Methods for treating tumors containing an unmethylated MGMT promoter

Abstract

The present disclosure relates to methods for treating a tumor (or cancer) in a subject in need thereof, the tumor comprising an unmethylated MGMT promoter. In some embodiments, the method comprises administering to the subject an anti-cancer treatment comprising an IL-7 protein, alone or in combination with an additional therapeutic agent (e.g., chemotherapy). Some embodiments of the present disclosure relate to methods for identifying a cancer subject (e.g., having glioblastoma) suitable for anti-cancer treatment. Provided herein is a method for treating a tumor in a subject in need thereof, the method comprising administering to the subject an anti-cancer treatment, wherein a tumor sample obtained from the subject contains unmethylated MGMT promoter. 6 -methylguanine-DNA methyltransferase (MGMT) promoter and the anti-cancer treatment comprises interleukin-7 (IL-7) protein.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This PCT application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 382,689, filed November 7, 2022, which is incorporated herein by reference in its entirety.

[0002] Reference to an electronically submitted sequence listing The contents of the Sequence Listing have been submitted electronically with this application (Name: 4241_038PC01_Seqlisting_ST26.xml; Size: 103,771 bytes; and Creation Date: November 6, 2023) and are incorporated herein by reference in their entirety.

[0003] The present disclosure generally relates to the detection of unmethylated O in a subject, e.g., a human subject. 6 The present invention relates to the use of an IL-7 protein (e.g., a long-acting IL-7 protein) to treat tumors containing the -methylguanine-DNA methyltransferase (MGMT) promoter. [Background technology]

[0004] Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults. Despite multimodality treatment with surgery, radiation therapy, and chemotherapy, the prognosis for GBM remains extremely poor. Specifically, studies have shown that these treatments can reduce the number of methylated O 6 -methylguanine DNA methyltransferase (MGMT) promoter has been found to have some efficacy in GBM. However, in patients with GBM that contain unmethylated MGMT promoter, this treatment (as well as other available treatment options available in the art) has minimal effect. Therefore, there is still a need for new treatment options with acceptable safety profile and high efficacy in cancer patients, especially in patients with tumors that contain unmethylated MGMT promoter. Summary of the Invention [Means for solving the problem]

[0005] Provided herein is a method of treating a tumor in a subject in need thereof, comprising administering to the subject an anti-cancer treatment, wherein a tumor sample obtained from the subject is found to contain unmethylated O 6 In some embodiments, the method further comprises determining the methylation status of the MGMT promoter in a tumor sample obtained from the subject prior to administering the anticancer treatment.

[0006] Provided herein is a method for identifying a subject suitable for anti-cancer treatment, comprising: detecting unmethylated O in a tumor sample obtained from the subject; 6 In some embodiments, the method further comprises administering the anti-cancer treatment to the subject identified as suitable for the anti-cancer treatment, the method comprising determining the methylation status of a -methylguanine-DNA methyltransferase (MGMT) promoter, and if the MGMT promoter is unmethylated, the subject is suitable for anti-cancer treatment, the anti-cancer treatment comprising an interleukin-7 (IL-7) protein.

[0007] Provided herein is a method for increasing an anti-tumor immune response in a subject in need thereof, comprising administering to the subject an anti-cancer treatment, wherein the subject is administered an anti-cancer treatment that includes administering to the subject an anti-tumor immune response ... 6Methods are provided for treating a subject having a tumor comprising a methylguanine-DNA methyltransferase (MGMT) promoter, wherein the anti-cancer treatment comprises an interleukin-7 (IL-7) protein. In some embodiments, the method comprises determining the methylation status of the MGMT promoter in a tumor sample obtained from the subject prior to administering. In some embodiments, after administration, the anti-tumor immune response in the subject is increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold compared to a reference subject (e.g., the subject prior to administration).

[0008] In any of the methods provided herein (e.g., the methods provided above), in some embodiments, after administering, the subject's median progression-free survival (mPFS) increases. In some embodiments, the subject's mPFS increases by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold compared to a reference subject (e.g., the subject before administering). In some embodiments, after administering, the subject's mPFS is at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or about 12 months.

[0009] In any of the methods provided herein (e.g., the methods provided above), in some embodiments, after administering, the subject's median overall survival (mPFS) increases. In some embodiments, the subject's mOS increases by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold compared to a reference subject (e.g., the subject before administering). In some embodiments, after administering, the subject's mOS is at least about 12 months, at least about 13 months, at least about 14 months, at least about 15 months, at least about 16 months, at least about 17 months, at least about 18 months, at least about 19 months, or at least about 20 months.

[0010] In any of the methods provided herein (e.g., those provided above), in some embodiments, after administering, the subject's absolute lymphocyte count (ALC) is increased. In some embodiments, the subject's ALC is increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold compared to a reference subject (e.g., the subject before administering). In some embodiments, the increase in ALC persists in the subject for at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 11 weeks, at least about 12 weeks, at least about 13 weeks, at least about 14 weeks, or at least about 15 weeks.

[0011] In any of the methods provided herein (e.g., those provided above), in some embodiments, after administering, the number of tumor-infiltrating T cells (TILs) in the subject's tumor is increased. In some embodiments, the number of TILs in the subject's tumor is increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold compared to a reference subject (e.g., the subject before administering).

[0012] In any of the methods provided herein, in some embodiments, the anti-cancer treatment comprises an additional therapeutic agent. In some embodiments, the additional therapeutic agent comprises standard of care (SOC). In some embodiments, the additional therapeutic agent comprises radiation therapy (RT), chemotherapy, hormone therapy, immunotherapy, photodynamic therapy, stem cell transplant, or a combination thereof. In some embodiments, the additional therapeutic agent comprises chemotherapy. In some embodiments, the chemotherapy comprises temozolomide (TMZ). In some embodiments, the immunotherapy comprises an immune checkpoint inhibitor, an immune checkpoint activator, adoptive cell therapy, or a combination thereof. In some embodiments, the immune checkpoint inhibitor comprises a CTLA-4 antagonist (e.g., an anti-CTLA-4 antibody), a PD-1 antagonist (e.g., an anti-PD-1 antibody, an anti-PD-L1 antibody), a TIM-3 antagonist (e.g., an anti-TIM-3 antibody), or a combination thereof. In some embodiments, the immune checkpoint activator comprises an OX40 agonist (e.g., an anti-OX40 antibody), a LAG-3 agonist (e.g., an anti-LAG-3 antibody), a 4-1BB (CD137) agonist (e.g., an anti-CD137 antibody), a GITR agonist (e.g., an anti-GITR antibody), or a combination thereof.

[0013] In any of the methods provided herein, in some embodiments, the tumor comprises a glioma. In some embodiments, the glioma comprises a high-grade glioma (HGG). In some embodiments, the HGG comprises a glioblastoma (GBM), an anaplastic astrocytoma, or both. In some embodiments, the tumor is newly diagnosed.

[0014] In any of the methods provided herein, in some embodiments, the methylation status of the MGMT promoter is determined by methylation-specific PCR (MS-PCR), pyrosequencing, high-resolution melting, microarray (e.g., Infinium MethylationEPIC), immunohistochemistry (IHC), multiplex ligation-dependent probe amplification (MPLA), or a combination thereof.

[0015] In some embodiments, the IL-7 protein is not a wild-type IL-7 protein. In some embodiments, the IL-7 protein comprises an oligopeptide consisting of 1 to 10 amino acid residues. In some embodiments, the oligopeptide is methionine (M), glycine (G), methionine-methionine (MM), glycine-glycine (GG), methionine-glycine (MG), glycine-methionine (GM), methionine-methionine-methionine (MMM), methionine-methionine-glycine (MMG), methionine-glycine-methionine (MGM), glycine-methionine-methionine (GMM), methionine-glycine-glycine (MGG), glycine-methionine-glycine (GMG), glycine-glycine-methionine (GGM), glycine-glycine-glycine (GGG), methionine-glycine-glycine-methionine (MGGM) (SEQ ID NO: 41), methionine-methionine-glycine-glycine (MMGG) (SEQ ID NO: 42), glycine-glycine-methionine-methionine (GGMM) (SEQ ID NO: 43), methionine-glycine-methionine-glycine (MGMG) (SEQ ID NO: 44), glycine-methionine-methionine-glycine (GMMG) (SEQ ID NO: 45). No. 45), glycine-glycine-glycine-methionine (GGGM) (SEQ ID NO: 46), methionine-glycine-glycine-glycine (MGGG) (SEQ ID NO: 47), glycine-methionine-glycine-glycine (GMGG) (SEQ ID NO: 48), glycine-glycine-methionine-glycine (GGMG) (SEQ ID NO: 49), glycine-glycine-methionine-methionine-methionine (GGMMM) (SEQ ID NO: 50), glycine-glycine-glycine-methionine-methionine (GGGMM) ( SEQ ID NO: 51), glycine-glycine-glycine-glycine-methionine (GGGGM) (SEQ ID NO: 52), methionine-glycine-methionine-methionine-methionine (MGMMM) (SEQ ID NO: 53), methionine-glycine-glycine-methionine-methionine (MGGMM) (SEQ ID NO: 54), methionine-glycine-glycine-glycine-methionine (MGGGM) (SEQ ID NO: 55), methionine-methionine-glycine-methionine-methionine (MMGMM) (SEQ ID NO: 56),Methionine-methionine-glycine-glycine-methionine (MMGGM) (SEQ ID NO: 57), methionine-methionine-glycine-glycine-glycine (MMGGG) (SEQ ID NO: 58), methionine-methionine-glycine-methionine (MMMGM) (SEQ ID NO: 59), methionine-glycine-methionine-glycine-methionine (MGMGM) (SEQ ID NO: 60), glycine-methionine-glycine-methionine-glycine (GMGMG) (SEQ ID NO: 61), glycine-methionine-methionine-methionine-glycine (GMMMG) (SEQ ID NO: 62), glycine-glycine-methionine-glycine-methionine (GGMGM) (SEQ ID NO: 63), glycine-glycine-methionine-methionine-glycine (GGMMG) (SEQ ID NO: 64), glycine- The oligopeptide may comprise methionine-methionine-glycine-methionine (GMMGM) (SEQ ID NO: 65), methionine-glycine-methionine-methionine-glycine (MGMMG) (SEQ ID NO: 66), glycine-methionine-glycine-glycine-methionine (GMGGM) (SEQ ID NO: 67), methionine-methionine-glycine-methionine-glycine (MMGMG) (SEQ ID NO: 68), glycine-methionine-methionine-glycine-glycine (GMMGG) (SEQ ID NO: 69), glycine-methionine-glycine-glycine-glycine (GMGGG) (SEQ ID NO: 70), glycine-glycine-methionine-glycine-glycine (GGMGG) (SEQ ID NO: 71), glycine-glycine-glycine-glycine-glycine (GGGGG) (SEQ ID NO: 72), or a combination thereof. In some embodiments, the oligopeptide is methionine-glycine-methionine (MGM).

[0016] In some embodiments, the IL-7 protein comprises a half-life extending moiety. In some embodiments, the half-life extending moiety comprises Fc, albumin, albumin-binding polypeptide, Pro / Ala / Ser (PAS), the C-terminal peptide of the beta subunit of human chorionic gonadotropin (CTP), polyethylene glycol (PEG), a long unstructured hydrophilic amino acid sequence (XTEN), hydroxyethyl starch (HES), an albumin-binding small molecule, or a combination thereof. In some embodiments, the half-life extending moiety is Fc. In some embodiments, the 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 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.

[0017] In some embodiments, 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 to SEQ ID NOs: 1-6, 80-85, and 15-27.

[0018] In some embodiments, the IL-7 protein is greater than about 30 μg / kg, greater than about 60 μg / kg, greater than about 90 μg / kg, greater than about 120 μg / kg, greater than about 150 μg / kg, greater than about 180 μg / kg, greater than about 210 μg / kg, greater than about 240 μg / kg, greater than about 270 μg / kg, greater than about 300 μg / kg, greater than about 400 μg / kg, greater than about 500 μg / kg, greater than about 600 μg / kg, greater than about 700 μg / kg, greater than about 800 μg / kg, greater than about 900 μg / kg, greater than about 1000 μg / kg, greater than about 1100 μg / kg, greater than about 1200 μg / kg, greater than about 1500 μg / kg, greater than about 180 μg / kg, greater than about 1500 μg / kg, greater than about 1800 μg / kg, greater than about 1900 μg / kg, greater than about 2100 μg / kg, greater than about 240 μg / kg, greater than about 270 μg / kg, greater than about 300 μg / kg, greater than about 400 μg / kg, greater than about 500 μg / kg, greater than about 600 μg / kg, greater than about 700 μg / kg, greater than about 800 μg / kg, greater than about 1000 μg / kg, greater than about 1200 μg / kg, greater than about 1500 μg / kg, greater than about 1800 μg / kg, greater than about 1900 μg / kg, greater than about 2000 μg / kg, greater than about 2100 μg / kg, greater than about 240 μ The dose is administered at a dose of greater than about 100 μg / kg, greater than about 900 μg / kg, greater than about 1,000 μg / kg, greater than about 1,100 μg / kg, greater than about 1,200 μg / kg, greater than about 1,300 μg / kg, greater than about 1,400 μg / kg, greater than about 1,500 μg / kg, greater than about 1,600 μg / kg, greater than about 1,700 μg / kg, greater than about 1,800 μg / kg, greater than about 1,900 μg / kg, or greater than about 2,000 μg / kg. In some embodiments, the IL-7 protein is administered at a concentration of about 60 μg / kg to about 1,200 μg / kg, about 120 μg / kg to about 1,200 μg / kg, about 240 μg / kg to about 1,200 μg / kg, about 540 μg / kg to about 1,200 μg / kg, about 610 μg / kg to about 1,200 μg / kg, about 650 μg / kg to about 1,200 μg / kg, or about 700 μg / kg. g ~ approx. 1,200 μg / kg, approx. 720 μg / kg ~ approx. 1,200 μg / kg, approx. 750 μg / kg ~ approx. 1,200 μg / kg, approx. 800 μg / kg ~ approx. 1,200 μg / kg, Approximately 850μg / kg to approximately 1,200μg / kg, approximately 900μg / kg to approximately 1,200μg / kg, approximately 950μg / kg to approximately 1,200μg / kg, approximately 960μg / kg to approximately 1,2 00μg / kg, approximately 1,000μg / kg to approximately 1,200μg / kg, 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 / kg, approximately 1,300μg / kg to approximately 2,000μg / kg, approximately 1,500μg / kg to approximately 2,000μg / kg, approximately 1,700μg / kg ~ approx. 2,000μg / kg, approx. 610μg / kg ~ approx. 1,000μg / kg, approx. 650μg / kg ~ approx. 1,000μg / kg, approx. 700μg / kg ~ approx. 1,000μg / k g, about 750μg / kg to about 1,000μg / kg, about 800μg / kg to about 1,000μg / kg, about 850μg / kg to about 1,000μg / kg, about 900μg / kg to about 1,In some embodiments, the IL-7 protein is administered at a dose of about 60 μg / kg to about 120 μg / kg, about 120 μg / kg to about 240 μg / kg, about 240 μg / kg to about 540 μg / kg, about 540 μg / kg to about 720 μg / kg, about 720 μg / kg to about 960 μg / kg, about 700 μg / kg to about 900 μg / kg, about 750 μg / kg / kg to about 950 μg / kg, about 700 μg / kg to about 850 μg / kg, about 750 μg / kg to about 850 μg / kg, about 700 μg / kg to about 800 μg / kg, about 800 μg / kg to about 900 μg / kg, about 750 μg / kg to about 850 μg / kg, or about 850 μg / kg to about 950 μg / kg. In some embodiments, the IL-7 protein is administered at a concentration of about 30 μg / kg, about 60 μg / kg, about 90 μg / kg, about 120 μg / kg, about 150 μg / kg, about 180 μg / kg, about 210 μg / kg, about 240 μg / kg, about 270 μg / kg, about 300 μg / kg, about 330 μg / kg, about 360 μg / kg, about 390 μg / kg, about 420 μg / kg, about 450 μg / kg / kg, approximately 480μg / kg, approximately 510μg / kg, approximately 540μg / kg, approximately 570μg / kg, approximately 600μg / kg, approximately 630μg / kg, approximately 650μg / kg, approximately 680μg / k g, about 700μg / kg, about 720μg / kg, about 740μg / kg, about 750μg / kg, about 760μg / kg, about 780μg / kg, about 800μg / kg, about 820μg / kg, about 840μg / kg, approximately 850μg / kg, approximately 860μg / kg, approximately 880μg / kg, approximately 900μg / kg, approximately 920μg / kg, approximately 940μg / kg, approximately 950μg / kg, approximately 96 0μg / 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 1,10 0μg / kg, approximately 1,120μg / kg, approximately 1,140μg / kg, approximately 1,160μg / kg, approximately 1,180μg / kg, approximately 1200μg / kg, approximately 1,220μg / kg, approximately 1,2 40μg / 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 1,1,54 0μg / 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 1,700μ g / kg, about 1,720 μg / kg, about 1,740 μg / kg, about 1,760 μg / kg, about 1,780 μg / kg, about 1,800 μg / kg, about 1,820 μg / kg, about 1,840 μg / kg, about 1,860 μg / kg, about 1,880 μg / kg, about 1,900 μg / kg, about 1,920 μg / kg, about 1,940 μg / kg, about 1,960 μg / kg, about 1,980 μg / kg, or about 2,000 μg / kg. In some embodiments, the IL-7 protein is at a concentration of about 0.01 nmol / kg, about 0.02 nmol / kg, about 0.03 nmol / kg, about 0.04 nmol / kg, about 0.05 nmol / kg, about 0.1 nmol / kg, about 0.2 nmol / kg, about 0.4 nmol / kg, about 0.6 nmol / kg, about 0.8 nmol / kg, about 1 nmol / kg, about 1.2 nmol / kg, about 1.4 nmol / kg, about 1.6 nmol / kg, about 1.8 nmol / kg, about 2 nmol / kg, about 2.2 nmol / kg, about 2.4 nmol / kg, about 2.6 nmol / kg, about 2.8 nmol / kg, about 3 nmol / kg, about 3.5 nmol / kg, about 4 nmol / kg, about 4.5 nmol / kg, about 5 nmol / kg, about 6 nmol / kg, about 7 nmol / kg, about 8 nmol / kg, about 9 nmol / kg, about 10 nmol / kg, about 11 nmol / kg, about 12 nmol / kg, about 13 nmol / kg, about 14 nmol / kg, about 15 nmol / kg, about 16 nmol / kg, about 17 nmol / kg, about 18 nmol / kg, about 19 nmol / kg, about 20 nmol / kg, about 21 nmol / kg, about 22 nmol / kg, about 23 nmol / kg, about 24 nmol / kg, about 25 nmol / kg, about 26 nmol / kg, about 27 nmol / kg, about 28 nmol / kg, about 29 nmol / kg, about 30 nmol / kg, about 31 nmol / kg, about 32 nmol / kg, about 33 nmol / kg, about 3 The compound is administered at a dose of about 10 nmol / kg, about 5 nmol / kg, about 6 nmol / kg, about 7 nmol / kg, about 8 nmol / kg, about 9 nmol / kg, about 10 nmol / kg, about 11 nmol / kg, about 12 nmol / kg, about 13 nmol / kg, about 14 nmol / kg, about 15 nmol / kg, about 16 nmol / kg, about 17 nmol / kg, about 18 nmol / kg, about 19 nmol / kg, about 20 nmol / kg, about 22 nmol / kg, about 24 nmol / kg, about 26 nmol / kg, about 28 nmol / kg, about 30 nmol / kg, about 32 nmol / kg, about 34 nmol / kg, about 36 nmol / kg, about 38 nmol / kg, or about 40 nmol / kg.

[0019] In some embodiments, the IL-7 protein is administered at a dosing frequency of about once per week, about once per 2 weeks, about once per 3 weeks, about once per 4 weeks, about once per 5 weeks, about once per 6 weeks, about once per 7 weeks, about once per 8 weeks, about once per 9 weeks, about once per 10 weeks, about once per 11 weeks, or about once per 12 weeks. In some embodiments, the IL-7 protein is administered to the subject at a dose of about 720 μg / kg and at a dosing frequency of about once per 12 weeks.

