX40 agonists (e.g., anti-OX40 antibodies), LAG-3 agonists (e.g., anti-LAG-3 antibodies), 4-1BB(CD137) agonists (e.g., anti-CD137 antibodies), GITR agonists (e.g., anti-GITR antibodies), or any combination thereof.

[0177] In some embodiments, the combination of the IL-7 protein and a second drug (e.g., an immune checkpoint inhibitor) described herein may be administered simultaneously as a single composition in a pharmaceutically acceptable carrier. In other embodiments, the combination of the IL-7 protein and a second drug (e.g., an immune checkpoint inhibitor) described herein may be administered simultaneously as separate compositions. In further embodiments, the combination of the IL-7 protein and a second drug (e.g., an immune checkpoint inhibitor) described herein may be administered sequentially. In some embodiments, the IL-7 protein is administered before the administration of the second drug (e.g., an immune checkpoint inhibitor).

[0178] IIa. IL-7 proteins useful in this disclosure This specification discloses IL-7 proteins that may be used in combination with immune checkpoint inhibitors to treat cancer (or tumors). In some embodiments, IL-7 proteins useful for this use may be wild-type IL-7 or modified IL-7 (i.e., non-wild-type IL-7 proteins) (e.g., IL-7 variants, IL-7 functional fragments, IL-7 derivatives, or any combination thereof, e.g., fusion proteins, chimeric proteins, etc.), provided that the IL-7 protein contains one or more of the biological activities of IL-7, e.g., binding to IL-7R, e.g., inducing early T cell development, promoting T cell homeostasis. ElKassar and See Gress.J Immunotoxicol.2010 Mar;7(1):1-7. In some embodiments, the IL-7 proteins of this disclosure are not wild-type IL-7 proteins (i.e., include one or more modifications). Non-limiting examples of such modifications may include oligopeptides and / or half-life extension moieties. See WO2016 / 200219 incorporated herein by reference in whole.

[0179] IL-7 binds to a receptor composed of two chains: IL-7Rα (CD127), which is shared with thymic stromal lymphocyte generating factor (TSLP) (Ziegler and Liu, 2006), and a γ chain (CD132), which is shared with IL-2, IL-15, IL-9, and IL-21. While γc is expressed in most hematopoietic cells, IL-7Rα is expressed almost exclusively in lymphoid cells. After binding to its receptor, IL-7 signals through two different pathways: Jak-Stat (Janus kinase-Signal transducer and activator of transcription) and PI3K / Akt, which are involved in differentiation and survival, respectively. Deficiency in IL-7 signaling is responsible for reduced thymocyte solidity, as observed in mice given anti-IL-7 neutralizing monoclonal antibodies (MAb) (Grabstein et al., 1993), IL-7- / - mice (von Freeden-Jeffry et al., 1995), IL-7Rα- / - mice (Peschon et al., 1994, Maki et al., 1996), γc- / - mice (Malissen et al., 1997), and Jak3- / - mice (Park et al., 1995). Mice lacking IL-7 signaling are deficient in T cells, B cells, and NK-T cells. IL-7α- / - mice (Peschon et al., 1994) are similar to IL-7- / - mice (von Freeden-Jeffry et al., 1995), but exhibit a more severe phenotype. This is thought to be because TSLP signaling is also suppressed in IL-7α- / - mice. IL-7 is necessary for the development of γδ cells (Maki et al., 1996) and NK-T cells (Boesteanu et al., 1997).

[0180] In some embodiments, the IL-7 protein useful in this disclosure comprises the amino acid sequence shown in any one of SEQ ID NOs: 1-6. In other embodiments, the IL-7 protein comprises an amino acid sequence having approximately 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or more than 99% sequence identity with the sequences of SEQ ID NOs: 1-6.

[0181] In some embodiments, the IL-7 protein comprises a modified IL-7 or a fragment thereof, which retains one or more levels of biological activity of wild-type IL-7. In some embodiments, the IL-7 protein may be derived from humans, rats, mice, monkeys, cattle, or sheep.

[0182] In some embodiments, human IL-7 may have the amino acid sequence represented by SEQ ID NO: 1 (Genbank acceptance number P13232), rat IL-7 may have the amino acid sequence represented by SEQ ID NO: 2 (Genbank acceptance number P56478), mouse IL-7 may have the amino acid sequence represented by SEQ ID NO: 3 (Genbank acceptance number P10168), monkey IL-7 may have the amino acid sequence represented by SEQ ID NO: 4 (Genbank acceptance number NP 001279008), bovine IL-7 may have the amino acid sequence represented by SEQ ID NO: 5 (Genbank acceptance number P26895), and sheep IL-7 may have the amino acid sequence represented by SEQ ID NO: 6 (Genbank acceptance number Q28540).

[0183] In some embodiments, the IL-7 protein useful in this disclosure includes an IL-7 fusion protein. In certain embodiments, the IL-7 fusion protein includes (i) an oligopeptide and (i) IL-7 or a variant thereof. In some embodiments, the oligopeptide is ligated to the N-terminal region of IL-7 or a variant thereof.

[0184] In some embodiments, the oligopeptides disclosed herein consist of 1 to 10 amino acids. In a particular embodiment, the oligopeptide consists of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or 10 amino acids. In some embodiments, one or more amino acids of the oligopeptide are selected from the group consisting of methionine, glycine, and combinations thereof. In a particular embodiment, the oligopeptide is selected from the group consisting of methionine, glycine, methionine-methionine, glycine-glycine, methionine-glycine, glycine-methionine-methionine, methionine-methionine-glycine, methionine-glycine-methionine, glycine-glycine-methionine, and glycine-glycine-glycine. In some forms, the oligopeptide is methionine-glycine-methionine.

[0185] In some embodiments, the IL-7 fusion protein comprises (i) IL-7 or a variant thereof, and (ii) a half-life extension portion. In some embodiments, the half-life extension portion extends the half-life of IL-7 or a variant thereof. In some embodiments, the half-life extension portion is ligated to the C-terminal region of IL-7 or a variant thereof.

[0186] In some embodiments, the IL-7 fusion protein comprises (i) IL-7 (first domain), (ii) a second domain comprising an amino acid sequence having 1 to 10 amino acid residues consisting of methionine, glycine, or a combination thereof, e.g., MGM, and (iii) a third domain comprising a half-life extension portion. In some embodiments, the half-life extension portion may be ligated to the N-terminus or C-terminus of the first or second domain. Furthermore, IL-7 comprising the first and second domains may be ligated to both ends of the third domain.

[0187] Non-limiting examples of the half-life extension moiety include Fc, albumin, albumin-binding polypeptide, Pro / Ala / Ser (PAS), the C-terminal peptide (CTP) of the β subunit of human chorionic gonadotropin, polyethylene glycol (PEG), long-chain unstructured hydrophilic amino acid sequence (XTEN), hydroxyethyl starch (HES), albumin-binding low molecule, and combinations thereof.

[0188] In some embodiments, the half-life extension moiety is Fc. In certain embodiments, the Fc is derived from a modified immunoglobulin in which antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) is attenuated by modification of the binding affinity to Fc receptors and / or complement. In some embodiments, the modified immunoglobulin can be selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and combinations thereof. In some embodiments, the Fc is a hybrid Fc ( "hFc" or "hyFc") that includes a hinge region, a CH2 domain, and a CH3 domain. In certain embodiments, the hinge region of the hybrid Fc disclosed herein includes the human IgD hinge region. In certain embodiments, the CH2 domain of the hybrid Fc includes a portion of the human IgD CH2 domain and a portion of the human IgG4 CH2 domain. In certain embodiments, the CH3 domain of the hybrid Fc includes a portion of the human IgG4 CH3 domain. Thus, in some embodiments, the hybrid Fc disclosed herein includes a hinge region, a CH2 domain, and a CH3 domain, the hinge region includes the human IgD hinge region, the CH2 domain includes a portion of the human IgD CH2 domain and a portion of the human IgG4 CH2 domain, and the CH3 domain includes a portion of the human IgG4 CH3 domain.

[0189] In some embodiments, the Fc disclosed herein may be Fc variants. As used herein, the term “Fc variant” means an Fc prepared by substituting some of the amino acids in an Fc region or by combining different types of Fc regions. Fc region variants can be prevented from being cleaved at the hinge region. In particular, in some embodiments, the Fc variants include modifications of the 144th and / or 145th amino acids of SEQ ID NO: 9. In certain embodiments, the 144th amino acid (K) and / or the 145th amino acid (K) are substituted with G or S.

