Methods of treating tumors with combination of IL-7 protein and VEGF antagonist

Abstract

The present disclosure relates to methods of treating cancer (or tumor) with an IL-7 protein in combination with a VEGF antagonist. In some aspects, the methods provided herein further comprise administering to the subject an additional therapeutic agent (e.g., an immune checkpoint inhibitor).

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This PCT application claims the benefit of priority to U.S. Provisional Application No. 63 / 295,336, filed on December 30, 2021, and Korean Patent Application No. 10-2022-0000051, filed on January 3, 2022, the entire contents of which are incorporated herein by reference in their entirety.

[0003] Reference to a sequence listing submitted electronically via EFS-WEB

[0004] The contents of the sequence listing submitted electronically in the .XML file (file name 4241_029PC02_SequenceListing_ST26; size: 122,317 bytes; creation date: December 30, 2022) submitted with this application are incorporated herein by reference in their entirety. Technical Field

[0005] The present disclosure generally relates to a combination therapy regimen that can be used to treat a tumor in a subject (e.g., a human subject). Specifically, the treatment regimen provided herein includes a combination of an IL-7 protein and a VEGF antagonist. In some aspects, the treatment regimen also includes additional therapeutic agents, such as immune checkpoint inhibitors. Provided herein is a method for treating a tumor using such a treatment regimen, which includes enhancing an anti-tumor immune response. Background Art

[0006] Human cancers contain many genetic and epigenetic changes that generate new antigens that can be recognized by the immune system. Sjoblom et al., Science 314:268-74 (2006). The adaptive immune system, which includes T lymphocytes and B lymphocytes, has a strong anti-cancer potential, with a wide range of response capabilities and high specificity to a variety of tumor antigens. In addition, the immune system exhibits considerable plasticity and memory elements. Successfully utilizing all of these attributes of the adaptive immune system will make immunotherapy unique among all cancer treatment modalities.

[0007] In recent years, cancer immunotherapy has been well established and has now become one of the more successful treatment options available for many cancer patients. Scott, AM, et al., Cancer Immun 12:14 (2012). In addition to targeting antigens involved in cancer cell proliferation and survival, antibodies can also activate or antagonize immune pathways that are important in cancer immune surveillance. And, after much effort, several immune checkpoint pathway inhibitors have been successfully developed, some of which have been approved by the Food and Drug Administration, such as the anti-CTLA-4 antibody: ipilimumab Anti-PD-1 antibody: nivolumab Pembrolizumab and anti-PD-L1 antibodies: atezolizumab Durvalumab Avelumab

[0008] Despite these advances, the prognosis for patients with certain malignancies (e.g., metastatic or refractory solid tumors) remains poor. Only a portion of these patients actually experience long-term cancer remission, and many patients do not respond to antibodies, or initially respond but eventually become resistant to them. Sharma, P., et al., Cell 168(4):707-723(2017). In addition, many cancer patients have lymphopenia, as many available standard of care cancer treatments (e.g., chemotherapy and radiotherapy) are known to cause lymphopenia. Grossman, SA, et al., J Natl Compr Canc Netw 13(10):1225-31(2015). Checkpoint inhibitors, such as anti-PD-1 antibodies, have been shown to have limited efficacy in such cancer patients. Yarchoan, M., et al., J Clin Oncol 35:e14512(2017). Therefore, for cancer patients, including patients with lymphopenia, new treatment options with acceptable safety profiles and high efficacy are still needed. Summary of the invention

[0009] Provided herein is a method of treating a tumor in a subject in need thereof, comprising administering to the subject a combination of an IL-7 protein and an antagonist of vascular endothelial growth factor ("VEGF") ("VEGF antagonist").

[0010] In some aspects, the tumor volume of the subject is reduced after administration. In some aspects, the tumor volume is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% after administration.

[0011] Also provided herein is a method of enhancing an anti-tumor immune response in a subject in need thereof, comprising administering to the subject a combination of an IL-7 protein and a vascular endothelial growth factor ("VEGF") antagonist ("VEGF antagonist").

[0012] In some aspects, the anti-tumor immune response of the subject increases at least about 1 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 40 times or at least about 50 times after administration. In some aspects, the increased anti-tumor immune response includes: (i) tumor volume is reduced; (ii) the effector activity of tumor-specific T cells is increased; (iii) the number of tumor-specific effector T cells is increased; (iv) the number of tumor-infiltrating lymphocytes (TIL) in the tumor is increased; (v) the number of myeloid-derived suppressor cells (MDSC) in the tumor is reduced; (vi) the number of regulatory T cells (Treg) in the tumor is reduced; (vii) the subject's survival duration is extended; (viii) the expression of TOX on CD8+T cells in tumors, tumor-draining lymph nodes (TDLN) or both is reduced; (ix) the number of stem cell-like T cells in tumors, TDLN or both is increased; or (x) a combination thereof.

[0013] In some aspects, the IL-7 protein and the VEGF antagonist are administered to the subject simultaneously. In some aspects, the IL-7 protein and the VEGF antagonist are administered to the subject sequentially.

[0014] In some aspects, an additional therapeutic agent is administered to the subject. In some aspects, the additional therapeutic agent comprises an immune checkpoint inhibitor, an immune checkpoint activator, a standard of care treatment, or a combination thereof.

[0015] In some aspects, immune checkpoint inhibitors include CTLA-4 antagonists (e.g., anti-CTLA-4 antibodies), PD-1 antagonists (e.g., anti-PD-1 antibodies, anti-PD-L1 antibodies), TIM-3 antagonists (e.g., anti-TIM-3 antibodies) or combinations thereof. In some aspects, immune checkpoint activators include OX40 agonists (e.g., anti-OX40 antibodies), LAG-3 agonists (e.g., anti-LAG-3 antibodies), 4-1BB (CD137) agonists (e.g., anti-CD137 antibodies), GITR agonists (e.g., anti-GITR antibodies) or combinations thereof. In some aspects, standard care treatment includes chemotherapy, radiation, or both.

[0016] In some aspects, the IL-7 protein is not wild-type IL-7.

[0017] In some aspects, the IL-7 protein comprises an oligopeptide consisting of 1 to 10 amino acid residues. In some aspects, the oligopeptide comprises 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). NO:44), glycine-methionine-methionine-glycine (GMMG) (SEQ ID 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), 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-methionine-glycine-methionine (MMMGM) (SEQ ID NO:60), methionine-methionine-glycine-glycine-methionine (MMMGM) (SEQ ID NO:61), methionine-methionine-glycine-glycine-methionine (MMMGM) (SEQ ID NO:62), methionine-methionine-glycine-glycine-methionine (MMMGM) (SEQ ID NO:63), methionine-methionine-glycine-glycine-methionine (MMMGM) (SEQ ID NO:64), methionine-methionine-glycine-methionine (MMMGM) (SEQ ID NO:65), methionine-methionine-glycine-methionine (MMMGM) (SEQ ID NO:66), methionine-methionine-glycine-glycine-methionine (MMMGM) (SEQ ID NO:67), methionine-methionine-glycine-glycineNO: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-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), and methionine-methionine-glycine-methionine-glycine (MMGMG) (SEQ ID NO:69). In some aspects, the oligopeptide is methionine-glycine-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 aspects, the oligopeptide is methionine-glycine-methionine (MGM).

[0018] In some aspects, the IL-7 protein comprises a half-life extension portion. In some aspects, the half-life extension portion includes Fc, albumin, albumin binding polypeptide, Pro / Ala / Ser (PAS), C-terminal peptide (CTP) of human chorionic gonadotropin β subunit, polyethylene glycol (PEG), long unstructured hydrophilic amino acid sequence (XTEN), hydroxyethyl starch (HES), albumin binding small molecule or its combination. In some aspects, wherein the half-life extension portion is Fc.

[0019] In some aspects, 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, wherein the CH2 domain comprises a portion of a human IgD CH2 domain and a portion of a human IgG4 CH2 domain, and wherein the CH3 domain comprises a portion of a human IgG4 CH3 domain.

[0020] In some aspects, 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 the sequence set forth in any one of SEQ ID NOs: 1-6, 15-26, and 80-85.

[0021] In some aspects, the VEGF antagonist comprises an antibody or antigen-binding fragment thereof that specifically binds to VEGF ("anti-VEGF antibody"), a polynucleotide encoding an anti-VEGF antibody, an antisense oligonucleotide, siRNA, shRNA, miRNA, a dsRNA targeting VEGF, an aptamer, a PNA, or a vector comprising the same. In some aspects, the VEGF antagonist is an anti-VEGF antibody. In some aspects, the anti-VEGF antibody includes aflibercept ( and )、bevacizumab ( and ), ranibizumab or a combination thereof.

[0022] In some aspects, tumors are derived from cancer, and cancer includes breast cancer, head and neck cancer, uterine cancer, brain cancer, skin cancer, renal cancer, lung cancer, colorectal cancer, prostate cancer, liver cancer, bladder cancer, kidney cancer, pancreatic cancer, thyroid cancer, esophageal cancer, eye cancer, stomach (stomach) cancer, gastrointestinal cancer, ovarian cancer, carcinoma, sarcoma, leukemia, lymphoma, myeloma or its combination. In some aspects, brain cancer includes glioblastoma. In some aspects, glioblastoma includes recurrent glioblastoma. In some aspects, glioblastoma is refractory to standard care therapy. In some aspects, skin cancer includes Merkel cell carcinoma (Merkel cell carcinoma, MCC), basal cell carcinoma (BCC), cutaneous squamous cell carcinoma (cSCC), melanoma or its combination.

[0023] In some aspects, the IL-7 protein is administered to the subject intramuscularly, parenterally, subcutaneously, ocularly, intravenously, intraperitoneally, intradermally, intraorbitally, intracerebrally, intracranially, intraspinally, intraventricularly, intrathecally, intracisternally, intracapsularly or intratumorally. In some aspects, the VEGF antagonist is administered to the subject intravenously, parenterally, intramuscularly, subcutaneously, ocularly, intraperitoneally, intradermally, intraorbitally, intracerebrally, intracranially, intracisternally, intracerebroventricularly, intrathecally, intracisternally, intracapsularly or intratumorally. In some aspects, the additional therapeutic agent is administered to the subject parenterally, intravenously, intramuscularly, subcutaneously, ocularly, intraperitoneally, intradermally, intraorbitally, intracerebrally, intracranially, intracisternally, intracerebroventricularly, intrathecally, intracisternally, intracapsularly or intratumorally.

[0024] In some aspects, the IL-7 protein is administered 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. In some aspects, the IL-7 protein is present at between about 610 μg / kg and about 1,200 μg / kg, between about 650 μg / kg and about 1,200 μg / kg, between about 700 μg / kg and about 1,200 μg / kg, between about 750 μg / kg and about 1,200 μg / kg, between about 800 μg / kg and about 1,200 μg / kg, between about 850 μg / kg and about 1, 200 μg / kg, between about 900 μg / kg and about 1,200 μg / kg, between about 950 μg / kg and about 1,200 μg / kg, between about 1,000 μg / kg and about 1,200 μg / kg, between about 1,050 μg / kg and about 1,200 μg / kg, between about 1,100 μg / kg and about 1,200 μg / kg, about 1,200 μg / kg kg to about 2,000 μg / kg, about 1,300 μg / kg to about 2,000 μg / kg, about 1,500 μg / kg to about 2,000 μg / kg, about 1,700 μg / kg to about 2,000 μg / kg, about 610 μg / kg to about 1,000 μg / kg, about 650 μg / kg to about 1,000 μg / kg, about 70 The dosage is between 0 μg / kg and about 1,000 μg / kg, between about 750 μg / kg and about 1,000 μg / kg, between about 800 μg / kg and about 1,000 μg / kg, between about 850 μg / kg and about 1,000 μg / kg, between about 900 μg / kg and about 1,000 μg / kg, or between about 950 μg / kg and about 1,000 μg / kg. In some aspects, the IL-7 protein is administered at a dose of between about 700 μg / kg and about 900 μg / kg, between about 750 μg / kg and about 950 μg / kg, between about 700 μg / kg and about 850 μg / kg, between about 750 μg / kg and about 850 μg / kg, between about 700 μg / kg and about 800 μg / kg, between about 800 μg / kg and about 900 μg / kg, between about 750 μg / kg and about 850 μg / kg, or between about 850 μg / kg and about 950 μg / kg.In some aspects, the IL-7 protein is administered at about 650 μg / kg, about 680 μg / kg, 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 820 μg / kg, about 840 μg / kg, about 850 μg / kg, about 860 μg / kg, about 880 μg / kg, about 900 μg / kg, about 920 μg / kg, about 940 μg / kg, about 950 μg / kg, about 960 μg / kg, about 980 μg / kg, about 1,000μg / kg, about 1,020μg / kg, about 1,040μg / kg, about 1,060μg / kg, about 1,080μg / kg, about 1,100μg / kg, about 1,120μg / kg, about 1,140μg / kg, about 1,160μg / kg, about 1,180μg / kg, about 1,200μg / kg, about 1,220μg / kg, about 1,240μg / kg, about 1,260μg / kg, about 1,280μg / kg, about 1,300μg / kg, about 1,320μg / kg g, 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 1,540 μg / kg, about 1,560 μg / kg, about 1,580 μg / kg, about 1,600 μg / kg, about 1,620 μg / kg, about 1,640 μg / kg, about 1,660 μg / kg, about or about 2,000 μg / kg.

[0025] In some aspects, the IL-7 protein is administered at a dosing frequency of once a week, once in two weeks, once in three weeks, once in four weeks, once in five weeks, once in six weeks, once in seven weeks, once in eight weeks, once in nine weeks, once in 10 weeks, once in 11 weeks, or once in 12 weeks. In some aspects, the IL-7 protein is administered at a dosing frequency of once in eight weeks.

[0026] In some aspects, the VEGF antagonist is administered to a subject at a dose of about 0.1 mg / kg to about 20 mg / kg. In some aspects, the VEGF antagonist is administered once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every 10 weeks, once every 11 weeks, or once every 12 weeks. In some aspects, the VEGF antagonist is administered at a dosing frequency of once every two weeks.

[0027] In some aspects, the anti-VEGF antibody is bevacizumab. In some aspects, bevacizumab is administered at a dose of about 5 mg / kg to about 15 mg / kg. In some aspects, bevacizumab is administered at a dose of about 5 mg / kg, about 7.5 mg / kg, about 10 mg / kg, about 12.5 mg / kg, or about 15 mg / kg.

[0028] In some aspects, the anti-VEGF antibody is aflibercept. In some aspects, aflibercept is administered at a dose of about 4 mg / kg to about 15 mg / kg. In some aspects, aflibercept is administered at a dose of about 4 mg / kg. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1A and 1B The anti-tumor effect of combined treatment of IL-7 protein and VEGF antagonist in an animal model of colon adenocarcinoma was shown. Figure 1A A schematic diagram of the schedule for tumor inoculation and treatment administration is provided. Figure 1B The tumor volumes (mm) of animals in different treatment groups at different time points after tumor inoculation are provided. 3 ). Treatment groups included: (1) IL-7 formulation buffer + isotype control antibody ("control"); (2) IL-7 protein + isotype control antibody ("IL-7"); (3) IL-7 formulation buffer + VEGF antagonist (e.g., anti-VEGF antibody) ("a-VEGF"); and (4) IL-7 protein + VEGF antagonist ("IL-7 + a-VEGF"). Data are shown as mean ± SEM. Statistical analysis (one-way ANOVA) was performed using GraphPad Prism 9.1.0 software. "**" = p < 0.01; and "****" = p < 0.0001.

[0030] Figure 2 A schematic diagram of the administration schedule for the Phase 2 studies described in Examples 3 and 4 is provided.

[0031] Figure 3 A general overview of the Phase 2 studies described in Examples 3 and 4 is provided.

[0032] Figure 4A and 4BInterim results from the Phase 2 study described in Examples 3 and 4 are provided. Figure 4A The current status of 20 subjects participating in the Phase 2 study is shown. Specifically, the following statuses of the subjects are provided: (1) alive (grey bar), (2) dead (dark grey bar), (3) partially responded to treatment (black circle), (4) stable disease (black triangle), (5) unconfirmed disease progression (open square), (6) confirmed disease progression (black square), and (7) end of treatment (black diamond). Figure 4B Absolute lymphocyte counts at week 0 and week 4 after initial administration of treatment are shown. (*) indicates a statistically significant difference between week 4 and week 0 (baseline) by Wilcoxan matched-pairs signed-rank test. DETAILED DESCRIPTION

[0033] The present disclosure provides a combination regimen comprising a combination of an IL-7 protein and a VEGF antagonist, which can be used to treat tumors. As demonstrated herein, administration of a combination of an IL-7 protein and a VEGF antagonist can result in greatly improved anti-tumor immunity compared to a single IL-7 protein or a VEGF antagonist. Therefore, in some aspects, the combination regimen described herein provides an effective and feasible treatment option for many cancers. Additional aspects of the present disclosure are provided throughout the present application.

[0034] Before describing the present disclosure in more detail, it should be understood that the present disclosure is not limited to the specific compositions or methods described, which compositions or methods may of course vary. It will be apparent to those skilled in the art upon reading the present disclosure that each individual aspect described and illustrated herein has discrete components and features that can be easily separated or combined with the features of any other several aspects without departing from the scope or spirit of the present disclosure. Any narrated method can be performed in the order of the events narrated or in any other order that is logically possible.