[0020] In any of the methods provided herein, the anti-cancer treatment is administered to the subject at a dosing frequency of about once per week, about once per two weeks, about once per three weeks, about once per four weeks, or about once per five weeks.

[0021] In some embodiments, the IL-7 protein and the additional therapeutic agent are administered to the subject simultaneously. In some embodiments, the IL-7 protein and the additional therapeutic agent are administered to the subject sequentially. In some embodiments, the additional agent is administered to the subject after the IL-7 protein.

[0022] In some embodiments, the subject is administered at least about two doses, at least about three doses, at least about four doses, or at least about five doses of the IL-7 protein. In some embodiments, the subject is administered at least about two doses, at least about three doses, at least about four doses, at least about five doses, at least about six doses, or at least about seven doses of the additional therapeutic agent. In some embodiments, the subject is administered at least about four doses of the IL-7 protein and at least about six doses of the additional agent. In some embodiments, the IL-7 protein is administered to the subject about once every 12 weeks, and the additional agent is administered to the subject about once every four weeks. In some embodiments, one or more doses of the additional agent are administered to the subject over about one day, over about two consecutive days, over about three consecutive days, over about four consecutive days, over about five consecutive days, over about six consecutive days, or over about seven consecutive days. In some embodiments, the additional therapeutic agent is administered to the subject on days 1-5 of a 28-day cycle.

[0023] In any of the methods provided herein, in some embodiments, the subject has not previously received anti-tumor therapy. In some embodiments, the subject has previously received standard of care (SOC). In some embodiments, the anti-tumor therapy comprises radiation therapy (RT), chemotherapy, hormonal therapy, immunotherapy, photodynamic therapy, stem cell transplant, or a combination thereof. In some embodiments, the anti-tumor therapy comprises both RT and chemotherapy. In some embodiments, the chemotherapy comprises temozolomide (TMZ).

[0024] In some embodiments, the IL-7 protein is administered intramuscularly, parenterally, subcutaneously, intraocularly, intravenously, intraperitoneally, intradermally, intraorbitally, intracerebrally, intracranially, intraspinally, intraventricularly, intrathecally, intracisternally, intracapsularly, or intratumorally to the subject. In some embodiments, an additional therapeutic agent is administered intramuscularly, parenterally, subcutaneously, intraocularly, intravenously, intraperitoneally, intradermally, intraorbitally, intracerebrally, intracranially, intraspinally, intraventricularly, intrathecally, intracisternally, or intratumorally to the subject. [Brief explanation of the drawings]

[0025] [Figure 1] 1 shows a schematic diagram of the Phase I study described in Example 1.

[0026] [Figure 2]

[0023] Figure 1 shows absolute lymphocyte counts (ALC) in cancer patients after 4 weeks of treatment with one of the following: (a) placebo plus TMZ adjuvant alone (i.e., no steroids); (b) placebo plus TMZ adjuvant after steroid administration; (c) long-acting IL-7 protein plus TMZ adjuvant alone (i.e., no steroids); or (d) long-acting IL-7 protein plus TMZ adjuvant after steroid administration. A shows the ALC at baseline (i.e., before treatment) ("BSL") and 4 weeks after treatment ("W4"). B shows the fold change in ALC between baseline and 4 weeks after treatment. In each figure, black circles represent patients who did not receive steroids, and gray circles represent patients who received steroids.

[0027] [Figure 3]

[0023] Figure 1 shows whether steroid administration influences the IL-7 protein-mediated increase in ALC observed in cancer patients. A shows a comparison of ALC at baseline (i.e., before treatment) ("BSL") and 4 weeks after treatment with one of the following: (a) steroid administration followed by placebo and TMZ adjuvant, or (b) steroid administration followed by long-acting IL-7 protein and TMZ adjuvant. B shows a comparison of ALC at baseline (i.e., before treatment) ("BSL") and 4 weeks after treatment with one of the following: (a) placebo and TMZ adjuvant alone (i.e., no steroid), or (c) long-acting IL-7 protein and TMZ adjuvant alone (i.e., no steroid).

[0028] [Figure 4]Figures 4A and 4B show a comparison of the effects of IL-7 protein administration on ALC in cancer patients with methylated and unmethylated MGMT promoters, respectively. Patients with both methylated (A) and unmethylated (B) MGMT promoters were treated with either placebo (alone or in combination with TMZ adjuvant) or long-acting IL-7 protein (alone or in combination with TMZ adjuvant). ALC was measured both at baseline (i.e., before treatment) ("BSL") and 4 weeks after treatment ("W4"). DETAILED DESCRIPTION OF THE INVENTION

[0029] The present disclosure relates generally to treating certain tumors with anti-cancer therapies that include IL-7 proteins. As demonstrated herein, such anti-cancer therapies target unmethylated O 6 These compounds are particularly useful for treating tumors that contain the -methylguanine-DNA methyltransferase (MGMT) promoter (e.g., high-grade gliomas). Further aspects of the disclosure are provided throughout the application.

[0030] Before describing the present disclosure in more detail, it is to be understood that the present disclosure is not limited to the particular compositions or methods described, which may, of course, vary. As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has distinct components and features that can be readily separated from or combined with the features of any of the other embodiments without departing from the scope or spirit of the present disclosure. Any method described can be carried out in the order of events described or in any other order that is logically possible.

[0031] The headings provided herein are not limitations of the various aspects of the disclosure, which may be defined by reference to the specification as a whole. It should also be understood that the terminology used herein is for the purpose of describing particular aspects only, and not for the purpose of limitation, since the scope of the disclosure is limited only by the appended claims.

[0032] I. Definition In order that this disclosure may be more readily understood, certain terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms shall have the meaning indicated below. Additional definitions are set forth throughout this application.

[0033] The term "a" or "an" entity refers to one or more of that entity; for example, "a nucleotide sequence" is understood to refer to one or more nucleotide sequences. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0034] Furthermore, as used herein, "and / or" should be understood as a specific disclosure of each of the two particular features or elements, with or without the other. Thus, the term "and / or" 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" used in phrases such as "A, B, and / or C" is intended to encompass 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).

[0035] When an embodiment is described herein using the phrase "comprising," it is understood that similar embodiments separately described with "consisting of" and / or "consisting essentially of" are also provided. "Comprising" is used interchangeably with "including," "containing," or "characterized by" and is an inclusive or open-ended phrase that does not exclude other unrecited elements or method steps. As used herein, "consisting of" excludes elements, steps, or ingredients not specified in the claim. The phrase "consisting essentially of" limits the scope of a claim to the specified materials or steps, as well as those that do not materially affect the basic and novel characteristics of the claimed subject matter. This disclosure contemplates each embodiment of the disclosed compositions and methods that falls within the scope of each of these phrases. Thus, a composition or method that includes recited elements or steps contemplates specific embodiments in which the composition or method consists essentially of or consists of those elements or steps.

[0036] Unless otherwise defined, all technical and scientific terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 5th ed., 2013, Academic Press; and Oxford Dictionary of Biochemistry and Molecular Biology, 2nd ed., 2008, Oxford University Press provide those skilled in the art with a general dictionary of many of the terms used in this disclosure.

[0037] Units, prefixes, and symbols are written in the format accepted by the International System of Units (SI). Numerical ranges are intended to be inclusive of the numbers defining the range. When a range of values ​​is recited, it is understood that each intervening integer and each fractional part between the recited upper and lower limits of that range is also expressly disclosed, along with each subrange between such values. The upper and lower limits of any range may independently be included or excluded from the range, and each range including either, neither, or both of the upper and lower limits is encompassed within the present disclosure. Thus, ranges recited herein are understood to be shorthand for all values ​​within the range, including the recited endpoints. For example, a range of 1 to 10 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0038] Where a value is explicitly recited, it is understood that values ​​that are about the same number or amount (e.g., ±10%) as the recited value (e.g., 10) are also included within the scope of the disclosure. Where a combination is disclosed, each subcombination of the elements of that combination is also specifically disclosed and is included within the scope of the disclosure. Conversely, where different elements or groups of elements are individually disclosed, combinations thereof are also disclosed. Where any element of a disclosure is disclosed as having multiple alternatives, examples of that disclosure in which each alternative is excluded, alone or in any combination with the other alternatives, are also disclosed herein; more than one element of a disclosure may have such an exclusion, and all combinations of elements with such exclusions are disclosed herein.

[0039] The term "about" is used herein to mean approximately, roughly, roughly, or within a range. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the stated numerical values. In general, the term "about" can modify a numerical value to be above or below the stated value by, for example, a variance of 10 percent above or below (higher or lower).

[0040] As used herein, the term "interleukin-7" or "IL-7" refers to IL-7 polypeptides and derivatives and analogs thereof that have substantial amino acid sequence identity to wild-type adult mammalian IL-7 and have substantially equivalent biological activity, e.g., in standard bioassays or assays of IL-7 receptor binding affinity. For example, IL-7 refers to the amino acid sequence of a recombinant or non-recombinant polypeptide having the amino acid sequence of: i) a native or naturally occurring allelic 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) a biologically active variant of an IL-7 polypeptide. IL-7 polypeptides of the present disclosure can be obtained from any species, e.g., human, bovine, or ovine. The nucleic acid and amino acid sequences of IL-7 are well known in the art. For example, the human IL-7 amino acid sequence has the Genbank accession number of P13232 (SEQ ID NO: 1), the mouse IL-7 amino acid sequence has the Genbank accession number of P10168 (SEQ ID NO: 3), the rat IL-7 amino acid sequence has the Genbank accession number of P56478 (SEQ ID NO: 2), the monkey IL-7 amino acid sequence has the Genbank accession number of NP_001279008 (SEQ ID NO: 4), the bovine IL-7 amino acid sequence has the Genbank accession number of P26895 (SEQ ID NO: 5), and the ovine IL-7 amino acid sequence has the Genbank accession number of Q28540 (SEQ ID NO: 6). Protein sequences of exemplary IL-7 proteins are shown in Table 2 (below). In some aspects, the IL-7 polypeptides of the present disclosure are variants of IL-7 proteins.

[0041] As used herein, "administering" refers to the physical introduction of a therapeutic agent or a composition containing a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Various routes of administration for the therapeutic agents described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, or other parenteral routes of administration, such as by injection or infusion. The phrase "parenteral administration," as used herein, refers to modes of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, intratracheal, pulmonary, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraventricular, intravitreal, epidural, and intrasternal injection and infusion, and in vivo electroporation. Alternatively, the therapeutic agents described herein can be administered via a non-parenteral route, e.g., a topical, epidermal, or mucosal route of administration, e.g., intranasally, orally, vaginally, rectally, sublingually, or topically. Administration can also be, for example, single, multiple, and / or over one or more extended periods of time.

[0042] As used herein, the term "naturally occurring," when applied to an object, refers to the object being found in nature. For example, a polypeptide or polynucleotide sequence that can be isolated from a natural source and exists in an organism (including a virus) that has not been intentionally modified by man in a laboratory is naturally occurring.

[0043] A "polypeptide" refers to a chain comprising at least two consecutively linked amino acid residues, with no upper limit to the length of the chain. One or more amino acid residues in a protein may contain modifications, such as, but not limited to, glycosylation, phosphorylation, or disulfide bond formation. A "protein" may include one or more polypeptides. Unless otherwise specified, the terms "protein" and "polypeptide" may be used interchangeably.

[0044] As used herein, the term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. A nucleic acid molecule may be single-stranded or double-stranded, and may be cDNA.

[0045] A "conservative amino acid substitution" means that an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In some aspects, 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 conservative nucleotide and amino acid substitutions that do not abolish antigen binding are well known in the art (see, e.g., 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)).

[0046] In the context of nucleic acids, the term "substantial homology" indicates that two nucleic acids, or their designated sequences, when optimally aligned and compared, are identical in at least about 80%, at least about 90%-95%, or at least about 98%-99.5% of the nucleotides, with appropriate nucleotide insertions or deletions. Alternatively, substantial homology exists when the segments will hybridize under selective hybridization conditions to the complement of the strand.

[0047] In the context of polypeptides, the term "substantial homology" indicates that two polypeptides, or their designated sequences, when optimally aligned and compared, are identical in at least about 80% of the amino acids, at least about 90%-95%, or at least about 98%-99.5% of the amino acids, with appropriate amino acid insertions or deletions.

[0048] The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = number of identical positions / total number of positions x 100), 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. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, for example, as described in the non-limiting examples below.

[0049] The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at worldwideweb.gcg.com) using the NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. The percent identity between two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) as incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Additionally, percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm 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, with a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0050] The nucleic acid and protein sequences described herein can also be used as "query sequences" to search public databases, for example, to identify related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, word length = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed with the XBLAST program, score = 50, word length = 3, to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See worldwideweb.ncbi.nlm.nih.gov.

[0051] Nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is "isolated" or "substantially purified" if it has been purified from other cellular components or other contaminants, such as other cellular nucleic acids (e.g., other parts of chromosomes) or proteins, by standard techniques, including alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other techniques well known in the art. See F. Ausubel, et al., eds., Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).

[0052] Nucleic acids, e.g., cDNA, can be mutated according to standard techniques to produce gene sequences. In the case of coding sequences, these mutations can affect the amino acid sequence as desired. In particular, DNA sequences that are substantially homologous to or derived from naturally occurring V, D, J, constant sequences, switch, and other such sequences described herein are contemplated (where "derived" indicates that the sequence is identical to or modified from another sequence).

[0053] As used herein, the term "vector" is intended to mean a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. Because plasmids are the most commonly used form of vector, "plasmid" and "vector" can be used interchangeably herein. However, also included are other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0054] As used herein, the term "recombinant host cell" (or simply "host cell") is intended to mean a cell containing a nucleic acid that does not naturally occur in the cell, and may be a cell into which a recombinant expression vector has been introduced. It is understood that such terms are intended to refer not only to the particular subject cell but also to the progeny of such a cell. Because certain modifications may occur in successive generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0055] As used herein, the term "linked" refers to the attachment of two or more molecules. The attachment may be covalent or non-covalent. The attachment may also be genetic (i.e., recombinantly fused). Such attachment may be achieved using a wide range of art-recognized techniques, such as chemical conjugation and recombinant protein production.

[0056] "Cancer" refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division and growth result in the formation of malignant tumors that invade adjacent tissues and may metastasize to distant parts of the body via the lymphatic system or bloodstream. As used herein, "cancer" refers to primary cancers, metastatic cancers, and recurrent cancers. As is evident from this disclosure, cancers that can be treated by the present disclosure include tumors with an unmethylated O6-methylguanine-DNA methyltransferase (MGMT) promoter. Non-limiting examples of suitable cancers that can be treated by the present disclosure are provided elsewhere in this disclosure.

[0057] The term "fusion protein" refers to a protein created by the joining of two or more genes that originally encoded separate proteins. Translation of the fusion gene results in a single polypeptide or multiple polypeptides with functional properties derived from each of the original proteins. In some embodiments, the two or more genes may contain substitutions, deletions, and / or additions in their nucleotide sequences.

[0058] An "Fc receptor" or "FcR" is a receptor that binds to the Fc region of an immunoglobulin. FcRs that bind IgG antibodies include receptors of the FcγR family, including allelic variants and alternatively spliced ​​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 FcγRs are known in the art. While most innate effector cell types co-express one or more activating FcγRs and the inhibitory FcγRIIB, natural killer (NK) cells selectively express one activating Fc receptor (FcγRIII in mice and FcγRIIIA in humans) but do not selectively express the inhibitory FcγRIIB in mice or humans. Human IgG1 binds to most human Fc receptors and is considered equivalent to mouse IgG2a with respect to the type of activating Fc receptor it binds.

[0059] "Fc region" (Fragment crystallizable region) or "Fc domain" or "Fc" refers to the C-terminal region of an antibody heavy chain that mediates immunoglobulin binding to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or the first component (C1q) of the classical complement system. Thus, the Fc region includes the constant region of an antibody excluding the first constant region immunoglobulin domain (e.g., CH1 or CL). In IgG, IgA, and IgD antibody isotypes, the Fc region comprises two identical protein fragments derived from the second (CH2) and third (CH3) constant domains of the antibody's two heavy chains, while the Fc regions of IgM and IgE comprise three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. In the case of IgG, the Fc region includes immunoglobulin domains CH2 and CH3, as well as the hinge between the CH1 and CH2 domains. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, as defined herein, the human IgG heavy chain Fc region is defined to stretch from amino acid residues D221 for IgG1, V222 for IgG2, L221 for IgG3, and P224 for IgG4, to the carboxy-terminus of the heavy chain, where numbering is according to the EU index as in 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 C-terminal to the CH2 domain within 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, an Fc region may be a native-sequence Fc, including any allotypic variants, or a variant Fc (e.g., a non-naturally occurring Fc). Fc may refer to this region alone or in the context of a protein polypeptide that contains Fc, such as an "Fc region-containing binding protein," also referred to as an "Fc fusion protein" (e.g., an antibody or immunoadhesin).

[0060] A "native sequence Fc region" or "native sequence Fc" comprises an amino acid sequence identical to that of an Fc region found in nature. Native sequence human Fc regions include native sequence human IgG1 Fc region, native sequence human IgG2 Fc region, native sequence human IgG3 Fc region, and native sequence human IgG4 Fc region, as well as naturally occurring variants thereof. Native sequence Fc includes the various allotypes of Fc (see, e.g., Jefferis et al. (2009) mAbs 1:1).

[0061] Furthermore, the Fc (native or variant) of the present disclosure may have native, more abundant, or fewer glycosylated forms than the native form, or may be deglycosylated. The Fc glycosylation of an immunoglobulin can be modified by conventional methods, such as chemical, enzymatic, and microbial genetic engineering. Removal of glycosylation from the Fc fragment significantly reduces the binding affinity to the C1q portion of the first component of complement C1, reducing or eliminating ADCC or CDC, thereby preventing the induction of unwanted immune responses in vivo. In this regard, a deglycosylated or nonglycosylated form of an immunoglobulin Fc region may be more suitable for use as a drug carrier in the present disclosure. As used herein, the term "deglycosylated" refers to an Fc region from which glycosylation has been enzymatically removed from the Fc fragment. Furthermore, the term "nonglycosylated" refers to an Fc fragment produced in a nonglycosylated form by a prokaryote, preferably in E. coli.

[0062] As used herein, the term "immune response" refers to a biological response in a vertebrate to foreign agents, which protects the organism from these agents and the diseases they cause. As used herein, the term "anti-tumor immune response" refers to an immune response to tumor antigens. Immune responses are mediated by the action of cells of the immune system (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 that selectively target, bind to, damage, destroy, and / or eliminate from the vertebrate body invading pathogens, pathogen-infected cells or tissues, cancer or other abnormal cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues. Immune responses include, for example, T cells, e.g., effector T cells or Th cells, e.g., CD4 + or CD8 + Examples include activation or inhibition of T cells, or inhibition of Treg cells.

[0063] The term "immunotherapy" refers to the treatment of a subject suffering from a disease or at risk of suffering from or having a recurrence of a disease by methods that involve inducing, enhancing, suppressing, or otherwise modifying the immune response. "Treatment" or "therapy" of a subject means any type of intervention or process performed on a subject, or the administration of an active agent to a subject, for the purpose of reversing, alleviating, ameliorating, inhibiting, slowing, or preventing the onset, progression, development, severity, or recurrence of symptoms, complications, or pathology, or biochemical manifestations associated with a disease.

[0064] The term "effector T cells" (Teff) refers to T cells with cytolytic activity (e.g., CD4 + and CD8 + This refers to T cells, which secrete cytokines to activate and guide other immune cells, as well as T helper (Th) cells, but does not include regulatory T cells (Treg cells).

[0065] As used herein, the term "regulatory T cells" (Tregs) refers to a population of T cells that have the ability to reduce or suppress the induction and proliferation of effector T cells, thereby regulating an immune response. In some embodiments, Tregs can suppress an immune response by secreting anti-inflammatory cytokines such as IL-10, TGF-β, and IL-35, which can prevent 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., develop in the thymus). In some embodiments, regulatory T cells are induced regulatory T cells (iTregs) (i.e., naive T cells that differentiate into Tregs in peripheral tissues upon exposure to specific stimuli). Methods for identifying Tregs are well known in the art. For example, Tregs express certain 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 + T cells.