[0190] In some embodiments, the Fc or Fc variant disclosed herein can be represented by the following formula: N'-(Z1)pY-Z2-Z3-Z4-C, where, N' includes the N-terminus. Z1 contains an amino acid sequence consisting of 5 to 9 consecutive amino acid residues from the amino acid residue at position 98 toward the N-terminus, among the amino acid residues at positions 90 to 98 of SEQ ID NO: 7. Y contains an amino acid sequence among the amino acid residues at positions 99-162 of SEQ ID NO: 7, with 5-64 consecutive amino acid residues from the amino acid residue at position 162 toward the N-terminus. Z2 contains an amino acid sequence among the amino acid residues at positions 163-199 of SEQ ID NO: 7, which has 4 to 37 consecutive amino acid residues from the amino acid residue at position 163 toward the C-terminus. Z3 contains an amino acid sequence consisting of 71 to 106 consecutive amino acid residues from the amino acid residue at position 220 toward the N-terminus, among the amino acid residues at positions 115 to 220 of SEQ ID NO: 8. Z4 contains an amino acid sequence consisting of 80 to 107 consecutive amino acid residues from the amino acid residue at position 221 toward the C-terminus, among the amino acid residues at positions 221 to 327 of Sequence ID No. 8.

[0191] In some embodiments, the Fc region disclosed herein may include the amino acid sequence of SEQ ID NO: 9 (hyFc), SEQ ID NO: 10 (hyFcM1), SEQ ID NO: 11 (hyFcM2), SEQ ID NO: 12 (hyFcM3), or SEQ ID NO: 13 (hyFcM4). In some embodiments, the Fc region may include the amino acid sequence of SEQ ID NO: 14 (insoluble mouse Fc).

[0192] Other non-limiting examples of the Fc area that can be used in this disclosure are described in U.S. Patent No. 7,867,491, which is incorporated herein by reference in its entirety.

[0193] In some embodiments, the IL-7 fusion proteins disclosed herein include both an oligopeptide and a half-life extension moiety.

[0194] In some embodiments, the IL-7 protein may be fused to albumin, its variants, or fragments. An example of an IL-7-albumin fusion protein can be found in International Patent Publication WO2011 / 124718A1. In some embodiments, the IL-7 protein is fused to pre-pro-B cell growth stimulant (PPBSF) by an optional mobile linker. See US2002 / 0058791A1. In other embodiments, the IL-7 protein useful in this disclosure is an IL-7 conformational isomer having a specific three-dimensional structure. See US2005 / 0249701A1. In some embodiments, the IL-7 protein may be fused to an Ig chain, where amino acid residues 70 and 91 of the IL-7 protein are glycosylated and amino acid residue 116 of the IL-7 protein is not glycosylated. See US7,323,549B2. In some embodiments, IL-7 proteins that do not contain potential T cell epitopes (and thus reduce the anti-IL-7 T cell response) may also be used in this disclosure. See US2006 / 0141581A1. In other embodiments, IL-7 proteins having one or more amino acid residue mutations in the carboxy-terminal helix D region may also be used in this disclosure. IL-7 variants may act as partial agonists of IL-7R despite having low receptor binding affinity. See US2005 / 0054054A1. Any IL-7 proteins described in the aforementioned patents or publications are incorporated herein by reference as a whole.

[0195] Furthermore, non-limiting examples of additional IL-7 proteins useful in this disclosure include US7708985, US8034327, US8153114, US7589179, US7323549, US7960514, US8338575, US7118754, US7488482, US7670607, US6730512, WO0017362, GB2434578A, WO2010 / 020766A2, WO91 / 01143, Beq et al. This is described in *Blood*, vol.114(4), 816, 23 July 2009, *Kang et al.*, *J.Virol*, Doi:10.1128 / JVI.02768-15, *Martin et al.*, *Blood*, vol.121(22), 4484, May 30, 2013, *McBride et al.*, *Acta Oncologica*, 34:3, 447-451, July 8, 2009, and *Xu et al.*, *Cancer Science*, 109:279-288, 2018, which are incorporated herein by reference as a whole.

[0196] This disclosure relates to a method for treating a tumor (or cancer) in a subject requiring such treatment, comprising administering to the subject an effective amount of interleukin-7 (IL-7) protein in combination with an effective amount of an immune checkpoint inhibitor. Non-limiting examples of immune checkpoint inhibitors that can be used in the method of the present invention include anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, and combinations thereof.

[0197] In some embodiments, the oligopeptides disclosed herein are directly linked to the N-terminal region of IL-7 or a variant thereof. In other embodiments, the oligopeptides are linked to the N-terminal region via a linker. In some embodiments, the half-life extension portions disclosed herein are directly linked to the C-terminal region of IL-7 or a variant thereof. In certain embodiments, the half-life extension portions are linked to the C-terminal region via a linker. In some embodiments, the linker is a peptide linker. In certain embodiments, the peptide linker contains a peptide of 10 to 20 amino acid residues consisting of Gly and Ser residues. In some embodiments, the linker is an albumin linker. In some embodiments, the linker is a chemical bond. In certain embodiments, the chemical bond includes disulfide bonds, diamine bonds, sulfide-amine bonds, carboxy-amine bonds, ester bonds, covalent bonds, or combinations thereof. When the linker is a peptide linker, in some embodiments, the bond can occur at any linking region. These can be bonded using crosslinking agents known in the art. In some embodiments, examples of crosslinking agents include, but are not limited to, N-hydroxysuccinimid esters such as 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, and 4-azidosalicylic acid; imide esters containing disuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate); and bifunctional maleimides such as bis-N-maleimide-1,8-octane.

[0198] In some embodiments, the IL-7 (or variant thereof) portion of the IL-7 fusion protein disclosed herein includes an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98%, or at least 99% identical to the amino acid sequences shown in SEQ ID NOs. 15-20. In certain embodiments, the IL-7 (or variant thereof) portion of the IL-7 fusion protein disclosed herein includes an amino acid sequence shown in SEQ ID NOs. 15-20.

[0199] In some embodiments, the IL-7 fusion protein comprises a first domain containing a polypeptide having IL-7 activity or similar activity, a second domain containing an amino acid sequence having 1 to 10 amino acid residues consisting of methionine, glycine, or a combination thereof, and a third domain which is the Fc region of a modified immunoglobulin bound to the C-terminus of the first domain.

[0200] In some embodiments, the IL-7 fusion protein that can be used in the method of the present invention comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98%, or at least 99% identical to the amino acid sequences shown in SEQ ID NOs. 21-25. In certain embodiments, the IL-7 fusion protein of this disclosure comprises the amino acid sequences shown in SEQ ID NOs. 21-25. In further embodiments, the IL-7 fusion protein disclosed herein comprises the amino acid sequences shown in SEQ ID NOs. 26 and 27.

[0201] In some embodiments, the IL-7 protein useful in this disclosure, when administered to a subject, can increase the absolute number of lymphocytes in that subject. In certain embodiments, the subject has a disease or disorder described herein (e.g., cancer). In other embodiments, the subject is a healthy individual (e.g., not having a disease or disorder described herein, e.g., cancer). In certain embodiments, the absolute number of lymphocytes increases by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% or more compared to a reference (e.g., the corresponding level in a subject that did not receive the IL-7 protein).

[0202] In some embodiments, the IL-7 protein disclosed herein is used to promote T cell proliferation in a subject (e.g., CD8 + T cells can be increased. In certain embodiments, the increase in T cell proliferation occurs peripherally (e.g., outside of tumors). In certain embodiments, T cell proliferation increases by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% or more compared to a reference (e.g., the corresponding level in a subject that did not receive IL-7 protein). In certain embodiments, T cells that proliferate in response to IL-7 administration (e.g., CD8 + T cells express one or more of the following markers: Eomesodermin (Eomes), granzyme B, CXCR3, IFN-γ, or a combination thereof.

[0203] In some embodiments, the IL-7 protein of this disclosure is used to target effector T cells (e.g., cytotoxic CD8) to tumors. + The recruitment of T lymphocytes can be increased. In certain embodiments, the recruitment of effector T cells to a tumor is increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% or more compared to a reference (e.g., the corresponding level in a subject that did not receive the IL-7 protein).

[0204] In some embodiments, the IL-7 protein of this disclosure may reduce the number and / or proportion of myeloid-derived suppressor cells (MDSCs) in a tumor of interest. In certain embodiments, the number and / or proportion of MDSCs in a tumor may be reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% compared to a reference (e.g., the corresponding level in a subject that did not receive the IL-7 protein).