[0035] The headings provided herein are not limitations of the various aspects of the disclosure, which can 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 is not intended to be limiting, as the scope of the disclosure will be limited only by the appended claims.

[0036] I. Definitions

[0037] In order to more easily understand the present disclosure, some terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms shall have the meaning described below. Other definitions are set forth throughout this application.

[0038] The term "a / an" entity refers to one or more of the entity; for example, "a nucleotide sequence" should be understood to mean one or more nucleotide sequences. Therefore, the terms "a" or "an", "one or more" and "at least one" are used interchangeably herein.

[0039] In addition, "and / or" as used herein should be considered as a specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" as used in phrases herein (e.g., "A and / or B") is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to include each of the following aspects: 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).

[0040] It should be understood that wherever aspects are described herein with the phrase "comprising," other similar aspects described with "consisting of" and / or "consisting essentially of" are also provided.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure relates. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 5th edition, 2013, Academic Press; and Oxford Dictionary Of Biochemistry And Molecular Biology, 2nd edition, 2008, Oxford University Press provide a comprehensive dictionary of many of the terms used in the present disclosure to the skilled person.

[0042] Units, prefixes and symbols are expressed in the form accepted by the International System of Units (SI). Numerical ranges include numbers that limit the range. When enumerating the range of values, it should be understood that each intermediate integer value and each fraction thereof between the upper and lower limits of the range are also specifically disclosed, as well as each subrange between such values. The upper and lower limits of any range can be independently included in the range or excluded from the range, and each range including any one limit, both limits do not include or include both limits is also included in the present disclosure. Therefore, the ranges listed herein should be understood as a shorthand form of all values ​​within the range, including the endpoints listed. For example, a range of 1 to 10 is understood to include any numerical value, numerical combination or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.

[0043] Where a value is explicitly enumerated (e.g., 10), it is understood that values ​​of approximately the same quantity or amount as the enumerated value (e.g., ±10%) are also within the scope of the present disclosure. Where a combination is disclosed, each subcombination of the elements of the combination is also specifically disclosed and is within the scope of the present disclosure. Conversely, where different elements or groups of elements are disclosed separately, their combinations are also disclosed. Where any element of the present disclosure is disclosed as having multiple alternatives, embodiments of the present disclosure in which each alternative is excluded, either individually or in any combination with other alternatives, are also hereby disclosed; more than one element of the present disclosure may have such exclusions, and all combinations of elements with such exclusions are hereby disclosed.

[0044] The term "about" is used herein to mean approximately, roughly, about, or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values ​​set forth. Generally, the term "about" can modify numerical values ​​above and below the stated value by, for example, a 10% variation up or down (higher or lower).

[0045] As used herein, the term "interleukin-7" or "IL-7" refers to IL-7 polypeptides and derivatives and analogs thereof, e.g., having substantial amino acid sequence identity and substantially equivalent biological activity to wild-type mature mammalian IL-7 in standard bioassays or assays for IL-7 receptor binding affinity. For example, IL-7 refers to the amino acid sequence of a recombinant or non-recombinant polypeptide having the following amino acid sequence: 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. The IL-7 polypeptides of the present disclosure can be obtained from any species, e.g., humans, cows, or sheep. IL-7 nucleic acid and amino acid sequences are well known in the art. For example, the Genbank accession number of the amino acid sequence of human IL-7 is P13232 (SEQ ID NO: 1); the Genbank accession number of the amino acid sequence of mouse IL-7 is P10168 (SEQ ID NO: 3); the Genbank accession number of the amino acid sequence of rat IL-7 is P56478 (SEQ ID NO: 2); the Genbank accession number of the amino acid sequence of monkey IL-7 is NP_001279008 (SEQ ID NO: 4); the Genbank accession number of the amino acid sequence of cow IL-7 is P26895 (SEQ ID NO: 5); and the Genbank accession number of the amino acid sequence of sheep IL-7 is Q28540 (SEQ ID NO: 6). In some aspects, the IL-7 polypeptides of the present disclosure are variants of the IL-7 protein. It is apparent from the present disclosure that in some aspects, the IL-7 polypeptides useful in the present disclosure do not comprise a signal peptide (see, e.g., Table 1A).

[0046] Table 1A. Sequences of exemplary IL-7 proteins

[0047]

[0048]

[0049]

[0050] As used herein, the term "vascular endothelial growth factor" or "VEGF" refers to a protein that is produced by many cells and stimulates blood vessel formation (i.e., angiogenesis). VEGF is part of a protein family that includes five major members: VEGF-A (also known as "VEGF"), placental growth factor (PGF), VEGF-B, VEGF-C, and VEGF-D. Unless otherwise indicated, the term "VEGF" refers to VEGF-A family members, including all isoforms (see, e.g., Table 1B) and variants thereof. Due to alternative promoter use, alternative splicing, and / or alternative initiation, there are at least 17 known human VEGF isoforms (i.e., VEGF-A). The sequences of different VEGF isoforms are provided in Table 1B (as follows).

[0051] Table 1B: Human VEGF sequences

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062] As used herein, "administering" refers to using any of the various methods and delivery systems known to those skilled in the art to physically introduce a therapeutic agent or a composition comprising a therapeutic agent into a subject. The different routes of administration of therapeutic agents described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal or other parenteral routes of administration, such as by injection or infusion. As used herein, the phrase "parenteral administration" means an administration mode other than enteral and topical administration, usually by injection, and includes but is not limited to intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, intratracheal, lung, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraventricular, intravitreal, epidural and intrasternal injection and infusion, and in vivo electroporation. Alternatively, therapeutic agents described herein can be administered via non-parenteral routes, such as local, epidermal or mucosal administration routes, such as intranasal, oral, vaginal, rectal, sublingual or topical administration. Administration can also be performed, for example, once, multiple times, and / or over one or more extended periods of time.

[0063] The term "antibody" or "antibodies" is a term of the art and is used interchangeably herein and refers to a molecule having an antigen binding site that specifically binds to an antigen. The term as used herein includes a complete antibody and any antigen binding fragment thereof (i.e., "antigen binding portion") or a single chain. In one aspect, an "antibody" refers to a glycoprotein or an antigen binding portion thereof that comprises at least two heavy chains (H) and two light chains (L) and is interconnected by a disulfide bond. In some aspects, an "antibody" refers to a single-chain antibody comprising a single variable domain (e.g., a VHH domain). Each heavy chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. In some naturally occurring antibodies, the heavy chain constant region comprises three domains, CH1, CH2, and CH3. In some naturally occurring antibodies, each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one domain, CL.

[0064] The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL contains three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various immune system cells (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0065] Antibodies typically bind specifically to their cognate antigens with high affinity, which is reflected in the -5 Up to 10 -11 M or less dissociation constant (K D ) above. It is generally believed that any -4 M of K D Indicates non-specific binding. As used herein, an antibody that "specifically binds" to an antigen refers to an antibody that binds to the antigen and substantially the same antigen with high affinity, but does not bind to unrelated antigens with high affinity, high affinity meaning K D For 10 -7 M or smaller, 10 -8 M or smaller, 5x 10 -9 M or smaller, or within 10 -8 M and 10 -10 M or less. An antigen is "substantially identical" to a given antigen if it has a high degree of sequence identity with the given antigen, such as at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with the sequence of the given antigen. For example, in some aspects, an antibody that specifically binds to VEGF may also have cross-reactivity with VEGF antigens from certain primate species (e.g., cynomolgus monkey anti-VEGF antibodies), but may not cross-react with VEGF molecules from other species or with molecules other than VEGF.

[0066] Immunoglobulin can be derived from any known isotype, including but not limited to IgA, secretory IgA, IgG and IgM. IgG subclasses are also well known to those skilled in the art, and include but are not limited to human IgG1, IgG2, IgG3 and IgG4. "Isotype" refers to the antibody class or subclass (e.g., IgM or IgG1) encoded by the heavy chain constant region gene. In some aspects, one or more amino acids of the isotype can be mutated to change the effector function. For example, the term "antibody" includes naturally occurring and non-naturally occurring Ab; monoclonal and polyclonal Ab; chimeric and humanized Ab; human or non-human Ab; fully synthetic Ab; and single-chain antibodies. Non-human antibodies can be humanized by recombinant methods to reduce their immunogenicity in the human body. In the absence of explicit instructions, and unless otherwise indicated by the context, the term "antibody" also includes an antigen-binding fragment or antigen-binding portion of any of the above-mentioned immunoglobulins, and includes monovalent and divalent fragments or portions, and single-chain antibodies.

[0067] An "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds VEGF is substantially free of antibodies that specifically bind antigens other than VEGF). However, an isolated antibody that specifically binds VEGF may have cross-reactivity with other antigens (such as VEGF molecules from different species). In addition, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0068] The term "monoclonal antibody" ("mAb") refers to a non-naturally occurring preparation of an antibody molecule of single molecular composition, i.e., an antibody molecule whose primary sequence is substantially identical and which exhibits a single binding specificity and affinity for a particular epitope. mAbs are examples of isolated antibodies. mAbs can be produced by hybridoma, recombinant, transgenic, or other techniques known to those skilled in the art.

[0069] "HuMAb" refers to an antibody with a variable region, wherein the framework region and the CDR region are both derived from human germline immunoglobulin sequences. In addition, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutations in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (such as a mouse) have been grafted onto human framework sequences. The terms "human" antibody and "fully human" antibody are used synonymously.

[0070] "Humanized antibody" refers to an antibody in which some, most or all of the amino acids outside the CDR domain of a non-human antibody are replaced by the corresponding amino acids derived from human immunoglobulin. In one aspect of a humanized form of an antibody, some, most or all of the amino acids outside the CDR domain have been replaced by amino acids from human immunoglobulin, while some, most or all of the amino acids in one or more CDR regions have not changed. A small amount of amino acid addition, deletion, insertion, substitution or modification is allowed as long as they do not eliminate the ability of the antibody to bind to a specific antigen. "Humanized" antibodies retain antigenic specificity similar to the original antibody.

[0071] A "chimeric antibody" refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species, such as an antibody in which the variable region is derived from a mouse antibody and the constant region is derived from a human antibody.

[0072] An "anti-antigen" antibody refers to an antibody that specifically binds to an antigen. For example, an anti-PD-1 antibody specifically binds to PD-1, and an anti-VEGF antibody specifically binds to VEGF.

[0073] An "antigen-binding portion" of an antibody (also referred to as an "antigen-binding fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind to the antigen to which the entire antibody binds.

[0074] As used herein, the terms "specific binding", "selective binding", "selectively binds" and "specifically binds" refer to the binding of an antibody to an epitope on a predetermined antigen. TM The results are as follows: The surface plasmon resonance (SPR) technique using a predetermined antigen as the analyte and an antibody as the ligand, or the Scatchard analysis of antibody binding to antigen-positive cells, is less than 10 -7 M, such as approximately less than 10 -8 M, 10 -9 M or 10 -10 M or even smaller equilibrium dissociation constant (K D ) and (ii) binds to the predetermined antigen with an affinity at least two times greater than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely related antigen.

[0075] As used herein, the term "naturally occurring" when applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence present in an organism (including a virus) is naturally occurring, which can be isolated from a natural source and cannot be intentionally modified by man in the laboratory.

[0076] "Polypeptide" refers to a chain of 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 contain one or more polypeptides. Unless otherwise indicated, the terms "protein" and "polypeptide" are used interchangeably.

[0077] As used herein, the term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. The nucleic acid molecule can be single-stranded or double-stranded, and can be a cDNA.

[0078] "Conservative amino acid substitution" refers to the replacement of an amino acid residue by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include: amino acids with basic side chains (e.g., lysine, arginine, histidine); amino acids with acidic side chains (e.g., aspartic acid, glutamic acid); amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan); amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine); amino acids with β-branched side chains (e.g., threonine, valine, isoleucine); and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In some aspects, the predicted non-essential amino acid residues in the antibody are replaced by another amino acid residue from the same side chain family. Methods for identifying conservative substitutions of nucleotides and amino acids 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)).

[0079] For nucleic acids, the term "substantial homology" indicates that two nucleic acids, or designated sequences thereof, are identical in at least about 80% of the nucleotides, at least about 90% to 95%, or at least about 98% to 99.5% of the nucleotides, with appropriate nucleotide insertions or deletions, when optimally aligned and compared. Alternatively, substantial homology exists when the fragments hybridize under selective hybridization conditions to the complement of a strand.

[0080] For polypeptides, the term "substantial homology" indicates that two polypeptides, or designated sequences thereof, when optimally aligned and compared, are identical in at least about 80% of their amino acids, at least about 90% to 95%, or at least about 98% to 99.5% of their amino acids, with appropriate amino acid insertions or deletions.

[0081] The identity percentage between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of spaces that need to be introduced to achieve the optimal comparison of the two sequences and the length of each space (i.e., the total number of homology %=number of identical positions / positions x 100). The comparison of sequences and the determination of the identity percentage between the two sequences can be accomplished using a mathematical algorithm, for example, as described in the following non-limiting examples.

[0082] The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available from 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)) which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12, a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. (48): 444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available from worldwideweb.gcg.com), using either the Blossum 62 matrix or the PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0083] The nucleic acid and protein sequences described herein can further be used as "query sequences" for searching against public databases to, for example, 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. In order to obtain gapped alignments for comparison purposes, gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25 (17): 3389-3402. When using BLAST and gapped BLAST programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used. See worldwideweb.ncbi.nlm.nih.gov.

[0084] Nucleic acids can be present in intact cells, in cell lysates, or in partially purified or substantially pure form. Nucleic acids are "isolated" or "rendered substantially pure" when 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 alkali / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other techniques well known in the art. See F. Ausubel, et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).

[0085] Nucleic acids, such as cDNAs, can be mutated according to standard techniques for providing gene sequences. For coding sequences, these mutations can affect the amino acid sequence as desired. Specifically, DNA sequences that are substantially homologous to or derived from natural V, D, J, constant, switch and other such sequences described herein are contemplated (wherein "derived from" means that the sequence is identical to or modified from another sequence).

[0086] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid connected thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop to which an additional DNA segment can be connected. Another type of vector is a viral vector, in which an additional DNA segment can be connected to a viral genome. Some vectors can replicate autonomously in the host cell into which they are introduced (e.g., bacterial vectors with bacterial replication origins and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, and thus replicated with the host genome. In addition, some vectors can guide the expression of the genes to which they are operably connected. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors useful in recombinant DNA technology are generally in the form of plasmids. In this specification, "plasmid" and "vector" are used interchangeably because plasmids are the most commonly used vector forms. However, other forms of expression vectors providing equivalent functions are also included, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses).

[0087] As used herein, the term "recombinant host cell" (or simply "host cell") is intended to refer to a cell that contains 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 should be understood that such terms refer not only to a specific subject cell, but also to the progeny of such a cell. Because some modifications may appear in the offspring due to mutations or environmental influences, such progeny may not actually be identical to the parent cell, but are still included in the scope of the term "host cell" as used herein.

[0088] As used herein, the term "linked" or "conjugated" refers to the association of two or more molecules. The connection can be covalent or non-covalent. The connection can also be genetic (i.e., recombinant fusion). Such connection can be achieved using a variety of art-recognized techniques, such as chemical conjugation and recombinant protein production.

[0089] "Cancer" refers to a wide variety of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth lead to the formation of malignant tumors that invade adjacent tissues and may also metastasize to distant parts of the body via the lymphatic system or bloodstream. "Cancer" as used herein refers to primary, metastatic, and recurrent cancers. Non-limiting examples of suitable cancers that can be treated with the present disclosure are provided elsewhere in the present disclosure.

[0090] The term "fusion protein" refers to a protein produced by joining two or more genes that originally encoded separate proteins. Translation of this fusion gene produces a single polypeptide or multiple polypeptides with functional properties derived from each original protein. In some aspects, the two or more genes may contain substitutions, deletions and / or additions in their nucleotide sequences.

[0091] "Fc receptor" or "FcR" is a receptor that binds to the Fc region of an immunoglobulin. FcRs that bind to 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. Most innate effector cell types co-express one or more activating FcγRs and inhibitory FcγRIIB, while natural killer (NK) cells selectively express one activating Fc receptor (FcγRIII in mice and FcγRIIIA in humans), but do not express inhibitory FcγRIIB in mice and humans. Human IgG1 binds to most human Fc receptors, and is considered equivalent to mouse IgG2a in terms of the type of activating Fc receptor it binds.

[0092] "Fc region" (fragment crystallizable region) or "Fc domain" or "Fc" refers to the C-terminal region of an antibody heavy chain that mediates the binding of the immunoglobulin 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 comprises the constant region of the antibody except for 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 two heavy chains of the antibody; IgM and IgE Fc regions comprise three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. For IgG, the Fc region comprises immunoglobulin domains CH2 and CH3 and a hinge between the CH1 and CH2 domains. Although the definition of the Fc region boundaries of immunoglobulin heavy chains may vary, as defined herein, the human IgG heavy chain Fc region is defined as extending from amino acid residue D221 of IgG1, amino acid residue V222 of IgG2, amino acid residue L221 of IgG3, and amino acid residue P224 of IgG4 to the carboxyl terminus of the heavy chain, wherein numbering is performed 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 on the C-terminal side of the CH2 domain in the Fc region, i.e., it extends from amino acid 341 of IgG to amino acid 447 or 446 (if there is no C-terminal lysine residue) or 445 (if there is no C-terminal glycine and lysine residue). As used herein, the Fc region can be a native sequence Fc, including any allotype variants, or a variant Fc (e.g., a non-naturally occurring Fc). Fc may also refer to this region either alone or in the context of a protein polypeptide comprising an Fc, such as an "Fc region-containing binding protein," also referred to as an "Fc fusion protein" (eg, an antibody or immunoadhesion).