[0066] As used herein, the term "tumor-infiltrating lymphocytes" or "TILs" refers to lymphocytes (e.g., effector T cells) that have migrated from the periphery (e.g., from the blood) into a tumor. In some embodiments, tumor-infiltrating lymphocytes are CD4+ TILs. In some embodiments, tumor-infiltrating lymphocytes are CD8+ TILs.

[0067] The increased ability to stimulate an immune response or the immune system may be due to improved agonist activity of T cell costimulatory receptors and / or improved antagonist activity of inhibitory receptors. The increased ability to stimulate an immune response or the immune system may be reflected in a fold increase in EC50 or maximal activity level in assays that measure immune responses, such as cytokine or chemokine release, cytolytic activity (determined directly on target cells or indirectly by detecting CD107a or granzymes), and changes in proliferation. The ability to stimulate an immune response or immune system activity may be improved by at least 10%, 30%, 50%, 75%, 2-fold, 3-fold, 5-fold, or more.

[0068] For example, a "variant" of an IL-7 protein is defined as an amino acid sequence in which one or more amino acids have been altered. A variant may have "conservative" changes, such as replacing a leucine with an isoleucine, where the substituted amino acid has similar structural or chemical properties. Less commonly, a variant may have "non-conservative" changes, such as replacing a glycine with a tryptophan. Similar minor changes can also include amino acid deletions or insertions, or both. Guidance for determining the type and number of amino acid residues that can be substituted, inserted, or deleted without impairing biological activity can be found using computer programs well known in the art, such as software for molecular modeling or alignment generation. For example, variant IL-7 proteins included in the present disclosure include IL-7 proteins that retain IL-7 activity. Variants that also contain additions, substitutions, or deletions are also included within the present disclosure, so long as the variants retain substantially the same biological activity as the corresponding non-variant (e.g., wild-type) version. For example, truncated forms of IL-7 that retain biological activity equivalent to the full-length form of the IL-7 protein are encompassed by the present disclosure. The activity of the therapeutic agents described herein (e.g., IL-7 protein and / or additional therapeutic agents) can be measured using any suitable method known in the art.

[0069] To determine the percent identity of two amino acid sequences or two nucleic acids, the sequences are aligned for optimal comparison (e.g., gaps can be introduced into a first amino acid sequence or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = number of identical positions / total number of positions × 100). Determining the percent homology between two sequences can be achieved using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm used to compare two sequences is the algorithm of 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 an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, word length = 12. BLAST protein searches can be performed with the XBLAST program, score = 50, word length = 3. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Research 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0070] A "subject" includes any human or non-human animal. The term "non-human animal" includes, but is not limited to, vertebrates, such as non-human primates, sheep, dogs, and rodents, such as mice, rats, and guinea pigs. In some embodiments, the subject is a human. The terms "subject" and "patient" are used interchangeably herein. Unless otherwise specified, the subjects described herein are afflicted with one or more of the indications (e.g., tumors) described herein.

[0071] The term "therapeutically effective amount" or "therapeutically effective dosage" refers to that amount of an agent that produces a desired biological, therapeutic, and / or prophylactic result. The result can be reduction, amelioration, palliation, attenuation, delay, and / or alleviation of one or more of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. With respect to solid tumors, an effective amount includes an amount sufficient to shrink the tumor and / or reduce the rate of tumor growth (e.g., inhibit tumor growth) or prevent or delay other unwanted cell proliferation. In some embodiments, an effective amount is an amount sufficient to delay tumor progression. In some embodiments, an effective amount is an amount sufficient to prevent or delay tumor recurrence. An effective amount can be administered in one or more administrations. An effective amount of a drug or composition can (i) reduce the number of cancer cells, (ii) reduce tumor size, (iii) inhibit, delay, slow to some extent, or even stop cancer cell invasion of peripheral organs, (iv) inhibit (i.e., slow to some extent, or even stop) tumor metastasis, (v) inhibit tumor growth, (vi) prevent or delay tumor onset and / or recurrence, and / or (vii) alleviate to some extent one or more symptoms associated with cancer. The ability of a therapeutic agent to promote disease regression can be evaluated using a variety of methods known to the skilled practitioner, for example, by assaying the activity of the agent in human subjects in clinical trials, in animal model systems predictive of efficacy in humans, or in in vitro assays.

[0072] The term "dosing frequency" or "dosing interval" refers to the number of times a therapeutic agent (e.g., an IL-7 protein and / or an additional therapeutic agent) is administered to a subject within a particular period of time. Dosing frequency can be expressed as the number of doses per given period of time, such as once daily, once weekly, or once every two weeks. As used herein, "dosing frequency" is applicable when a subject receives multiple (or repeated) doses of a therapeutic agent. Suitable dosing frequencies for any of the IL-7 protein and additional therapeutic agents described herein are set forth elsewhere in this disclosure.

[0073] As used herein, the term "standard of care" refers to a treatment that is accepted by medical professionals as an appropriate treatment for a particular type of disease and is widely used by medical professionals. The term may be used interchangeably with any of the following terms: "best practice," "standard medical care," and "standard therapy."

[0074] By way of example, an "anti-cancer drug" promotes cancer regression or prevents further tumor growth in a subject. In some embodiments, a therapeutically effective amount of a drug promotes cancer regression to the point where the cancer disappears. "Promoting cancer regression" means that administering an effective amount of the drug, alone or in combination with an anti-tumor drug, reduces tumor growth or size, causes tumor necrosis, reduces the severity of at least one disease symptom, prolongs the frequency and duration of disease-free periods, or prevents functional impairment or disability due to disease affliction. Furthermore, the terms "effective" and "efficacy" in relation to treatment include both pharmacological effectiveness and physiological safety. Pharmacological effectiveness refers to the ability of a drug to promote cancer regression in a patient. Physiological safety refers to the level of toxicity or other adverse physiological effects (adverse effects) at the cellular, organ, and / or organismal level resulting from the administration of a drug.

[0075] By way of example, in the treatment of tumors, a therapeutically effective amount of an anticancer agent can inhibit cell proliferation 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 untreated subjects, or in some embodiments, compared to patients treated with standard therapeutic regimens. In some embodiments, tumor regression can be observed and may continue for a period of at least about 20 days, at least about 40 days, or at least about 60 days. Regardless of these ultimate measures of therapeutic efficacy, evaluation of immunotherapeutic agents must also take into account "immune-related" response patterns.

[0076] As used herein, the term "immune checkpoint inhibitor" refers to a molecule that completely or partially reduces, inhibits, prevents, or modulates one or more immune checkpoint proteins. Checkpoint proteins control T cell activation or function. Numerous checkpoint proteins are known, such as 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 costimulatory or inhibitory interactions in T cell responses. Immune checkpoint proteins control and maintain self-tolerance and the duration and magnitude of physiological immune responses. Immune checkpoint inhibitors include or are derived from antibodies. Suitable immune checkpoint inhibitors useful in the present disclosure are set forth elsewhere in this disclosure.

[0077] As used herein, the term "reference subject" refers to a corresponding subject (e.g., a cancer subject) who has not been administered an anti-cancer treatment described herein (e.g., an IL-7 protein alone or in combination with an additional therapeutic agent). The term "reference subject" can also refer to the same subject (i.e., a subject treated with the methods provided herein), but prior to administration of the anti-cancer treatment combination described herein. In some embodiments, the term "reference subject" refers to the average of a population of subjects (e.g., cancer subjects).

[0078] As used herein, the terms "ug" and "uM" are used interchangeably with "μg" and "μM," respectively.

[0079] The various aspects described herein are described in further detail in the following subsections.

[0080] II. Methods of the Disclosure The present disclosure relates generally to treating tumors (or cancers) with anti-cancer therapies that include interleukin (IL-7) proteins. As demonstrated herein, such anti-cancer therapies can be used to treat tumors (or cancers) by targeting unmethylated O 6 These methods are particularly useful for treating tumors containing an -methylguanine-DNA methyltransferase (MGMT) promoter. Accordingly, some embodiments of the present disclosure relate to methods for treating a tumor in a subject in need thereof, the method comprising administering an anti-cancer treatment to the subject, wherein the anti-cancer treatment comprises an interleukin-7 (IL-7) protein. In some embodiments, the treatment methods provided herein comprise confirming the methylation status of the MGMT promoter of the tumor (i.e., determining whether the MGMT promoter is methylated or unmethylated) before administering the anti-cancer treatment to the subject. For example, in some embodiments, provided herein are methods for treating a tumor in a subject in need thereof, the methods comprising determining the methylation status of the MGMT promoter in a tumor sample obtained from the subject, and administering an anti-cancer treatment comprising an IL-7 protein to the subject if the MGMT promoter is unmethylated.

[0081] As is apparent from the present disclosure, in some embodiments, by determining the methylation status of the MGMT promoter, it may be possible to assess whether a subject having a tumor (also referred to herein as a "cancer subject") will respond to an anti-cancer treatment provided herein. Accordingly, some embodiments of the present disclosure relate to a method for identifying a cancer subject suitable for anti-cancer treatment comprising an IL-7 protein, the method comprising determining the methylation status of the MGMT promoter in a tumor sample obtained from the subject, wherein the subject is suitable for the anti-cancer treatment if the MGMT promoter is unmethylated. In some embodiments, the method further comprises administering the anti-cancer treatment to the subject identified as suitable for the anti-cancer treatment.

[0082] When the methods provided herein involve determining the methylation status of the MGMT promoter, any suitable method known in the art can be used to determine the methylation status. Non-limiting examples of such methods include methylation-specific PCR (MS-PCR), pyrosequencing, high-resolution melting, microarrays (e.g., Infinium MethylationEPIC), immunohistochemistry (IHC), multiplex ligation-dependent probe amplification (MPLA), or a combination thereof. Further disclosures regarding useful methods for determining the methylation status of MGMT are provided in Brandner et al., Neuro Oncol 23(9):1457-1469 (Sep. 2021) and U.S. Patent No. 10,053,724, each of which is incorporated herein by reference in its entirety.

[0083] As demonstrated and described herein, the anti-cancer treatments provided herein (i.e., comprising IL-7 protein) exhibit enhanced therapeutic efficacy compared to treatments known in the art. Accordingly, some embodiments of the present disclosure relate to methods for enhancing anti-tumor immune responses in a subject in need thereof, comprising administering to the subject an anti-cancer treatment comprising IL-7 protein, wherein the subject has a tumor comprising an unmethylated MGMT promoter. In some embodiments, such methods comprise determining the methylation status of the MGMT promoter in a tumor sample obtained from the subject prior to administering the anti-cancer treatment.

[0084] In some embodiments, after administration, the subject's anti-tumor immune response is increased 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%, or at least about 100% compared to the corresponding value in a reference subject (e.g., the subject before administration of the anti-cancer treatment and / or a matched subject who did not receive the anti-cancer treatment). In some embodiments, after administration, the subject's anti-tumor immune response is increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, or at least about 50-fold compared to the corresponding value in the reference subject.

[0085] In some embodiments, an increased anti-tumor immune response may be associated with one or more of the following therapeutic effects: (i) a decrease in tumor volume and / or growth, (ii) an increase in the effector activity of tumor-specific T cells (e.g., the ability to kill tumor cells), (iii) an increase in the number of tumor-specific effector T cells, e.g., in the tumor, (iv) an increase in the number of tumor-infiltrating lymphocytes (TILs), e.g., in the tumor, or (v) any combination thereof. Unless otherwise specified, the term "number" refers to both absolute numbers and percentages (%). Any of the therapeutic effects described above or elsewhere in this disclosure can be determined using any of the appropriate methods provided herein and / or known in the art.

[0086] In some embodiments, after administration of an anti-cancer treatment provided herein (i.e., comprising an IL-7 protein) to a subject having a tumor comprising an unmethylated MGMT promoter, the subject's tumor volume is reduced compared to the corresponding value in a reference subject (e.g., the subject prior to administration and / or a corresponding subject not treated with the anti-cancer treatment). In some embodiments, after administration, the subject's 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% compared to the corresponding value in the reference subject. In some embodiments, after administration of the anti-cancer treatment, tumor growth in the subject is reduced compared to the corresponding value in the reference subject. In some embodiments, after administration, tumor growth in the subject 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% compared to the corresponding value in a reference subject.

[0087] In some embodiments, after administration of an anti-cancer treatment (i.e., comprising an IL-7 protein), tumor-specific T cells (e.g., CD8 + T cells and / or CD4 +In some embodiments, effector activity of T cells (e.g., CD8 T cells) is increased compared to a corresponding value in a reference subject. In some embodiments, effector activity in the subject is decreased 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%, or about 100% compared to a corresponding value in a reference subject. In some embodiments, effector activity in the subject is increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, or at least about 50-fold compared to a corresponding value in a reference subject. In some embodiments, effector activity includes T cells (e.g., CD8 T cells) that may be useful in the treatment of cancer. + T cells and / or CD4 + Effector activities include any cytolytic activity of T cells. Non-limiting examples of such effector activities include the ability to produce effector molecules (e.g., cytokines, perforin, and / or granzymes) upon antigenic stimulation, the ability to recognize and kill tumor cells, or both.

[0088] In some embodiments, following administration of an anti-cancer treatment provided herein (e.g., comprising an IL-7 protein) to a subject having a tumor comprising an unmethylated MGMT promoter, the number of tumor-specific effector T cells (e.g., CD8 + and / or CD4 +) is increased. In some embodiments, after administration, the number of tumor-specific effector T cells in the subject is increased 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%, or at least about 100% compared to the corresponding value in a reference subject. In some embodiments, the number of tumor-specific effector T cells in the subject is increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, or at least about 50-fold compared to the corresponding value in a reference subject.

[0089] Without being bound by any one theory, in some embodiments, the increase in number is due to an increase in proliferation of tumor-specific effector T cells. In some embodiments, following administration of an anticancer treatment provided herein to a subject having a tumor comprising an unmethylated MGMT promoter, the rate of proliferation of tumor-specific effector T cells 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%, or at least about 100% compared to the corresponding value in a reference subject. In some embodiments, following administration of an anti-cancer treatment, the proliferation rate of tumor-specific effector T cells increases by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, or at least about 50-fold compared to the corresponding value in a reference subject. In some embodiments, the increase in the number of tumor-specific effector T cells is due to the ability of the T cells to survive and / or persist. In some embodiments, following administration of an anti-cancer treatment (e.g., comprising an IL-7 protein) to a subject having a tumor comprising an unmethylated MGMT promoter, the ability of tumor-specific effector T cells to survive and / or persist in the subject is increased. In some embodiments, the ability of tumor-specific effector T cells to survive and / or persist in a subject is increased 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%, or at least about 100% compared to the corresponding value in a reference subject.In some embodiments, the ability of tumor-specific effector T cells to survive and / or persist in the subject is increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, or at least about 50-fold compared to the corresponding value in a reference subject. In some embodiments, both the proliferation rate and survival / persistence rate of tumor-specific effector T cells are increased in the subject following administration of the anti-cancer treatment.

[0090] In some embodiments, tumor-specific effector T cells are tumor-infiltrating lymphocytes (TILs). Thus, in some embodiments, after administering an anti-cancer treatment (e.g., comprising IL-7 protein) described herein to a subject with a tumor comprising an unmethylated MGMT promoter, the number of TILs in the subject's tumor increases compared to the corresponding value in a reference subject. In some embodiments, after administering an anti-cancer treatment, the number of TILs in the subject's tumor decreases 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%, or about 100% compared to the corresponding value in a reference subject. In some embodiments, after administration of the anti-cancer treatment, the number of TILs in the subject's tumor is increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, or at least about 50-fold compared to the corresponding value in a reference subject.

[0091] In some embodiments, the anti-cancer treatments provided herein (i.e., comprising an IL-7 protein) can increase the proliferation, survival, and / or persistence rates of tumor-specific T cells, which may also increase overall lymphocyte counts (i.e., "absolute lymphocyte counts"). Accordingly, in some embodiments, following administration of an anti-cancer treatment provided herein (e.g., comprising an IL-7 protein) to a subject having a tumor comprising an unmethylated MGMT promoter, the subject's absolute lymphocyte count is increased compared to the corresponding value in a reference subject. In some embodiments, the subject's absolute lymphocyte count is decreased 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%, or about 100% compared to the corresponding value in a reference subject. In some embodiments, after administration, the subject's absolute white blood cell count increases by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, or at least about 50-fold compared to the corresponding value in a reference subject.

[0092] In some embodiments, following administration of an anti-cancer treatment, the increase in the subject's absolute lymphocyte count is maintained significantly longer compared to the corresponding value in a reference subject. In some embodiments, the increase in absolute lymphocyte count is maintained 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% longer compared to the reference subject. In some embodiments, the increase in absolute lymphocyte count is maintained at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, or at least about 50-fold longer compared to the corresponding value in the reference subject. In some embodiments, following administration of the anti-cancer treatment, the increase in absolute white blood cell count in the subject is maintained for at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 11 weeks, at least about 12 weeks, at least about 13 weeks, at least about 14 weeks, or at least about 15 weeks.

[0093] As will be apparent to those skilled in the art, in some embodiments, one or more of the above therapeutic effects can lead to an increase in overall survival. In some embodiments, the increase in overall survival includes progression-free survival. Thus, in some embodiments, after administration of an anti-cancer treatment provided herein (i.e., comprising an IL-7 protein) to a subject having a tumor comprising an unmethylated MGMT promoter, the subject's overall survival is extended. In some embodiments, the subject's overall survival is increased 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%, or at least about 100% compared to the corresponding value of a reference subject. In some embodiments, after administration, the subject's overall survival length is increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, or at least about 50-fold compared to the corresponding value in a reference subject. In some embodiments, after administration of the anti-cancer treatment, the subject's overall survival length is increased 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, at least about 12 months, at least about 13 months, at least about 14 months, at least about 15 months, at least about 16 months, at least about 17 months, at least about 18 months, at least about 19 months, at least about 20 months, at least about 21 months, at least about 22 months, at least about 23 months, or at least 24 months. In some embodiments, after administration, the subject's survival length is at least about 12 months, at least about 13 months, at least about 14 months, at least about 15 months, at least about 16 months, at least about 17 months, at least about 18 months, at least about 19 months, or at least about 20 months.

[0094] In some embodiments, after administration of an anti-cancer treatment (e.g., comprising an IL-7 protein) to a subject having a tumor comprising an unmethylated MGMT promoter, the length of the subject's progression-free survival increases. For example, in some embodiments, the subject's progression-free survival 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%, or at least about 100% compared to the corresponding value of a reference subject. In some embodiments, after administration, the subject's progression-free survival increases by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, or at least about 50-fold compared to the corresponding value of a reference subject. In some embodiments, after administration of the anti-cancer treatment, the subject's progression-free survival is increased 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, at least about 12 months, at least about 13 months, at least about 14 months, at least about 15 months, at least about 16 months, at least about 17 months, at least about 18 months, at least about 19 months, at least about 20 months, at least about 21 months, at least about 22 months, at least about 23 months, or at least 24 months. In some embodiments, after administration of the anti-cancer treatment, the subject's progression-free survival is at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or about 12 months.

[0095] 1. Combination treatment As described herein, the anti-cancer treatment useful in the present disclosure comprises an IL-7 protein. In some embodiments, the anti-cancer treatment comprises one or more therapeutic agents. Thus, in some embodiments, administering an anti-cancer treatment to a subject (e.g., having a tumor, including an unmethylated MGMT tumor) comprises administering an IL-7 protein to the subject in combination with an additional therapeutic agent. Unless otherwise specified, the additional therapeutic agent can include any agent known in the art that may be useful in treating the tumors described herein.