[0205] In some embodiments, the IL-7 protein that can be used in this disclosure, when administered to a subject, affects CD8 in tumors. + The ratio of TIL to MDSC can be increased. In certain embodiments, CD8 +The ratio of TIL to MDSC increases after administration by at least approximately 5%, at least approximately 10%, at least approximately 20%, at least approximately 30%, at least approximately 40%, at least approximately 50%, at least approximately 60%, at least approximately 70%, at least approximately 80%, at least approximately 90%, at least approximately 100%, at least approximately 125%, at least approximately 150%, at least approximately 200%, at least approximately 250%, or at least approximately 300%, compared to a reference (e.g., the corresponding level in a subject that did not receive IL-7 protein).

[0206] IIb. PD-1 Antagonist In some embodiments, the Disclosure provides methods for treating tumors in a subject requiring such treatment, comprising administering to the subject an effective amount of IL-7 protein in combination with an effective amount of a PD-1 pathway antagonist ("PD-1 antagonist"). As used herein, the term "PD-1 antagonist" may be used synonymously with the term "PD-1 pathway inhibitor," but does not include, PD-1 conjugates, PD-L1 conjugates, and PD-L2 conjugates. PD-1 conjugates include antibodies that specifically bind to PD-1. PD-L1 and PD-L2 conjugates include antibodies that specifically bind to PD-L1 and / or PD-L2, as well as soluble PD-1 polypeptides that bind to PD-L1 and / or PD-L2.

[0207] Anti-PD-1 antibody In some embodiments, the PD-1 antagonists that can be used in this disclosure are anti-PD-1 antibodies. Antibodies that specifically bind to PD-1 with high affinity (e.g., human antibodies) are disclosed in U.S. Patents 8,008,449 and 8,779,105, which are incorporated herein by reference, respectively. Other anti-PD-1 mAbs are described, for example, in U.S. Patents 6,808,710, 7,488,802, 8,168,757, and 8,354,509, and PCT Publication WO2012 / 145493, which are incorporated herein by reference, respectively. Each of the anti-PD-1 HuMAb disclosed in U.S. Patent 8,008,449 has been shown to exhibit one or more of the following characteristics: (a) 1 × 10⁻¹⁶ when determined by surface plasmon resonance using a Biacore biosensor system -7 K below M D (b) binds to human PD-1; (c) substantially does not bind to human CD28, CTLA-4, or ICOS; (d) increases T cell proliferation in a mixed lymphocyte reaction (MLR) assay; (e) increases interferon-γ production in an MLR assay; (f) binds to human PD-1 and cynomolgus monkey PD-1; (g) inhibits the binding of PD-L1 and / or PD-L2 to PD-1; (h) stimulates an antigen-specific memory response; (i) stimulates an Ab response; and (j) inhibits tumor cell growth in vivo. Anti-PD-1 antibodies useful in the present invention include mAbs that specifically bind to human PD-1 and exhibit at least one, preferably at least five, of the above characteristics.

[0208] In some embodiments, the anti-PD-1 antibody is nivolumab. Nivolumab (also known as "OPDIVO®"; 5C4, BMS-936558, MDX-1106, or ONO-4538) is a fully human IgG4(S228P) PD-1 immune checkpoint inhibitor antibody that selectively prevents interaction with PD-1 ligands (PD-L1 and PD-L2), thereby inhibiting the downregulation of antitumor T cell function (U.S. Patent No. 8,008,449, Wang et al., 2014 Cancer Immunol Res. 2(9):846-56, these are incorporated herein by reference, respectively). In some embodiments, the anti-PD-1 antibody or a fragment thereof cross-competes with nivolumab. In other embodiments, the anti-PD-1 antibody or a fragment thereof binds to the same epitope as nivolumab. In certain embodiments, the anti-PD-1 antibody has the same CDR as nivolumab. In some embodiments, an anti-PD-1 antibody (e.g., nivolumab) is administered to the subject at a constant dose of approximately 240 mg every two weeks or approximately 480 mg every four weeks (e.g., in combination with the IL-7 protein disclosed herein). In certain embodiments, an anti-PD-1 antibody (e.g., nivolumab) is administered at a weight-based dose of approximately 3 mg / kg every two weeks.

[0209] Suitable anti-PD-1 antibodies (or VH and / or VL domains derived therefrom) for use in the present invention can be produced using methods well known in the art. Alternatively, anti-PD-1 antibodies recognized in the art may be used. For example, monoclonal antibodies 5C4 (referred herein as nivolumab or BMS-936558), 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4, whose teachings are incorporated herein by reference, can be used. Other known PD-1 antibodies include lambrolizumab (MK-3475), described in WO2008 / 156712, and AMP-514, described in WO2012 / 145493, whose teachings are incorporated herein by reference. Further known anti-PD-1 antibodies and other PD-1 inhibitors include those described in WO2009 / 014708, WO03 / 099196, WO2009 / 114335, and WO2011 / 161699, the teachings of which are incorporated herein by reference. Another known anti-PD-1 antibody is pidilizumab (CT-011). Antibodies or antigen-binding fragments that compete with any of these antibodies or inhibitors for binding to PD-1 may also be used.

[0210] In some embodiments, the anti-PD-1 antibody or its antigen-binding fragment cross-compete with pembrolizumab. In some embodiments, the anti-PD-1 antibody or its antigen-binding fragment binds to the same epitope as pembrolizumab. In certain embodiments, the anti-PD-1 antibody or its antigen-binding fragment has the same CDR as pembrolizumab. In another embodiment, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab (also known as "KEYTRUDA®", lambrolizumab, and MK-3475) is a humanized IgG4 monoclonal antibody against the human cell surface receptor PD-1 (programmed death-1 or programmed cell death-1). Pembrolizumab is described, for example, in U.S. Patent Nos. 8,354,509 and 8,900,587. See also worldwideweb.cancer.gov / drugdictionary?cdrid=695789 (last accessed May 25, 2017). These are incorporated herein by reference, respectively. Pembrolizumab is approved by the FDA for the treatment of relapsed or refractory melanoma. In some embodiments, an anti-PD-1 antibody (e.g., pembrolizumab) is administered to the subject at a constant dose of approximately 200 mg every three weeks (e.g., in combination with the IL-7 protein disclosed herein). In certain embodiments, an anti-PD-1 antibody (e.g., pembrolizumab) is administered at a weight-based dose of approximately 2 mg / kg every three weeks.

[0211] In other embodiments, the anti-PD-1 antibody or its antigen-binding fragment cross-compete with MEDI0608. In yet another embodiment, the anti-PD-1 antibody or its antigen-binding fragment binds to the same epitope as MEDI0608. In certain embodiments, the anti-PD-1 antibody has the same CDR as MEDI0608. In other embodiments, the anti-PD-1 antibody is the monoclonal antibody MEDI0608 (formerly known as AMP-514). MEDI0608 is described, for example, in U.S. Patent No. 8,609,089 or worldwideweb.cancer.gov / drugdictionary?cdrid=756047 (last accessed May 25, 2017), which are incorporated herein by reference, respectively.

[0212] In other embodiments, the anti-PD-1 antibody or its antigen-binding fragment cross-compete with BGB-A317. In some embodiments, the anti-PD-1 antibody or its antigen-binding fragment binds to the same epitope as BGB-A317. In certain embodiments, the anti-PD-1 antibody or its antigen-binding fragment has the same CDR as BGB-A317. In certain embodiments, the anti-PD-1 antibody or its antigen-binding fragment is the humanized monoclonal antibody BGB-A317. BGB-A317 is described in U.S. Publication No. 2015 / 0079109, which is incorporated herein by reference.

[0213] In some embodiments, an antibody or antigen-binding fragment that cross-competes with nivolumab for binding to human PD-1, or that binds to the same human PD-1 epitope region as nivolumab, is an mAb. For administration to human subjects, these cross-competitive antibodies may be chimeric antibodies, humanized antibodies, or human antibodies. Such chimeric mAbs, humanized mAbs, or human mAbs can be prepared and isolated by methods well known in the art.

[0214] An anti-PD-1 antibody or its antigen-binding fragment suitable for use in this disclosure is an antibody that binds to PD-1 with high specificity and affinity, blocks the binding of PD-L1 and / or PD-L2, and inhibits the immunosuppressive effect of the PD-1 signaling pathway. In certain embodiments, the anti-PD-1 antibody or its antigen-binding moiety cross-competes with nivolumab for binding to human PD-1. In other embodiments, the anti-PD-1 antibody or its antigen-binding moiety is a chimeric antibody, a humanized antibody, or a human monoclonal antibody, or a portion thereof. In certain embodiments, the antibody is a humanized antibody. In other embodiments, the antibody is a human antibody. Antibodies of the IgG1, IgG2, IgG3, or IgG4 isotype can be used.