[0093] A "native sequence Fc region" or "native sequence Fc" comprises an amino acid sequence identical to the amino acid sequence of a naturally occurring Fc region. Native sequence human Fc regions include native sequence human IgG1 Fc regions, native sequence human IgG2 Fc regions, native sequence human IgG3 Fc regions, and native sequence human IgG4 Fc regions, as well as naturally occurring variants thereof. Native sequence Fc includes various allotypes of Fc (see, e.g., Jefferis et al. (2009) mAbs 1:1).

[0094] In addition, the Fc (native or variant) of the present disclosure may be in the form of native sugar chains, increased sugar chains or reduced sugar chains compared to the native form, or may be in a deglycosylated form. Immunoglobulin Fc sugar chains may be modified by conventional methods, such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms. Removing sugar chains from the Fc fragment results in a sharp decrease in binding affinity with the C1q portion of the first complement component C1, as well as a decrease or loss of ADCC or CDC, thereby not inducing any unnecessary immune response in vivo. In this regard, a deglycosylated or aglycosylated form of an immunoglobulin Fc region may be more suitable for the purpose of the present disclosure as a drug carrier. As used herein, the term "deglycosylated" refers to an Fc region in which sugars are enzymatically removed from the Fc fragment. In addition, the term "aglycosylated" refers to an Fc fragment produced by a prokaryotic organism in a non-glycosylated form, and preferably produced in Escherichia coli.

[0095] As used herein, the term "immune response" refers to a biological response to an exogenous agent in a vertebrate, which protects the organism against these agents and the diseases caused by them. For example, the term "anti-tumor immune response" refers to an immune response against tumor antigens. The immune response is 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, and the immune response causes selective targeting, binding, damage, destruction of invading pathogens, cells or tissues infected with pathogens, cancer cells or other abnormal cells or (in the case of autoimmunity or pathological inflammation) normal human cells or tissues and / or eliminates them from the vertebrate body. The immune response includes, for example, the activation or inhibition of T cells, such as effector T cells or Th cells, such as CD4 + or CD8 + T cells, or Treg cells.

[0096] The term "immunotherapy" refers to the treatment of a subject having a disease or at risk of disease or recurrence by methods including inducing, enhancing, suppressing or altering an immune response. "Treatment" or "therapy" of a subject refers to any type of intervention or procedure performed on a subject, or the administration of an active agent to a subject, with the purpose of reversing, alleviating, ameliorating, suppressing, slowing down or preventing the onset, progression, development, severity or recurrence of symptoms, complications or illnesses or biochemical markers associated with a disease.

[0097] The term "effector T cells" (Teff) refers to T cells with cytolytic activity (e.g., CD4 + and CD8 +T cells) and helper T (Th) cells (which secrete cytokines and activate and direct other immune cells) but do not include regulatory T cells (Treg cells). The combination of IL-7 protein and VEGF antagonist (e.g., anti-VEGF antibody) activates and / or increases Teff cells (e.g., CD4 + and CD8 + T cell) frequency.

[0098] As used herein, the term "regulatory T cells" (Treg) refers to a T cell group with the ability to reduce or suppress the induction and proliferation of effector T cells and thus regulate immune response. In some aspects, Treg can suppress immune response by secreting anti-inflammatory cytokines such as IL-10, TGF-β and IL-35, which can interfere with the activation of naive T cells and the differentiation of naive T cells to effector T cells. In some aspects, Treg can also produce cytolytic molecules such as granzyme B, and cytolytic molecules can induce apoptosis of effector T cells. In some aspects, regulatory T cells are natural regulatory T cells (nTreg) (i.e., developing in the thymus). In some aspects, regulatory T cells are inductive regulatory T cells (iTreg) (i.e., differentiated into the naive T cells of Treg in peripheral tissues after being exposed to certain stimuli). Methods for identifying Treg are known in the art. For example, Treg expression can use certain phenotypic markers (e.g., CD25, Foxp3 or CD39) measured by flow cytometry. See, for example, International Publication No. WO 2017 / 062035 A1; Gu J, et al., Cell Mol Immunol 14(6):521-528 (2017). In some aspects, Treg is CD45RA - CD39 + T cells.

[0099] As used herein, the term "tumor infiltrating lymphocyte" or "TIL" refers to lymphocytes (e.g., effector T cells) that have migrated from the periphery (e.g., from the blood) into a tumor. In some aspects, the tumor infiltrating lymphocyte is a CD4+TIL. In some aspects, the tumor infiltrating lymphocyte is a CD8+TIL.

[0100] The ability to stimulate immune response or immune system may be enhanced by the agonistic activity enhancement of T cell costimulatory receptors and / or the antagonistic activity enhancement of inhibitory receptors. The increase in the ability to stimulate immune response or immune system can be reflected by the multiple increase of EC50 or the maximum activity level in the determination of measuring immune response (for example, measuring cytokine or chemokine release, cytolytic activity (directly measured on target cells or indirectly measured by detecting CD107a or granzyme) and the change of propagation). The ability to stimulate immune response or immune system activity can be enhanced by at least 10%, 30%, 50%, 75%, 2 times, 3 times, 5 times or more.

[0101] For example, a "variant" of an IL-7 protein is defined as an amino acid sequence that is altered by one or more amino acids. Variants may have "conservative" changes, in which the substituted amino acids have similar structures or chemical properties, such as replacing leucine with isoleucine. More rarely, variants may have "non-conservative" changes, such as replacing glycine with tryptophan. Similar minor changes may also include amino acid deletions or insertions, or both. Using computer programs well known in the art, such as software for molecular modeling or generating alignments, guidance for determining which and how many amino acid residues can be substituted, inserted or deleted without losing biological activity can be found. For example, variant IL-7 proteins included in the present disclosure include IL-7 proteins that retain IL-7 activity. Similarly, variants of VEGF antagonists described herein refer to variants that retain antagonistic activity to VEGF. Variants that also contain additions, substitutions or deletions are also included in the present disclosure, as long as the variants retain biological activity that is substantially equivalent to the non-variant (e.g., wild-type) counterpart. For example, truncations of IL-7 that retain biological activity comparable to the full-length form of the IL-7 protein are included in the present disclosure. The activity of the therapeutic agents described herein (eg, IL-7 proteins and / or VEGF antagonists) can be measured using any suitable method known in the art.

[0102] To determine the percent identity of two amino acid sequences or two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence to achieve optimal alignment with a second amino acid or nucleic acid sequence). The percent identity between the 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). The determination of the percent homology between two sequences can be accomplished using a mathematical algorithm. A preferred non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68, as modified in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. This 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 with score = 100 and word length = 12. BLAST protein searches can be performed with the XBLAST program with score = 50 and 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.

[0103] "Subject" includes any human or non-human animal, the term "non-human animal" including, but not limited to, vertebrates, such as non-human primates, sheep, dogs, and rodents, such as mice, rats, and guinea pigs. In some aspects, the subject is a human. The terms "subject" and "patient" are used interchangeably herein.

[0104] The term "therapeutically effective amount" or "therapeutically effective dose" refers to the amount of an agent that provides a desired biological, therapeutic and / or preventive result. The result can be a reduction, improvement, alleviation, reduction, delay and / or alleviation of one or more signs, symptoms or causes of a disease, or any other desired change in a biological system. With respect to solid tumors, an effective amount comprises an amount sufficient to cause tumor shrinkage and / or sufficient to reduce tumor growth rate (e.g., inhibit tumor growth) or sufficient to prevent or delay other undesirable cell proliferation. In some aspects, an effective amount is an amount sufficient to delay tumor development. In some aspects, 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 down and may prevent cancer cells from infiltrating peripheral organs to a certain extent; (iv) inhibit (i.e., slow down and may prevent tumor metastasis to a certain extent; (v) inhibit tumor growth; (vi) prevent or delay the occurrence and / or recurrence of tumors; and / or (vii) alleviate to a certain extent one or more symptoms associated with cancer. In some aspects, a "therapeutically effective amount" is a combination of an amount of IL-7 protein and an amount of a VEGF antagonist (e.g., an anti-VEGF antibody) that has been clinically demonstrated to achieve a significant reduction in cancer or a reduction in the progression (regression) of cancer (e.g., advanced solid tumors). The ability of a therapeutic agent to promote disease regression can be assessed using a variety of methods known to skilled practitioners, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by measuring the activity of the agent in in vitro assays.

[0105] The term "dosing frequency" refers to the number of times a therapeutic agent (e.g., IL-7 protein and / or VEGF antagonist) is administered to a subject within a specific time period. The dosing frequency can be expressed as the number of doses per given time, for example, once a day, once a week, or once every two weeks. As used herein, "dosing frequency" applies to situations where a subject receives multiple (or repeated) administrations of a therapeutic agent. Suitable dosing frequencies for any IL-7 protein and VEGF antagonist described herein are provided elsewhere in the present disclosure.

[0106] As used herein, the term "standard of care" refers to a treatment that is accepted by medical experts as an appropriate treatment for a certain type of disease and is widely used by health care professionals. The term can be used interchangeably with any of the following terms: "best practice", "standard medical care" and "standard therapy". As further described elsewhere in this disclosure, in some aspects, standard of care includes chemotherapy (e.g., temozolomide). In some aspects, standard of care includes radiotherapy. In some aspects, standard of care includes radiotherapy and chemotherapy (e.g., temozolomide).

[0107] As used herein, the term "drug" refers to any biologically active agent (e.g., IL-7 protein and / or VEGF antagonist) intended to be administered to a human or non-human mammal to prevent or treat a disease or other undesirable condition. Drugs include hormones, growth factors, proteins, peptides, and other compounds. In some aspects, the drugs disclosed herein are anticancer agents. Unless otherwise indicated, the term "drug" can be used interchangeably with the term "therapeutic agent".

[0108] For example, "anticancer agents" promote the regression of cancer in subjects or prevent further tumor growth. In some aspects, a therapeutically effective amount of a drug promotes the regression of cancer to the extent of eliminating cancer. "Promoting cancer regression" means that an effective amount of a drug is administered alone or in combination with an anti-tumor agent, resulting in a reduction in tumor growth or size, tumor necrosis, a reduction in the severity of at least one disease symptom, an increase in the frequency and duration of the disease asymptomatic period, or prevention of damage or disability caused by disease torture. In addition, the terms "effective" and "effectiveness" for treatment include pharmacological effectiveness and physiological safety. Pharmacological effectiveness refers to the ability of a drug to promote the regression of cancer in patients. Physiological safety refers to the level of toxicity or other adverse physiological reactions (adverse reactions) at the cell, organ and / or organism level caused by drug administration.

[0109] For example, for the treatment of tumors, a therapeutically effective amount of an anticancer agent can inhibit cell growth or tumor growth by at least about 10%, at least about 20%, at least about 40%, at least about 60%, or at least about 80%, relative to untreated subjects, or in some aspects, relative to patients treated with standard of care therapy. In some aspects, tumor regression can be observed and persists for a period of at least about 20 days, at least about 40 days, or at least about 60 days. While these ultimate measures of therapeutic effectiveness are made, the evaluation of immunotherapy drugs must also take into account "immune-related" response patterns.

[0110] As used herein, the term "immune checkpoint inhibitor" refers to a molecule that completely or partially reduces, inhibits, interferes with, or modulates one or more checkpoint proteins. Checkpoint proteins regulate T cell activation or function. Many 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 the co-stimulatory or inhibitory interactions of T cell responses. Immune checkpoint proteins regulate and maintain self-tolerance and the duration and amplitude of physiological immune responses. Immune checkpoint inhibitors include antibodies or are derived from antibodies. Suitable immune checkpoint inhibitors that can be used in the present disclosure are provided elsewhere in the present disclosure.

[0111] As used herein, the term "reference subject" refers to a corresponding subject (e.g., a cancer subject) who has not received the combination therapy described herein (e.g., a combination of an IL-7 protein and a VEGF antagonist). In some aspects, the reference subject refers to a corresponding subject who has received a single IL-7 protein or a single VEGF antagonist. In some aspects, the reference subject has neither received an IL-7 protein nor a VEGF antagonist. The term "reference subject" may also refer to the same subject (i.e., a subject treated with the methods provided herein), but before administering a combination of an IL-7 protein and a VEGF antagonist. In some aspects, the term "reference subject" refers to an average value of a subject population (e.g., a cancer subject).

[0112] As used herein, the terms "ug" and "uM" are used interchangeably with "μg" and "μΜ", respectively.

[0113] Various aspects described herein are described in more detail in the following subsections.

[0114] II. Methods of the Disclosure

[0115] Provided herein is a method of treating a tumor (or cancer) in a subject in need thereof, comprising administering to the subject a combination of an interleukin-7 (IL-7) protein and a vascular endothelial growth factor ("VEGF") antagonist ("VEGF antagonist"). Non-limiting examples of IL-7 proteins and VEGF antagonists that can be used in the methods are provided elsewhere in this disclosure. As demonstrated herein, a treatment regimen comprising an IL-7 protein and a VEGF antagonist (such as those described herein) can be associated with significantly improved anti-tumor immunity compared to either therapeutic agent alone.

[0116] Therefore, in some aspects, the administration of the combination therapy described herein (e.g., a combination of IL-7 protein and VEGF antagonists) can increase the anti-tumor immune response of the subject. In some aspects, compared with the corresponding value of the reference subject, after administration, the anti-tumor immune response increases by at least about 0.5 times, at least about 1 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 40 times or at least about 50 times. For example, in some aspects, compared with the anti-tumor immune response of the subject before the administration of the combination therapy, the anti-tumor immune response of the subject increases. In some aspects, compared with the corresponding subject receiving the administration of a single IL-7 protein or a single VEGF antagonist, the anti-tumor immune response of the subject increases. In some aspects, compared with the corresponding subject not receiving IL-7 protein or VEGF antagonist, the anti-tumor immune response of the subject increases.

[0117] In some aspects, the increase in anti-tumor immune response can be associated with one or more of the following therapeutic effects: (i) tumor volume and / or growth is reduced; (ii) the effector activity of tumor-specific T cells (e.g., the ability to kill tumor cells) increases; (iii) for example, the number of tumor-specific effector T cells in a tumor increases; (iv) for example, the number of tumor-infiltrating lymphocytes (TILs) in a tumor increases; (v) for example, the number of myeloid-derived suppressor cells (MDSCs) in a tumor decreases; (vi) for example, the number of regulatory T cells (Tregs) in a tumor decreases; (vii) the subject's survival duration is prolonged; (viii) for example, the expression of TOX on CD8+T cells in a tumor, a tumor-draining lymph node (TDLN), or both decreases; (ix) for example, the number of stem-like T cells in a tumor, a TDLN, or both increases; or (x) any combination thereof. Unless otherwise indicated, the term "numerical value" refers to both absolute numbers and percentages. Any therapeutic effect described above or elsewhere in the present disclosure can be determined using any suitable method provided herein and / or known in the art.

[0118] In some aspects, the combination therapy described herein (e.g., a combination of IL-7 protein and VEGF antagonists) reduces the tumor volume of the subject. In some aspects, compared with the corresponding value of the reference subject, the tumor volume is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 100%. In some aspects, the combination therapy described herein (e.g., a combination of IL-7 protein and VEGF antagonists) can reduce the growth of tumors in subjects ("tumor growth"). In some aspects, compared with the corresponding value of the reference subject, 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 at least about 100%.

[0119] In some aspects, administration of a combination therapy described herein (e.g., a combination of an IL-7 protein and a VEGF antagonist) can increase tumor-specific T cells (e.g., CD8 + T cells and / or CD4 +In some aspects, the effector activity is increased by at least about 0.5 times, at least about 1 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 40 times, or at least about 50 times, compared to the corresponding value of a reference subject. In some aspects, the effector activity includes T cells (e.g., CD8 + T cells and / or CD4 + Any cytolytic activity of a T cell (T cell). Non-limiting examples of such effector activities include the ability to produce effector molecules (e.g., cytokines, perforins, and / or granzymes) upon antigen stimulation; the ability to recognize and kill tumor cells; or both.

[0120] In some aspects, administration of a combination therapy described herein (e.g., a combination of an IL-7 protein and a VEGF antagonist) can increase tumor-specific effector T cells (e.g., CD8 + and / or CD4 + In some aspects, the number of tumor-specific effector T cells is increased by at least about 0.5-fold, 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.