[0096] In some embodiments, the additional therapeutic agent comprises standard of care (SOC). Thus, in some embodiments, the methods provided herein comprise administering an anti-cancer treatment to a subject (e.g., having a tumor comprising an unmethylated MGMT promoter), wherein the anti-cancer treatment comprises an IL-7 protein and an SOC. In some embodiments, the SOC comprises surgery, chemotherapy, radiation therapy, or a combination thereof. For example, in the case of glioma, the first stage of SOC generally comprises surgery to remove as much of the tumor as possible from the subject. After surgery, the SOC generally comprises radiation therapy, chemotherapy, or both. Thus, in some embodiments, an anti-cancer treatment useful in the present disclosure comprises an IL-7 protein and an SOC, wherein the SOC comprises surgery. In some embodiments, the anti-cancer treatment comprises an IL-7 protein and an SOC, wherein the SOC comprises radiation therapy. In some embodiments, the anti-cancer treatment comprises an IL-7 protein and an SOC, wherein the SOC comprises chemotherapy. In some embodiments, anti-cancer treatments useful in the present disclosure include an IL-7 protein and an SOC, wherein the SOC comprises surgery and radiation therapy. In some embodiments, anti-cancer treatments useful in the present disclosure include an IL-7 protein and an SOC, wherein the SOC comprises surgery and chemotherapy. In some embodiments, anti-cancer treatments useful in the present disclosure include an IL-7 protein and an SOC, wherein the SOC comprises radiation therapy and chemotherapy. In some embodiments, anti-cancer treatments useful in the present disclosure include an IL-7 protein and an SOC, wherein the SOC comprises surgery, radiation therapy, and chemotherapy. For any anti-cancer treatment described herein that includes chemotherapy, in some embodiments, the chemotherapy includes temozolomide (TMZ) (see Tan et al., CA Cancer J Clin 70(4): 299-312 (Jul. 2020)). Thus, in some embodiments, the methods provided herein include administering to a subject (e.g., having a tumor containing an unmethylated MGMT promoter) an anti-cancer treatment, wherein the anti-cancer treatment comprises an IL-7 protein and SOC, and the SOC comprises concurrent radiation therapy in combination with TMZ.In some embodiments, anti-cancer treatments useful in the present disclosure include an IL-7 protein and an SOC, wherein the SOC includes concurrent radiation therapy in combination with TMZ and an additional adjuvant. In some embodiments, the additional adjuvant TMZ is administered to the subject after the concurrent radiation therapy in combination with TMZ has been stopped.

[0097] In some embodiments, additional therapeutic agents that can be used in combination with the IL-7 proteins described herein include additional radiation therapy, additional chemotherapy, hormone therapy, immunotherapy, photodynamic therapy, stem cell transplantation, or a combination thereof. Thus, in some embodiments, an anti-cancer treatment useful in the present disclosure includes an IL-7 protein and radiation therapy. In some embodiments, an anti-cancer treatment useful in the present disclosure includes an IL-7 protein and chemotherapy (e.g., TMZ). In some embodiments, an anti-cancer treatment includes an IL-7 protein and hormone therapy. In some embodiments, an anti-cancer treatment includes an IL-7 protein and photodynamic therapy. In some embodiments, an anti-cancer treatment includes an IL-7 protein and stem cell transplantation. In some embodiments, an anti-cancer treatment includes an IL-7 protein and immunotherapy. Non-limiting examples of immunotherapies that can be used include immune checkpoint inhibitors, immune checkpoint activators, adoptive cell therapy, or a combination thereof. For example, in some embodiments, anti-cancer therapies useful in the present disclosure comprise an IL-7 protein and an immune checkpoint inhibitor, where the immune checkpoint inhibitor comprises a CTLA-4 antagonist (e.g., an anti-CTLA-4 antibody), a PD-1 antagonist (e.g., an anti-PD-1 antibody, an anti-PD-L1 antibody), a TIM-3 antagonist (e.g., an anti-TIM-3 antibody), or a combination thereof. Non-limiting examples of such immune checkpoint inhibitors are well known in the art. For example, suitable anti-PD-1 antagonists are described in, e.g., U.S. Patent Nos. 6,808,710, 7,488,802, 8,008,449, 8,168,757, 8,354,509, 8,609,089, 8,779,105, 9,217,034, 7,635,757, 7,943,743, 8,217,149, 8,981,063, and 9,624,298, each of which is incorporated by reference in its entirety.Suitable anti-CTLA-4 antagonists are described, for example, in U.S. Patent Nos. 5,977,318, 6,051,227; 6,682,736, 6,984,720, 7,034,121, 7,605,238, 10,479,833, and International Publication No. WO2007 / 113648, each of which is incorporated herein by reference in its entirety. Suitable TIM-3 antagonists are described, for example, in U.S. Patent Nos. 10,508,149, 10,533,052, and 10,894,830, each of which is incorporated herein by reference in its entirety. In some embodiments, the anti-cancer treatment comprises an IL-7 protein and an immune checkpoint activator, wherein the immune checkpoint activator comprises an OX40 agonist (e.g., an anti-OX40 antibody), a LAG-3 agonist (e.g., an anti-LAG-3 antibody), a 4-1BB (CD137) agonist (e.g., an anti-CD137 antibody), a GITR agonist (e.g., an anti-GITR antibody), or a combination thereof.

[0098] When the anti-cancer treatment includes both an IL-7 protein and an additional therapeutic agent (e.g., as described above and elsewhere in this application), in some embodiments, the IL-7 protein and the additional therapeutic agent are administered to the subject simultaneously. For example, in some embodiments, the IL-7 protein and the additional therapeutic agent can be administered simultaneously as a single composition. In some embodiments, the IL-7 protein and the additional therapeutic agent can be administered simultaneously as separate compositions. In some embodiments, the IL-7 protein and the additional therapeutic agent can be administered sequentially to the subject. In some embodiments, the IL-7 protein is administered to the subject before administration of the additional therapeutic agent. In some embodiments, the IL-7 protein is administered to the subject after administration of the additional therapeutic agent.

[0099] As demonstrated herein, in some embodiments, administering an IL-7 protein in combination with an additional therapeutic agent (e.g., those described herein) can result in a greater therapeutic effect compared to administering either therapeutic agent alone. Thus, in some embodiments, administering an anti-cancer treatment comprising both an IL-7 protein and an additional therapeutic agent (i.e., a "combination treatment") to a subject (e.g., having a tumor containing an unmethylated MGMT promoter) can increase the anti-tumor immune response compared to a corresponding subject who did not receive the combination treatment (e.g., who received the IL-7 protein alone or the additional therapeutic agent alone). In some embodiments, following administration of the combination treatment, the subject's anti-tumor immune response is increased 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%, or at least about 100% compared to a corresponding subject who did not receive the combination treatment. In some embodiments, after administration of the combination treatment, the subject's anti-tumor immune response is increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, or at least about 50-fold compared to a corresponding subject who did not receive the combination treatment.

[0100] In some embodiments, administration of a combination therapy provided herein (e.g., comprising an IL-7 protein and an additional therapeutic agent) can reduce tumor volume compared to a corresponding subject who did not receive the combination therapy (e.g., who received an IL-7 protein alone or who received an additional therapeutic agent alone). In some embodiments, after administration of the combination therapy, the subject's 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 about 100% compared to a corresponding subject who did not receive the combination therapy. In some embodiments, after administration of the combination therapy, the subject's tumor growth 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 corresponding subject who did not receive the combination therapy.

[0101] In some embodiments, administering a combination treatment provided herein (e.g., comprising an IL-7 protein and an additional therapeutic agent) to a subject (e.g., having a tumor comprising an unmethylated MGMT promoter) results in an increase in tumor-specific T cells (e.g., CD8 + T cells and / or CD4 +The combination therapy can increase effector activity of tumor-specific T cells (T cells). In some embodiments, effector activity in a subject is increased 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%, or at least about 100% compared to a corresponding subject who did not receive the combination therapy. In some embodiments, effector activity in a subject is increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, or at least about 50-fold compared to a corresponding subject who did not receive the combination therapy. Non-limiting examples of tumor-specific T cells are provided elsewhere in this disclosure.

[0102] In some embodiments, administering a combination treatment provided herein (e.g., comprising an IL-7 protein and an additional therapeutic agent) to a subject (e.g., having a tumor comprising an unmethylated MGMT promoter) results in an increase in tumor-specific effector T cells (e.g., CD8 + and / or CD4 +In some embodiments, after administration of the combination treatment, the number of tumor-specific effector T cells in the subject is increased 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%, or at least about 100%, compared to a corresponding subject that did not receive the combination treatment. In some embodiments, the number of tumor-specific effector T cells in the subject is increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, or at least about 50-fold, compared to a corresponding subject that did not receive the combination treatment.

[0103] In some embodiments, administering a combination treatment provided herein (e.g., comprising an IL-7 protein and an additional therapeutic agent) to a subject (e.g., having a tumor comprising an unmethylated MGMT promoter) can increase the rate of proliferation of tumor-specific effector T cells in the subject, as compared to a corresponding subject who did not receive the combination treatment (e.g., who was administered the IL-7 protein alone or the additional therapeutic agent alone). In some embodiments, after administration of the combination treatment, the rate of proliferation of tumor-specific effector T cells is increased 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%, or at least about 100%, as compared to a corresponding subject who did not receive the combination treatment. In some embodiments, after administration of the combination treatment, the rate of proliferation of tumor-specific effector T cells increases by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, or at least about 50-fold compared to a corresponding subject who did not receive the combination treatment.

[0104] In some embodiments, administering a combination treatment provided herein (e.g., comprising an IL-7 protein and an additional therapeutic agent) to a subject (e.g., having a tumor comprising an unmethylated MGMT promoter) can increase the ability of tumor-specific effector T cells to survive and / or persist in the subject, as compared to a corresponding subject that did not receive the combination treatment (e.g., administered the IL-7 protein alone or the additional therapeutic agent alone). In some embodiments, following administration of the combination treatment, the ability of tumor-specific effector T cells to survive and / or persist in the subject is increased 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%, or at least about 100%, as compared to a corresponding subject that did not receive the combination treatment. In some embodiments, following administration of the combination treatment, the ability of tumor-specific effector T cells to survive and / or persist in the subject is increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, or at least about 50-fold, compared to a corresponding subject that did not receive the combination treatment. In some embodiments, following administration of the combination treatment, both the proliferation rate and survival / persistence rate of tumor-specific effector T cells in the subject are increased, compared to a corresponding subject that did not receive the combination treatment.

[0105] In some embodiments, administering a combination treatment provided herein (e.g., comprising an IL-7 protein and an additional therapeutic agent) to a subject (e.g., having a tumor comprising an unmethylated MGMT promoter) can increase the number of TILs in the subject's tumor, compared to a corresponding subject who did not receive the combination treatment (e.g., who was administered the IL-7 protein alone or the additional therapeutic agent alone). In some embodiments, after administration of the combination treatment, the number of TILs in the subject's tumor is increased 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%, or at least about 100%, compared to a corresponding subject who did not receive the combination treatment. In some embodiments, after administration of the combination treatment, the number of TILs in the subject's tumor is increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, or at least about 50-fold compared to a corresponding subject who did not receive the combination treatment.

[0106] In some embodiments, administering a combination treatment provided herein (e.g., comprising an IL-7 protein and an additional therapeutic agent) to a subject (e.g., having a tumor comprising an unmethylated MGMT promoter) can increase the subject's absolute lymphocyte count, as compared to a corresponding subject who did not receive the combination treatment (e.g., who was administered the IL-7 protein alone or the additional therapeutic agent alone). In some embodiments, after administration of the combination treatment, the subject's absolute lymphocyte 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%, or at least about 100%, as compared to a corresponding subject who did not receive the combination treatment. In some embodiments, after administration of the combination treatment, the subject's absolute lymphocyte count increases by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, or at least about 50-fold compared to a corresponding subject who did not receive the combination treatment.

[0107] In some embodiments, administering a combination treatment provided herein (e.g., comprising an IL-7 protein and an additional therapeutic agent) to a subject (e.g., having a tumor comprising an unmethylated MGMT promoter) can increase the subject's overall survival length, as compared to a corresponding subject who did not receive the combination treatment (e.g., who was administered the IL-7 protein alone or the additional therapeutic agent alone). In some embodiments, after administration of the combination treatment, the subject's overall survival length is increased 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%, or at least about 100%, as compared to a corresponding subject who did not receive the combination treatment. In some embodiments, after administration of the combination treatment, the subject's overall survival length is increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, or at least about 50-fold, compared to a corresponding subject who did not receive the combination treatment.

[0108] In some embodiments, administering a combination treatment provided herein (e.g., comprising an IL-7 protein and an additional therapeutic agent) to a subject (e.g., having a tumor comprising an unmethylated MGMT promoter) can increase the subject's progression-free survival compared to a corresponding subject who did not receive the combination treatment (e.g., who was administered the IL-7 protein alone or the additional therapeutic agent alone). In some embodiments, after administration of the combination treatment, the subject's progression-free survival is increased 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%, or at least about 100% compared to a corresponding subject who did not receive the combination treatment. In some embodiments, after administration of the combination treatment, the subject's progression-free survival is increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, or at least about 50-fold, compared to a corresponding subject who did not receive the combination treatment.

[0109] In some embodiments, any of the therapeutic effects described herein may occur under lymphopenic conditions. For example, many cancer patients (e.g., those with brain tumors, such as GBM) exhibit edema (e.g., cerebral edema). Accordingly, steroids (e.g., corticosteroids) can be administered to cancer patients to treat such symptoms. While steroids can be useful in alleviating certain symptoms, they are known to reduce immune system activity and function, including causing lymphopenia when administered to humans (see, e.g., Patrick Roth et al., Neurooncol Pract 2(1): 6-12 (Mar. 2015)). For example, in some embodiments, anti-cancer treatments described herein (e.g., including an IL-7 protein alone or in combination with an additional therapeutic agent) can enhance anti-tumor immune responses in subjects treated with steroids (e.g., those that cause lymphopenia). In some embodiments, the anti-tumor immune response in the subject is increased 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%, or at least about 100% compared to a corresponding subject who did not receive the anti-cancer treatment (i.e., a subject treated with a steroid). In some embodiments, the anti-tumor immune response in the subject is increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, or at least about 50-fold compared to a corresponding subject who did not receive the anti-cancer treatment (i.e., a subject treated with a steroid).

[0110] In some embodiments, anti-cancer treatments described herein (e.g., comprising an IL-7 protein alone or in combination with an additional therapeutic agent) can reduce tumor volume and / or tumor growth rate in a subject treated with a steroid (such as one that causes lymphopenia). In some embodiments, tumor volume in the subject 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% compared to a corresponding subject who did not receive the anti-cancer treatment (i.e., a subject treated with a steroid). In some embodiments, tumor growth rate in the subject 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% compared to a corresponding subject who did not receive the anti-cancer treatment (i.e., a subject treated with a steroid).

[0111] In some embodiments, the anti-cancer treatments described herein (e.g., comprising an IL-7 protein alone or in combination with an additional therapeutic agent) may increase tumor-specific effector T cells (e.g., CD8 T cells) in subjects treated with steroids (such as those that cause lymphopenia). + T cells and / or CD4 +The effector activity of T cells (T cells) can be increased. In some embodiments, effector activity in the subject is increased 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%, or at least about 100% compared to a corresponding subject who did not receive the anti-cancer treatment (i.e., a subject treated with a steroid). In some embodiments, effector activity in the subject is increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, or at least about 50-fold compared to a corresponding subject who did not receive the anti-cancer treatment (i.e., a subject treated with a steroid).

[0112] In some embodiments, the anti-cancer treatments described herein (e.g., comprising an IL-7 protein alone or in combination with an additional therapeutic agent) may increase tumor-specific effector T cells (e.g., CD8 T cells) in subjects treated with steroids (such as those that cause lymphopenia). + T cells and / or CD4 +The number of tumor-specific effector T cells (T cells) can be increased 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%, or at least about 100% in a subject compared to a corresponding subject who did not receive the anti-cancer treatment (i.e., a subject treated with a steroid). In some embodiments, the number of tumor-specific effector T cells in a subject is increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, or at least about 50-fold in a subject compared to a corresponding subject who did not receive the anti-cancer treatment (i.e., a subject treated with a steroid).

[0113] In some embodiments, an anti-cancer treatment described herein (e.g., comprising an IL-7 protein alone or in combination with an additional therapeutic agent) can increase the proliferation rate of tumor-specific effector T cells in a subject treated with a steroid (such as one that causes lymphopenia). In some embodiments, the proliferation rate of tumor-specific effector T cells is increased 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%, or at least about 100% compared to a corresponding subject that did not receive the anti-cancer treatment (i.e., a subject treated with a steroid). In some embodiments, the proliferation rate of tumor-specific effector T cells is increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, or at least about 50-fold compared to a corresponding subject that did not receive the anti-cancer treatment (i.e., a subject treated with a steroid).

[0114] In some embodiments, an anti-cancer treatment described herein (e.g., comprising an IL-7 protein alone or in combination with an additional therapeutic agent) can enhance the ability of tumor-specific effector T cells to survive and / or persist in a subject treated with a steroid (such as one that causes lymphopenia). In some embodiments, the ability of tumor-specific effector T cells to survive and / or persist in a subject is increased 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%, or at least about 100% compared to a corresponding subject that did not receive the anti-cancer treatment (i.e., a subject treated with a steroid). In some embodiments, the ability of tumor-specific effector T cells to survive and / or persist in the subject is increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, or at least about 50-fold, compared to a corresponding subject that did not receive the anti-cancer treatment (i.e., a subject that was treated with a steroid). In some embodiments, both the proliferation rate and survival / persistence rate of tumor-specific effector T cells in the subject are increased, compared to a corresponding subject that did not receive the anti-cancer treatment (i.e., a subject that was treated with a steroid).

[0115] In some embodiments, an anti-cancer treatment described herein (e.g., comprising an IL-7 protein alone or in combination with an additional therapeutic agent) can increase the number of TILs in a subject treated with a steroid (such as one that causes lymphopenia). In some embodiments, the number of TILs in the subject's tumor is increased 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%, or at least about 100% compared to a corresponding subject who did not receive the combination treatment (i.e., a subject treated with a steroid). In some embodiments, the number of TILs in the subject's tumor is increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, or at least about 50-fold compared to a corresponding subject that did not receive the combination treatment (i.e., a subject treated with a steroid).

[0116] In some embodiments, an anti-cancer treatment described herein (e.g., comprising an IL-7 protein alone or in combination with an additional therapeutic agent) can increase absolute lymphocyte count in a subject treated with a steroid (such as one that causes lymphopenia). In some embodiments, the absolute lymphocyte count in the subject is increased 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%, or at least about 100% compared to a corresponding subject that did not receive the anti-cancer treatment (i.e., a subject treated with a steroid). In some embodiments, the absolute lymphocyte count in the subject is increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, or at least about 50-fold compared to a corresponding subject that did not receive the anti-cancer treatment (i.e., a subject that was treated with a steroid).

[0117] In some embodiments, the anti-cancer treatments described herein (e.g., comprising an IL-7 protein alone or in combination with an additional therapeutic agent) can increase the overall survival length of a subject treated with a steroid (such as one that causes lymphopenia). In some embodiments, the overall survival length of the subject is increased 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%, or at least about 100% compared to a corresponding subject that did not receive the anti-cancer treatment (i.e., a subject treated with a steroid). In some embodiments, the subject's overall survival length is increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, or at least about 50-fold compared to a corresponding subject who did not receive the anti-cancer treatment (i.e., a subject treated with a steroid).

[0118] In some embodiments, an anti-cancer treatment described herein (e.g., comprising an IL-7 protein alone or in combination with an additional therapeutic agent) can increase progression-free survival in a subject treated with a steroid (such as one that causes lymphopenia). In some embodiments, the subject's progression-free survival is increased 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%, or at least about 100% compared to a corresponding subject who did not receive the anti-cancer treatment (i.e., a subject treated with a steroid). In some embodiments, the subject's progression-free survival is increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, or at least about 50-fold compared to a corresponding subject who did not receive the anti-cancer treatment (i.e., a subject treated with a steroid).

[0119] In some embodiments, the anti-cancer treatments described herein (e.g., comprising an IL-7 protein, alone or in combination with an additional therapeutic agent) can be used in combination with one or more additional cancer therapies. Non-limiting examples of such additional cancer therapies include chemotherapeutic agents, small molecule drugs, radiation, immune checkpoint inhibitors, immune checkpoint activators, or combinations thereof. Accordingly, in some embodiments, provided herein are methods of treating a tumor (e.g., including unmethylated MGMT tumors) in a subject in need thereof, comprising administering to the subject (i) an anti-cancer treatment comprising an IL-7 protein, alone or in combination with an additional therapeutic agent (e.g., chemotherapy, e.g., TMZ), and (ii) the additional cancer therapy.Non-limiting examples of such combinations include therapies that enhance tumor antigen presentation (e.g., dendritic cell vaccines, GM-CSF-secreting cellular vaccines, CpG oligonucleotides, imiquimod); therapies that inhibit negative immune regulation, for example, by inhibiting the CTLA-4 and / or PD1 / PD-L1 / PD-L2 pathway and / or depleting or blocking Tregs or other immune suppressive cells (e.g., myeloid-derived suppressor cells); therapies that stimulate positive immune regulation, for example, by stimulating the CD-137, OX-40, and / or CD40 or GITR pathways and / or agonists that stimulate T cell effector function; therapies that systemically increase the frequency of anti-tumor T cells; for example, by using antagonists of CD25 (e.g., daclizumab) or ex These include therapies that deplete or inhibit Tregs, such as Tregs in tumors, by in vivo anti-CD25 bead depletion; therapies that affect the function of suppressor myeloid cells in tumors; therapies that improve 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 exhaustion; therapies that induce innate immune activation and / or inflammation at the tumor site; administration of immune-stimulating cytokines; or blockade of immune-suppressing cytokines.