[0215] In certain embodiments, the anti-PD-1 antibody or its antigen-binding fragment comprises a heavy chain constant region which is a human IgG1 or IgG4 isotype. In certain other embodiments, the sequence of the IgG4 heavy chain constant region of the anti-PD-1 antibody or its antigen-binding fragment comprises an S228P mutation which replaces a serine residue in the hinge region with a proline residue which is normally found at the corresponding position in the IgG1 isotype antibody. This mutation is present in nivolumab and maintains a low affinity for the activated Fc receptor associated with wild-type IgG4 antibody while preventing Fab arm exchange with endogenous IgG4 antibody (Wang et al., 2014). In yet another embodiment, the antibody comprises a light chain constant region which is a human kappa or lambda constant region. In other embodiments, the anti-PD-1 antibody or its antigen-binding fragment is an mAb or its antigen-binding portion. In certain embodiments of any of the therapeutic methods described herein that involve administration of an anti-PD-1 antibody, the anti-PD-1 antibody is nivolumab. In other embodiments, the anti-PD-1 antibody is pembrolizumab. In other embodiments, the anti-PD-1 antibody is selected from the human antibodies 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4 described in U.S. Patent No. 8,008,449, which is incorporated herein by reference. In yet another embodiment, the anti-PD-1 antibody is MEDI0608 (formerly AMP-514), AMP-224, or pidilizumab (CT-011).

[0216] Anti-PD-L1 antibody In some embodiments, the PD-1 antagonists that can be used in this disclosure are anti-PD-L1 antibodies. Suitable anti-human PD-L1 antibodies (or VH and / or VL domains derived therefrom) for use in the present invention can be produced using methods well known in the art. Alternatively, anti-PD-L1 antibodies recognized in the art may be used. For example, human anti-PD-L1 antibodies disclosed in U.S. Patent No. 7,943,743, the contents of which are incorporated herein by reference, may be used. Such anti-PD-L1 antibodies include 3G10, 12A4 (also known as BMS-936559), 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4. Other anti-PD-L1 antibodies recognized in the art that may be used include, for example, those described in U.S. Patents 7,635,757 and 8,217,149, U.S. Publication 2009 / 0317368, and PCT Publications WO2011 / 066389 and WO2012 / 145493, the teachings of which are also incorporated herein by reference. Other examples of anti-PD-L1 antibodies include atezolizumab (TECENTRIQ®; RG7446) or durvalumab (IMFINZI®; MEDI4736). Antibodies or antigen-binding fragments that compete with any of these art-recognized antibodies or inhibitors for binding to PD-L1 may also be used. In some embodiments, an anti-PD-L1 antibody (e.g., atezolizumab) is administered to the subject at a dose of approximately 1200 mg every three weeks (e.g., in combination with the IL-7 protein disclosed herein). In some embodiments, an anti-PD-L1 antibody (e.g., durvalumab) is administered at a dose of approximately 10 mg / kg every two weeks (e.g., in combination with the IL-7 protein disclosed herein).

[0217] In certain embodiments, the anti-PD-L1 antibody is BMS-936559 (formerly known as 12A4 or MDX-1105) (see, for example, U.S. Patent No. 7,943,743 and WO2013 / 173223, both of which are incorporated herein by reference). In other embodiments, the anti-PD-L1 antibody is MPDL3280A (also known as RG7446 and atezolizumab) (see, e.g., Herbst et al. 2013 J Clin Oncol 31(suppl):3000, U.S. Patent No. 8,217,149, both incorporated herein by reference), MEDI4736 (Khleif, 2013, In: Proceedings from the European Cancer Congress 2013; September 27-October 1, 2013; Amsterdam, The Netherlands. Abstract 802, incorporated herein by reference), or MSB0010718C (also known as avelumab; see, U.S. 2014 / 0341917, incorporated herein by reference). In certain embodiments, antibodies that cross-compete with the PD-L1 antibody referenced above for binding to human PD-L1, or that bind to the same human PD-L1 epitope region as the PD-L1 antibody referenced above, are mAbs. For administration to human subjects, these cross-competing antibodies may be chimeric antibodies, humanized antibodies, or human antibodies. Such chimeric mAbs, humanized mAbs, or human mAbs can be prepared and isolated by methods well known in the art. In some embodiments, an anti-PD-L1 antibody (e.g., avelumab) is administered to a subject at a dose of approximately 800 mg every two weeks (e.g., in combination with the IL-7 protein disclosed herein).

[0218] IIc.CTLA-4 Antagonist In some embodiments, the disclosure also provides a method for treating a tumor in a subject requiring it, comprising administering to the subject an effective amount of IL-7 protein in combination with an effective amount of a CTLA-4 pathway antagonist ("CTLA-4 antagonist"). In some embodiments, the CTLA-4 antagonist is an anti-CTLA-4 antibody.

[0219] HuMAbs that specifically bind to CTLA-4 with high affinity are disclosed in U.S. Patents 6,984,720 and 7,605,238, which are incorporated herein by reference, respectively. Other anti-CTLA-4 mAbs are described, for example, in U.S. Patents 5,977,318, 6,051,227, 6,682,736 and 7,034,121, which are incorporated herein by reference, respectively. The anti-CTLA-4 HuMAbs disclosed in U.S. Patents 6,984,720 and 7,605,238 (both incorporated herein by reference) have been shown to exhibit one or more of the following characteristics: (a) at least about 10 when determined by Biacore analysis 7 M -1 , or about 10 9 M -1 , or about 10 10 M -1 ~10 11 M -1 The equilibrium association constant (K) a (b) Binding affinity reflected in the following, specifically binding to human CTLA-4; (b) at least about 10 3 , about 10 4 , or about 10 5 m -1 s -1 The dynamic synodic constant (k a (c) at least about 10 3 , about 10 4 , or about 10 5 m -1 s -1 The dynamical dissociation constant (k d(d) inhibits the binding of CTLA-4 to B7-1 (CD80) and B7-2 (CD86). Anti-CTLA-4 antibodies useful in the present invention include mAbs that specifically bind to human CTLA-4 and exhibit at least one, at least two, or at least three of the above-mentioned features. An exemplary clinical anti-CTLA-4 antibody is human mAb 10D1 (now known as ipilimumab and marketed as YERVOY®), disclosed in U.S. Patent No. 6,984,720, incorporated herein by reference. Ipilimumab is an anti-CTLA-4 antibody used in the manner disclosed herein. Ipilimumab is a fully human IgG1 monoclonal antibody that blocks CTLA-4 from binding to its B7 ligand, thereby stimulating T cell activation and improving overall survival (OS) in patients with advanced melanoma. In some embodiments, an anti-CTLA-4 antibody (e.g., ipilimumab) is administered to the subject (e.g., in combination with the IL-7 protein disclosed herein) at a dose of approximately 3 mg / kg every 3 weeks (e.g., to treat unresectable or metastatic melanoma). In some embodiments, an anti-CTLA-4 antibody (e.g., ipilimumab) is administered to the subject (e.g., in combination with the IL-7 protein disclosed herein) at four doses of approximately 10 mg / kg every 3 weeks, followed by a dose of 10 mg / kg every 12 weeks for up to 3 years (e.g., for adjuvant treatment of melanoma).

[0220] Another anti-CTLA-4 antibody useful in the methods of the present invention is tremelimumab (also known as tisilimumb and CP-675,206). Tremelimumab is a human IgG2 monoclonal anti-CTLA-4 antibody. Tremelimumab is described in WO / 2012 / 122444, U.S. Publication 2012 / 263677, and WO Publication 2007 / 113648A2, which are incorporated herein by reference, respectively. Other non-limiting examples of anti-CTLA-4 antibodies useful in this disclosure include MK-1308 (Merck) and AGEN-1884 (Agenus Inc.; see WO2016 / 196237).

[0221] The anti-CTLA-4 antibodies useful in this disclosure also include isolated antibodies that specifically bind to human CTLA-4 and cross-compete with ipilimumab, tremelimumab, MK-1308, or AGEN-1884 for binding to human CTLA-4, or that bind to the same human CTLA-4 epitope region as ipilimumab, tremelimumab, MK-1308, or AGEN-18844. In certain embodiments, antibodies that cross-compete with ipilimumab, tremelimumab, MK-1308, or AGEN-18844 for binding to human CTLA-4, or that bind to the same human CTLA-4 epitope region to which ipilimumab, tremelimumab, MK-1308, or AGEN-18844, are antibodies containing the heavy chain of a human IgG1 isotype. For administration to human subjects, these cross-competing antibodies are chimeric antibodies, or humanized antibodies or human antibodies. The antigen-binding portion of the above antibody, for example, Fab, F(ab')2, Fd, or Fv fragment, may be used in the method of the present invention.