[0121] Without being bound by any theory, in some aspects, the increase in number is due to an increase in tumor-specific effector T cell proliferation. Thus, in some aspects, after administration of a combination therapy described herein (e.g., a combination of an IL-7 protein and a VEGF antagonist), the proliferation of tumor-specific effector T cells increases by at least about 0.5 times, at least about 1 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 40 times, or at least about 50 times, compared to the corresponding value of a reference subject. In some aspects, the increase in the number of tumor-specific effector T cells is due to the ability of T cells to survive and / or persist. In some aspects, the ability of tumor-specific effector T cells to survive and / or persist in a subject after administration is increased by at least about 0.5 times, at least about 1 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 40 times, or at least about 50 times, compared to the corresponding values ​​in a reference subject. In some aspects, the combination therapy described herein (e.g., a combination of an IL-7 protein and a VEGF antagonist) can increase the proliferation and survival / persistence of tumor-specific effector T cells when administered to a subject.

[0122] In some aspects, tumor-specific effector T cells are tumor infiltrating lymphocytes (TIL).Therefore, in some aspects, applying combined therapy as described herein (for example, the combination of IL-7 protein and VEGF antagonist) can increase the TIL quantity in subject tumor.In some aspects, compared with the corresponding value of reference subject, the TIL quantity in subject tumor increases at least about 0.5 times, at least about 1 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 40 times or at least about 50 times.In some aspects, the increase of TIL quantity in subject tumor may be related to the increase of TIL from the periphery to tumor raising. In some aspects, the recruitment of TILs in tumors is increased by at least about 0.5-fold, 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.

[0123] In some aspects, administration of a combination therapy described herein (e.g., a combination of an IL-7 protein and a VEGF antagonist) can reduce the number of regulatory T cells (Tregs) in a subject's tumor. In some aspects, the regulatory T cells are CD4 + Regulatory T cells. In some aspects, the regulatory T cells are Foxp3 + In some aspects, the number and / or percentage of regulatory T cells in a tumor is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% compared to the corresponding value in a reference subject.

[0124] In some aspects, administering a combination therapy as described herein (e.g., a combination of IL-7 protein and a VEGF antagonist) can reduce the number of myeloid-derived suppressor cells (MDSC) in a subject's tumor. As used herein, the term "myeloid-derived suppressor cells" (MDSC) refers to a heterogeneous population of immune cells, which is defined by myeloid, immature state, and the ability to effectively suppress T cell responses. It is known that they can be amplified under certain pathological conditions, such as chronic infection and cancer. In some aspects, MDSC is a mononuclear cell MDSC (M-MDSC). In some aspects, MDSC is a polymorphonuclear MDSC (PMN-MDSC). In some aspects, compared with the corresponding value of a reference subject, after administration, the MDSC quantity in the tumor is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100%.

[0125] It will be apparent to those skilled in the art that in some aspects, one or more of the therapeutic effects described above can result in (i) an increase in the survival rate (e.g., progression-free survival) of the subject, (ii) an increase in the response rate, (iii) a slowing of disease progression, or (iv) a combination thereof. Thus, in some aspects, administration of a combination therapy described herein (e.g., a combination of an IL-7 protein and a VEGF antagonist) can prolong the survival duration of a subject (e.g., a subject with a tumor). In some aspects, the survival duration is extended by at least about 0.5 times, at least about 1 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 40 times, or at least about 50 times, compared to the corresponding value of a reference subject. In some aspects, compared to a reference subject, after administration, the subject's survival duration is extended by at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 1 year or longer. In some aspects, after administration, the level of the subject's survival duration extension is higher than the reference subject's survival duration (higher by about one month, higher by about two months, higher by about three months, higher by about four months, higher by about five months, higher by about six months, higher by about seven months, higher by about eight months, higher by about nine months, higher by about ten months, higher by about eleven months, or higher by about one year).

[0126] In some aspects, administration of a combination therapy described herein (e.g., a combination of an IL-7 protein and a VEGF antagonist) can prolong the progression-free survival duration of a subject (e.g., a cancer patient). For example, compared to a reference subject, the progression-free survival of the subject is extended by at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 1 year.

[0127] In some aspects, the combination therapy described herein (e.g., a combination of IL-7 protein and a VEGF antagonist) can be used to increase the response rate of a group of subjects. For example, the response rate of a group of subjects is increased by at least about 0.5 times, at least about 1 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 40 times, or at least about 50 times compared to the corresponding value of a reference subject.

[0128] In some aspects, the combination therapy described herein (e.g., a combination of IL-7 protein and a VEGF antagonist) can be used to slow the progression of cancer. In some aspects, the progression of the disease 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 the corresponding value of a reference subject after administration.

[0129] In some aspects, administration of a combination therapy described herein (e.g., a combination of IL-7 protein and a VEGF antagonist) can reduce, for example, the expression of TOX in CD8+T cells present in tumors, tumor-draining lymph nodes, or both. In some aspects, after administration, compared to the corresponding value of a reference subject, the expression of TOX in CD8+T cells 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%. Without being bound by any theory, at least on CD8+T cells, TOX is believed to play an important role in the induction of T cell exhaustion. See, for example, Khan et al., Nature 571: 211-218 (2019). Therefore, in some aspects, the combination therapy described herein (e.g., a combination of IL-7 protein and a VEGF antagonist) can help reduce T cell exhaustion when administered to a subject.

[0130] In some aspects, the combination therapy described herein can be used to increase the number of stem cell-like T cells present in, for example, tumors, tumor-draining lymph nodes, or both. As used herein, "stem cell-like T cells" refer to T cells with a low degree of differentiation but retaining effector activity (e.g., the ability to kill tumor cells). Stem cell-like T cells can be identified by their phenotypic expression using any suitable method known in the art (e.g., flow cytometry). In some aspects, stem cell-like T cells that can be used for the present disclosure are TCF1+ and TOX-. In some aspects, stem cell-like T cells are TIM3- in addition. In some aspects, compared with the corresponding values ​​of reference subjects, after administration, stem cell-like T cell number increases by at least about 0.5 times, at least about 1 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 40 times, or at least about 50 times.

[0131] In some aspects, the subject treated in the method is a non-human animal, such as a rat or mouse. In some aspects, the subject treated in the method is a human.

[0132] As is clear from this disclosure, the combination therapies described herein (eg, a combination of an IL-7 protein and a VEGF antagonist) can be used to treat any suitable cancer known in the art.Non-limiting examples of cancers (or tumors) that can be treated with the disclosure provided herein include squamous cell carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer, squamous non-small cell lung cancer (NSCLC), non-squamous NSCLC, gastrointestinal cancer, renal cancer (e.g., clear cell carcinoma), ovarian cancer, liver cancer (e.g., hepatocellular carcinoma), colorectal cancer, endometrial cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), prostate cancer (e.g., hormone-refractory prostate adenocarcinoma), thyroid cancer, pancreatic cancer, cervical cancer, stomach cancer, bladder cancer, liver cancer, breast cancer, colon cancer, and head and neck cancer (or carcinoma), gastric cancer (e.g., ovarian cancer, liver cancer, hepatocellular carcinoma), colorectal cancer, endometrial cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), prostate cancer (e.g., hormone-refractory prostate adenocarcinoma), thyroid cancer, pancreatic cancer, cervical cancer, stomach cancer, bladder cancer, liver cancer, breast cancer, colon cancer, and head and neck cancer (or carcinoma), gastric cancer ( cancer), germ cell tumors, pediatric sarcomas, sinonasal natural killer cells, melanoma (e.g., metastatic malignant melanoma, such as cutaneous or intraocular malignant melanoma), bone cancer, skin cancer, uterine cancer, cancer of the anal region, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer (e.g., esophagogastric junction cancer), small intestine cancer, endocrine system cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, solid tumors in children, ureteral cancer, renal pelvic cancer, tumor angiogenesis, pituitary adenoma, Kaposi Sarcoma (Kaposi's sarcoma), epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers (including those induced by asbestos), virus-related cancers or cancers of viral origin (e.g. human papillomavirus (HPV-related or derived tumors)) and hematological malignancies derived from the two major blood cell lineages, namely the myeloid cell lineage (which produces granulocytes, erythrocytes, platelets, macrophages and mast cells) or the lymphocyte lineage (which produces B cells, T cells, NK cells and plasma cells), such as all types Leukemias, lymphomas and myelomas of the invention, such as acute, chronic, lymphocytic and / or myeloid leukemias, such as acute leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) and chronic myeloid leukemia (CML), undifferentiated AML (MO), myeloblastic leukemia (M1), myeloblastic leukemia (M2; with cell maturation), promyelocytic leukemia (M3 or M3 variant [M3V]), myelomonocytic leukemia (M4 or with eosinophilia), myelomonocytic leukemia (M5 or with eosinophilia), myelomonocytic leukemia (M6 or with eosinophilia), myelocytic leukemia (M7 or with eosinophilia), myelomonocytic leukemia (M8 or with eosinophilia), myeloblastic leukemia (M9 or with eosinophilia), myeloblastic leukemia (M1), myeloblastic leukemia (M2; with cell maturation), promyelocytic leukemia (M3 or M3 variant [M3V]), myelomonocytic leukemia (M4 or with eosinophilia), myelomonocytic leukemia (M5 or with eosinophilia), myelomonocytic leukemia (M6 or with eosinophilia), myelomonocytic leukemia (M8 or with eosinophilia), myeloblastic leukemia (M9 or with eosinophilia), myeloblastic leukemia (M1), myeloblastic leukemia (M2; with cell maturation), promyelocytic leukemia (M3 or M3 variant [M3V]), myelomonocytic leukemia (M4 or with eosinophilia), myelomon M4 variant [M4E]), monocytic leukemia (M5), erythroleukemia (M6), megakaryocytic leukemia (M7), isolated granulocytic sarcoma and chloroma; lymphomas, such as Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), B-cell hematological malignancies (e.g., B-cell lymphoma), T-cell lymphoma, lymphoplasmacytoid lymphoma, monocytic B-cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic (e.g., Ki1. +) large cell lymphoma, adult T-cell lymphoma / leukemia, mantle cell lymphoma, angioimmunoblastic T-cell lymphoma, angiocentric lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, precursor T-lymphoblastic lymphoma, T-lymphoblastic; and lymphoma / leukemia (T-Lbly / T-ALL), peripheral T-cell lymphoma, lymphoblastic lymphoma, post-transplant lymphoproliferative disorder, true histiocytic lymphoma, primary effusion lymphoma, B-cell lymphoma, lymphoblastic lymphoma (LBL), hematopoietic neoplasms of the lymphoid lineage, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma (Burkitt's lymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma, precursor B lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTLC) (also called mycosis fungoides or Sezary syndrome), and lymphoplasmacytoid lymphoma (LPL) with Waldenstrom's macroglobulinemia. macroglobulinemia); myelomas, such as IgG myeloma, light chain myeloma, non-secretory myeloma, smoldering myeloma (also known as indolent myeloma), solitary plasmacytoma and multiple myeloma, chronic lymphocytic leukemia (CLL), hairy cell lymphoma; hematopoietic tumors of myeloid lineage, tumors of mesenchymal origin (including fibrosarcoma and rhabdomyosarcoma); seminoma, teratocarcinoma, tumors of mesenchymal origin (including fibrosarcoma, rhabdomyoscaroma and osteosarcoma); and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, follicular thyroid carcinoma and teratocarcinoma, hematopoietic tumors of lymphoid lineage, such as T-cell tumors and B-cell tumors, including but not limited to T-cell disorders, such as T-prolymphocytic leukemia (T-PLL), including tumors of small cell and brain cell types; large granular lymphocytic leukemia (LGL) of the T-cell type; a / d T-NHL hepatosplenic lymphoma; peripheral / post-thymic T-cell lymphoma (pleomorphic and immunoblastic subtypes); angiocentric (nasal) T-cell lymphoma; head or neck cancer, kidney cancer, rectal cancer, thyroid cancer; acute myeloid lymphoma, and any combination thereof.

[0133] In some aspects, the cancer (or tumor) that can be treated includes breast cancer, head and neck cancer, uterine cancer, brain cancer, skin cancer, kidney cancer, lung cancer, colorectal cancer, prostate cancer, liver cancer, bladder cancer, kidney cancer, pancreatic cancer, thyroid cancer, esophageal cancer, eye cancer, stomach (stomach) cancer, gastrointestinal cancer, carcinoma, sarcoma, leukemia, lymphoma, myeloma or its combination. In some aspects, the cancer (tumor) that can be treated with the disclosure includes pancreatic cancer. In some aspects, the cancer (tumor) that can be treated includes brain cancer. In some aspects, brain cancer includes glioma. In some aspects, brain cancer includes glioblastoma. In some aspects, glioblastoma includes primary glioblastoma. In some aspects, glioblastoma includes secondary glioblastoma. In some aspects, the glioblastoma that can be treated with the disclosure is refractory (for example, for previous cancer therapy, for example, standard care including radiotherapy and / or chemotherapy, such as temozolomide). In some aspects, the cancer (tumor) that can be treated includes skin cancer. Non-limiting examples of skin cancer include: Merkel cell carcinoma (MCC), basal cell carcinoma (BCC), cutaneous squamous cell carcinoma (cSCC), melanoma, or a combination thereof.

[0134] In some aspects, the disclosure provided herein (e.g., treatment comprising a combination of IL-7 protein and VEGF antagonists) can also be used to treat metastatic cancer, unresectable refractory cancer (e.g., previous cancer treatment, such as cancer refractory to immunotherapy and / or standard care) and / or recurrent cancer. In some aspects, previous cancer therapy includes chemotherapy. In some aspects, chemotherapy includes platinum-based therapy. In some aspects, platinum-based therapy includes anti-tumor drugs based on platinum selected from the group consisting of: cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, satraplatin and any combination thereof. In some aspects, platinum-based therapy includes cisplatin. In some aspects, platinum-based therapy includes carboplatin.

[0135] In some aspects, the subject treated with the disclosure provided herein has received one, two, three, four, five or more previous cancer treatments. In some aspects, the subject is untreated (i.e., never received previous cancer treatment). In some aspects, the subject has made progress in other cancer treatments. In some aspects, previous cancer treatments include immunotherapy. In some aspects, previous cancer treatments include chemotherapy. In some aspects, previous cancer treatments include radiotherapy. In some aspects, previous cancer treatments include standard care, such as chemotherapy (e.g., temozolomide) and / or radiotherapy. In some aspects, the tumor has recurred. In some aspects, the tumor is metastatic. In some aspects, the tumor is non-metastatic. Therefore, as further described elsewhere in the disclosure, in some aspects, the subject treated with the disclosure suffers from cancer that is refractory to previous cancer treatments (e.g., standard care). In some aspects, the subject treated with the disclosure suffers from glioblastoma that is refractory to standard care (e.g., radiotherapy and / or temozolomide).

[0136] In some aspects, the unit dose of IL-7 protein disclosed herein (e.g., for human use) can be in the range of about 0.001 mg / kg to about 10 mg / kg. In some aspects, the unit dose of IL-7 protein is in the range of about 0.01 mg / kg to about 2 mg / kg. In some aspects, the unit dose is in the range of about 0.02 mg / kg to about 1 mg / kg.

[0137] In some aspects, the IL-7 protein disclosed herein can be administered to a subject at a dosage based on weight. In some aspects, the IL-7 protein can be administered at a dosage based on weight between about 20 μg / kg and about 600 μg / kg. In some aspects, the IL-7 protein disclosed herein can be administered at a dosage based on weight 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 aspects, the IL-7 protein is administered to a subject at a dosage of 60 μg / kg. In some aspects, the IL-7 protein is administered to a subject at a dosage of 120 μg / kg. In some aspects, the IL-7 protein is administered to a subject at a dosage of 240 μg / kg.

[0138] In some aspects, the IL-7 protein disclosed herein can be administered to a subject at a dose of greater than about 600 μg / kg. In some aspects, the IL-7 protein is administered to a 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.

[0139] In some aspects, the IL-7 protein of the present disclosure is between 610 μg / kg and about 1,200 μg / kg, between 650 μg / kg and about 1,200 μg / kg, between about 700 μg / kg and about 1,200 μg / kg, between about 750 μg / kg and about 1,200 μg / kg, between about 800 μg / kg and about 1,200 μg / kg, between about 850 μg / kg and about 1,200 μg / kg, between about 900 μg / kg and about 1,200 μg / kg, between about 950 μg / kg and about 1,200 μg / kg, between about 1,000 μg / kg and about 1,200 μg / kg, between about 1,050 μg / kg and about 1,200 μg / kg, between about 1,100 μg / kg and about 1,200 μg / kg, about 1,200 μg / kg / kg to about 2,000 μg / kg, about 1,300 μg / kg to about 2,000 μg / kg, about 1,500 μg / kg to about 2,000 μg / kg, about 1,700 μg / kg to about 2,000 μg / kg, about 610 μg / kg to about 1,000 μg / kg, about 650 μg / kg to about 1,000 μg / kg, about 7 In some embodiments, the present invention is administered at a dose of between about 100 μg / kg and about 1,000 μg / kg, between about 750 μg / kg and about 1,000 μg / kg, between about 800 μg / kg and about 1,000 μg / kg, between about 850 μg / kg and about 1,000 μg / kg, between about 900 μg / kg and about 1,000 μg / kg, or between about 950 μg / kg and about 1,000 μg / kg.