[0120] 2. Tumor As is evident from the present disclosure, the anti-cancer treatments provided herein (e.g., comprising an IL-7 protein alone or in combination with an additional therapeutic agent) may be useful in treating a wide range of cancers. As demonstrated and described herein, the anti-cancer treatments provided herein are particularly effective in treating tumors with an unmethylated MGMT promoter. 6Methylation of the -methylguanine DNA methyltransferase (MGMT) promoter has been shown to be a strong prognostic factor in the treatment of GBM (Ferdheim et al., Cancers (Beel) 11(12):1837 (Dec. 2019)). For example, individuals with GBM with a methylated MGMT promoter show improved response to treatment (e.g., chemotherapy with temozolomide) and prolonged overall survival (Alnahhas et al., Neurooncol Adv 2(1):vdaa082 (Jan.-Dec. 2020)). Unfortunately, when the MGMT promoter is unmethylated, individuals with such GBM are less responsive to treatment and have a much poorer prognosis.

[0121] Therefore, target tumors that can be treated with the present disclosure include any tumor containing an unmethylated MGMT promoter. In some embodiments, tumors containing an unmethylated MGMT promoter include gliomas. As used herein, the term "glioma" refers to a type of tumor that originates in the brain and spinal cord (i.e., arises from glial cells or glial cell precursors in the brain or spinal cord). There are four major types of gliomas: (1) astrocytoma (the most common type of glioma in both adults and children), (2) ependymoma, (3) oligodendroglioma, and (4) mixed glioma. Gliomas can be classified according to their location as intratentorial (i.e., located in the lower part of the brain and mainly found in pediatric patients) or supratentorial (i.e., located in the upper part of the brain and mainly found in adult patients).

[0122] Gliomas can be further classified according to their malignancy, determined by pathological evaluation of the tumor. The World Health Organization (WHO) has developed a grading system ranging from Grade I gliomas, which tend to be the least aggressive, to Grade IV gliomas, which tend to be the most aggressive and malignant. Non-limiting examples of low-grade gliomas include pilocytic astrocytoma (also known as juvenile pilocytic astrocytoma), fibrous astrocytoma, pleomorphic xanthoastrocytoma, and dysembryoplastic neuroepithelial tumor. Non-limiting examples of high-grade gliomas include anaplastic astrocytoma (Grade III) and glioblastoma multiforme (Grade IV). Among different gliomas, Grade IV gliomas have the poorest prognosis, with a median survival time of approximately 12 months or less.

[0123] In some embodiments, gliomas treatable with the present disclosure include high-grade gliomas. In some embodiments, gliomas include grade III gliomas. In some embodiments, gliomas include grade IV gliomas. Thus, in some embodiments, provided herein is a method of treating a tumor in a subject in need thereof comprising an unmethylated MGMT promoter, the method comprising administering to the subject an anti-cancer treatment, wherein the anti-cancer treatment is interleukin-7 (IL-7) protein, and the tumor comprises a high-grade glioma. Non-limiting examples of such high-grade gliomas include glioblastoma multiforme (GBM), anaplastic astrocytoma, or both. In some embodiments, provided herein is a method of treating GBM in a subject in need thereof, the method comprising administering to the subject an anti-cancer treatment comprising IL-7 protein, and wherein the GBM has an unmethylated MGMT promoter. In some embodiments, provided herein is a method for treating GBM in a subject in need thereof, comprising administering to the subject an anti-cancer treatment comprising an IL-7 protein and an additional therapeutic agent (e.g., as described herein), wherein the GBM has an unmethylated MGMT promoter. Although the present disclosure generally relates to treating tumors with an unmethylated MGMT promoter, it will be apparent to those skilled in the art that the disclosure provided herein may also be useful for treating other types of tumors. For example, in some embodiments, the present disclosure may be useful for treating high-risk grade II gliomas.

[0124] In some embodiments, tumors that can be treated by the present disclosure contain an unmethylated MGMT promoter and are metastatic, unresectable, refractory (e.g., to a previous cancer therapy, such as immunotherapy with an immune checkpoint inhibitor), and / or recurrent. In some embodiments, the tumor contains an unmethylated MGMT promoter and is metastatic. In some embodiments, the tumor contains an unmethylated MGMT promoter and is unresectable. In some embodiments, the tumor contains an unmethylated MGMT promoter and is refractory. In some embodiments, the tumor contains an unmethylated MGMT promoter and is recurrent. In some embodiments, the tumor is newly diagnosed (i.e., not previously treated with an anti-cancer therapy).

[0125] 3. Targeted Unless otherwise specified, any subject with tumor that comprises unmethylated MGMT promoter can be treated by the present disclosure.In some embodiments, the subject is a non-human animal, such as rat or mouse.In some embodiments, the subject that can be treated by the present disclosure includes human.

[0126] In some aspects, subjects that can be treated with the present disclosure have been newly diagnosed with a tumor described herein (e.g., comprising an unmethylated MGMT promoter). In some aspects, the subject is treatment-naive (i.e., has not previously undergone cancer treatment). In some aspects, the subject has progressed to other cancer treatments. Thus, in some aspects, subjects that can be treated with the present disclosure have previously undergone cancer treatment. In some aspects, the previous cancer treatment included immunotherapy (e.g., with an anti-PD-1 antibody). In some aspects, the previous cancer treatment included chemotherapy. In some aspects, the chemotherapy includes a platinum-based therapy. In some aspects, the platinum-based therapy includes a platinum-based antineoplastic agent selected from the group consisting of cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, satraplatin, and any combination thereof.

[0127] In some embodiments, subjects that can be treated with the present disclosure have grade III gliomas as determined by the WHO classification system. In some embodiments, subjects have grade IV gliomas as determined by the WHO classification system. In some embodiments, subjects have high-risk grade II gliomas as determined by the WHO classification system. As is apparent from the present disclosure, subjects that can be treated with the present disclosure have gliomas containing an unmethylated MGMT promoter. In some embodiments, the subject has undergone one or more postoperative treatments. Non-limiting examples of such postoperative treatments include radiation, chemotherapy (e.g., TMZ or carmustine (also known as GLIADEL Wafer)), glucocorticoid therapy, tumor treating fields (TTF) (OPTUNE®), or a combination thereof. In some embodiments, the postoperative treatment included both radiation and TMZ. In some embodiments, subjects that can be treated with the present disclosure have adequate organ and bone marrow function as defined below: (1) absolute neutrophil count ≥ 1,000 / mcL, (2) platelets ≥ 75,000 / mcL, (3) hemoglobin ≥ 8 g / dL, (4) total bilirubin ≤ 3.0 times the upper limit of normal in the institution, and (5) AST (SGOT) / ALT (SGPT) ≤ 3.0 times the upper limit of normal in the institution. In some embodiments, subjects that can be treated with the present disclosure have an absolute lymphocyte count (ALC) ≥ 600 mcL. In some embodiments, subjects that can be treated with the present disclosure do not have an active viral infection. In some embodiments, subjects that can be treated with the present disclosure do not have an active autoimmune disease or syndrome. In some embodiments, subjects that can be treated with the present disclosure have not received a live attenuated vaccine 30 days prior to administration of the first dose of an anti-cancer treatment described herein (i.e., comprising an IL-7 protein alone or in combination with an additional therapeutic agent).

[0128] 4. Dosage regimen As is apparent from the present disclosure, the methods provided herein (e.g., methods for treating tumors containing an unmethylated MGMT promoter) include administering to a subject one or more doses of IL-7 protein. In some embodiments, a unit dose (e.g., for use in humans) of the IL-7 protein disclosed herein can range from about 0.001 mg / kg to about 10 mg / kg. In some embodiments, the unit dose of IL-7 protein ranges from about 0.01 mg / kg to about 2 mg / kg. In some embodiments, the unit dose ranges from about 0.02 mg / kg to about 1 mg / kg.

[0129] In some embodiments, the IL-7 protein disclosed herein can be administered to a subject at a body weight dose. In some embodiments, the IL-7 protein can be administered at a body weight dose of about 20 μg / kg to about 600 μg / kg. In some embodiments, the IL-7 protein of the present disclosure can be administered at a body weight dose 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. In some embodiments, the IL-7 protein is administered to a subject at a dose of 60 μg / kg. In some embodiments, the IL-7 protein is administered to a subject at a dose of 120 μg / kg. In some embodiments, the IL-7 protein is administered to a subject at a dose of 240 μg / kg.

[0130] In some embodiments, the IL-7 proteins disclosed herein can be administered to a subject at a dose of greater than about 30 μg / kg, greater than about 60 μg / kg, greater than about 90 μg / kg, greater than about 120 μg / kg, greater than about 150 μg / kg, greater than about 180 μg / kg, greater than about 210 μg / kg, greater than about 240 μg / kg, greater than about 270 μg / kg, greater than about 300 μg / kg, greater than about 400 μg / kg, greater than about 500 μg / kg, or greater than about 600 μg / kg. In some embodiments, the IL-7 protein is administered to the subject at a dose of greater than about 600 μg / kg, greater than about 700 μg / kg, greater than about 800 μg / kg, greater than about 900 μg / kg, greater than about 1,000 μg / kg, greater than about 1,100 μg / kg, greater than about 1,200 μg / kg, greater than about 1,300 μg / kg, greater than about 1,400 μg / kg, greater than about 1,500 μg / kg, greater than about 1,600 μg / kg, greater than about 1,700 μg / kg, greater than about 1,800 μg / kg, greater than about 1,900 μg / kg, or greater than about 2,000 μg / kg.

[0131] In some embodiments, the IL-7 protein of the present disclosure is administered at a concentration of about 60 μg / kg to about 1,200 μg / kg, about 120 μg / kg to about 1,200 μg / kg, about 240 μg / kg to about 1,200 μg / kg, about 540 μg / kg to about 1,200 μg / kg, 610 μg / kg to about 1,200 μg / kg, 650 μg / kg to about 1,200 μg / kg, or about 700 μg / kg. / kg ~ approx. 1,200μg / kg, approx. 750μg / kg ~ approx. 1,200μg / kg, approx. 800μg / kg ~ approx. 1,200μg / kg, approx. 850μg / kg ~ approx. 1,200μg / k g, approximately 900μg / kg to approximately 1,200μg / kg, approximately 950μg / kg to approximately 1,200μg / kg, approximately 1,000μg / kg to approximately 1,200μg / kg, approximately 1,050μg / kg ~1,200μg / kg, approximately 1,100μg / kg~1,200μg / kg, approximately 1,200μg / kg~2,000μg / kg, approximately 1,300μg / kg~2,000μ g / kg, approximately 1,500μg / kg to approximately 2,000μg / kg, approximately 1,700μg / kg to approximately 2,000μg / kg, approximately 610μg / kg to approximately 1,000μg / kg, approximately 650μ The drug is administered at a dose of about 700 μg / kg to about 1,000 μg / kg, about 750 μg / kg to about 1,000 μg / kg, about 800 μg / kg to about 1,000 μg / kg, about 850 μg / kg to about 1,000 μg / kg, about 900 μg / kg to about 1,000 μg / kg, or about 950 μg / kg to about 1,000 μg / kg.

[0132] In some embodiments, the IL-7 protein is administered at a dose of about 60 μg / kg to about 1,200 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 120 μg / kg to about 1,200 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 240 μg / kg to about 1,200 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 540 μg / kg to about 1,200 μg / kg. In some embodiments, the IL-7 protein of the present disclosure is administered at a dose of 610 μg / kg to about 1,200 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of 650 μg / kg to about 1,200 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 700 μg / kg to about 1,200 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 750 μg / kg to about 1,200 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 800 μg / kg to about 1,200 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 850 μg / kg to about 1,200 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 900 μg / kg to about 1,200 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 950 μg / kg to about 1,200 μg / kg. In some embodiments, the IL-7 protein disclosed herein is administered at a dose of about 1,000 μg / kg to about 1,200 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,050 μg / kg to about 1,200 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,100 μg / kg to about 1,200 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,200 μg / kg to about 2,000 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,300 μg / kg to about 2,000 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,500 μg / kg to about 2,000 μg / kg.In some embodiments, the IL-7 protein is administered at a dose of about 1,700 μg / kg to about 2,000 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 610 μg / kg to about 1,000 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 650 μg / kg to about 1,000 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 700 μg / kg to about 1,000 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 750 μg / kg to about 1,000 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 800 μg / kg to about 1,000 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 850 μg / kg to about 1,000 μg / kg. In some embodiments, the IL-7 protein of the present disclosure is administered at a dose of about 900 μg / kg to about 1,000 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 950 μg / kg to about 1,000 μg / kg.

[0133] In some embodiments, the IL-7 protein is administered at a dose of about 700 μg / kg to about 900 μg / kg, about 750 μg / kg to about 950 μg / kg, about 700 μg / kg to about 850 μg / kg, about 750 μg / kg to about 850 μg / kg, about 700 μg / kg to about 800 μg / kg, about 800 μg / kg to about 900 μg / kg, about 750 μg / kg to about 850 μg / kg, or about 850 μg / kg to about 950 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 700 μg / kg to about 900 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 750 μg / kg to about 950 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 700 μg / kg to about 850 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 750 μg / kg to about 850 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 700 μg / kg to about 800 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 800 μg / kg to about 900 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 750 μg / kg to about 850 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 850 μg / kg to about 950 μg / kg.

[0134] In some embodiments, the IL-7 protein is administered at a concentration of about 30 μg / kg, about 60 μg / kg, about 90 μg / kg, about 120 μg / kg, about 150 μg / kg, about 180 μg / kg, about 210 μg / kg, about 240 μg / kg, about 270 μg / kg, about 300 μg / kg, about 330 μg / kg, about 360 μg / kg, about 390 μg / kg, about 420 μg / kg, about 450 μg / kg, about 480 μg / kg, about 510 μg / kg, about 540 μg / kg, about 570 μg / kg, about 600 μg / kg, about 630 μg / kg, about 650 μg / kg, about 680 μg / kg, Approximately 700μg / kg, approximately 720μg / kg, approximately 740μg / kg, approximately 750μg / kg, approximately 760μg / kg, approximately 780μg / kg, approximately 800μg / kg, approximately 820μg / kg, approximately 840μg / kg, approximately 850μg / kg, approximately 860μg / kg, approximately 880μg / 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 1,000 μg / kg, approximately 1,020 μg / kg, approximately 1,020 μg / kg, approximately 1,040 μg / kg, approximately 1,060 μg / kg, approximately 1,080 μg / kg kg, approximately 1,100μg / kg, approximately 1,120μg / kg, approximately 1,140μg / kg, approximately 1,160μg / kg, approximately 1,180μg / kg, approximately 1200μg / 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 μg / 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 1,700μg / k g, approximately 1,720μg / kg, approximately 1,740μg / kg, approximately 1,760μg / kg, approximately 1,780μg / kg, approximately 1,800μg / kg, approximately 1,820μg / kg, approximately 1,840μg / kg, approximately 1,860μg / kg, approximately 1,880μg / kg, approximately 1,900μg / kg, approximately 1,It is administered at a dose of 920 μg / kg, about 1,940 μg / kg, about 1,960 μg / kg, about 1,980 μg / kg, or about 2,000 μg / kg.

[0135] In some embodiments, the IL-7 protein is administered at a dose of about 30 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 60 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 90 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 120 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 150 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 180 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 210 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 240 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 270 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 300 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 330 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 360 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 390 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 420 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 450 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 480 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 510 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 540 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 570 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 600 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 630 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 650 μg / kg. In some embodiments, the IL-7 protein disclosed herein is administered at a dose of about 680 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 700 μg / kg.In some embodiments, the IL-7 protein is administered at a dose of about 720 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 740 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 750 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 760 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 780 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 800 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 820 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 840 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 850 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 860 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 880 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 900 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 920 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 940 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 950 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 960 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 980 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,000 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,020 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,040 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,060 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,080 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,100 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,120 μg / kg.In some embodiments, the IL-7 protein is administered at a dose of about 1,140 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,160 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,180 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,200 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,220 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,240 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,260 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,280 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,300 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,320 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,340 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,360 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,380 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,400 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,420 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,440 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,460 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,480 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,500 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,520 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,540 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,560 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,580 μg / kg.In some embodiments, the IL-7 protein is administered at a dose of about 1,600 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,620 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,640 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,660 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,680 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,700 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,720 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,740 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,760 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,780 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,800 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,820 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,840 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,860 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,880 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,900 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,920 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,940 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,960 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,980 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 2,000 μg / kg.

[0136] In some embodiments, the IL-7 protein described herein is administered to a subject at a dose of about 0.01 nmol / kg, about 0.02 nmol / kg, about 0.03 nmol / kg, about 0.04 nmol / kg, about 0.05 nmol / kg, about 0.1 nmol / kg, about 0.2 nmol / kg, about 0.4 nmol / kg, about 0.6 nmol / kg, about 0.8 nmol / kg, about 1 nmol / kg, about 1.2 nmol / kg, about 1.4 nmol / kg, about 1.6 nmol / kg, about 1.8 nmol / kg, about 2 nmol / kg, about 2.2 nmol / kg, about 2.4 nmol / kg, about 2.6 nmol / kg, about 2.8 nmol / kg, about 3 nmol / kg, about 3.5 nmol / kg, about 4 nmol / kg , about 4.5 nmol / kg, about 5 nmol / kg, about 6 nmol / kg, about 7 nmol / kg, about 8 nmol / kg, about 9 nmol / kg, about 10 nmol / kg, about 11 nmol / kg, about 12 nmol / kg, about 13 nmol / kg, about 14 nmol / kg, about 15 nmol / kg, about 16 nmol / kg, about 17 nmol / kg, about 18 nmol / kg, about 19 nmol / kg, about 20 nmol / kg, about 22 nmol / kg, about 24 nmol / kg, about 26 nmol / kg, about 28 nmol / kg, about 30 nmol / kg, about 32 nmol / kg, about 34 nmol / kg, about 36 nmol / kg, about 38 nmol / kg, or about 40 nmol / kg.

[0137] In some embodiments, the IL-7 protein can be administered at a fixed dose of about 0.25 mg to about 9 mg. In some embodiments, the IL-7 protein can be administered at a fixed dose of about 0.25 mg, about 1 mg, about 3 mg, about 6 mg, or about 9 mg.

[0138] In some embodiments, a subject disclosed herein receives a single dose of IL-7 protein at any of the doses described above. In some embodiments, an IL-7 protein disclosed herein is administered to a subject in multiple doses (i.e., repeated administrations). In some embodiments, an IL-7 protein is administered to a subject at least about two times, at least about three times, at least about four times, at least about five times, at least about six times, at least about seven times, at least about eight times, at least about nine times, or at least about ten times or more. In some embodiments, an IL-7 protein is administered to a subject about two times. In some embodiments, an IL-7 protein is administered to a subject about three times. In some embodiments, an IL-7 protein is administered to a subject about four times. In some embodiments, an IL-7 protein is administered to a subject about five times. In some embodiments, an IL-7 protein is administered to a subject about six times. In some embodiments, an IL-7 protein is administered to a subject about seven times. In some embodiments, an IL-7 protein is administered to a subject about eight times. In some embodiments, the IL-7 protein is administered to the subject about 9 times. In some embodiments, the IL-7 protein is administered to the subject about 10 times.