[0222] III. Nucleic Acids, Vectors, and Host Cells Further embodiments described herein relate to one or more nucleic acid molecules encoding therapeutic agents described herein (e.g., IL-7 protein). Nucleic acids may be present in whole cells, in cell lysates, or in partially purified or substantially pure forms. A nucleic acid is considered "isolated" or "substantially pure" if it has been purified from other cellular components or other contaminants, e.g., other cellular nucleic acids (e.g., other chromosomal DNA, e.g., chromosomal DNA naturally linked to isolated DNA) or proteins by standard techniques including alkali / SDS treatment, CsCl banding, column chromatography, restriction enzymes, agarose gel electrophoresis, and other techniques well known in the art. See F. Ausubel, et al., ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. The nucleic acids described herein may be, for example, DNA or RNA, and may or may not contain intron sequences. In certain embodiments, the nucleic acid is a cDNA molecule. The nucleic acids described herein can be obtained using standard molecular biology techniques known in the art.

[0223] Certain nucleic acid molecules disclosed herein encode the IL-7 protein (e.g., those disclosed herein). Exemplary nucleic acid sequences encoding the IL-7 protein disclosed herein are shown in SEQ ID NOs. 29-39.

[0224] In some embodiments, the disclosure provides vectors comprising isolated nucleic acid molecules encoding therapeutic agents disclosed herein (e.g., IL-7 protein). In some embodiments, the vectors may be used in gene therapy.

[0225] When used as gene therapy (e.g., in humans), the nucleic acids encoding the therapeutic agents disclosed herein (e.g., IL-7 protein) may be administered in doses ranging from 0.1 mg to 200 mg. In certain embodiments, the dose is in the range of 0.6 mg to 100 mg. In further embodiments, the dose is in the range of 1.2 mg to 50 mg.

[0226] Preferred vectors in this disclosure include expression vectors, viral vectors, and plasmid vectors. In some embodiments, the vector is a viral vector.

[0227] As used herein, an expression vector means any nucleic acid construct that, when introduced into a suitable host cell, contains the elements necessary for the transcription and translation of an inserted coding sequence, or, in the case of an RNA viral vector, the elements necessary for replication and translation. Expression vectors may include plasmids, phagemids, viruses, and their derivatives.

[0228] As used herein, viral vectors include, but are not limited to, the following viruses: retroviruses such as Moloney's mouse leukemia virus, Harvey's mouse sarcoma virus, mouse mammary tumor virus, and Rous sarcoma virus; lentiviruses; adenoviruses; adeno-associated viruses; SV40 virus; polyomavirus; Epstein-Barr virus; papillomavirus; herpesvirus; vaccinia virus; poliovirus; and nucleic acid sequences derived from RNA viruses such as retroviruses. Other vectors known in the art may also be readily used. Certain viral vectors are based on non-cellularly invasive eukaryotic viruses in which non-essential genes are replaced with genes of interest. Non-cellularly invasive viruses include retroviruses, the life cycle of which involves the reverse transcription of genomic viral RNA into DNA, after which the provirus is incorporated into host cell DNA.

[0229] In some embodiments, the vector is derived from an adeno-associated virus. In other embodiments, the vector is derived from a lentivirus. Examples of lentiviral vectors are disclosed in WO9931251, WO9712622, WO9817815, WO9817816, and WO9818934, which are each incorporated herein by reference as a whole.

[0230] Other vectors include plasmid vectors. Plasmid vectors are widely reported in the art and are well known to those skilled in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. In recent years, plasmid vectors have been found to be particularly advantageous for delivering genes to cells in vivo because they do not replicate within the host genome and are not integrated into the host genome. However, these plasmids, which have promoters that are compatible with host cells, can express peptides from genes operably encoded within the plasmid. Some commonly used plasmids available from commercial suppliers include pBR322, pUC18, pUC19, various pcDNA plasmids, pRC / CMV, various pCMV plasmids, pSV40, and pBlueScript. Further examples of specific plasmids include pcDNA3.1, catalog number V79020; pcDNA3.1 / hygro, catalog number V87020; pcDNA4 / myc-His, catalog number V86320; and pBudCE4.1, catalog number V53220, all of which are from Invitrogen (Carlsbad, CA). Other plasmids are also well known to those skilled in the art. Furthermore, plasmids can be custom designed using standard molecular biology techniques to remove and / or add specific DNA fragments.

[0231] This disclosure also includes methods for producing the therapeutic agents (e.g., IL-7 protein) disclosed herein. In some embodiments, such methods may include expressing the therapeutic agent (e.g., IL-7 protein) in cells containing nucleic acid molecules encoding the therapeutic agent, e.g., SEQ ID NOs. 29-39. Further details on the methods for producing the IL-7 protein disclosed herein are provided, for example, in WO2016 / 200219, which is incorporated herein by reference in whole. Host cells containing these nucleotide sequences are included herein. Non-limiting examples of host cells that can be used include immortal hybridoma cells, NS / 0 myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, human amniotic fluid-derived cells (CapT cells), COS cells, or combinations thereof.

[0232] IV. Pharmaceutical Compositions Furthermore, this specification provides compositions comprising one or more therapeutic agents (e.g., IL-7 protein and / or immune checkpoint inhibitors) having a physiologically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences (1990), Mack Publishing Co., Easton, PA) of a desired purity. In some embodiments, the compositions disclosed herein comprise either IL-7 protein or an immune checkpoint inhibitor. As disclosed herein, such compositions may be used in combination (e.g., a first composition comprising IL-7 protein and a second composition comprising an immune checkpoint inhibitor). In other embodiments, the compositions disclosed herein may comprise both IL-7 protein and an immune checkpoint inhibitor.

[0233] Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dosage and concentration used and include buffers such as phosphoric acid, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol alcohol, butyl alcohol, or benzyl alcohol; alkylparabens such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol, etc.); low molecular weight (less than approximately 10 residues) polypeptides; Proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, such as glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0234] In some embodiments, the compositions disclosed herein (e.g., those comprising IL-7 protein or immune checkpoint inhibitors) comprise one or more additional components selected from bulking agents, stabilizers, surfactants, buffers, or combinations thereof.

[0235] Useful buffers in this disclosure may be weak acids or weak bases used to maintain the acidity (pH) of a solution near a selected value after the addition of another acid or base. Suitable buffers can maximize the stability of pharmaceutical compositions by maintaining pH control of the composition. Suitable buffers can also ensure physiological compatibility or optimize solubility. Rheology, viscosity, and other properties may also depend on the pH of the composition. Common buffers include, but are not limited to, Tris buffer, Tris-Cl buffer, histidine buffer, TAE buffer, HEPES buffer, TBE buffer, sodium phosphate buffer, MES buffer, ammonium sulfate buffer, potassium phosphate buffer, potassium thiocyanate buffer, succinate buffer, tartaric acid buffer, DIPSO buffer, HEPPSO buffer, POPSO buffer, PIPES buffer, PBS buffer, MOPS buffer, acetate buffer, phosphate buffer, cacodylate buffer, glycine buffer, sulfur This includes acid buffers, imidazole buffers, guanidine hydrochloride buffers, phosphate-citrate buffers, borate buffers, malonic acid buffers, 3-picoline buffers, 2-picoline buffers, 4-picoline buffers, 3,5-lutidine buffers, 3,4-lutidine buffers, 2,4-lutidine buffers, Aces, diethyl malonate buffers, N-methylimidazole buffers, 1,2-dimethylimidazole buffers, TAPS buffers, bis-tris buffers, L-arginine buffers, lactate buffers, glycolate buffers, or combinations thereof.

[0236] In some embodiments, the compositions disclosed herein further include bulking agents. Bulking agents may be added to pharmaceutical products to facilitate their accurate measurement and handling by adding volume and mass to the product. Bulking agents that may be used in this disclosure include, but are not limited to, sodium chloride (NaCl), mannitol, glycine, alanine, or combinations thereof.

[0237] In some embodiments, the compositions disclosed herein may include stabilizers. Non-limiting examples of stabilizers that may be used in this disclosure include sucrose, trehalose, raffinose, arginine, or combinations thereof.

[0238] In some embodiments, the compositions disclosed herein include a surfactant. In certain embodiments, the surfactant may be selected from alkyl ethoxylates, nonylphenol ethoxylates, amine ethoxylates, polyethylene oxides, polypropylene oxides, fatty alcohols such as cetyl alcohol or oleyl alcohol, cocamide MEA, cocamide DEA, polysorbate, dodecyldimethylamine oxide, or combinations thereof. In some embodiments, the surfactant is polysorbate 20 or polysorbate 80.