[0140] In some aspects, the IL-7 protein of the present disclosure is administered at a dose between 610 μg / kg and about 1,200 μg / kg. In some aspects, the IL-7 protein is administered at a dose between 650 μg / kg and about 1,200 μg / kg. In some aspects, the IL-7 protein is administered at a dose between about 700 μg / kg and about 1,200 μg / kg. In some aspects, the IL-7 protein is administered at a dose between about 750 μg / kg and about 1,200 μg / kg. In some aspects, the IL-7 protein is administered at a dose between about 800 μg / kg and about 1,200 μg / kg. In some aspects, the IL-7 protein is administered at a dose between about 850 μg / kg and about 1,200 μg / kg. In some aspects, the IL-7 protein is administered at a dose between about 900 μg / kg and about 1,200 μg / kg. In some aspects, the IL-7 protein is administered at a dose between about 950 μg / kg and about 1,200 μg / kg. In some aspects, the IL-7 protein disclosed herein is administered at a dose between about 1,000 μg / kg and about 1,200 μg / kg. In some aspects, the IL-7 protein is administered at a dose between about 1,050 μg / kg and about 1,200 μg / kg. In some aspects, the IL-7 protein is administered at a dose between about 1,100 μg / kg and about 1,200 μg / kg. In some aspects, the IL-7 protein is administered at a dose between about 1,200 μg / kg and about 2,000 μg / kg. In some aspects, the IL-7 protein is administered at a dose between about 1,300 μg / kg and about 2,000 μg / kg. In some aspects, the IL-7 protein is administered at a dose between about 1,500 μg / kg and about 2,000 μg / kg. In some aspects, the IL-7 protein is administered at a dose between about 1,700 μg / kg and about 2,000 μg / kg. In some aspects, the IL-7 protein is administered at a dose between about 610 μg / kg and about 1,000 μg / kg. In some aspects, the IL-7 protein is administered at a dose between about 650 μg / kg and about 1,000 μg / kg. In some aspects, the IL-7 protein is administered at a dose between about 700 μg / kg and about 1,000 μg / kg. In some aspects, the IL-7 protein is administered at a dose between about 750 μg / kg and about 1,000 μg / kg. In some aspects, the IL-7 protein is administered at a dose between about 800 μg / kg and about 1,000 μg / kg. In some aspects, the IL-7 protein is administered at a dose between about 850 μg / kg and about 1,000 μg / kg. In some aspects, the IL-7 protein of the present disclosure is administered at a dose between about 900 μg / kg and about 1,000 μg / kg. In some aspects, the IL-7 protein is administered at a dose between about 950 μg / kg and about 1,000 μg / kg.

[0141] In some aspects, 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 aspects, the IL-7 protein is administered at a dose of about 700 μg / kg to about 900 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 750 μg / kg to about 950 μg / kg. In some aspects, the IL-7 protein is administered at a dosage between about 700 μg / kg and about 850 μg / kg. In some aspects, the IL-7 protein is administered at a dosage between about 750 μg / kg and about 850 μg / kg. In some aspects, the IL-7 protein is administered at a dosage between about 700 μg / kg and about 800 μg / kg. In some aspects, the IL-7 protein is administered at a dosage between about 800 μg / kg and about 900 μg / kg. In some aspects, the IL-7 protein is administered at a dosage between about 750 μg / kg and about 850 μg / kg. In some aspects, the IL-7 protein is administered at a dosage between about 850 μg / kg and about 950 μg / kg.

[0142] In some aspects, the IL-7 protein is at about 650 μg / kg, about 680 μg / kg, 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 820 μg / kg, about 840 μg / kg, about 850 μg / kg, about 860 μg / kg, about 880 μg / kg, about 900 μg / kg, about 920 μg / kg, about 940 μg / kg, about 950 μg / kg, about 960 μg / kg, about 980 μg / kg. kg, about 1,000 μg / kg, about 1,020 μg / kg, about 1,020 μg / kg, about 1,040 μg / kg, about 1,060 μg / kg, about 1,080 μg / kg, about 1,100 μg / kg, about 1,120 μg / 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, about 1,240 μg / kg, about 1,260 μg / kg, about 1,280 μg / kg, about 1,300 μg / kg, about 1,3 20μg / kg, about 1,340μg / kg, about 1,360μg / kg, about 1,380μg / kg, about 1,400μg / kg, about 1,420μg / kg, about 1,440μg / kg, about 1,460μg / kg, about 1,480μg / kg, about 1,500μg / kg, about 1,520μg / kg, about 1,540μg / kg, about 1,560μg / kg, about 1,580μg / kg, about 1,600μg / kg, about 1,620μg / kg, about 1,640μg / kg, about 1,660μg / kg The invention relates to the administration of an amount of about 1,600 μg / kg, about 1,700 μg / 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.

[0143] In some aspects, the IL-7 protein is administered at a dose of about 650 μg / kg. In some aspects, the IL-7 protein disclosed herein is administered at a dose of about 680 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 700 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 720 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 740 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 750 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 760 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 780 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 800 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 820 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 840 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 850 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 860 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 880 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 900 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 920 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 940 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 950 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 960 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 980 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 1,000 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 1,020 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 1,040 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 1,060 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 1,080 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 1,100 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 1,120 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 1,140 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 1,160 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 1,180 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 1,200 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 1,220 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 1,240 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 1,260 μg / kg.In some aspects, the IL-7 protein is administered at a dose of about 1,280 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 1,300 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 1,320 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 1,340 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 1,360 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 1,380 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 1,400 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 1,420 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 1,440 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 1,460 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 1,480 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 1,500 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 1,520 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 1,540 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 1,560 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 1,580 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 1,600 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 1,620 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 1,640 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 1,660 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 1,680 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 1,700 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 1,720 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 1,740 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 1,760 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 1,780 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 1,800 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 1,820 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 1,840 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 1,860 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 1,880 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 1,900 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 1,920 μg / kg.In some aspects, the IL-7 protein is administered at a dose of about 1,940 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 1,960 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 1,980 μg / kg. In some aspects, the IL-7 protein is administered at a dose of about 2,000 μg / kg.

[0144] In some aspects, the IL-7 protein can be administered at a fixed dose. In some aspects, the IL-7 protein can be administered at a fixed dose of about 0.25 mg to about 9 mg. In some aspects, 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.

[0145] In some aspects, the subject disclosed herein receives a single administration of any of the above-mentioned doses of IL-7 protein. In some aspects, the IL-7 protein disclosed herein is administered to the subject in multiple doses (i.e., repeated administration). In some aspects, the subject is administered IL-7 protein at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, or at least ten times or more.

[0146] In the case where a subject (e.g., a subject with a tumor) receives multiple IL-7 protein administrations, in some aspects, the IL-7 protein is administered at a frequency of about once a week, about once every two weeks, about once every three weeks, about once every four weeks, about once every five weeks, about once every six weeks, about once every seven weeks, about once every eight weeks, about once every nine weeks, about once every 10 weeks, about once every 11 weeks, or about once every 12 weeks, for example, with one of the above-mentioned dosages. In some aspects, the IL-7 protein is administered at a frequency of about once every 10 days, about once every 20 days, about once every 30 days, about once every 40 days, about once every 50 days, about once every 60 days, about once every 70 days, about once every 80 days, about once every 90 days, or about once every 100 days. In some aspects, the IL-7 protein is administered once in three weeks. In some aspects, the IL-7 protein is administered once a week. In some aspects, the IL-7 protein is administered once in two weeks. In some aspects, the IL-7 protein is administered once around four weeks. In some aspects, the IL-7 protein is administered once in six weeks. In some aspects, the IL-7 protein is administered once every eight weeks. In some aspects, the IL-7 protein is administered once every nine weeks. In some aspects, the IL-7 protein is administered once every 12 weeks. In some aspects, the IL-7 protein is administered once every 10 days. In some aspects, the IL-7 protein is administered once every 20 days. In some aspects, the IL-7 protein is administered once every 30 days. In some aspects, the IL-7 protein is administered once every 40 days. In some aspects, the IL-7 protein is administered once every 50 days. In some aspects, the IL-7 protein is administered once every 60 days. In some aspects, the IL-7 protein is administered once every 70 days. In some aspects, the IL-7 protein is administered once every 80 days. In some aspects, the IL-7 protein is administered once every 90 days. In some aspects, the IL-7 protein is administered once every 100 days. As described herein, in some aspects, the IL-7 protein is administered at a dosage of about 1,200 μg / kg and a dosing interval of about 8 weeks.

[0147] In some aspects, VEGF antagonists (e.g., in combination with IL-7 proteins as described herein) can be administered to a subject at any suitable dose and / or dosing frequency to treat a tumor as described herein. In some aspects, a VEGF antagonist is administered to a subject at a dose of about 0.001 mg / kg to about 20 mg / kg. In some aspects, a VEGF antagonist is administered at a dose of about 1 mg / kg to about 20 mg / kg. In some aspects, a VEGF antagonist (e.g., bevacizumab) is administered at a dose of about 5 mg / kg to about 15 mg / kg. In some aspects, a VEGF antagonist (e.g., bevacizumab) is administered at a dose of about 5 mg / kg, about 7.5 mg / kg, about 10 mg / kg, about 12.5 mg / kg, or about 15 mg / kg. In some aspects, a VEGF antagonist is administered to a subject at a dose of about 5 mg / kg. In some aspects, a VEGF antagonist is administered to a subject at a dose of about 7.5 mg / kg. In some aspects, the VEGF antagonist is administered to the subject at a dose of about 10 mg / kg. In some aspects, the VEGF antagonist is administered to the subject at a dose of about 12.5 mg / kg. In some aspects, the VEGF antagonist is administered to the subject at a dose of about 15 mg / kg. In some aspects, the VEGF antagonist (e.g., aflibercept) is administered to the subject at a dose of about 4 mg / kg to about 15 mg / kg. In some aspects, the VEGF antagonist (e.g., aflibercept) is administered to the subject at a dose of about 4 mg / kg.

[0148] In some aspects, the subject disclosed herein receives a single administration of any dose of a VEGF antagonist (e.g., in combination with an IL-7 protein) as described above. In some aspects, the VEGF antagonist disclosed herein is administered to a subject (e.g., administered alone or in combination with an IL-7 protein) at multiple doses (i.e., repeated administration). In some aspects, a VEGF antagonist is administered to a subject at least about twice, 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.

[0149] In the case where a subject (e.g., a subject with a tumor) receives multiple VEGF antagonists (e.g., alone or in combination with an IL-7 protein), in some aspects, the VEGF antagonist is administered at a dosage of about once a day, about once every two days, about once every three days, about once every four days, about once every five days, about once every six days, about once a week, about once every two weeks, about once every three weeks, about once every four weeks, about once every five weeks, about once every six weeks, about once every seven weeks, about once every eight weeks, about once every nine weeks, about once every 10 weeks, about once every 11 weeks, or about once every 12 weeks, for example, at one of the doses described herein. As described herein, in some aspects, a VEGF antagonist (e.g., bevacizumab) is administered at a dosage of about 10 mg / kg and a dosing interval of about 2 weeks.

[0150] Thus, in some aspects, a subject described herein (e.g., having a tumor) receives a single dose of an IL-7 protein (e.g., one of the doses described herein) and a single dose of a VEGF antagonist (e.g., one of the doses described herein). In these aspects, the IL-7 protein and the VEGF antagonist can be administered to the subject simultaneously. For example, in some aspects, the IL-7 protein and the VEGF antagonist can be administered simultaneously as a single composition in a pharmaceutically acceptable carrier. In some aspects, the IL-7 protein and the VEGF antagonist can be administered simultaneously as separate compositions. In some aspects, the IL-7 protein and the VEGF antagonist are administered to the subject sequentially. In the case of sequentially administering IL-7 protein and VEGF antagonist to a subject, in some aspects, the duration between administering IL-7 protein and administering VEGF antagonist is at least about one day, at least about two days, at least about three days, at least about four days, at least about five days, at least about six days, at least about one week, at least about two weeks, at least about three weeks, at least about four weeks, at least about five weeks, at least about six weeks, at least about seven weeks, at least about eight weeks, at least about nine weeks, at least about ten weeks, at least about 11 weeks, at least about 12 weeks or longer. In some aspects, IL-7 protein is administered to a subject before administering VEGF antagonist. In some aspects, IL-7 protein is administered to a subject after administering VEGF antagonist.

[0151] In some aspects, the subject described herein (e.g., having a tumor) receives a single dose of an IL-7 protein (e.g., one of the doses described herein) and multiple doses (e.g., at least about 2, at least about 3, or at least about 4 doses) of a VEGF antagonist. In some aspects, the subject described herein (e.g., having a tumor) receives multiple doses of an IL-7 protein (e.g., one of the doses described herein) and a single dose of a VEGF antagonist (e.g., one of the doses described herein). In some aspects, the subject described herein (e.g., having a tumor) receives multiple doses of an IL-7 protein (e.g., one of the doses described herein) and multiple doses of a VEGF antagonist (e.g., one of the doses described herein).

[0152] In the case of administering multiple doses of IL-7 protein and / or VEGF antagonist to a subject, in some aspects, at least one dose of IL-7 protein and at least one dose of VEGF antagonist are administered to the subject simultaneously (e.g., as a single composition or two separate compositions). In some aspects, each of the multiple doses of IL-7 protein and each of the multiple doses of VEGF antagonist are administered to the subject simultaneously (e.g., as a single composition or two separate compositions). In some aspects, one or more of the multiple doses of IL-7 protein and one or more of the multiple doses of VEGF antagonist are administered to the subject sequentially. In some aspects, (i) at least one of the multiple doses of IL-7 protein and at least one of the multiple doses of VEGF antagonist are administered simultaneously (e.g., as a single composition or two separate compositions), and (ii) at least one of the multiple doses of IL-7 protein and at least one of the multiple doses of VEGF antagonist are administered sequentially (e.g., on separate days).

[0153] It is clear from the present disclosure that in some aspects, the subject receives multiple cycles of the treatment regimen described herein (combination of IL-7 protein and VEGF antagonist). In some aspects, the subject receives at least about two, at least about three, at least about four, at least about five, at least about six, at least about seven, at least about eight, at least about nine, at least about 10 cycles of treatment regimen. In some aspects, the subject receives two cycles of treatment regimen. In some aspects, the subject receives three cycles of treatment regimen. In some aspects, the subject receives four cycles of treatment regimen. In some aspects, the subject receives five cycles of treatment regimen. In some aspects, the subject receives six cycles of treatment regimen. In some aspects, the subject receives seven cycles of treatment regimen. In some aspects, the subject receives eight cycles of treatment regimen. In some aspects, the subject receives nine cycles of treatment regimen. In some aspects, the subject receives 10 cycles of treatment regimen. In the case of applying multiple cycles of treatment regimen to the subject, in some aspects, each treatment cycle is the same. In some aspects, one or more treatment cycles are different (e.g., different dosages and / or dosing intervals).

[0154] In some aspects, each treatment cycle is about one week, about two weeks, about three weeks, about four weeks, about five weeks, about six weeks, about seven weeks, about eight weeks, about nine weeks, about 10 weeks, about 11 weeks or about 12 weeks long. As described herein, in some aspects, each treatment cycle is about 8 weeks long. Unless otherwise indicated, each treatment cycle includes at least one dose of IL-7 protein and at least one dose of VEGF antagonist. In some aspects, each treatment cycle includes one dose of IL-7 protein and one dose of VEGF antagonist. In some aspects, each treatment cycle includes one dose of IL-7 protein and two doses of VEGF antagonist. In some aspects, each treatment cycle includes one dose of IL-7 protein and three doses of VEGF antagonist. In some aspects, each treatment cycle includes one dose of IL-7 protein and four doses of VEGF antagonist. In some aspects, each treatment cycle includes one dose of IL-7 protein and five doses of VEGF antagonist. As demonstrated herein (see, e.g., Example 3), in some aspects, each treatment cycle includes one dose of IL-7 protein and four doses of VEGF antagonist, administered at a two-week dosing interval. For example, in some aspects, an IL-7 protein and a VEGF antagonist are administered to a subject on day 1 of a treatment cycle, and then additional doses of a VEGF antagonist are administered on days 15, 29, and 43 of the treatment cycle.

[0155] In some aspects, the combination therapy of IL-7 protein and VEGF antagonist described herein can be used together with one or more additional therapeutic agents. Non-limiting examples of such additional therapeutic agents include: chemotherapeutic drugs, small molecule drugs, radiation, antibodies that stimulate immune responses to a given cancer, or a combination thereof. Therefore, in some aspects, there is provided herein a method for treating a tumor in a subject in need thereof, comprising administering to the subject a combination of IL-7 protein with a VEGF antagonist and an additional therapeutic agent. Non-limiting examples of such combinations may include: therapies that enhance tumor antigen presentation (e.g., dendritic cell vaccines, cellular vaccines that secrete GM-CSF, CpG oligonucleotides, imiquimod); therapies that inhibit negative immune regulation, such as by inhibiting the CTLA-4 and / or PD1 / PD-L1 / PD-L2 pathways and / or depleting or blocking Treg or other immunosuppressive cells (e.g., myeloid-derived suppressor cells); therapies that stimulate positive immune regulation, such as with agonists that stimulate the CD-137, OX-40 and / or CD40 or GITR pathways and / or stimulate T cell effector function; therapies that systemically increase the frequency of anti-tumor T cells; therapies that, for example, use antagonists of CD25 (e.g., daclizumab); depleting or inhibiting Tregs, such as Tregs in tumors, by anti-CD25 (daclizumab) or by ex vivo anti-CD25 bead depletion; therapies that affect the function of suppressive myeloid cells in tumors; therapies that enhance the immunogenicity of tumor cells (e.g., anthracyclines); adoptive T cell or NK cell transfer, including genetically modified cells, such as cells modified by chimeric antigen receptors (CAR-T therapy); therapies that inhibit metabolic enzymes, such as indoleamine dioxidase (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 immunostimulatory cytokines; or blockade of immunosuppressive cytokines.