[0139] When IL-7 protein is administered multiple times to a subject (e.g., having a tumor containing an unmethylated MGMT promoter), in some embodiments, the IL-7 protein is administered, e.g., at one of the doses described above, at a dosing frequency of about once per week, about once per 2 weeks, about once per 3 weeks, about once per 4 weeks, about once per 5 weeks, about once per 6 weeks, about once per 7 weeks, about once per 8 weeks, about once per 9 weeks, about once per 10 weeks, about once per 11 weeks, or about once per 12 weeks. In some embodiments, the IL-7 protein is administered at a dosing frequency of about once per 10 days, about once per 20 days, about once per 30 days, about once per 40 days, about once per 50 days, about once per 60 days, about once per 70 days, about once per 80 days, about once per 90 days, or about once per 100 days. In some embodiments, the IL-7 protein is administered once every 3 weeks. In some embodiments, the IL-7 protein is administered once per week. In some embodiments, the IL-7 protein is administered once per two weeks. In some embodiments, the IL-7 protein is administered once per four weeks. In some embodiments, the IL-7 protein is administered once per six weeks. In some embodiments, the IL-7 protein is administered once per eight weeks. In some embodiments, the IL-7 protein is administered once per nine weeks. In some embodiments, the IL-7 protein is administered once per twelve weeks. In some embodiments, the IL-7 protein is administered once per ten days. In some embodiments, the IL-7 protein is administered once per twenty days. In some embodiments, the IL-7 protein is administered once per thirty days. In some embodiments, the IL-7 protein is administered once per forty days. In some embodiments, the IL-7 protein is administered once per fifty days. In some embodiments, the IL-7 protein is administered once per sixty days. In some embodiments, the IL-7 protein is administered once every 70 days. In some embodiments, the IL-7 protein is administered once every 80 days. In some embodiments, the IL-7 protein is administered once every 90 days. In some embodiments, the IL-7 protein is administered once every 100 days.

[0140] In some embodiments of the present disclosure, administering an anti-cancer therapy to a subject (e.g., having a tumor, including an unmethylated MGMT tumor) comprises administering an IL-7 protein in combination with an additional therapeutic agent to the subject. In some embodiments, the additional therapeutic agent comprises chemotherapy. In some embodiments, the chemotherapy comprises TMZ. Non-limiting examples of other additional therapeutic agents that can be used are provided elsewhere in this disclosure.

[0141] In some embodiments, a subject treatable with the present disclosure receives a single dose of the additional therapeutic agent. In some embodiments, the additional therapeutic agent is administered to the subject in multiple doses (i.e., repeated administrations). In some embodiments, the additional therapeutic agent (e.g., chemotherapy, e.g., TMZ) is administered to the subject at least about two times, at least about three times, at least about four times, at least about five times, at least about six times, at least about seven times, at least about eight times, at least about nine times, or at least about ten times or more. In some embodiments, the additional therapeutic agent (e.g., chemotherapy, e.g., TMZ) is administered to the subject about two times. In some embodiments, the additional therapeutic agent (e.g., chemotherapy, e.g., TMZ) is administered to the subject about three times. In some embodiments, the additional therapeutic agent (e.g., chemotherapy, e.g., TMZ) is administered to the subject about four times. In some embodiments, the additional therapeutic agent (e.g., chemotherapy, e.g., TMZ) is administered to the subject about five times. In some embodiments, the additional therapeutic agent (e.g., chemotherapy, e.g., TMZ) is administered to the subject about 6 times. In some embodiments, the additional therapeutic agent (e.g., chemotherapy, e.g., TMZ) is administered to the subject about 7 times. In some embodiments, the additional therapeutic agent (e.g., chemotherapy, e.g., TMZ) is administered to the subject about 8 times. In some embodiments, the additional therapeutic agent (e.g., chemotherapy, e.g., TMZ) is administered to the subject about 9 times. In some embodiments, the additional therapeutic agent (e.g., chemotherapy, e.g., TMZ) is administered to the subject about 10 or more times.

[0142] When an additional therapeutic agent is administered multiple times to a subject (e.g., having a tumor containing an unmethylated MGMT promoter), in some embodiments, the additional therapeutic agent is administered, e.g., at one of the doses described above, at a dosing frequency of about once per week, about once per 2 weeks, about once per 3 weeks, about once per 4 weeks, about once per 5 weeks, about once per 6 weeks, about once per 7 weeks, about once per 8 weeks, about once per 9 weeks, about once per 10 weeks, about once per 11 weeks, or about once per 12 weeks. In some embodiments, the additional therapeutic agent is administered once per 3 weeks. In some embodiments, the additional therapeutic agent is administered once per week. In some embodiments, the additional therapeutic agent is administered once per 2 weeks. In some embodiments, the additional therapeutic agent is administered once per 4 weeks. In some embodiments, the additional therapeutic agent is administered once per 6 weeks. In some embodiments, the additional therapeutic agent is administered once per 8 weeks. In some embodiments, the additional therapeutic agent is administered once per 9 weeks. In some embodiments, the additional therapeutic agent is administered once every 12 weeks.

[0143] In some embodiments, a subject (e.g., having a tumor containing an unmethylated MGMT promoter) receives multiple administrations of IL-7 protein and multiple administrations of an additional therapeutic agent (e.g., chemotherapy, e.g., TMZ). In some embodiments, the subject is administered (i) at least about 2 doses, at least about 3 doses, at least about 4 doses, or at least about 5 doses of IL-7 protein and (ii) at least about 2 doses, at least about 3 doses, at least about 4 doses, at least about 5 doses, at least about 6 doses, or at least about 7 doses of an additional agent. In some embodiments, the subject is administered at least about 4 doses of IL-7 protein and at least about 6 doses of an additional agent. In some embodiments, the IL-7 protein is administered to the subject about once every 12 weeks, and the additional agent is administered to the subject about once every 4 weeks. In some embodiments, the subject is administered about 4 doses of IL-7 protein and about 6 doses of the additional agent, wherein the IL-7 protein is administered to the subject about once every 12 weeks and the additional agent is administered to the subject about once every 4 weeks.

[0144] In some embodiments, one or more doses of an additional agent (e.g., chemotherapy, e.g., TMZ) are administered to the subject over about 1 day, over about 2 consecutive days, over about 3 consecutive days, over about 4 consecutive days, over about 5 consecutive days, over about 6 consecutive days, or over about 7 consecutive days. In some embodiments, one or more doses of an additional agent are administered to the subject over about 1 day. In some embodiments, one or more doses of an additional agent are administered to the subject over about 2 consecutive days. In some embodiments, one or more doses of an additional agent are administered to the subject over about 3 consecutive days. In some embodiments, one or more doses of an additional agent are administered to the subject over about 4 consecutive days. In some embodiments, one or more doses of an additional agent are administered to the subject over about 5 consecutive days. In some embodiments, one or more doses of an additional agent are administered to the subject over about 6 consecutive days. In some embodiments, one or more doses of an additional agent are administered to the subject over about 7 consecutive days. In some embodiments, the additional therapeutic agent is administered to the subject on days 1-5 of a 28-day cycle.

[0145] 5. IL-7 Proteins Useful in the Present Disclosure Disclosed herein are IL-7 proteins that can be used to treat tumors described herein (e.g., containing an unmethylated MGMT promoter). In some embodiments, the IL-7 proteins useful herein may be wild-type IL-7 or modified IL-7 (i.e., not a wild-type IL-7 protein) (e.g., an IL-7 variant, a functional fragment of IL-7, an IL-7 derivative, or any combination thereof, e.g., a fusion protein, a chimeric protein, etc.), so long as they have one or more biological activities of IL-7, such as, for example, being capable of binding to IL-7R and being capable of, for example, inducing early T cell development and promoting T cell homeostasis (see ElKassar and Gress. J Immunotoxicol. 2010 Mar;7(1):1-7). In some embodiments, the IL-7 proteins of the present disclosure are not wild-type IL-7 proteins (i.e., contain one or more modifications). Non-limiting examples of such modifications may include oligopeptides and / or half-life extending moieties (see, e.g., WO2016 / 200219, which is incorporated herein by reference in its entirety).

[0146] IL-7 binds to its receptor, which consists of two chains: IL-7Rα (CD127), which is shared with thymic stromal lymphopoietin (TSLP) (Ziegler and Liu, 2006), and the γ chain (CD132), which is shared with IL-2, IL-15, IL-9, and IL-21. While γc is expressed on most hematopoietic cells, IL-7Rα is expressed almost exclusively on lymphoid cells. After binding to its receptor, IL-7 transduces signals via two distinct pathways: Jak-Stat (Janus kinase-signal transducer and activator of transcription) and PI3K / Akt, which are responsible for differentiation and survival, respectively. The absence of IL-7 signaling is responsible for the reduced thymic cellularity observed in mice treated with anti-IL-7 neutralizing monoclonal antibodies (MAbs; 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). In the absence of IL-7 signaling, mice lack T cells, B cells, and NK-T cells. IL-7α- / - mice (Peschon et al., 1994) have a similar but more severe phenotype to IL-7- / - mice (von Freeden-Jeffry et al., 1995), likely because TSLP signaling is also abrogated in IL-7α- / - mice. IL-7 is required for the development of γδ cells (Maki et al., 1996) and NK-T cells (Boesteanu et al., 1997).

[0147] In some embodiments, an IL-7 protein useful in the present disclosure comprises the amino acid sequence set forth in any one of SEQ ID NOS: 1-6 and 80-85. In some embodiments, an IL-7 protein comprises an amino acid sequence having about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% or more sequence identity to the sequence of SEQ ID NOS: 1-6. In some embodiments, an IL-7 protein that can be used with the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 80 (i.e., the amino acid sequence set forth in SEQ ID NO: 1, but without the signal peptide). In some embodiments, an IL-7 protein that can be used with the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 81 (i.e., the amino acid sequence set forth in SEQ ID NO: 2, but without the signal peptide). In some embodiments, an IL-7 protein that can be used with the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 82 (i.e., the amino acid sequence set forth in SEQ ID NO: 3, but without the signal peptide). In some embodiments, an IL-7 protein that can be used with the present disclosure comprises the amino acid sequence set forth in SEQ ID NO:83 (i.e., the amino acid sequence set forth in SEQ ID NO:4, but without the signal peptide). In some embodiments, an IL-7 protein that can be used with the present disclosure comprises the amino acid sequence set forth in SEQ ID NO:84 (i.e., the amino acid sequence set forth in SEQ ID NO:5, but without the signal peptide). In some embodiments, an IL-7 protein that can be used with the present disclosure comprises the amino acid sequence set forth in SEQ ID NO:85 (i.e., the amino acid sequence set forth in SEQ ID NO:6, but without the signal peptide).

[0148] In some embodiments, the IL-7 protein comprises a modified IL-7 or a fragment thereof, wherein the modified IL-7 or fragment retains one or more biological activities of wild-type IL-7. In some embodiments, the IL-7 protein may be derived from a human, rat, mouse, monkey, cow, or sheep.

[0149] In some embodiments, human IL-7 may have the amino acid sequence represented by SEQ ID NO: 1 (Genbank Accession No. P13232), rat IL-7 may have the amino acid sequence represented by SEQ ID NO: 2 (Genbank Accession No. P56478), mouse IL-7 may have the amino acid sequence represented by SEQ ID NO: 3 (Genbank Accession No. P10168), monkey IL-7 may have the amino acid sequence represented by SEQ ID NO: 4 (Genbank Accession No. NP001279008), bovine IL-7 may have the amino acid sequence represented by SEQ ID NO: 5 (Genbank Accession No. P26895), and ovine IL-7 may have the amino acid sequence represented by SEQ ID NO: 6 (Genbank Accession No. Q28540).

[0150] In some embodiments, IL-7 proteins useful in the present disclosure include IL-7 fusion proteins. In some embodiments, the IL-7 fusion protein comprises (i) an oligopeptide and (i) IL-7 or a variant thereof. In some embodiments, the oligopeptide is linked to the N-terminal region of IL-7 or a variant thereof.

[0151] In some embodiments, the oligopeptides disclosed herein consist of 1 to 10 amino acids. In some embodiments, the oligopeptides consist 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, methionine-methionine, glycine-glycine, methionine-glycine, glycine-methionine, methionine-methionine-methionine, methionine-methionine-glycine, methionine-glycine-methionine, glycine-methionine-methionine, methionine-glycine-glycine, glycine-methionine-glycine, glycine-glycine-methionine, and glycine-glycine-glycine. In some embodiments, the oligopeptide is methionine-glycine-methionine.

[0152] In some embodiments, the IL-7 fusion protein comprises (i) IL-7 or a variant thereof and (ii) a half-life extending moiety. In some embodiments, the half-life extending moiety extends the half-life of IL-7 or its variant. In some embodiments, the half-life extending moiety is linked to the C-terminal region of IL-7 or its variant.

[0153] In some embodiments, the IL-7 fusion protein comprises (i) IL-7 (a first domain), (ii) a second domain comprising an amino acid sequence of 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 extending moiety. In some embodiments, the half-life extending moiety may be linked to the N-terminus or C-terminus of the first domain or the second domain. Furthermore, the IL-7 comprising the first domain and the second domain may be linked to both termini of the third domain.

[0154] Non-limiting examples of half-life extending moieties include Fc, albumin, albumin-binding polypeptide, Pro / Ala / Ser (PAS), C-terminal peptide of the beta subunit of human chorionic gonadotropin (CTP), polyethylene glycol (PEG), long unstructured hydrophilic amino acid sequences (XTEN), hydroxyethyl starch (HES), albumin-binding small molecules, and combinations thereof.

[0155] In some embodiments, the half-life extending moiety is an Fc. In some embodiments, the Fc is derived from an engineered immunoglobulin with attenuated antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) due to altered binding affinity with Fc receptors and / or complement. In some embodiments, the engineered 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") comprising a hinge region, a CH2 domain, and a CH3 domain. In some embodiments, the hinge region of a hybrid Fc disclosed herein comprises a human IgD hinge region. In some embodiments, the CH2 domain of the hybrid Fc comprises a portion of a human IgD CH2 domain and a portion of a human IgG4 CH2 domain. In some embodiments, the CH3 domain of the hybrid Fc comprises a portion of a human IgG4 CH3 domain. Thus, in some aspects, the hybrid Fc disclosed herein comprises 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 a human IgD CH2 domain and a portion of a human IgG4 CH2 domain, and the CH3 domain comprises a portion of a human IgG4 CH3 domain.

[0156] In some embodiments, the Fc disclosed herein may be an Fc variant. As used herein, the term "Fc variant" refers to an Fc prepared by substituting a portion of amino acids in the Fc region or by combining different types of Fc regions. The Fc region variant can prevent cleavage at the hinge region. Specifically, in some embodiments, the Fc variant comprises a modification of amino acid 144 and / or amino acid 145 of SEQ ID NO: 9. In some embodiments, amino acid 144 (K) and / or amino acid 145 (K) are substituted with G or S.

[0157] In some embodiments, the Fc or Fc variant disclosed herein can be represented by the formula: N'-(Z1)pY-Z2-Z3-Z4-C, wherein: N' includes the N-terminus, Z1 comprises an amino acid sequence having 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 comprises an amino acid sequence having 5 to 64 consecutive amino acid residues from the amino acid residue at position 162 toward the N-terminus among the amino acid residues at positions 99 to 162 of SEQ ID NO: 7, Z2 comprises an amino acid sequence having 4 to 37 consecutive amino acid residues from the amino acid residue at position 163 toward the C-terminus among the amino acid residues at positions 163 to 199 of SEQ ID NO: 7, Z3 comprises an amino acid sequence having 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 comprises an amino acid sequence having 80 to 107 consecutive amino acid residues from the amino acid residue at position 221 of amino acid residues 221 to 327 of SEQ ID NO:8 toward the C-terminus.

[0158] In some embodiments, the Fc region disclosed herein may comprise 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 comprise the amino acid sequence of SEQ ID NO:14 (non-lytic mouse Fc). Non-limiting examples of Fc sequences are shown in Table 2 (below).

[0159] Other non-limiting examples of Fc regions that can be used in the present disclosure are described in US Pat. No. 7,867,491, which is incorporated by reference herein in its entirety.

[0160] In some embodiments, the IL-7 fusion proteins disclosed herein comprise both an oligopeptide and a half-life extending moiety.

[0161] In some embodiments, the IL-7 protein may be fused to albumin, a variant, or a fragment thereof. Examples of IL-7-albumin fusion proteins can be found in International Application Publication No. WO2011 / 124718A1, the entire contents of which are incorporated herein by reference. In some embodiments, the IL-7 protein is fused to prepro-B cell growth stimulating factor (PPBSF), optionally via a flexible linker. See, for example, US 2002 / 0058791A1, the entire contents of which are incorporated herein by reference. In some embodiments, the IL-7 protein useful in the present disclosure is a conformer of IL-7 having a specific three-dimensional structure. See, for example, US 2005 / 0249701A1, the entire contents of which are incorporated herein by reference. In some embodiments, the IL-7 protein may be fused to an Ig chain, and amino acid residues 70 and 91 of the IL-7 protein are glycosylated, while amino acid residue 116 of the IL-7 protein is not glycosylated. See, e.g., US 7,323,549 / B2, incorporated herein by reference in its entirety. In some embodiments, IL-7 proteins that do not contain potential T cell epitopes (thus reducing anti-IL-7 T cell responses) can also be used in the present disclosure. See, e.g., US 2006 / 0141581 A1, incorporated herein by reference in its entirety. In some embodiments, IL-7 proteins with one or more amino acid residue mutations in the carboxy-terminal helix D region can be used in the present disclosure. IL-7 mutants can act as IL-7R partial agonists despite having lower binding affinity for the receptor. See, e.g., US 2005 / 0054054 A1, incorporated herein by reference in its entirety. Any IL-7 proteins described in the above patents or publications are incorporated herein by reference in their entirety.

[0162] Further, non-limiting examples of additional IL-7 proteins useful in the present disclosure include those described in US7708985, US8034327, US8153114, US7589179, US7323549, US7960514, US8338575, US7118754, US7488482, US7670607, US6730512, WO0017362, GB2434578A, WO2010 / 020766A2, WO91 / 01143, Beqet al., Blood, vol.114(4), 816, 23 July 2009, Kang et al., J. Virol. Doi:10.1128 / JVI.02768-15, Martin et al. 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.

[0163] In some embodiments, the oligopeptide disclosed herein is directly linked to the N-terminal region of IL-7 or a variant thereof. In some embodiments, the oligopeptide is linked to the N-terminal region via a linker. In some embodiments, the half-life extending moiety disclosed herein is directly linked to the C-terminal region of IL-7 or a variant thereof. In some embodiments, the half-life extending moiety is linked to the C-terminal region via a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker comprises 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 some embodiments, the chemical bond comprises a disulfide bond, a diamine bond, a sulfide-amine bond, a carboxy-amine bond, an ester bond, a covalent bond, or a combination thereof. When the linker is a peptide linker, in some embodiments, the connection can occur at any linkage region. These can be conjugated using cross-linking agents well known in the art. In some embodiments, exemplary cross-linking agents can include, but are not limited to, N-hydroxysuccinimide esters such as 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, and 4-azidosalicylic acid; imidoesters including disuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate); and bifunctional maleimides such as bis-N-maleimide-1,8-octane.

[0164] In some embodiments, the IL-7 (or variant thereof) portion of an IL-7 fusion protein disclosed herein comprises an amino acid sequence 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 sequence set forth in SEQ ID NOs: 15-20. In some embodiments, the IL-7 (or variant thereof) portion of an IL-7 fusion protein disclosed herein comprises the amino acid sequence set forth in SEQ ID NOs: 15-20.

[0165] In some embodiments, the IL-7 fusion protein comprises a first domain comprising a polypeptide having an activity of IL-7 or an activity similar thereto; a second domain comprising 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 an Fc region of a modified immunoglobulin linked to the C-terminus of the first domain.

[0166] In some embodiments, IL-7 fusion proteins that can be used in the methods comprise an amino acid sequence 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 sequence set forth in SEQ ID NOs: 21-25. In some embodiments, IL-7 fusion proteins of the present disclosure comprise the amino acid sequence set forth in SEQ ID NOs: 21-25. In some embodiments, IL-7 fusion proteins disclosed herein comprise the amino acid sequence set forth in SEQ ID NOs: 26 and 27. Sequences of exemplary IL-7 proteins / fusion proteins are shown in Table 2 (below).