[0239] In some embodiments, compositions comprising the IL-7 protein may be formulated using the same formulations as the immune checkpoint inhibitors disclosed herein (for example, used in combination with the IL-7 protein). In other embodiments, the IL-7 protein and the immune checkpoint inhibitor are formulated using different formulations.

[0240] In some embodiments, the IL-7 protein disclosed herein is incorporated into a composition comprising (a) a basal buffer, (b) a sugar, and (c) a surfactant. In certain embodiments, the basal buffer comprises histidine acetate or sodium citrate. In some embodiments, the basal buffer is at a concentration of about 10 to about 50 nM. In some embodiments, the sugar comprises sucrose, trehalose, dextrose, or a combination thereof. In some embodiments, the sugar is present at a concentration of about 2.5 to about 5.0 w / v%. In further embodiments, the surfactant is selected from polysorbate, polyoxyethylene alkyl ether, polyoxyethylene stearate, alkyl sulfate, polyvinylpyridone, poloxamer, or a combination thereof. In some embodiments, the surfactant is at a concentration of about 0.05% to about 6.0 w / v%.

[0241] In some embodiments, the composition containing IL-7 further comprises amino acids. In certain embodiments, the amino acids are selected from arginine, glutamic acid, glycine, histidine, or combinations thereof. In certain embodiments, the composition further comprises sugar alcohols. Non-limiting examples of sugar alcohols include sorbitol, xylitol, maltitol, mannitol, or combinations thereof.

[0242] In some embodiments, the IL-7 protein disclosed herein is incorporated into a composition comprising (a) sodium citrate (e.g., about 20 mM), (b) sucrose (e.g., about 5%), (c) sorbitol (e.g., about 1.5%), and (d) Tween80 (e.g., about 0.05%).

[0243] In some embodiments, the IL-7 protein of this disclosure is formulated as described in WO2017 / 078385A1, which is incorporated herein as a whole.

[0244] In some embodiments, compositions that can be used with the IL-7 protein disclosed herein include (i) nivolumab (OPDIVO®) (e.g., about 10 mg), (ii) mannitol (e.g., about 30 mg), (iii) pentetic acid (e.g., about 0.008 mg), (iv) polysorbate 80 (e.g., about 0.2 mg), (v) sodium chloride (e.g., about 2.92 mg), and (vi) anhydrous sodium citrate (e.g., about 5.88 mg). In certain embodiments, the composition may further contain hydrochloric acid and / or sodium hydroxide to adjust the pH of the composition to about 6.

[0245] In some embodiments, compositions that can be used with the IL-7 protein disclosed herein include (i) pembrolizumab (KEYTRUDA®) (e.g., about 25 mg), (ii) L-histidine (e.g., about 1.55 mg), (iii) polysorbate 80 (e.g., about 0.2 mg), and (iv) sucrose (e.g., about 70 mg). In certain embodiments, the composition may further contain hydrochloric acid and / or sodium hydroxide to adjust the pH to about 5.5.

[0246] In some embodiments, compositions that can be used with the IL-7 protein disclosed herein include (i) atezolizumab (TECENTRIQ®) (e.g., about 60 mg), (ii) glacial acetic acid (e.g., about 16.5 mg), (iii) L-histidine (e.g., about 62 mg), (iv) sucrose (e.g., about 821.6 mg), and (v) polysorbate 20 (e.g., about 8 mg). In certain embodiments, the composition includes hydrochloric acid and / or sodium hydroxide to adjust the pH to about 5.8.

[0247] In some embodiments, compositions that can be used with the IL-7 protein disclosed herein include (i) durvalumab (IMFINZI®) (e.g., about 50 mg), (ii) L-histidine (e.g., about 2 mg), (iii) L-histidine hydrochloride monohydrate (e.g., about 2.7 mg), (iv) α,α-trehalose dihydrate (e.g., about 104 mg), and (v) polysorbate 80 (e.g., about 0.2 mg).

[0248] In some embodiments, compositions that can be used with the IL-7 protein disclosed herein include (i) ipilimumab (YERVOY®) (e.g., 5 mg), (ii) diethylenetriaminepentaacetic acid (DTPA) (e.g., about 0.04 mg), (iii) mannitol (e.g., about 10 mg), (iv) polysorbate 80 (plant-derived) (e.g., about 0.1 mg), (v) sodium chloride (e.g., about 5.85 mg), and (vi) tris hydrochloride (e.g., about 3.15 mg).

[0249] In some embodiments, compositions that can be used with the IL-7 protein disclosed herein include (i) avelumab (BAVENCIO®) (e.g., about 20 mg), (ii) D-mannitol (e.g., about 51 mg), (iii) glacial acetic acid (e.g., about 0.6 mg), (iv) polysorbate 20 (e.g., about 0.5 mg), and (v) sodium hydroxide (e.g., about 0.3 mg).

[0250] Pharmaceutical compositions may be formulated for any route of administration to the target. Specific examples of routes of administration include intramuscular, subcutaneous, ophthalmic, intravenous, intraperitoneal, intradermal, orbital, intracerebral, intracranial, intraspinal, intraventricular, intrathecal, intracisional, intrasacral, or intratumoral. Parenteral administration, characterized by subcutaneous, intramuscular, or intravenous injection, is also envisioned herein. Injectables may be prepared in conventional forms, either as a liquid solution or suspension, a solid form suitable for solution or suspension in liquid before injection, or an emulsion. Injectables, solutions, and emulsions also contain one or more excipients. Suitable excipients include, for example, water, saline, dextrose, glycerol, or ethanol. Furthermore, if desired, the administered pharmaceutical composition may also contain trace amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, pH buffers, stabilizers, solubility enhancers, and other such agents, such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, and cyclodextrin.

[0251] Pharmaceutically acceptable carriers used in parenteral preparations include aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspensions and dispersants, emulsifiers, chelating agents or sequestering agents, and other pharmaceutically acceptable substances. Examples of aqueous vehicles include sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, dextrose, and lactated Ringer's injection. Examples of non-aqueous parenteral vehicles include plant-derived fixative oils, cottonseed oil, corn oil, sesame oil, and peanut oil. Antimicrobial agents in bacteriostatic or fungiostatic concentrations may be added to parenteral preparations packaged in multi-dose containers containing phenol or cresol, mercury preparations, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoates, thimerosal, benzalkonium chloride, and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. The buffer contains phosphates and citrates. The antioxidant contains sodium bisulfate. The local anesthetic contains procaine hydrochloride. The suspending and dispersing agents contain sodium carboxymethylcellulose, hydroxypropyl methylcellulose, and polyvinylpyrrolidone. The emulsifier contains polysorbate 80 (TWEEN® 80). The metal ion sequestering or chelating agent contains EDTA. The pharmaceutical carrier also contains ethyl alcohol, polyethylene glycol, and propylene glycol for water-miscible vehicles, as well as sodium hydroxide, hydrochloric acid, citric acid, or lactic acid for pH adjustment.

[0252] Preparations for parenteral administration include sterile solutions ready for injection, sterile, dried, soluble products that are combined with a solvent immediately before use, such as lyophilized powders, subcutaneous tablets, sterile suspensions ready for injection, sterile, dried, insoluble products that are combined with a vehicle immediately before use, and sterile emulsions. The solutions may be aqueous or non-aqueous.

[0253] When administered intravenously, suitable carriers include physiological saline or phosphate-buffered saline (PBS), as well as solutions containing thickeners and solubilizers (such as glucose, polyethylene glycol, and polypropylene glycol, and mixtures thereof).

[0254] Topical mixtures containing antibodies are prepared as described for topical and systemic administration. The resulting mixtures may be solutions, suspensions, emulsions, etc., and may be formulated as creams, gels, ointments, emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes, foams, aerosols, irrigators, sprays, suppositories, bandages, skin patches, or any other formulation suitable for topical administration.

[0255] The antibodies or antigen-binding moieties described herein may be formulated as aerosols for topical application, such as by inhalation (see, for example, U.S. Patents 4,044,126, 4,414,209, and 4,364,923, which describe aerosols for the delivery of steroids useful in the treatment of inflammatory diseases, particularly asthma). These formulations for administration to the airways may be in the form of aerosols or solutions for nebulizers, or as ultrafine powders for blowing, either alone or in combination with an inert carrier such as lactose. In such cases, the particles of the formulation have a diameter of less than 50 microns in one form and less than 10 microns in another form.

[0256] The therapeutic agents disclosed herein (e.g., IL-7 protein) may be in the form of gels, creams, and lotions for topical or local application, e.g., to the skin and mucous membranes, e.g., to the eyes, and may be formulated for application to the eyes, and for application into the cisterna magna or spinal cord. Local administration is envisioned by transdermal delivery, administration to the eyes or mucous membranes, or inhalation therapy. Antibodies may also be administered as nasal drops alone or in combination with other pharmaceutically acceptable excipients.