[0156] In some aspects, additional therapeutic agents that can be used with the combination therapy of IL-7 protein and VEGF antagonists include immune checkpoint inhibitors (i.e., blocking signal transduction through specific immune checkpoint pathways). Therefore, in some aspects, the present disclosure provides a method for treating a tumor in a subject in need, comprising administering a combination of IL-7 protein and VEGF antagonists and immune checkpoint inhibitors to the subject. Non-limiting examples of immune checkpoint inhibitors that can be used for the present method include CTLA-4 antagonists (e.g., anti-CTLA-4 antibodies), PD-1 antagonists (e.g., anti-PD-1 antibodies, anti-PD-L1 antibodies), TIM-3 antagonists (e.g., anti-TIM-3 antibodies) or combinations thereof. Non-limiting examples of such immune checkpoint inhibitors are known in the art. For example, suitable anti-PD-1 antagonists are described in, e.g., U.S. Pat. Nos. 6,808,710; 7,488,802; 8,008,449; 8,168,757; 8,354,509; 8,609,089; 8,779,105; and 9,217,034; and U.S. Pat. Nos. 7,635,757; 7,943,743; 8,217,149; 8,981,063; and 9,624,298, each of which is incorporated herein by reference in its entirety. Suitable anti-CTLA-4 antagonists are described in, for example, 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. WO 2007 / 113648, each of which is incorporated herein by reference in its entirety. Suitable TIM-3 antagonists are described in, for example, U.S. Patent Nos. 10,508,149; 10,533,052; 10,894,830, each of which is incorporated herein by reference in its entirety.

[0157] In some aspects, additional therapeutic agents that can be used include immune checkpoint activators (i.e., promoting signaling through specific immune checkpoint pathways). In some aspects, immune checkpoint activators include OX40 agonists (e.g., anti-OX40 antibodies), LAG-3 agonists (e.g., anti-LAG-3 antibodies), 4-1BB (CD137) agonists (e.g., anti-CD137 antibodies), GITR agonists (e.g., anti-GITR antibodies) or any combination thereof.

[0158] In some aspects, the treatment regimens provided herein (combination of IL-7 protein and VEGF antagonist) are not used in combination with one or more other anti-cancer treatments. Thus, in some aspects, subjects being treated with a combination of IL-7 protein and VEGF antagonist do not receive one or more of the following anti-cancer treatments: (1) any concomitant therapy approved or experimental for the treatment of cancer, including chemotherapy, hormone therapy, immunotherapy, radiotherapy, research drugs or herbal treatments; (2) immunosuppressants, including but not limited to cyclophosphamide, azathioprine, methotrexate and thalidomide; (3) granulocyte colony stimulating factor and granulocyte macrophage colony stimulating factor (e.g., pegfilgrastim); and (4) immunostimulants (e.g., IFN-α, IFN-γ and IL-2).

[0159] II.A. IL-7 Proteins Useful in the Disclosure

[0160] Disclosed herein is an IL-7 protein that can be used in combination with a VEGF antagonist for the treatment of cancer (or tumor). In some aspects, the IL-7 protein that can be used for the purposes of the present invention can be a wild-type IL-7 or a modified IL-7 (i.e., a non-wild-type IL-7 protein) (e.g., an IL-7 variant, an IL-7 functional fragment, an IL-7 derivative, or any combination thereof, such as a fusion protein, a chimeric protein, etc.), as long as the IL-7 protein contains one or more biological activities of IL-7, such as being able to bind to IL-7R, such as inducing early T cell development and promoting T cell homeostasis. See ElKassar and Gress. J Immunotoxicol. March 2010; 7(1): 1–7. In some aspects, the IL-7 protein of the present disclosure is not a wild-type IL-7 protein (i.e., comprising one or more modifications). Non-limiting examples of such modifications may include oligopeptides and / or half-life extension moieties. See WO 2016 / 200219, which is incorporated herein by reference in its entirety.

[0161] IL-7 binds to its receptor, which consists of two chains IL-7Rα (CD127) shared with thymic stromal lymphopoietin (TSLP) (Ziegler and Liu, 2006), and a common γ chain (CD132) for IL-2, IL-15, IL-9 and IL-21. γc is expressed by most hematopoietic cells, while IL-7Rα is almost exclusively expressed on lymphoid cells. After binding to its receptor, IL-7 signals through two different pathways: Jak-Stat (Janus kinase signal transducer and activator of transcription) and PI3K / Akt, which are responsible for differentiation and survival, respectively. The absence of IL-7 signaling leads to a decrease in thymocytes, as observed in mice that have received 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), probably because TSLP signaling is also abolished 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).

[0162] In some aspects, the IL-7 protein useful in the present disclosure comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 1 to 6. In some aspects, the 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 sequences of SEQ ID NOs: 1 to 6. In some aspects, the IL-7 protein comprises an amino acid sequence having at least 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% sequence identity to the sequence shown in SEQ ID NO: 1. In some aspects, the IL-7 protein comprises an amino acid sequence that is at least 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% identical to the sequence shown in SEQ ID NO: 2. In some aspects, the IL-7 protein comprises an amino acid sequence that is at least 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% identical to the sequence shown in SEQ ID NO: 3. In some aspects, the IL-7 protein comprises an amino acid sequence that is at least 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% identical to the sequence shown in SEQ ID NO: 4. In some aspects, the IL-7 protein comprises an amino acid sequence that is at least 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% identical to the sequence shown in SEQ ID NO: 5. In some aspects, the IL-7 protein comprises an amino acid sequence that is at least 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% identical to the sequence shown in SEQ ID NO:6.

[0163] In some aspects, the IL-7 protein includes 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. Therefore, in some aspects, the IL-7 protein that can be used in the present disclosure comprises a fragment of the amino acid sequence shown in any one of SEQ ID NO: 1-6, wherein the fragment retains one or more biological activities of the wild-type IL-7 protein. In some aspects, the IL-7 protein can be derived from humans, rats, mice, monkeys, cows or sheep.

[0164] In some aspects, human IL-7 may have an amino acid sequence represented by SEQ ID NO: 1 (Genbank Accession No. P13232); rat IL-7 may have an amino acid sequence represented by SEQ ID NO: 2 (Genbank Accession No. P56478); mouse IL-7 may have an amino acid sequence represented by SEQ ID NO: 3 (Genbank Accession No. P10168); monkey IL-7 may have an amino acid sequence represented by SEQ ID NO: 4 (Genbank Accession No. NP 001279008); cow IL-7 may have an amino acid sequence represented by SEQ ID NO: 5 (Genbank Accession No. P26895), and sheep IL-7 may have an amino acid sequence represented by SEQ ID NO: 6 (Genbank Accession No. Q28540). It will be apparent to those skilled in the art that the IL-7 protein provided in any of SEQ ID NOs: 1-6 has a single peptide, but the mature form of the IL-7 protein does not include a signal peptide. Therefore, it is clear from this disclosure that in some aspects, the IL-7 proteins that can be used with the methods provided herein lack a signal peptide. The amino acid sequences of such exemplary IL-7 proteins are provided in SEQ ID NOs: 80 to 85 (see also Table 1A).

[0165] Thus, in some aspects, the IL-7 proteins useful for the present disclosure comprise 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 compared to the amino acid sequence shown in any one of SEQ ID NOs: 80 to 85. In some aspects, the IL-7 protein comprises an amino acid sequence having at least 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% sequence identity to the sequence shown in SEQ ID NO: 80. In some aspects, the IL-7 protein comprises the amino acid sequence shown in SEQ ID NO: 80. In some aspects, the IL-7 protein comprises an amino acid sequence that is at least 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% identical to the sequence shown in SEQ ID NO: 81. In some aspects, the IL-7 protein comprises the amino acid sequence shown in SEQ ID NO: 81. In some aspects, the IL-7 protein comprises an amino acid sequence that is at least 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% identical to the sequence shown in SEQ ID NO: 82. In some aspects, the IL-7 protein comprises the amino acid sequence shown in SEQ ID NO: 82. In some aspects, the IL-7 protein comprises an amino acid sequence that is at least 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% identical to the sequence shown in SEQ ID NO: 83. In some aspects, the IL-7 protein comprises the amino acid sequence shown in SEQ ID NO. 83. In some aspects, the IL-7 protein comprises an amino acid sequence that is at least 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% identical to the sequence shown in SEQ ID NO: 84. In some aspects, the IL-7 protein comprises the amino acid sequence shown in SEQ ID NO: 84. In some aspects, the IL-7 protein comprises an amino acid sequence that is at least 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% identical to the sequence shown in SEQ ID NO:85.In some aspects, the IL-7 protein comprises the amino acid sequence shown in SEQ ID NO:85.

[0166] In some aspects, the IL-7 protein that can be used in the present disclosure includes an IL-7 fusion protein. In some aspects, the IL-7 fusion protein comprises (i) an oligopeptide and (i) IL-7 or a variant thereof (e.g., an IL-7 protein shown in any one of SEQ ID NOs: 1-6 and 80-85). In some aspects, the oligopeptide is connected to the N-terminal region of IL-7 or a variant thereof.

[0167] In some aspects, the oligopeptides disclosed herein are composed of 1 to 10 amino acids. In some aspects, the oligopeptides are composed 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 aspects, one or more amino acids of the oligopeptides are selected from the group consisting of methionine, glycine and a combination thereof. In some aspects, the oligopeptides 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-methionine-glycine, glycine-methionine-glycine, glycine-glycine-methionine and glycine-glycine-glycine. In some aspects, the oligopeptides are methionine-glycine-methionine.

[0168] In some aspects, the IL-7 fusion protein comprises (i) IL-7 or its variant, and (ii) a half-life extension portion. In some aspects, the half-life extension portion extends the half-life of IL-7 or its variant. In some aspects, the half-life extension portion is connected to the C-terminal region of IL-7 or its variant.

[0169] In some aspects, the IL-7 fusion protein comprises: (i) IL-7 (first domain); (ii) a second domain comprising an amino acid sequence having 1 to 10 amino acid residues consisting of methionine, glycine, or a combination thereof (e.g., MGM); and (iii) a third domain comprising a half-life extending portion. In some aspects, the half-life extending portion can be connected to the N-terminus or C-terminus of the first domain or the second domain. In addition, the IL-7 comprising the first domain and the second domain can be connected to both ends of the third domain.

[0170] Non-limiting examples of half-life extending moieties include Fc, albumin, albumin binding polypeptides, Pro / Ala / Ser (PAS), C-terminal peptide (CTP) of the beta subunit of human chorionic gonadotropin, polyethylene glycol (PEG), long unstructured hydrophilic amino acid sequences (XTEN), hydroxyethyl starch (HES), albumin binding small molecules, and combinations thereof.

[0171] In some aspects, the half-life extension part is Fc. In some aspects, Fc is from a modified immunoglobulin, wherein antibody-dependent cellular toxicity (ADCC) or complement-dependent cellular toxicity (CDC) is weakened due to the modification of the binding affinity with Fc receptors and / or complement. In some aspects, the modified immunoglobulin can be selected from the group consisting of: IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE and a combination thereof. In some aspects, Fc is a hybrid Fc ("hFc" or "hyFc"), which includes a hinge region, a CH2 domain and a CH3 domain. In some aspects, the hinge region of the hybrid Fc disclosed herein includes a human IgD hinge region. In some aspects, the CH2 domain of the hybrid Fc includes a part of the human IgD CH2 domain and a part of the human IgG4 CH2 domain. In some aspects, the CH3 domain of the hybrid Fc includes a part of the human IgG4 CH3 domain. Thus, in some aspects, a 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, wherein the CH2 domain comprises a portion of a human IgD CH2 domain and a portion of a human IgG4 CH2 domain, and wherein the CH3 domain comprises a portion of a human IgG4 CH3 domain.

[0172] In some aspects, the Fc disclosed herein can be an Fc variant. As used herein, the term "Fc variant" refers to an Fc prepared by replacing a portion of amino acids in the Fc region or by combining different types of Fc regions. Fc region variants can prevent being cut off at the hinge region. Specifically, in some aspects, the Fc variant comprises a modification of the 144th amino acid and / or the 145th amino acid of SEQ ID NO:9. In some aspects, the 144th amino acid (K) and / or the 145th amino acid (K) are replaced by G or S.

[0173] In some aspects, the Fc or Fc variant disclosed herein can be represented by the following formula: N'-(Z1)p-Y-Z2-Z3-Z4-C, wherein:

[0174] N' includes the N terminus;

[0175] Z1 comprises an amino acid sequence having 5 to 9 consecutive amino acid residues from the amino acid residue at position 98 to the N-terminus among the amino acid residues at positions 90 to 98 of SEQ ID NO:7;

[0176] Y comprises an amino acid sequence having 5 to 64 consecutive amino acid residues from the amino acid residue at position 162 to the N-terminus among the amino acid residues at positions 99 to 162 of SEQ ID NO:7;

[0177] Z2 comprises an amino acid sequence having 4 to 37 consecutive amino acid residues from the amino acid residue at position 163 to the C-terminus among the amino acid residues at positions 163 to 199 of SEQ ID NO:7;

[0178] Z3 comprises an amino acid sequence having 71 to 106 consecutive amino acid residues from the amino acid residue at position 220 to the N-terminus among the amino acid residues at positions 115 to 220 of SEQ ID NO:8; and

[0179] Z4 comprises an amino acid sequence having 80 to 107 consecutive amino acid residues from the amino acid residue at position 221 to the C-terminus among the amino acid residues at positions 221 to 327 of SEQ ID NO:8.

[0180] In some aspects, 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 aspects, the Fc region may comprise the amino acid sequence of SEQ ID NO: 14 (non-lytic mouse Fc).

[0181] Other non-limiting examples of Fc regions that can be used with the present disclosure are described in U.S. Pat. No. 7,867,491, which is herein incorporated by reference in its entirety.

[0182] In some aspects, the IL-7 fusion proteins disclosed herein comprise both an oligopeptide and a half-life extending moiety.

[0183] In some aspects, the IL-7 protein can be fused with albumin, its variant or fragment. Examples of IL-7-albumin fusion proteins can be found in International Application Publication No. WO 2011 / 124718 A1. In some aspects, the IL-7 protein is optionally fused with a progenitor B cell growth stimulating factor (PPBSF) via a flexible linker. See US2002 / 0058791A1. In some aspects, the IL-7 protein that can be used in the present disclosure is an IL-7 conformer with a specific three-dimensional structure. See US 2005 / 0249701A1. In some aspects, the IL-7 protein can be fused with an Ig chain, wherein amino acid residues 70 and 91 in the IL-7 protein are glycosylated, and amino acid residue 116 in the IL-7 protein is not glycosylated. See US 7,323,549 B2. In some aspects, an IL-7 protein that does not contain a potential T cell epitope (thereby reducing anti-IL-7 T cell response) can also be used in the present disclosure. See US2006 / 0141581 A1. In some aspects, IL-7 proteins having one or more amino acid residue mutations in the carboxyl terminal helix D region can be used in the present disclosure. Despite having a lower binding affinity to the receptor, the IL-7 mutants can still act as IL-7R partial agonists. See US2005 / 0054054A1. Any IL-7 protein described in the patents or publications listed above is incorporated herein by reference in its entirety.

[0184] In addition, non-limiting examples of additional IL-7 proteins that can be used in the present disclosure are described in: US 7708985; US 8034327; US 8153114; US 7589179; US 7323549; US 7960514; US 8338575; US 7118754; US 7488482; US 7670607; US 6730512; WO0017362; GB2434578A; WO 2010 / 020766 A2; WO91 / 01143; Beq et al., Blood, Vol. 114(4), 816, July 23, 2009; Kang et al., J. Virol. Doi: 10.1128 / JVI.02768-15; Martin et al., Blood, Vol. 121(22), 4484, May 30, 2013; McBride et al., Acta Oncologica, 34:3, 447-451, July 8, 2009; and Xu et al., Cancer Science, 109:279-288, 2018, the references are incorporated herein by reference in their entirety.

[0185] In some aspects, the oligopeptide disclosed herein is directly connected to the N-terminal region of IL-7 or its variant. In some aspects, the oligopeptide is connected to the N-terminal region through a linker. In some aspects, the half-life extension portion disclosed herein is directly connected to the C-terminal region of IL-7 or its variant. In some aspects, the half-life extension portion is connected to the C-terminal region through a linker. In some aspects, the linker is a peptide linker. In some aspects, the peptide linker comprises a peptide of 10 to 20 amino acid residues composed of Gly and Ser residues. In some aspects, the linker is an albumin linker. In some aspects, the linker is a chemical bond. In some aspects, the chemical bond includes a disulfide bond, a diamine bond, a sulfide-amine bond, a carboxyl-amine bond, an ester bond, a covalent bond or a combination thereof. When the linker is a peptide linker, in some aspects, the connection can occur in any connection region. They can be coupled using cross-linking agents known in the art. In some aspects, examples of cross-linking agents may include N-hydroxysuccinimide esters, such as 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, and 4-azidosalicylic acid; imide esters, including disuccinimide esters, such as 3,3'-dithiobis(succinimidyl propionate), and difunctional maleimides, such as bis-N-maleimido-1,8-octane, but are not limited thereto.