[0167] Unless otherwise specified, the IL-7 proteins (including fusion proteins) described herein can be administered to a subject (e.g., in combination with an additional therapeutic agent, e.g., chemotherapy, e.g., TMZ) using any suitable route of administration. In some embodiments, the IL-7 protein is administered to a subject intravenously, parenterally, intramuscularly, subcutaneously, intraocularly, intraperitoneally, intradermally, intraorbitally, intracerebrally, intracranially, intraspinally, intraventricularly, intrathecally, intracisternally, intracisternally, or intratumorally.

[0168] III. Nucleic Acids, Vectors, and Host Cells Some embodiments described herein relate to one or more nucleic acid molecules encoding a therapeutic agent described herein (e.g., an IL-7 protein and / or an additional therapeutic agent). The nucleic acid may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is "isolated" or "substantially pure" when purified from other cellular components or other contaminants, e.g., other cellular nucleic acids (e.g., other chromosomal DNA, e.g., chromosomal DNA linked to naturally isolated DNA) or proteins, by standard techniques, including alkaline / SDS treatment, CsCl banding, column chromatography, restriction enzymes, agarose gel electrophoresis, and others 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 intronic sequences. In some embodiments, the nucleic acid is a cDNA molecule. The nucleic acids described herein can be obtained using standard molecular biology techniques well known in the art.

[0169] Some nucleic acid molecules disclosed herein encode IL-7 proteins (e.g., as disclosed herein) that can be used alone or in combination with additional therapeutic agents to treat tumors, including, for example, unmethylated MGMT tumors. Exemplary nucleic acid sequences encoding the IL-7 proteins disclosed herein are set forth in SEQ ID NOs: 29-39 (see, e.g., Table 2 below).

[0170] In some aspects, the present disclosure provides vectors comprising an isolated nucleic acid molecule encoding a therapeutic agent (e.g., an IL-7 protein and / or an additional therapeutic agent) disclosed herein. In some aspects, the vectors can be used in gene therapy.

[0171] When used as a gene therapy (e.g., in humans), nucleic acids encoding a therapeutic agent (e.g., an IL-7 protein and / or an additional therapeutic agent) disclosed herein can be administered at a dosage ranging from 0.1 mg to 200 mg. In some embodiments, the dosage ranges from 0.6 mg to 100 mg. In some embodiments, the dosage ranges from 1.2 mg to 50 mg.

[0172] Suitable vectors for the present disclosure include expression vectors, viral vectors, and plasmid vectors. In some embodiments, the vector is a viral vector.

[0173] As used herein, an expression vector refers to any nucleic acid construct that, upon introduction into an appropriate host cell, contains the necessary elements for the transcription and translation of an inserted coding sequence, or, in the case of RNA viral vectors, the elements necessary for replication and translation. Expression vectors can include plasmids, phagemids, viruses, and their derivatives.

[0174] As used herein, viral vectors include, but are not limited to, nucleic acid sequences derived from retroviruses, such as Moloney murine leukemia virus, Harvey murine sarcoma virus, mouse mammary tumor virus, and Rous sarcoma virus, lentiviruses, adenoviruses, adeno-associated viruses, SV40 viruses, polyomaviruses, Epstein-Barr viruses, papillomaviruses, herpesviruses, vaccinia viruses, and polioviruses, as well as RNA viruses, such as retroviruses. Those skilled in the art can readily use other vectors known to those skilled in the art. Some viral vectors are based on non-cytopathic eukaryotic viruses in which nonessential genes have been replaced with a gene of interest. Non-cytopathic viruses include retroviruses, whose life cycle involves reverse transcription of genomic viral RNA into DNA followed by proviral integration into host cell DNA.

[0175] In some embodiments, the vector is derived from an adeno-associated virus. In some embodiments, the vector is derived from a lentivirus. Examples of lentiviral vectors are disclosed in WO9931251, WO9712622, WO9817815, WO9817816, and WO9818934, each of which is incorporated herein by reference in its entirety.

[0176] Other vectors include plasmid vectors. Plasmid vectors have been widely described 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 the past few years, plasmid vectors have been found to be particularly advantageous for delivering genes to cells in vivo because they cannot replicate or integrate into the host genome. However, these plasmids have promoters compatible with the host cell and can express peptides from genes functionally encoded within the plasmid. Some commonly used plasmids available from commercial sources include pBR322, pUC18, pUC19, various pcDNA plasmids, pRC / CMV, various pCMV plasmids, pSV40, and pBlueScript. Further examples of specific plasmids include pcDNA3.1 (Catalog No. V79020), pcDNA3.1 / hygro (Catalog No. V87020), pcDNA4 / myc-His (Catalog No. V86320), and pBudCE4.1 (Catalog No. V53220), all commercially available from Invitrogen (Carlsbad, Calif.). Other plasmids will be known to those of skill in the art. Additionally, plasmids can be custom designed to remove and / or add specific DNA fragments using standard molecular biology techniques.

[0177] The present disclosure also encompasses methods of producing the therapeutic agents (e.g., IL-7 proteins) disclosed herein. In some embodiments, such methods may include expressing the therapeutic agent (e.g., IL-7 proteins) in cells containing a nucleic acid molecule encoding the therapeutic agent (e.g., SEQ ID NOS: 29-39). Further details regarding methods of producing the IL-7 proteins disclosed herein are provided, for example, in U.S. Pat. No. 11,041,007, the entire contents of which are incorporated herein by reference. Host cells comprising these nucleotide sequences are encompassed 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.

[0178] IV. Pharmaceutical Compositions Further provided herein are compositions comprising one or more therapeutic agents (e.g., an IL-7 protein and / or an additional therapeutic agent) at a desired purity and with a physiologically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA).

[0179] Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include buffers, e.g., phosphate, citric acid, and other organic acids, antioxidants including ascorbic acid and methionine, preservatives (e.g., octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl alcohol or benzyl alcohol, alkyl parabens, e.g., methyl paraben or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight (less than about 10 residues) polypeptides, proteins, e.g., For example, 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, including glucose, mannose, or dextrins; 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 non-ionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0180] In some embodiments, the compositions disclosed herein comprise one or more additional components selected from a bulking agent, a stabilizer, a surfactant, a buffer, or a combination thereof.

[0181] Buffers useful in the present disclosure can 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. An appropriate buffer can maximize the stability of the pharmaceutical compositions disclosed herein by maintaining pH control of the composition. A suitable buffer 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 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, sulfate buffer, imidazole buffer, etc. The buffer includes, but is not limited to, Tris buffer, guanidine hydrochloride buffer, phosphate-citrate buffer, borate buffer, malonate buffer, 3-picoline buffer, 2-picoline buffer, 4-picoline buffer, 3,5-lutidine buffer, 3,4-lutidine buffer, 2,4-lutidine buffer, Aces, diethyl malonate buffer, N-methylimidazole buffer, 1,2-dimethylimidazole buffer, TAPS buffer, bis-Tris buffer, L-arginine buffer, lactate buffer, glycolate buffer, or a combination thereof.

[0182] In some embodiments, the compositions disclosed herein further comprise a bulking agent. Bulking agents can be added to pharmaceutical products to add volume and mass to the product, facilitating its accurate measurement and handling. Bulking agents that can be used in the present disclosure include, but are not limited to, sodium chloride (NaCl), mannitol, glycine, alanine, or combinations thereof.

[0183] In some embodiments, the compositions disclosed herein may include a stabilizer. Non-limiting examples of stabilizers that can be used in the present disclosure include sucrose, trehalose, raffinose, arginine, or a combination thereof.

[0184] In some embodiments, the composition disclosed herein comprises a surfactant.In some embodiments, the surfactant can be selected from the following: alkyl ethoxylate, nonylphenol ethoxylate, amine ethoxylate, polyethylene oxide, polypropylene oxide, fatty alcohol such as cetyl alcohol or oleyl alcohol, cocamide MEA, cocamide DEA, polysorbate, dodecyl dimethylamine oxide, or a combination thereof.In some embodiments, the surfactant is polysorbate 20 or polysorbate 80.

[0185] In some embodiments, a composition comprising an IL-7 protein can be formulated using the same formulation as an additional therapeutic agent (e.g., used in combination with the IL-7 protein). In some embodiments, the IL-7 protein and the additional therapeutic agent are formulated using different formulations.

[0186] The pharmaceutical composition can be formulated for any route of administration to a subject. Specific examples of administration routes include intramuscular, subcutaneous, ocular, intravenous, intraperitoneal, intradermal, intraorbital, intracranial, intraspinal, intraventricular, intrathecal, intracapsular, intraarticular, or intratumoral. Parenteral administration, characterized by subcutaneous, intramuscular, or intravenous injection, is also contemplated herein. Injectables can be prepared in any conventional form, such as liquid solutions or suspensions, solid forms suitable for dissolution or suspension in liquid before injection, or emulsions. Injectables, solutions, and emulsions also contain one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol, or ethanol. In addition, if desired, the administered pharmaceutical composition may also contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, and other such agents, such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, and cyclodextrins.

[0187] Pharmaceutically acceptable carriers used in parenteral preparations include aqueous vehicles, non-aqueous vehicles, antibacterial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating 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. Non-aqueous parenteral vehicles include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, and peanut oil. Antibacterial agents in bacteriostatic or fungistatic concentrates may be added to parenteral preparations packaged in multi-dose containers, including phenols or cresols, mercury, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride, and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. Buffers include phosphate and citrate. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcellulose, hydroxypropyl methylcellulose, and polyvinylpyrrolidone. Emulsifying agents include polysorbate 80 (TWEEN® 80). Sequestering or chelating agents for metal ions include EDTA. Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol, and propylene glycol for water-miscible vehicles; and sodium hydroxide, hydrochloric acid, citric acid, or lactic acid for pH adjustment.

[0188] Preparations for parenteral administration include sterile solutions ready for injection, sterile dry soluble products such as lyophilized powders ready to be combined with a solvent immediately before use, including hypodermic tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle immediately before use, and sterile emulsions. Solutions can be either aqueous or non-aqueous.

[0189] If administered intravenously, suitable carriers include saline or phosphate buffered saline (PBS), as well as solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, and polypropylene glycol, and mixtures thereof.

[0190] Topical mixtures containing antibodies are prepared as described for local and systemic administration. The resulting mixture may be a solution, suspension, emulsion, etc., and may be formulated as a cream, gel, ointment, emulsion, solution, elixir, lotion, suspension, tincture, paste, foam, aerosol, irrigant, spray, suppository, bandage, skin patch, or any other formulation suitable for topical administration.

[0191] The antibodies or antigen-binding portions thereof described herein can be formulated as aerosols for topical application, such as by inhalation (see, e.g., U.S. Pat. Nos. 4,044,126, 4,414,209, and 4,364,923, which describe aerosols for administering steroids useful in the treatment of inflammatory diseases, particularly asthma). These formulations for administration to the respiratory tract can be in the form of an aerosol or solution for nebulization, or as a microfine powder for insufflation, either alone or in combination with an inert carrier such as lactose. In such cases, the particles of the formulation have diameters of less than 50 microns in one embodiment, and less than 10 microns in one embodiment.

[0192] The therapeutic agents disclosed herein can be formulated for topical or local application, e.g., topical application to the skin and mucous membranes, e.g., the eyes, in the form of gels, creams, and lotions, and for ophthalmic application, or for intravesical or intrathecal application. Topical administration is contemplated for transdermal delivery, as well as for administration to the eye or mucous membranes, or for inhalation therapy. Nasal solutions of antibodies alone or in combination with other pharmaceutically acceptable excipients can also be administered.

[0193] Transdermal patches, including iontophoretic and electrophoretic devices, are well known to those of skill in the art and can be used to administer antibodies. For example, such patches are disclosed in U.S. Patent Nos. 6,267,983, 6,261,595, 6,256,533, 6,167,301, 6,024,975, 6,010715, 5,985,317, 5,983,134, 5,948,433, and 5,860,957, each of which is incorporated herein by reference in its entirety.

[0194] In some aspects, pharmaceutical compositions containing the therapeutic agents described herein are lyophilized powders that can be reconstituted for administration as solutions, emulsions, and other mixtures. Pharmaceutical compositions can also be reconstituted and formulated as solids or gels. Lyophilized powders are prepared by dissolving an antibody or antigen-binding portion thereof, or a pharmaceutically acceptable derivative thereof, described herein in a suitable solvent. In some aspects, the lyophilized powder is sterilized. The solvent may contain excipients to improve stability or other pharmacological components of the powder, or a reconstituted solution prepared from the powder. Excipients that can be used 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, such as citric acid, sodium phosphate, or potassium phosphate, or other such buffers known to those of skill in the art, in one embodiment at about neutral pH. The solution is then sterile filtered under standard conditions known to those of skill in the art, followed by lyophilization, to yield the desired formulation. In some aspects, the resulting solution can be dispensed into vials for lyophilization. Each vial may contain a single dose or multiple doses of the compound. The lyophilized powder may be stored under appropriate conditions, such as at room temperature at about 4°C.

[0195] Reconstitution of this lyophilized powder with water for injection provides a formulation for use in parenteral administration. When reconstituted, the lyophilized powder is added to sterile water or other suitable carrier. The exact amount depends on the compound selected. Such amounts can be determined empirically.

[0196] The compositions provided herein can also be formulated to target specific tissues, receptors, or other areas of the body of the subject being treated.Many such targeting methods are well known to those skilled in the art.It is contemplated herein that all such targeting methods can be used with the compositions of the present invention.Non-limiting examples of targeting methods are described in, for example, U.S. Patent Nos. 6,316,652, 6,274,552, 6,271,359, 6,253,872, 6,139,865, 6,131,570, 6,120,751, 6,071,495, 6,071,576, and 6,071,576. See Nos. 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.

[0197] Compositions to be used for in vivo administration can be sterilized, which is readily accomplished, for example, by filtration through sterile filtration membranes.

[0198] The following examples are illustrative only and should not be construed as limiting the scope of the present disclosure in any way, as many variations and equivalents will become apparent to those skilled in the art upon reading this disclosure. [Example]

[0199] Example 1. Effect of IL-7 protein administration on the treatment of tumors with an unmethylated MGMT promoter To demonstrate the efficacy of the anticancer therapy provided herein in treating tumors containing an unmethylated MGMT promoter, a phase I clinical trial was conducted in patients with high-grade gliomas and anaplastic astrocytomas, including GBM. The primary inclusion criteria were absolute lymphocyte count ≥ 600 cells / mm 3 Additional inclusion and exclusion criteria are described elsewhere in this disclosure. A total of 19 patients were enrolled: median age = 58.0 years [25-78], median baseline ALC = 1000 cells / mm 3 [400-2,000], median baseline dexamethasone use = 0 mg / day [0-12]. 63% of enrolled patients had unmethylated MGMT promoter ("unmethylated GBM").

[0200] The overall experimental design is shown in Figure 1. As shown in the figure, patients were administered an initial dose of the IL-7 protein (long-acting) described herein within 2 weeks (i.e., week 1) after completion of concurrent radiation therapy (RT) and TMZ treatment. Additional doses of IL-7 protein were administered every 12 weeks (i.e., weeks 13, 25, and 37) for a total of four doses. IL-7 protein was administered to patients at escalating doses (60 μg / kg, 120 μg / kg, 240 μg / kg, 360 μg / kg, 540 μg / kg, 720 μg / kg, and 960 μg / kg) to determine the maximum tolerated dose (MTD). TMZ adjuvant was administered to patients for 5 consecutive days every 28 days for a total of six doses (i.e., weeks 4, 8, 12, 16, 20, and 24).

[0201] In general, the administered IL-7 protein was well tolerated, with grade 1 / 2 injection site reactions (42%) being the most common treatment-related adverse events (TRAEs). Due to two dose-limiting toxicities at 960 mcg / kg (grade 3 alanine aminotransferase elevation and grade 3 back pain), the MTD was 720 μg / kg. A dose-dependent increase in ALC was observed at 4 weeks (1.3-4.1-fold from baseline) and persisted through 12 weeks. As shown in Table 1, the median progression-free survival (mPFS) and median overall survival (mOS) for patients with methylated GBM were 19.1 months (95% CI: 15.1-NA) and 22 months (95% CI: 20.7-NA), respectively. In patients with unmethylated GBM, mPFS and mOS were 11.2 months (95% CI: 5.4-22.4) and 16 months (95% CI: 12.3-24.3), respectively. In patients with unmethylated GBM treated with radiation therapy with concurrent and adjuvant TMZ, mPFS and mOS were 4.99 months (95% CI: 4.25-5.72) and 14.11 months (95% CI: 13.18-15.04), respectively (Alnahhas et al., Neurooncol Adv 2(1): vdaa082 (Jan.-Dec. 2020)). [Table 1]

[0202] The above results demonstrate the therapeutic potential of the IL-7 proteins described herein. Specifically, the above results demonstrate that the combination of SOC with an IL-7 protein (such as those described herein) can significantly improve anti-tumor responses and improve survival rates.

[0203] Example 2. Effect of IL-7 protein administration on absolute lymphocyte counts To further demonstrate the therapeutic efficacy of the anti-cancer treatment provided herein (e.g., the IL-7 protein described herein alone or in combination with a TMZ adjuvant; see, e.g., Example 1), a randomized Phase II clinical trial was conducted in patients with high-grade gliomas, including GBM and anaplastic astrocytoma. The primary objective of this study was to evaluate whether administration of the IL-7 protein (long-acting) described herein results in an increase in absolute lymphocyte count (ALC) compared to placebo control. The effect on ALC was assessed 4 weeks after IL-7 protein administration. The general study design was similar to that shown in Figure 1. Some patients received steroid treatment (e.g., to treat cerebral edema) prior to the anti-cancer treatment described herein (long-acting IL-7 + TMZ). Thus, the different treatment groups were: (a) placebo plus TMZ adjuvant alone (i.e., no steroids), (b) placebo plus TMZ adjuvant followed by steroids, (c) long-acting IL-7 protein plus TMZ alone (i.e., no steroids), or (d) long-acting IL-7 protein plus TMZ followed by steroids.

[0204] As shown in Figures 2A and 2B, there was no significant difference in ALC between baseline and week 4 in the placebo control group. In contrast, a significant increase in ALC was observed in patients after IL-7 protein administration compared to baseline. Furthermore, as shown in Figures 3A and 3B, the positive effect on ALC was observed regardless of whether IL-7 protein was administered after or without steroids. Steroids are known to cause lymphopenia. These data indicate that the therapeutic effect of IL-7 protein administration is observed even under lymphopenic conditions (e.g., those induced by steroid administration). Furthermore, the greatest effect on ALC was observed in patients with an unmethylated MGMT promoter (see Figures 4A and 4B).

[0205] The above results further support the therapeutic efficacy of the IL-7 proteins described herein, and demonstrate that such proteins (e.g., in combination with SOC) inhibit the proliferation of unmethylated O 6 This suggests that this compound may be useful in treating tumors with the -methylguanine-DNA methyltransferase (MGMT) promoter.

[0206] Table 2 (below) provides exemplary sequences relevant to this application. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10] [Table 2-11] [Table 2-12] [Table 2-13] Table 2-14 Table 2-15 Table 2-16 Table 2-17 Table 2-18

Claims

1. 1. A method of treating a tumor in a subject in need thereof, comprising administering to said subject an anti-cancer treatment, wherein a tumor sample obtained from said subject contains unmethylated O ６ -methylguanine-DNA methyltransferase (MGMT) promoter and the anti-cancer treatment comprises interleukin-7 (IL-7) protein.

2. 10. The method of claim 1, further comprising, prior to said administering, determining the methylation status of the MGMT promoter in said tumor sample obtained from said subject.

3. 1. A method for identifying a subject suitable for anti-cancer treatment, comprising: ６ determining the methylation status of a methylguanine-DNA methyltransferase (MGMT) promoter, wherein if the MGMT promoter is unmethylated, the subject is suitable for the anti-cancer treatment, and the anti-cancer treatment comprises an interleukin-7 (IL-7) protein.

4. 4. The method of claim 3, further comprising administering the anti-cancer treatment to the subject identified as suitable for the anti-cancer treatment.

5. 1. A method of increasing an anti-tumor immune response in a subject in need thereof, comprising administering to said subject an anti-cancer treatment, wherein said subject has unmethylated O ６ -methylguanine-DNA methyltransferase (MGMT) promoter-containing tumors, and the anti-cancer treatment comprises interleukin-7 (IL-7) protein.

6. 6. The method of claim 5, comprising, prior to said administering, determining the methylation status of the MGMT promoter in a tumor sample obtained from said subject.