[0257] Transdermal patches, including ionography and electrophoresis devices, are well known to those skilled in the art and can be used to administer antibodies. For example, such patches are disclosed in U.S. Patents 6,267,983, 6,261,595, 6,256,533, 6,167,301, 6,024,975, 6,010,715, 5,985,317, 5,983,134, 5,948,433, and 5,860,957, each incorporated herein by reference as a whole.

[0258] In certain embodiments, a pharmaceutical composition comprising a therapeutic agent described herein (e.g., IL-7 protein or immune checkpoint inhibitor) is a lyophilized powder that can be reconstituted for administration as a solution, emulsion, and other mixture. It may also be reconstituted and formulated as a solid or gel. The lyophilized powder is prepared by dissolving the antibody or its antigen-binding moiety, or a pharmaceutically acceptable derivative thereof, in a suitable solvent. In some embodiments, the lyophilized powder is sterile. The solvent may contain excipients or other pharmacological components of the powder to improve stability, or a reconstituted solution prepared from the powder. Possible excipients include, but are not limited to, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable agents. The solvent may also contain a buffer, e.g., citric acid, sodium phosphate, or potassium phosphate, or other such buffers known to those skilled in the art, in one embodiment at a nearly neutral pH. The solution is then sterile filtered under standard conditions known to those skilled in the art, followed by lyophilization to obtain the desired formulation. In some embodiments, the resulting solution can be dispensed into vials for lyophilization. Each vial may contain a single dose or a multi-dose dose of the compound. The lyophilized powder can be stored under suitable conditions, such as from about 4°C to room temperature.

[0259] Reconstituting this lyophilized powder with sterile water for injection yields a formulation for parenteral administration. During reconstitution, the lyophilized powder is added to sterile water or another suitable carrier. The exact amount depends on the selected compound. Such amounts may be determined empirically.

[0260] The compositions provided herein may be formulated to target specific tissues, receptors, or other areas of the body being treated. Many such targeting methods are well known to those skilled in the art. All such targeting methods are assumed in the use of the compositions of the present invention. Non-limiting examples of targeting methods include, for example, U.S. Patents 6,316,652, 6,274,552, 6,271,359, 6,253,872, 6,139,865, 6,131,570, 6,120,751, 6,071,495, and 6,0 See issues 60,082, 6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, 5,900,252, 5,840,674, 5,759,542, and 5,709,874.

[0261] The composition used for in vivo administration may be sterile. This can be easily done, for example, by filtration through a sterile filtration membrane.

[0262] The following embodiments are merely illustrative and should not be construed as limiting the scope of the disclosure in any way, as many variations and equivalents will be apparent to those skilled in the art upon reading the disclosure. [Examples]

[0263] Example 1: Effect of combination therapy with IL-7 protein and PD-1 pathway inhibitors on tumor volume To evaluate the effect of the combination of IL-7 protein and PD-1 pathway inhibitors on tumor volume, an animal model of colon adenocarcinoma was used. Briefly, MC-38 colon adenocarcinoma tumor cells (1 × 10⁶) 5Tumors were transplanted (subcutaneously) into each C57BL / 6 mouse. Four days after tumor dissemination, the animals were subcutaneously administered IL-7 protein (1.25 mpk or 25 μg / mouse) or IL-7 buffer. See Figure 1A. Subsequently, on days 12, 15, and 18 after tumor dissemination, the animals were intraperitoneally administered anti-PD-1 antibody (5 mpk or 100 μg / mouse) or isotype control antibody. Tumor volume was measured on days 8, 11, 13, 15, 18, and 20 after tumor dissemination. Figure 1A is a graph of the drug administration schedule, and Table 1 (below) shows each treatment group. [Table 1]

[0264] Figures 1B and 1C present the results of two different studies. As shown, animals treated with combination therapy (IL-7 protein + anti-PD-1 antibody) had significantly reduced tumor volume compared not only to control animals (i.e., those given only IL-7 combined buffer) but also to animals treated with either IL-7 protein or anti-PD-1 antibody alone.

[0265] Example 2: Effects of combination therapy with IL-7 protein and PD-1 pathway inhibitors on tumor-infiltrating lymphocytes To further evaluate the antitumor effect of the combination of IL-7 protein and PD-1 pathway inhibitors, MC-38 colon adenocarcinoma tumor cells (1 × 10⁶) were used. 5 The tumors (subcutaneously) were transplanted into C57BL / 6 mice. Four days after tumor dissemination, the animals were treated with a single dose of IL-7 protein (1.25 mpk or 25 μg / mouse, subcutaneously) or IL-7 buffer. See Figure 2A. Subsequently, on days 9 and 12 after tumor dissemination, the animals were treated with anti-PD-1 antibody (5 mpk or 100 μg / mouse, intraperitoneally) or isotype control antibody. The animals were sacrificed on day 14 after tumor dissemination, and tumor-infiltrating lymphocytes in each animal were analyzed by flow cytometry.

[0266] As shown in Figure 2B, in animals treated with either anti-PD-1 antibody or IL-7 protein alone, approximately 5 - 7% of CD45 + cells in the tumors of the animals were CD4 + TILs. This ratio was similar to that observed in control animals. However, in animals treated with combination therapy (IL-7 protein + anti-PD-1 antibody), a significant increase in the number of CD4 + TILs in the tumors was observed (approximately 10 - 12% of CD45 + cells in the tumors). As shown in Figure 2C, in the case of CD8 + TILs, treatment with IL-7 protein alone increased the number of CD8 + TILs moderately compared to both control animals and animals treated with anti-PD-1 antibody alone. When animals were treated with both IL-7 protein and anti-PD-1 antibody, a further increase in the number of CD8 + TILs in CD45 + cells was also observed.

[0267] In summary, the above two results of Examples 1 and 2 indicate that a treatment regimen combining IL-7 protein and a PD-1 pathway inhibitor (e.g., anti-PD-1 antibody) can effectively treat cancer.

[0268] Example 3 Effects of the triple combination of cyclophosphamide (CPA), IL-7 protein, and PD-1 pathway inhibitor on tumor volume and survival period Next, the antitumor effect of combination therapy with IL-7 protein, a PD-1 pathway inhibitor, and a chemotherapeutic agent (e.g., CPA) was evaluated in a murine model of colon adenocarcinoma. Briefly, MC-38 colon adenocarcinoma tumor cells (1×10 5Tumors were transplanted (subcutaneously) into C57BL / 6 mice. Subsequently, 10 days after tumor dissemination, a single dose of CPA (100 mpk or 2 mg / mouse) or PBS was administered intraperitoneally to the animals. Two days after CPA administration, the animals were subcutaneously administered IL-7 protein (10 mpk or 200 μg / mouse) or IL-7 combined buffer. Starting on day 6 after CPA administration, anti-PD-1 antibody (5 mpk or 100 μg / mouse), anti-PD-L1 antibody (5 mpk or 100 μg / mouse), or isotype control antibody was administered intraperitoneally to the animals every 3 days for a total of 5 doses (i.e., on days 6, 9, 12, 15, and 18 after CPA induction). See Figure 3A. Tumor volume was measured on days 0, 1, 4, 6, 8, 11, 13, 15, 18, and 20 after CPA induction.

[0269] As shown in Figure 3B, tumor volume was significantly reduced in animals treated with CPA and IL-7 protein ("3") compared to animals treated with PBS ("1") or CPA alone ("2"). Further reductions in tumor volume occurred in animals ("4" and "5", respectively) when either anti-PD1 antibody or anti-PD-L1 antibody was added to CPA and IL-7 protein. As shown in Figure 3C, increased tumor volume reduction correlated with extended survival.

[0270] The above results suggest that combination therapy with IL-7 protein and PD-1 pathway inhibitors can be effectively used in combination with other anticancer agents such as cyclophosphamide.