[0186] In some aspects, the IL-7 (or variant thereof) portion of the IL-7 fusion protein disclosed herein comprises an amino acid sequence having 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% identity to the amino acid sequence shown in SEQ ID NO: 15-20. In some aspects, the IL-7 (or variant thereof) portion of the IL-7 fusion protein disclosed herein comprises an amino acid sequence shown in SEQ ID NO: 15-20.

[0187] In some aspects, the IL-7 fusion protein comprises: a first domain comprising a polypeptide having IL-7 activity 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 coupled to the C-terminus of the first domain, which is a modified immunoglobulin Fc region.

[0188] In some aspects, the IL-7 fusion protein that can be used with the present method comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98%, or at least 99% identical to the amino acid sequence shown in SEQ ID NO: 21-25. In some aspects, the IL-7 fusion protein of the present disclosure comprises the amino acid sequence shown in SEQ ID NO: 21-25. In some aspects, the IL-7 fusion protein disclosed herein comprises the amino acid sequence shown in SEQ ID NO: 26 and 27.

[0189] Unless otherwise indicated, the IL-7 proteins described herein (including fusion proteins) can be administered to a subject using any suitable route of administration (e.g., in combination with a VEGF antagonist). In some aspects, the IL-7 protein is administered to a subject intravenously, parenterally, intramuscularly, subcutaneously, ocularly, intraperitoneally, intradermally, intraorbitally, intracerebrally, intracranially, intraspinally, intraventricularly, intrathecally, intracisternalally, intracapsules, or intratumors. In some aspects, the IL-7 protein is administered intramuscularly to a subject.

[0190] II.B. VEGF antagonists

[0191] In some aspects, the present disclosure provides a method for treating a tumor in a subject in need thereof, comprising administering to the subject a combination of an IL-7 protein and a VEGF antagonist. As used herein, the term "VEGF antagonist" refers to any molecule that can neutralize, block, inhibit, eliminate, reduce and / or interfere with VEGF activity, including its binding to one or more VEGF receptors. VEGF antagonists include molecules that interfere with the interaction between VEGF and natural VEGF receptors, such as molecules that bind to VEGF or VEGF receptors and prevent or otherwise hinder the interaction between VEGF and VEGF receptors. Therefore, in some aspects, the VEGF antagonists described herein can specifically target VEGF itself. In some aspects, the VEGF antagonists described herein specifically target one or more VEGF receptors.

[0192] Unless otherwise indicated, the VEGF antagonists useful in the present disclosure may be proteins, peptides, nucleic acids, small molecules, or combinations thereof. In some aspects, the VEGF antagonists useful in the present disclosure include antisense oligonucleotides, siRNA, shRNA, miRNA, dsRNA, aptamers, PNA (peptide nucleic acid) or vectors comprising them that target VEGF. In some aspects, the VEGF antagonists include antibodies or antigen-binding fragments thereof that specifically bind to VEGF or one or more VEGF receptors (VEGFR) ("anti-VEGF antibodies" and "anti-VEGFR antibodies," respectively), polynucleotides encoding anti-VEGF antibodies and / or anti-VEGFR antibodies, or vectors comprising their polynucleotides.

[0193] Non-limiting examples of suitable VEGF antagonists are known in the art and include, for example, aflibercept ( and ), bevacizumab ( and ), Ranibizumab Ramucirumab Sorafenib Sunitinib Pazopanib Dasatinib Regorafenib Cabozantinib Lenvatinib Ponatinib Axitinib Tivozanib Vandetanib or any combination thereof.

[0194] In some aspects, the VEGF antagonist that can be used in the present disclosure is an anti-VEGF antibody. In some aspects, the VEGF antagonist is bevacizumab, for example, as described in U.S. Publication No. 2015 / 0147317, which is incorporated herein by reference in its entirety. In some aspects, the VEGF antagonist is a variant of bevacizumab. For example, in some aspects, this variant cross-competes with bevacizumab. In some aspects, bevacizumab variants bind to the same epitope as bevacizumab. In some aspects, bevacizumab variants have the same CDR as bevacizumab. Therefore, in some aspects, the methods provided herein (e.g., treating tumors) include administering a combination of IL-7 protein and bevacizumab (or its variants) to a subject in need. For these aspects, bevacizumab (or its variants) can be administered to a subject at any suitable dose known in the art (e.g., a dose approved by the FDA), such as a dose between about 5 mg / kg and about 15 mg / kg. For example, in some aspects, bevacizumab (or its variant) is administered in combination with IL-7 protein at a dosage of about 5mg / kg, about 7.5mg / kg, about 10mg / kg, about 12.5mg / kg or about 15mg / kg. In some aspects, bevacizumab (or its variant) is administered in combination with IL-7 protein at a dosage of about 10mg / kg. As described herein, in some aspects, this method also includes administering another therapeutic agent (e.g., immune checkpoint inhibitor) to the subject.

[0195] In some aspects, the VEGF antagonist that can be used in the present disclosure is aflibercept, for example, as described in U.S. Patent No. 11,135,266, which is incorporated herein by reference in its entirety. In some aspects, the VEGF antagonist is a variant of aflibercept. In some aspects, the aflibercept variant cross-competes with aflibercept. In some aspects, aflibercept binds to the same epitope as aflibercept. In some aspects, the aflibercept variant has the same CDR as aflibercept. Therefore, in some aspects, the methods provided herein (e.g., treating tumors) include administering a combination of IL-7 protein and aflibercept (or its variant) to a subject in need. For these aspects, aflibercept (or its variant) can be administered to a subject at any suitable dose known in the art (e.g., an FDA-approved dose), such as a dose between about 4 mg / kg and about 15 mg / kg. For example, in some aspects, aflibercept (or its variant) is administered in combination with IL-7 protein at a dose of about 4 mg / kg. As described herein, in some aspects, the method can further comprise administering to the subject an additional therapeutic agent (e.g., an immune checkpoint inhibitor).

[0196] In some aspects, the VEGF antagonist that can be used with the present disclosure is ranibizumab, for example, as described in U.S. Patent No. 7,169,901, which is incorporated herein by reference in its entirety. In some aspects, the VEGF antagonist is a variant of ranibizumab. In some aspects, the ranibizumab variant crosses competition with ranibizumab. In some aspects, the ranibizumab variant binds to the same epitope as ranibizumab. In some aspects, the ranibizumab variant has the same CDR as ranibizumab. Therefore, in some aspects, the method provided herein (e.g., treating tumors) includes administering a combination of IL-7 protein and ranibizumab (or its variant) to a subject in need. For these aspects, ranibizumab (or its variant) can be administered to a subject at any suitable dose known in the art (e.g., FDA-approved dose). In some aspects, this method also includes administering another therapeutic agent (e.g., immune checkpoint inhibitor) to the subject.

[0197] In some aspects, the VEGF antagonist is Ramucirumab, for example, as described in U.S. Patent No. 8,057,791, which is incorporated herein by reference in its entirety. In some aspects, the VEGF antagonist is a variant of Ramucirumab. In some aspects, Ramucirumab variants cross-compete with Ramucirumab. In some aspects, Ramucirumab variants bind to the same epitope as Ramucirumab. In some aspects, Ramucirumab has the same CDR as Ramucirumab. Therefore, in some aspects, the method provided herein (e.g., treating tumors) includes administering a combination of IL-7 protein and Ramucirumab (or its variants) to a subject in need. For such aspects, Ramucirumab (or its variants) can be administered to a subject at any suitable dose known in the art (e.g., FDA-approved dose). In some aspects, this method also includes administering another therapeutic agent (e.g., immune checkpoint inhibitor) to the subject.

[0198] Unless otherwise indicated, the VEGF antagonists described herein can be administered to a subject (e.g., in combination with an IL-7 protein) using any suitable route of administration. In some aspects, the VEGF antagonist is administered to a subject intravenously, parenterally, intramuscularly, subcutaneously, ocularly, intraperitoneally, intradermally, intraorbitally, intracerebrally, intracranially, intraspinally, intraventricularly, intrathecally, intracisternalally, intracapsules, or intratumors. In some aspects, the VEGF antagonist is administered intravenously to a subject. Therefore, in some aspects, the methods of treatment provided herein include administering at least one dose of IL-7 protein to a subject intramuscularly and administering at least one dose of VEGF antagonist intravenously.

[0199] III. Nucleic Acids, Vectors and Host Cells

[0200] Some aspects described herein relate to one or more nucleic acid molecules encoding therapeutic agents described herein (e.g., IL-7 proteins and / or VEGF antagonists). Nucleic acids can be present in intact cells, in cell lysates, or in partially purified or substantially pure form. Nucleic acids are "isolated" or "present as substantially pure" when purified from other cellular components or other contaminants such as other cellular nucleic acids (e.g., other chromosomal DNA, e.g., chromosomal DNA linked to DNA isolated in nature) or proteins by standard techniques (including alkaline / SDS treatment, CsCl banding, column chromatography, restriction endonucleases, agarose gel electrophoresis, and other methods well known in the art). See, F.Ausubel et al., ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. Nucleic acids described herein can be, for example, DNA or RNA, and may or may not contain intron sequences. In some aspects, nucleic acids are cDNA molecules. Nucleic acids described herein can be obtained using standard molecular biology techniques known in the art.

[0201] Some nucleic acid molecules disclosed herein are nucleic acid molecules encoding IL-7 proteins (e.g., disclosed herein), which can be used in combination with VEGF antagonists, e.g., for treating tumors. Exemplary nucleic acid sequences encoding IL-7 proteins disclosed herein are shown in SEQ ID NOs: 29-39.

[0202] In some aspects, the present disclosure provides a vector comprising an isolated nucleic acid molecule encoding a therapeutic agent disclosed herein (eg, an IL-7 protein and / or a VEGF antagonist). In some aspects, the vector can be used for gene therapy.

[0203] When used as a gene therapy (e.g., in humans), nucleic acids encoding therapeutic agents disclosed herein (e.g., IL-7 proteins and / or VEGF antagonists) can be administered at a dose ranging from 0.1 mg to 200 mg. In some aspects, the dose is in the range of 0.6 mg to 100 mg. In some aspects, the dose is in the range of 1.2 mg to 50 mg.

[0204] Suitable vectors for use in the present disclosure include expression vectors, viral vectors, and plasmid vectors. In some aspects, the vector is a viral vector.

[0205] As used herein, an expression vector refers to any nucleic acid construct that contains the necessary elements for transcription and translation of an inserted coding sequence, or for an RNA viral vector, the necessary elements for replication and translation when introduced into an appropriate host cell. Expression vectors may include plasmids, phagemids, viruses, and derivatives thereof.

[0206] As used herein, viral vectors include, but are not limited to, nucleic acid sequences from the following viruses: retroviruses, such as Moloney murine leukemia virus, Harvey murine sarcoma virus, mouse mammary tumor virus, and Rous sarcoma virus; lentivirus; adenovirus; adeno-associated virus; SV40 virus; polyoma virus; Epstein-Barr virus; papilloma virus; herpes virus; vaccinia virus; poliovirus; and RNA viruses, such as retroviruses. Other vectors well known in the art can be readily used. Some viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced by genes of interest. Non-cytopathic viruses include retroviruses, whose life cycle includes reverse transcription of genomic viral RNA into DNA, followed by integration of the provirus into host cell DNA.

[0207] In some aspects, the vector is derived from an adeno-associated virus. In some aspects, the vector is derived from a lentivirus. Examples of lentiviral vectors are disclosed in WO9931251, W09712622, W09817815, W09817816, and WO9818934, each of which is incorporated herein by reference in its entirety.

[0208] 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 recent years, it has been found that plasmid vectors are particularly beneficial for delivering genes to cells in vivo because they cannot be replicated in the host genome and integrated into the host genome. However, these plasmids with promoters compatible with host cells can express peptides from genes operably encoded in the plasmid. Some commonly used plasmids available from commercial suppliers include pBR322, pUC18, pUC19, various pcDNA plasmids, pRC / CMV, various pCMV plasmids, pSV40 and pBlueScript. Additional examples of specific plasmids include: pcDNA3.1, catalog number V79020; pcDNA3.1 / hygro, catalog number V87020; pcDNA4 / myc-His, catalog number V86320; and pBudCE4.1, catalog number V53220, all from Invitrogen (Carlsbad, CA.). Other plasmids are well known to those of ordinary skill in the art. In addition, plasmids can be custom designed using standard molecular biology techniques to remove and / or add specific DNA fragments.

[0209] The present disclosure also encompasses a method for preparing a therapeutic agent disclosed herein (e.g., IL-7 protein). In some aspects, this method may include expressing a therapeutic agent (e.g., IL-7 protein) in a cell, the cell comprising a nucleic acid molecule encoding the therapeutic agent, e.g., SEQ ID NO: 29-39. Other details about the method for preparing the IL-7 protein disclosed herein are provided in, e.g., U.S. Patent No. 11,041,007, which is incorporated herein by reference in its entirety. Host cells comprising these nucleotide sequences are contemplated 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 a combination thereof.

[0210] IV. Pharmaceutical Compositions

[0211] Also provided herein are compositions comprising one or more therapeutic agents (e.g., IL-7 protein and / or VEGF antagonist) of desired purity in a physiologically acceptable carrier, excipient or stabilizer (Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA). In some aspects, the compositions disclosed herein comprise IL-7 protein or VEGF antagonist. As disclosed herein, such compositions can be used in combination (e.g., a first composition comprising IL-7 protein and a second composition comprising VEGF antagonist). In some aspects, the compositions disclosed herein comprise IL-7 protein and VEGF antagonist.

[0212] Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphates, citrates, and other organic acids; antioxidants (including ascorbic acid and methionine); preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as or polyethylene glycol (PEG).

[0213] In some aspects, the compositions disclosed herein comprise one or more additional components selected from the group consisting of a bulking agent, a stabilizer, a surfactant, a buffer, or a combination thereof.

[0214] Buffers that can be used in the present disclosure can be weak acids or weak bases that are used to maintain the acidity (pH) of a solution near a selected value after adding another acid or base. Suitable buffers can maximize the stability of the pharmaceutical composition by maintaining pH control of the composition. Suitable buffers can also ensure physiological compatibility or optimize solubility. Rheology, viscosity and other properties can also depend on the pH of the composition. Common buffers include, but are not limited to, Tris buffers, Tris-Cl buffers, histidine buffers, TAE buffers, HEPES buffers, TBE buffers, sodium phosphate buffers, MES buffers, ammonium sulfate buffers, potassium phosphate buffers, potassium thiocyanate buffers, succinate buffers, tartrate buffers, DIPSO buffers, HEPPSO buffers, POPSO buffers, PIPES buffers, PBS buffers, MOPS buffers, acetate buffers, phosphate buffers, cacodylate buffers, glycine buffers, sulfate buffers. buffer, imidazole buffer, guanidine hydrochloride buffer, phosphate-citrate buffer, borate buffer, malonate buffer, 3-methylpyridine buffer, 2-methylpyridine buffer, 4-methylpyridine 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.

[0215] In some aspects, the compositions disclosed herein also include an extender. An extender can be added to a pharmaceutical product to increase the volume and quality of the product, thereby promoting its accurate measurement and processing. Extenders that can be used together with the present disclosure include but are not limited to sodium chloride (NaCl), mannitol, glycine, alanine or a combination thereof.

[0216] In some aspects, the compositions disclosed herein may further comprise a stabilizer. Non-limiting examples of stabilizers that can be used with the present disclosure include: sucrose, trehalose, raffinose, arginine, or a combination thereof.

[0217] In some aspects, the composition disclosed herein comprises a surfactant. In some aspects, the surfactant can be selected from the following: alkyl ethoxylates, nonylphenol polyoxyethylene ethers, amine ethoxylates, polyethylene oxide, polypropylene oxide, fatty alcohols such as hexadecanol or oleyl alcohol, cocamide MEA, cocamide DEA, polysorbate, dodecyl dimethylamine oxide or a combination thereof. In some aspects, the surfactant is polysorbate 20 or polysorbate 80.

[0218] In some aspects, the composition comprising the IL-7 protein can be formulated using the same formulation of the VEGF antagonist (e.g., it will be used in combination with the IL-7 protein). In some aspects, the IL-7 protein and the VEGF antagonist are formulated using different formulations.

[0219] The pharmaceutical composition can be formulated for any route of administration to a subject. Specific examples of route of administration include intramuscular, subcutaneous, ocular, intravenous, intraperitoneal, intradermal, intraorbital, intracerebral, intracranial, intraspinal, intraventricular, intrathecal, intracisternal, intracapsular or intratumoral. Parenteral administration characterized by subcutaneous, intramuscular or intravenous injection is also contemplated herein. Injections in conventional form can be prepared as liquid solutions or suspensions, solid forms suitable for dissolving or suspending in liquids before injection, or as emulsions. Injections, 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 pharmaceutical composition to be administered can also contain a small amount of nontoxic auxiliary substances, such as wetting agents or emulsifiers, pH buffers, stabilizers, solubilizers and other such agents, such as, for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate and cyclodextrin.