7. 7. The method of claim 6, wherein after said administering, the anti-tumor immune response in the subject is increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold compared to a reference subject (e.g., the subject before said administering).

8. 8. The method of any one of claims 1, 2, and 4-7, wherein after said administering, the subject's median progression-free survival (mPFS) is increased.

9. 9. The method of claim 8, wherein the mPFS of the subject is increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold compared to a reference subject (e.g., the subject prior to the administering).

10. 10. The method of claim 8 or 9, wherein after administering, the subject's mPFS is at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or about 12 months.

11. 11. The method of any one of claims 1, 2, and 4-10, wherein after said administering, the subject's median overall survival (mOS) is increased.

12. 12. The method of claim 11, wherein the mOS of the subject is increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold compared to a reference subject (e.g., the subject prior to the administering).

13. 13. The method of claim 11 or 12, wherein after administering, the mOS of the subject is at least about 12 months, at least about 13 months, at least about 14 months, at least about 15 months, at least about 16 months, at least about 17 months, at least about 18 months, at least about 19 months, or at least about 20 months.

14. 14. The method of any one of claims 1, 2, and 4-13, wherein after said administering, the subject's absolute lymphocyte count (ALC) is increased.

15. 15. The method of claim 14, wherein the ALC of the subject is increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold compared to a reference subject (e.g., the subject before the administration).

16. 16. The method of claim 14 or 15, wherein the increase in ALC persists in the subject for at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 11 weeks, at least about 12 weeks, at least about 13 weeks, at least about 14 weeks, or at least about 15 weeks.

17. 17. The method of any one of claims 1, 2, and 4-16, wherein after said administering, the number of tumor infiltrating lymphocytes (TILs) in the tumor of said subject is increased.

18. 18. The method of claim 17, wherein the number of TILs in the tumor of the subject is increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold compared to a reference subject (e.g., the subject before the administration).

19. The method of any one of claims 1 to 18, wherein the anti-cancer treatment comprises an additional therapeutic agent.

20. 20. The method of claim 19, wherein the additional therapeutic agent comprises standard of care (SOC).

21. 21. The method of claim 19 or 20, wherein the additional therapeutic agent comprises radiation therapy (RT), chemotherapy, hormone therapy, immunotherapy, photodynamic therapy, stem cell transplant, or a combination thereof.

22. 22. The method of claim 21, wherein the additional therapeutic agent comprises chemotherapy.

23. 23. The method of claim 21 or 22, wherein the chemotherapy comprises temozolomide (TMZ).

24. 24. The method of any one of claims 21 to 23, wherein the immunotherapy comprises an immune checkpoint inhibitor, an immune checkpoint activator, an adoptive cell therapy, or a combination thereof.

25. 25. The method of claim 24, wherein the immune checkpoint inhibitor comprises a CTLA-4 antagonist (e.g., an anti-CTLA-4 antibody), a PD-1 antagonist (e.g., an anti-PD-1 antibody, an anti-PD-L1 antibody), a TIM-3 antagonist (e.g., an anti-TIM-3 antibody), or a combination thereof.

26. 26. The method of claim 24 or 25, wherein the immune checkpoint activator comprises an OX40 agonist (e.g., an anti-OX40 antibody), a LAG-3 agonist (e.g., an anti-LAG-3 antibody), a 4-1BB (CD137) agonist (e.g., an anti-CD137 antibody), a GITR agonist (e.g., an anti-GITR antibody), or a combination thereof.

27. The method of any one of claims 1 to 26, wherein the tumor comprises a glioma.

28. 28. The method of claim 27, wherein the glioma comprises high-grade glioma (HGG).

29. 29. The method of claim 28, wherein the HGG comprises glioblastoma (GBM), anaplastic astrocytoma, or both.

30. The method of any one of claims 1 to 29, wherein the tumor is newly diagnosed.

31. 31. The method of any one of claims 2 to 4 and claims 6 to 30, wherein the methylation status of the MGMT promoter is determined by methylation-specific PCR (MS-PCR), pyrosequencing, high-resolution melting, microarray (e.g., Infinium Methylation EPIC), immunohistochemistry (IHC), multiplex ligation-dependent probe amplification (MPLA), or a combination thereof.

32. The method of any one of claims 1 to 13, wherein the IL-7 protein is not wild-type IL-7.

33. The method of any one of claims 1 to 32, wherein the IL-7 protein comprises an oligopeptide consisting of 1 to 10 amino acid residues.

34. The oligopeptide may be methionine (M), glycine (G), methionine-methionine (MM), glycine-glycine (GG), methionine-glycine (MG), glycine-methionine (GM), methionine-methionine-methionine (MMM), methionine-methionine-glycine (MMG), methionine-glycine-methionine (MGM), glycine-methionine-methionine (GMM), methionine-glycine-glycine (MGG), glycine-methionine-glycine (GMG), glycine-glycine-methionine (GGM), or glycine-methionine. Lysine-glycine-glycine (GGG), methionine-glycine-glycine-methionine (MGGM) (SEQ ID NO: 41), methionine-methionine-glycine-glycine (MMGG) (SEQ ID NO: 42), glycine-glycine-methionine-methionine (GGMM) (SEQ ID NO: 43), methionine-glycine-methionine-glycine (MGMG) (SEQ ID NO: 44), glycine-methionine-methionine-glycine (GMMG) (SEQ ID NO: 45), glycine-glycine-glycine-methionine (GGGM) (SEQ ID NO: 46), methionine-glycine Lysine-glycine-glycine (MGGG) (SEQ ID NO: 47), glycine-methionine-glycine-glycine (GMGG) (SEQ ID NO: 48), glycine-glycine-methionine-glycine (GGMG) (SEQ ID NO: 49), glycine-glycine-methionine-methionine-methionine (GGMMM) (SEQ ID NO: 50), glycine-glycine-glycine-methionine-methionine (GGGMM) (SEQ ID NO: 51), glycine-glycine-glycine-glycine-methionine (GGGGM) (SEQ ID NO: 52), methionine-glycine-methionine- methionine-methionine (MGMMM) (SEQ ID NO: 53), methionine-glycine-glycine-methionine-methionine (MGGMM) (SEQ ID NO: 54), methionine-glycine-glycine-glycine-methionine (MGGGM) (SEQ ID NO: 55), methionine-methionine-glycine-methionine-methionine (MMGMM) (SEQ ID NO: 56), methionine-methionine-glycine-glycine-methionine (MMGGM) (SEQ ID NO: 57), methionine-methionine-glycine-glycine-glycine (MMGGG) (SEQ ID NO: 58),Methionine-methionine-methionine-glycine-methionine (MMMGM) (SEQ ID NO: 59), methionine-glycine-methionine-glycine-methionine (MGMGM) (SEQ ID NO: 60), glycine-methionine-glycine-methionine-glycine (GMGMMG) (SEQ ID NO: 61), glycine-methionine-methionine-methionine-glycine (GMMMG) (SEQ ID NO: 62), glycine-glycine-methionine-glycine-methionine (GGMGM) (SEQ ID NO: 63), glycine-glycine-methionine-methionine-glycine (GGMMMG) (SEQ ID NO: 64), glycine-methionine-methionine-glycine-methionine (GMMGM) (SEQ ID NO: 65), methionine-glycine -methionine-methionine-glycine (MGMMG) (SEQ ID NO: 66), glycine-methionine-glycine-glycine-methionine (GMGGM) (SEQ ID NO: 67), methionine-methionine-glycine-methionine-glycine (MMGMG) (SEQ ID NO: 68), glycine-methionine-methionine-glycine-glycine (GMMGG) (SEQ ID NO: 69), glycine-methionine-glycine-glycine-glycine (GMGGG) (SEQ ID NO: 70), glycine-glycine-methionine-glycine-glycine (GGMGG) (SEQ ID NO: 71), glycine-glycine-glycine-glycine-glycine (GGGGGG) (SEQ ID NO: 72), or a combination thereof.

35. 35. The method of claim 34, wherein the oligopeptide is methionine-glycine-methionine (MGM).

36. 36. The method of any one of claims 1 to 35, wherein the IL-7 protein comprises a half-life extending moiety.

37. 37. The method of claim 36, wherein the half-life extending moiety comprises Fc, albumin, an albumin-binding polypeptide, Pro / Ala / Ser (PAS), the C-terminal peptide of the beta subunit of human chorionic gonadotropin (CTP), polyethylene glycol (PEG), a long unstructured hydrophilic amino acid sequence (XTEN), hydroxyethyl starch (HES), an albumin-binding small molecule, or a combination thereof.

38. 38. The method of claim 37, wherein the half-life extending moiety is an Fc.

39. the Fc is a hybrid Fc comprising a hinge region, a CH2 domain, and a CH3 domain; the hinge region comprises a human IgD hinge region; the CH2 domain comprises a portion of a human IgD CH2 domain and a portion of a human IgG4 CH2 domain; 39. The method of claim 38, wherein the CH3 domain comprises a portion of a human IgG4 CH3 domain.

40. 40. The method of any one of claims 1 to 39, 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 to SEQ ID NOs: 1-6, 80-85, and 15-27.

41. the IL-7 protein is greater than about 30 μg / kg, greater than about 60 μg / kg, greater than about 90 μg / kg, greater than about 120 μg / kg, greater than about 150 μg / kg, greater than about 180 μg / kg, greater than about 210 μg / kg, greater than about 240 μg / kg, greater than about 270 μg / kg, greater than about 300 μg / kg, greater than about 400 μg / kg, greater than about 500 μg / kg, greater than about 600 μg / kg, greater than about 700 μg / kg, greater than about 800 μg / kg, greater than about 900 μg / kg 41. The method of any one of claims 1, 2, and 4-40, wherein the patient is administered at a dose of greater than about 1,000 μg / kg, greater than about 1,100 μg / kg, greater than about 1,200 μg / kg, greater than about 1,300 μg / kg, greater than about 1,400 μg / kg, greater than about 1,500 μg / kg, greater than about 1,600 μg / kg, greater than about 1,700 μg / kg, greater than about 1,800 μg / kg, greater than about 1,900 μg / kg, or greater than about 2,000 μg / kg.

42. The IL-7 protein is from about 60 μg / kg to about 1,200 μg / kg, from about 120 μg / kg to about 1,200 μg / kg, from about 240 μg / kg to about 1,200 μg / kg, from about 540 μg / kg to about 1,200 μg / kg, from about 610 μg / kg to about 1,200 μg / kg, from about 650 μg / kg to about 1,200 μg / kg, from about 700 μg / kg to about 1,200 μg / kg, or from about 720 μg / kg ~About 1,200μg / kg, About 750μg / kg~About 1,200μg / kg, About 800μg / kg~About 1,200μg / kg, About 850μg / kg~About 1,200μg / kg, About 900μg / kg ~About 1,200μg / kg, About 950μg / kg~About 1,200μg / kg, About 960μg / kg~About 1,200μg / kg, About 1,000μg / kg~About 1,200μg / kg, About 1,050μ g / kg to about 1,200 μg / kg, about 1,100 μg / kg to about 1,200 μg / kg, about 1,200 μg / kg to about 2,000 μg / kg, about 1,300 μg / kg to about 2,000 μg / kg , about 1,500μg / kg to about 2,000μg / kg, about 1,700μg / kg to about 2,000μg / kg, about 610μg / kg to about 1,000μg / kg, about 650μg / kg to about 1,000μ 42. The method of any one of claims 1, 2, and 4-41, wherein the patient is administered at a dose of about 700 μg / kg to about 1,000 μg / kg, about 750 μg / kg to about 1,000 μg / kg, about 800 μg / kg to about 1,000 μg / kg, about 850 μg / kg to about 1,000 μg / kg, about 900 μg / kg to about 1,000 μg / kg, or about 950 μg / kg to about 1,000 μg / kg.

43. The IL-7 protein is from about 60 μg / kg to about 120 μg / kg, from about 120 μg / kg to about 240 μg / kg, from about 240 μg / kg to about 540 μg / kg, from about 540 μg / kg to about 720 μg / kg, from about 720 μg / kg to about 960 μg / kg, from about 700 μg / kg to about 900 μg / kg, from about 750 μg / kg to about 950 μg / kg, from about 70 43. The method of any one of claims 1, 2, and 4-42, wherein the patient is administered at a dose of 0 μg / kg to about 850 μg / kg, about 750 μg / kg to about 850 μg / kg, about 700 μg / kg to about 800 μg / kg, about 800 μg / kg to about 900 μg / kg, about 750 μg / kg to about 850 μg / kg, or about 850 μg / kg to about 950 μg / kg.

44. The IL-7 protein is at a concentration of about 30 μg / kg, about 60 μg / kg, about 90 μg / kg, about 120 μg / kg, about 150 μg / kg, about 180 μg / kg, about 210 μg / kg, about 240 μg / kg, about 270 μg / kg, about 300 μg / kg, about 330 μg / kg, about 360 μg / kg, about 390 μg / kg, about 420 μg / kg, about 450 μg / kg, about 480 μg / kg, about 510 μg / kg, about 540 μg / kg, about 570 μg / kg, about 600 μg / kg, about 630 μg / kg, about 650 μg / kg, about 680 μg / kg, or about 700 μg / kg , about 720 μg / kg, about 740 μg / kg, about 750 μg / kg, about 760 μg / kg, about 780 μg / kg, about 800 μg / kg, about 820μg / kg, about 840μg / kg, about 850μg / kg, about 860μg / kg, about 880μg / kg, about 900μg / kg, about 920 μg / kg, approximately 940 μg / kg, approximately 950 μg / kg, approximately 960 μg / kg, approximately 980 μg / kg, approximately 1,000 μg / kg, approximately 1.0 20μg / kg, about 1,040μg / kg, about 1,060μg / kg, about 1,080μg / kg, about 1,100μg / kg, about 1,120μ g / kg, about 1,140 μg / kg, about 1,160 μg / kg, about 1,180 μg / kg, about 1,200 μg / kg, about 1,220 μg / kg kg, about 1,240 μg / kg, about 1,260 μg / kg, about 1,280 μg / kg, about 1,300 μg / kg, about 1,320 μg / kg, About 1,340 μg / kg, about 1,360 μg / kg, about 1,380 μg / kg, about 1,400 μg / kg, about 1,420 μg / kg, about 1 , 440 μg / kg, about 1,460 μg / kg, about 1,480 μg / kg, about 1,500 μg / kg, about 1,520 μg / kg, about 1,54 0μg / kg, about 1,560μg / kg, about 1,580μg / kg, about 1,600μg / kg, about 1,620μg / kg, about 1,640μ g / kg, about 1,660 μg / kg, about 1,680 μg / kg, about 1,700 μg / kg, about 1,720 μg / kg, about 1,740 μg / k g, about 1,760 μg / kg, about 1,780 μg / kg, about 1,800 μg / kg, about 1,820 μg / kg, about 1,840 μg / kg, About 1,860 μg / kg, about 1,880 μg / kg, about 1,900 μg / kg, about 1,920 μg / kg, about 1,940 μg / kg, about 1,The method according to any one of claims 1, 2, and 4 to 43, wherein the compound is administered at a dose of about 960 μg / kg, about 1,980 μg / kg, or about 2,000 μg / kg.

45. the IL-7 protein is about 0.01 nmol / kg, about 0.02 nmol / kg, about 0.03 nmol / kg, about 0.04 nmol / kg, about 0.05 nmol / kg, about 0.1 nmol / kg, about 0.2 nmol / kg, about 0.4 nmol / kg, about 0.6 nmol / kg, about 0.8 nmol / kg, about 1 nmol / kg, about 1.2 nmol / kg, about 1.4 nmol / kg, about 1.6 nmol / kg, about 1.8 nmol / kg, about 2 nmol / kg, about 2.2 nmol / kg, about 2.4 nmol / kg, about 2.6 nmol / kg, about 2.8 nmol / kg, about 3 nmol / kg, about 3.5 nmol / kg, about 4 nmol / kg, about 4.5 nmol / kg, about 5 nmol / kg, about 45. The method of any one of claims 1, 2, and 4-44, wherein the medicament is administered at a dose of 6 nmol / kg, about 7 nmol / kg, about 8 nmol / kg, about 9 nmol / kg, about 10 nmol / kg, about 11 nmol / kg, about 12 nmol / kg, about 13 nmol / kg, about 14 nmol / kg, about 15 nmol / kg, about 16 nmol / kg, about 17 nmol / kg, about 18 nmol / kg, about 19 nmol / kg, about 20 nmol / kg, about 22 nmol / kg, about 24 nmol / kg, about 26 nmol / kg, about 28 nmol / kg, about 30 nmol / kg, about 32 nmol / kg, about 34 nmol / kg, about 36 nmol / kg, about 38 nmol / kg, or about 40 nmol / kg.

46. 46. ​​The method of any one of claims 1, 2, and 4-45, wherein the IL-7 protein is administered at a dosing frequency of about once per week, about once per 2 weeks, about once per 3 weeks, about once per 4 weeks, about once per 5 weeks, about once per 6 weeks, about once per 7 weeks, about once per 8 weeks, about once per 9 weeks, about once per 10 weeks, about once per 11 weeks, or about once per 12 weeks.

47. 47. The method of claim 46, wherein the IL-7 protein is administered to the subject at a dose of about 720 μg / kg and at a dosing frequency of about once every 12 weeks.

48. 48. The method of any one of claims 19-47, wherein the additional agent is administered to the subject at a dosing frequency of about once per week, about once per two weeks, about once per three weeks, about once per four weeks, or about once per five weeks.

49. 49. The method of any one of claims 19 to 48, wherein the IL-7 protein and the additional therapeutic agent are administered to the subject simultaneously.

50. 49. The method of any one of claims 19 to 48, wherein the IL-7 protein and the additional therapeutic agent are administered to the subject sequentially.

51. 51. The method of claim 50, wherein the additional agent is administered to the subject after the IL-7 protein.

52. 52. The method of any one of claims 1, 2, and 4-51, wherein the subject is administered at least about 2 doses, at least about 3 doses, at least about 4 doses, or at least about 5 doses of the IL-7 protein.

53. 53. The method of any one of claims 1, 2, and 4-52, wherein the subject is administered at least about 2 doses, at least about 3 doses, at least about 4 doses, at least about 5 doses, at least about 6 doses, or at least about 7 doses of the additional therapeutic agent.

54. 54. The method of claim 53, wherein the subject is administered at least about 4 doses of the IL-7 protein and at least about 6 doses of the additional agent.

55. 55. The method of any one of claims 52-54, wherein the IL-7 protein is administered to the subject about once every 12 weeks, and the additional agent is administered to the subject about once every 4 weeks.

56. 56. The method of any one of claims 53-55, wherein one or more doses of the additional agent are administered to the subject for about 1 day, for about 2 consecutive days, for about 3 consecutive days, for about 4 consecutive days, for about 5 consecutive days, for about 6 consecutive days, or for about 7 consecutive days.

57. 57. The method of claim 56, wherein the additional agent is administered to the subject on days 1-5 of a 28-day cycle.

58. 58. The method of any one of claims 1 to 57, wherein the subject has not previously received anti-tumor therapy.

59. The method of any one of claims 1 to 56, wherein the subject has previously undergone anti-tumor therapy.

60. 60. The method of claim 58 or 59, wherein the anti-tumor therapy comprises standard of care (SOC).

61. 61. The method of claim 59 or 60, wherein the anti-tumor therapy comprises radiation therapy (RT), chemotherapy, hormone therapy, immunotherapy, photodynamic therapy, stem cell transplantation, or a combination thereof.

62. 62. The method of claim 61, wherein the anti-tumor therapy comprises both RT and chemotherapy.

63. 63. The method of claim 62, wherein the chemotherapy comprises temozolomide (TMZ).

64. 64. The method of any one of claims 1, 2, and 4-63, wherein the IL-7 protein is administered to the subject intramuscularly, parenterally, subcutaneously, intraocularly, intravenously, intraperitoneally, intradermally, intraorbitally, intracerebrally, intracranially, intraspinally, intraventricularly, intrathecally, intracisternally, intracapsularly, or intratumorally.

65. 65. The method of any one of claims 19-64, wherein the additional agent is administered to the subject intramuscularly, parenterally, subcutaneously, intraocularly, intravenously, intraperitoneally, intradermally, intraorbitally, intracerebrally, intracranially, intraspinally, intraventricularly, intrathecally, intracisternally, intracisternally, or intratumorally.

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