[0271] Example 4: Effect of combination therapy with IL-7 protein and PD-1 pathway inhibitors on tumor volume in lymphopenia induced by thymectomy. As mentioned above, many anticancer drugs (e.g., chemotherapy or radiotherapy) can cause lymphopenia in cancer patients. Therefore, thymectomized mice were used to evaluate the antitumor effect of a combination of IL-7 protein and PD-1 pathway inhibitors in a lymphopenic state. Briefly, C57BL / 6 mice were anesthetized and fixed to a dissection board. The airway was opened by using a rubber band to tilt the mouse's head backward. Rolled tissue pads were placed under both shoulders of the mouse to facilitate contact and help push the heart and thymus forward. For sterilization, the neck and upper chest of the mouse were wiped with 70% ethanol. A 1.5–2 cm incision was made in the midline longitudinal direction of the skin from the neck notch toward the chest. Scissors were inserted substernally and the first sternum was cut. The chest was opened by spreading forceps. After tearing the infrahyoid muscle, the thymus was carefully grasped and then removed from the chest. The skin was quickly closed along the midline longitudinal using appliers and clips used for animal skin suturing. The time from first sternal resection to skin closure was less than 1 minute. The animals were allowed to recover from the surgery for approximately 5 weeks. Subsequently, MC-38 colon adenocarcinoma tumor cells (1 × 10⁶) were administered to the animals. 5 Tumors were seeded (subcutaneously). See Figure 4A. On day 5 after tumor seeding, animals were subcutaneously administered IL-7 protein (1.25 mpk or 25 μg / mouse) or IL-7 buffer. Anti-PD-1 antibody (5 mpk or 100 μg / mouse) or isotype control antibody was administered to animals on days 10, 13, and 16 after tumor seeding. Tumor volume was measured on days 10, 13, 16, 19, 21, and 23 after tumor seeding.

[0272] As shown in Figure 4B, thymectomized animals treated with a combination therapy regimen of IL-7 protein and anti-PD-1 antibody had significantly reduced tumor volume compared to other treatment groups (control, IL-7 protein alone, and anti-PD-1 antibody alone).

[0273] Example 5: Effects of combination therapy with IL-7 protein and PD-1 pathway inhibitors on tumor-infiltrating lymphocytes in lymphopenia caused by thymectomy. To evaluate whether the combination of IL-7 protein and PD-1 pathway inhibitors affects TIL in a lymphopenic environment, C57BL / 6 mice underwent thymectomy as described in Example 4. Five weeks after surgery, MC-38 colon adenocarcinoma tumor cells (1 × 10⁶) were observed. 5 Subcutaneous tumor cells were transplanted into animals. Four days after tumor dissemination, the animals were treated with a single dose of IL-7 protein (1.25 mpk or 25 μg / mouse, subcutaneous) or IL-7 buffer. See Figure 5A. Subsequently, on days 9 and 12 after tumor dissemination, the animals were treated with anti-PD-1 antibody (5 mpk or 100 μg / mouse, intraperitoneal) or isotype control antibody. The animals were sacrificed on day 14 after tumor dissemination, and tumor-infiltrating lymphocytes in each animal were analyzed by flow cytometry.

[0274] As observed in non-lymphocytopenic animals (see Figure 2B), treating thymectomized animals with both IL-7 protein and anti-PD-1 antibody resulted in increased CD4 levels in the tumors. + The rate of TILs increased significantly compared to other treatment groups. Figure 5B. CD8 + A significant increase in the proportion of TILs was also observed. As shown in Figure 5C, thymectomized animals treated with both IL-7 protein and anti-PD-1 antibody showed a significantly higher proportion of CD8 compared to both the control group and the anti-PD-1 antibody alone group. + TILs were present in the tumor. This increase was comparable to that observed in the IL-7 protein monotherapy group.

[0275] In summary, the results described above (i.e., Examples 4 and 5) demonstrate that combination therapy with IL-7 protein and PD-1 pathway inhibitors can effectively treat cancer even in lymphopenic conditions.

[0276] Example 6: Effects of IL-7 protein on T cell proliferation and activation To better understand the antitumor effect of the IL-7 protein disclosed in this specification, the effect of the IL-7 protein on the proliferation and activation of T cells was first evaluated in normal mice. Briefly, C57BL / 6 mice were treated by subcutaneous administration of 10 mg / kg of the IL-7 protein. Control animals were given only buffer. Blood samples of the animals were taken at various time points after administration (i.e., day 2, day 4, day 5, day 6, day 8, day 10, day 12, and day 14), and the proportions of various CD8+ T cell populations were evaluated using flow cytometry. On day 5 after treatment, some animals were sacrificed, and the CD8+ T cells in the spleen were evaluated for the expression of various activation markers (T-bet, Eomes, PD-1, granzyme B (GzmB), CXCR3) and cytokine production (IFN-γ, TNF-α, and IL-2). Intracellular cytokine staining was used to evaluate cytokine production after PMA / ionomycin stimulation ex vivo.

[0277] As shown in Figure 6A, administration of the IL-7 protein to normal mice increased the proliferation of CD8+ T cells (as evident from the increased expression of Ki-67) compared to control animals. The greatest effect was observed in the CD44+ (central memory) CD8+ T cell population. In addition to the increased proliferation, the splenic CD8+ T cells of the IL-7 protein-treated animals also expressed higher levels of T-bet, Eomes, PD-1, granzyme B, and CXCR3, suggesting that these cells were more activated compared to cells from control animals (see Figure 6B). Also, a higher proportion of cells produced IFN-γ, TNF-α, and IL-2 after ex vivo stimulation.

[0278] Next, to further evaluate the effect of the IL-7 protein on T cells, naive T cells (10 6 cells / mouse) or central memory T cells (5 × 10 5Cells (from mice) were labeled with CELLTRACE® Violet (CTV) and transfe...

Claims

1. A combination of pharmaceuticals for the treatment of colorectal cancer, comprising interleukin-7 (IL-7) protein and anti-PD-1 antibody, The IL-7 protein comprises an oligopeptide consisting of methionine-glycine-methionine (MGM), human IL-7, and a half-life extension portion. The half-life extension portion is a hybrid Fc comprising a hinge region, a CH2 domain, and a CH3 domain, wherein the hinge region comprises a human IgD hinge region, the CH2 domain comprises a portion of the human IgD CH2 domain and a portion of the human IgG4 CH2 domain, and the CH3 domain comprises a portion of the human IgG4 CH3 domain, and A combination of pharmaceuticals in which the IL-7 protein is administered at a dose of 600 μg / kg to 2,000 μg / kg.

2. The pharmaceutical combination according to claim 1, wherein the pharmaceutical combination also treats lymphopenia caused by or related to colorectal cancer, or caused by or related to past treatment for colorectal cancer.

3. The pharmaceutical combination according to claim 1 or 2, wherein the IL-7 protein comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:

24.

4. The pharmaceutical combination according to claim 1, wherein the anti-PD-1 antibody comprises nivolumab, pembrolizumab, MEDI0680, PDR001, BGB-A317, or any combination thereof.

5. The pharmaceutical combination according to any one of claims 1 to 4, wherein the IL-7 protein and the anti-PD-1 antibody are administered simultaneously or sequentially, or the IL-7 protein is administered before the anti-PD-1 antibody is administered.

6. The IL-7 protein is administered in doses of 600 μg / kg to 1,200 μg / kg, 600 μg / kg to 1,440 μg / kg, 600 μg / kg to 1,700 μg / kg, 600 μg / kg to 2,000 μg / kg, 720 μg / kg to 1,200 μg / kg, 720 μg / kg to 1,440 μg / kg, 720 μg / kg to 1,700 μg / kg, and 720 μg / kg to 2,000 μg / kg. The pharmaceutical combination according to any one of claims 1 to 5, wherein the IL-7 protein is administered at a frequency of once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, once every eleven weeks, once every twelve weeks, or at intervals of 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 days.

7. The anti-PD-1 antibody is nivolumab, and the nivolumab is administered at a constant dose of 240 mg every two weeks or 480 mg every four weeks, or at a weight-based dose of 3 mg / kg every two weeks; or The pharmaceutical combination according to claim 4, wherein the anti-PD-1 antibody is pembrolizumab, and the pembrolizumab is administered at a constant dose of 200 mg every three weeks, or at a weight-based dose of 2 mg / kg every three weeks.

8. The pharmaceutical combination according to any one of claims 1 to 7, wherein the IL-7 protein is administered parenterally, intramuscularly, subcutaneously, ophthalmicly, intravenously, intraperitoneally, intradermally, intraorbitally, intracerebrally, intracranially, intraspinally, intracardiacly, intrathecally, intracisionally, intracisionally, intracapsularly, or intratumorally.

9. The pharmaceutical combination according to any one of claims 1 to 8, wherein the anti-PD-1 antibody is administered parenterally, intramuscularly, subcutaneously, intravenously, or intraperitoneally.

10. The pharmaceutical combination according to claim 2, wherein the pharmaceutical combination treats a colorectal cancer (patient) with lymphopenia by increasing the number of tumor-infiltrating lymphocytes (TILs) in the colorectal cancer after administration of the IL-7 protein, wherein the TILs are CD4+ TILs or CD8+ TILs.

11. The pharmaceutical combination according to claim 10, wherein the number of TILs increases by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 125%, at least about 150%, at least about 200%, at least about 250%, or at least about 300% after administration.

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