[0220] Pharmaceutically acceptable carriers for parenteral preparations include aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending agents and dispersants, emulsifiers, chelating agents (sequestering / chelating agent) and other pharmaceutically acceptable substances. The example of aqueous vehicles includes sodium chloride injection, Ringer's (Ringers) injection, isotonic dextrose injection, sterile water injection, glucose and lactated Ringer's injection. Non-aqueous parenteral vehicles include fixed oils, cottonseed oil, corn oil, sesame oil and peanut oil of plant origin. Antimicrobial agents of antibacterial or fungistatic concentrations can be added to parenteral preparations packaged in multi-dose containers, and the multi-dose containers include phenol or cresol, mercury, benzyl alcohol, chlorobutanol, methyl and propyl parabens, thimerosal, benzalkonium chloride (benzalkonium chloride) and benzethonium chloride (benzethonium chloride). Isotonic agents include sodium chloride and dextrose. Buffers include phosphates and citrates. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcellulose, hydroxypropyl methylcellulose, and polyvinyl pyrrolidone. Emulsifiers include polysorbate 80 ( 80). Metal ion sequestrants or chelators include EDTA. Pharmaceutical carriers also include ethanol, polyethylene glycol and propylene glycol for water-miscible vehicles; and sodium hydroxide, hydrochloric acid, citric acid or lactic acid for pH adjustment.

[0221] Preparations for parenteral administration include sterile solutions ready for injection, sterile dry soluble products ready for mixing with solvents before use (such as lyophilized powders, including hypodermic tablets), sterile suspensions ready for injection, sterile dry insoluble products ready for mixing with vehicles before use, and sterile emulsions. Solutions may be aqueous or non-aqueous.

[0222] If administered intravenously, suitable carriers include physiological saline or phosphate buffered saline (PBS) and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol and polypropylene glycol and mixtures thereof.

[0223] The topical mixture containing the antibody is prepared as described for topical and systemic administration. The resulting mixture can be a solution, suspension, emulsion, etc., and can be formulated into a cream, gel, ointment, emulsion, solution, elixir, lotion, suspension, tincture, paste, foam, aerosol, rinse, spray, suppository, bandage, skin patch or any other formulation suitable for topical administration.

[0224] The antibodies or antigen-binding portions thereof described herein can be formulated as aerosols for topical application, for example by inhalation (see, for example, U.S. Pat. Nos. 4,044,126, 4,414,209 and 4,364,923, which describe aerosols for delivering steroids useful for treating inflammatory diseases, particularly asthma). These formulations for administration to the respiratory tract can be in the form of an aerosol or solution for a nebulizer, or as a fine powder for insufflation, alone or in combination with an inert carrier such as lactose. In this case, the diameter of the particles of the formulation will be less than 50 microns in one aspect, and less than 10 microns in one aspect.

[0225] The therapeutic agents disclosed herein (e.g., IL-7 proteins and / or VEGF antagonists) can be formulated in the form of gels, creams, and lotions for local or topical applications, such as topical applications to the skin and mucous membranes, such as in the eyes, and applications to the eyes or intracisternal or intraspinal applications. Topical administration is contemplated for transdermal delivery, and is also used for application to the eyes or mucous membranes, or for inhalation therapy. Nasal solutions of antibodies can also be administered alone or in combination with other pharmaceutically acceptable excipients.

[0226] Transdermal patches including iontophoresis and electrophoresis devices are well known to those skilled in the art and can be used to administer antibodies. For example, such patches are disclosed in U.S. 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.

[0227] In some aspects, the pharmaceutical composition comprising the therapeutic agent described herein (e.g., IL-7 protein and / or VEGF antagonist) is a lyophilized powder that can be reconstituted and administered in the form of a solution, emulsion, and other mixtures. It can also be reconstituted and formulated as a solid or gel. The lyophilized powder is prepared by dissolving the antibody or antigen-binding portion thereof described herein or a pharmaceutically acceptable derivative thereof in a suitable solvent. In some aspects, the lyophilized powder is sterile. The solvent may contain an excipient that improves the stability of the powder or the reconstituted solution prepared from the powder or other pharmacological components. Excipients that can be used include, but are not limited to, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose, or other suitable agents. The solvent may also contain a buffer, such as citrate, sodium phosphate or potassium phosphate or other such buffers known to those skilled in the art, in one aspect, at about neutral pH. The solution is then sterile filtered and then lyophilized under standard conditions known to those skilled in the art to provide 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, for example at about 4°C to room temperature.

[0228] Reconstitution of the lyophilized powder with water for injection provides a formulation for parenteral administration. For reconstitution, the lyophilized powder is added to sterile water or other suitable carrier. The exact amount depends on the selected compound. Such amounts can be determined empirically.

[0229] The compositions provided herein can also be formulated into specific tissues, receptors or other regions of the subject's body to be treated. Many such targeting methods are well known to those skilled in the art. All such targeting methods for the compositions of the present invention are contemplated herein. For non-limiting examples of targeting methods, see, 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, 6,060,082, 6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, 5,900,252, 5,840,674, 5,759,542 and 5,709,874.

[0230] Compositions to be used for in vivo administration may be sterile. This is easily achieved, for example, by filtration through sterile filtration membranes.

[0231] The following examples are merely illustrative and are not to be construed as limiting the scope of the present disclosure in any way, as numerous variations and equivalents will become apparent to those skilled in the art upon reading this disclosure.

[0232] Example

[0233] Example 1: Effect of combined treatment with IL-7 protein and VEGF antagonist on tumor volume

[0234] To evaluate the effect of the combination of IL-7 protein and VEGF antagonist on tumor volume, a colon adenocarcinoma animal model was used. Briefly, MC-38 colon adenocarcinoma tumor cells (5×10 5 cells, subcutaneously) were transplanted into each C57BL / 6 mouse. On day 8 after tumor inoculation (average tumor size was approximately 60 mm 3 ), animals were treated with IL-7-HyFc protein and antagonist anti-VEGF antibody (aflibercept) alone or in combination. Control animals were treated with IL-7 formulation buffer and isotype control antibody. IL-7 protein was administered once (i.e., day 8 after tumor inoculation) (intramuscularly; 10 mg / kg per mouse). Antagonist anti-VEGF antibody was administered twice a week (intravenously; 10 mg / kg per mouse per dose) for two weeks (i.e., days 8, 11, 15, and 18 after tumor inoculation). Animals were then monitored for tumor volume at different time points. Figure 1A A graphical depiction of the dosing regimen is provided, and Table 2 (below) provides the different treatment groups.

[0235] Table 2. Treatment groups

[0236]

[0237] like Figure 1B As shown, animals treated with IL-7 protein alone (i.e., Group 2) or anti-VEGF antibody alone (i.e., Group 3) showed a reduction in tumor volume compared to control animals (i.e., Group 1). However, in mice from the combination treatment group (i.e., Group 4), the reduction in tumor volume was more significant. On day 18 after tumor inoculation, the average tumor volumes from the different treatment groups were as follows: Group 1: 635.30 mm 3 ; Group 2: 269.46mm 3 ; Group 3: 257.99mm 3 ; and Group 4: 86.33mm 3 .

[0238] The above results indicate that the combination therapy described herein (the combination of IL-7 protein and VEGF antagonist) can be used to treat certain cancers. In addition, the data further show that the IL-7 protein described herein can enhance the anti-tumor effect of VEGF antagonists, such as those commercially available and / or previously shown to have anti-tumor effects.

[0239] Example 2: Analysis of the anti-tumor effect of triple combination therapy of IL-7 protein, VEGF antagonist and immune checkpoint inhibitor

[0240] In order to further evaluate the anti-tumor effect of the combination therapy described herein, a tumor animal model (e.g., a colon adenocarcinoma animal model) will be treated with a combination of IL-7 protein alone or with a VEGF antagonist (e.g., an anti-VEGF antibody). In some aspects, some animals will further receive the administration of immune checkpoint inhibitors. Therefore, in some aspects, some animals will be treated with a triple combination of IL-7 protein, VEGF antagonist, and immune checkpoint inhibitors. After administration, the anti-tumor immune response of the animal will be assessed, for example, by monitoring the tumor volume at different time points after treatment.

[0241] Example 3: Effect of combined treatment with IL-7 protein and VEGF antagonist in human glioblastoma subjects

[0242] To evaluate the efficacy and safety of the treatment regimen described herein, a Phase 2 study is ongoing in human subjects with recurrent glioblastoma. Figure 2As shown and further described below, eligible subjects (see, e.g., inclusion criteria and exclusion criteria further described below) have received administration of a combination of an IL-7 protein as described herein and bevacizumab (i.e., a VEGF antagonist) (also referred to herein as "study drug"). Eligible subjects will receive up to six cycles (one cycle = eight weeks) of combined treatment, or until disease progression or unacceptable toxicity occurs. Tumor assessments will be performed every eight weeks prior to treatment administration. Figure 3 A general overview of the study is provided.

[0243] Test subjects

[0244] Eligible subjects were diagnosed with glioblastoma (eg, recurrent glioblastoma) with documented disease progression following standard therapy (concurrent chemotherapy and / or temozolomide-adjuvant chemotherapy).

[0245] IL-7 protein administration

[0246] IL-7 protein is administered intramuscularly to qualified subjects at a dosage of 1,200 μg / kg and an 8-week dosing interval. As described above, subjects without severe toxicity and clinical clear evidence of disease progression will receive up to 6 cycles of IL-7 protein treatment (based on the clinical significance and risk assessment of the investigator). In each dose step, the amount of IL-7 protein administered to the subject can be determined based on the subject's weight on the first day of each cycle. There is no restriction on the dosage for overweight subjects.

[0247] Bevacizumab administration

[0248] Bevacizumab was administered to eligible subjects at a dose of 10 mg / kg by intravenous administration every 2 weeks until disease progression or unacceptable toxicity (as described elsewhere in this example).

[0249] Dosage Considerations

[0250] The dose of IL-7 protein may be reduced to 960 μg / kg at the investigator's discretion under the following conditions: adverse skin reactions at the injection site or systemically. If a subject experiences unacceptable toxicity (Grade 3 or higher CTCAE) following administration of IL-7 protein, administration of IL-7 protein should be discontinued. If the toxicity resolves within 28 days, IL-7 protein administration may be resumed. For subjects who experience clinically manageable toxicity (Grade 3 immune-related adverse events) but show clear signs of clinical benefit (e.g., tumor shrinkage or stable disease), administration of IL-7 protein may continue under close supervision. If after initial administration, the absolute lymphocyte count (ALC) (treatment-related) exceeds 20,000 cells / mm 3(corresponding to grade 3 NCI CTC), further administration should be temporarily withheld. If ALC decreases below the upper limit of normal levels (4,000 cells / mm 3 ), then the application can be resumed.

[0251] For VEGF antagonists, the dose may be adjusted when the subject's body weight changes by more than about 10%. Otherwise, dose adjustment is generally not recommended. If adverse events occur, administration of VEGF agonists may be temporarily or permanently discontinued at the investigator's discretion.

[0252] Injection site reactions

[0253] Common adverse reactions that may occur at the injection site include: allergic reactions (e.g., rash, urticaria, redness), itching, swelling, and erythema. Appropriate doses of steroids (e.g., prednisolone at a dose of about 5-7.5 mg) can be used as palliative treatment. Antihistamines can also be used for preventive purposes (e.g., administered to subjects at least 14 days prior to treatment).

[0254] Prohibited treatments

[0255] During this study, the following treatments were prohibited: (1) any concomitant treatment approved or experimental for the treatment of cancer, including chemotherapy, hormone therapy, immunotherapy, radiation therapy, investigational drugs, or herbal therapies; (2) immunosuppressants, including but not limited to cyclophosphamide, azathioprine, methotrexate, and thalidomide; (3) granulocyte colony-stimulating factor and granulocyte macrophage colony-stimulating factor (e.g., pegfilgrastim); and (4) immunostimulants (e.g., IFN-α, IFN-γ, and IL-2).

[0256] Inclusion criteria

[0257] Inclusion criteria used to determine subject eligibility included the following: (1) age ≥19 years; (2) patients with histologically diagnosed glioblastoma with confirmed disease progression after a trial of standard therapy (RT / CCRT and / or adjuvant chemotherapy (TMZ)); (3) Karnofsky Performance Status; KPS ≥60 or ECOG status 0-2; (4) life expectancy greater than 12 weeks; and (5) adequate hematologic and end-organ function.

[0258] Exclusion criteria

[0259] Subjects who exhibited one or more of the following conditions were excluded from this study: (1) malignancy other than the study disease within 5 years before the first dose of study drug; (2) receipt of bevacizumab or other VEGF antagonists before the current study; (3) body mass index (BMI) ≥ 30 kg / m2; (4) intracranial hemorrhage confirmed by plain CT or MRI; (5) clinically significant cardiovascular disease; (6) history of arterial or venous thromboembolism within 6 months before the current study; (7) uncontrolled hypertension (blood pressure ≥ 150 / 90 mmHg, receiving appropriate antihypertensive therapy); (8) hypertensive crisis or hypertensive cerebral infarction; medical history; (9) receiving therapeutic anticoagulation (except low molecular weight heparin or warfarin); (10) pregnancy or breastfeeding; (11) active viral infection; (12) autoimmune disease / syndrome; (13) administration of a live attenuated vaccine within 4 weeks prior to Day 1 of Cycle 1, or anticipation of requiring such a live attenuated vaccine during the current study; (14) history of severe allergic reaction, anaphylaxis, or other hypersensitivity reaction to chimeric or humanized antibodies or fusion proteins; (15) serious infection during the screening period, including but not limited to infectious complications, bacteremia, or severe pneumonia; and (16) previous allogeneic bone marrow transplantation or previous solid organ transplantation.

[0260] Example 4: Interim Results of a Phase 2 Study Involving Combination Treatment of IL-7 Protein and VEGF Antagonist in Human Glioblastoma Subjects

[0261] In addition to Example 3 provided above, Figure 4A and 4B The interim results of the study were presented. Twenty subjects were enrolled in the study and Figure 4A As shown, 14 subjects are still receiving treatment (patient numbers 2, 5, 6, 9, and 11-20) and four subjects have confirmed progression (patient numbers 1, 4, 7, and 10). Of the subjects with confirmed progression, two subjects have withdrawn from the study (patient numbers 4 and 7). Figure 4B As shown, 4 weeks after the initial administration, almost all subjects had a significant increase in absolute lymphocyte counts. Other outcomes (such as clinical response and survival status) are ongoing.

[0262] The above results demonstrate the therapeutic potential of the combination therapy described herein in the treatment of cancers such as glioblastoma.

Claims

1. A method of treating a tumor in a subject in need thereof, comprising administering to the subject a combination of an IL-7 protein and a vascular endothelial growth factor ("VEGF") antagonist ("VEGF antagonist").

2. The method of claim 1, wherein the subject's tumor volume is reduced after the administration.

3. The method of claim 2, wherein the tumor volume is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% after the administration.

4. A method of increasing an anti-tumor immune response in a subject in need thereof, comprising administering to the subject a combination of an IL-7 protein and a vascular endothelial growth factor ("VEGF") antagonist ("VEGF antagonist").

5. The method of claim 4, wherein the anti-tumor immune response 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 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 after the administration.

6. The method of claim 4 or 5, wherein the increased anti-tumor immune response comprises: (i) a reduction in tumor volume; (ii) an increase in the effector activity of tumor-specific T cells; (iii) an increase in the number of tumor-specific effector T cells; (iv) an increase in the number of tumor-infiltrating lymphocytes (TILs) in the tumor; (v) a decrease in the number of myeloid-derived suppressor cells (MDSCs) in the tumor; (vi) a decrease in the number of regulatory T cells (Tregs) in the tumor; (vii) an extended duration of survival of the subject; (viii) a decrease in the expression of TOX on CD8+ T cells in the tumor, tumor-draining lymph nodes (TDLN), or both; (ix) an increase in the number of stem-like T cells in the tumor, TDLN, or both; or (x) a combination thereof.

7. The method of any one of claims 1 to 6, wherein the IL-7 protein and the VEGF antagonist are administered to the subject simultaneously.

8. The method of any one of claims 1 to 6, wherein the IL-7 protein and the VEGF antagonist are administered to the subject sequentially.

9. The method of any one of claims 1 to 8, wherein an additional therapeutic agent is administered to the subject.

10. The method of claim 9, wherein the additional therapeutic agent comprises an immune checkpoint inhibitor, an immune checkpoint activator, a standard of care treatment, or a combination thereof.

11. The method of claim 10, 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.

12. The method of claim 10 or 11, 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.

13. The method of any one of claims 10 to 12, wherein the standard of care treatment comprises chemotherapy, radiation, or both.

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

15. The method of any one of claims 1 to 14, wherein the IL-7 protein comprises an oligopeptide consisting of 1 to 10 amino acid residues.

16. The method of claim 15, wherein the oligopeptide comprises 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). ID NO:44), glycine-methionine-methionine-glycine (GMMG) (SEQ ID 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), 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:51), methionine-methionine-glycine-methionine-glycine-methionine (MGMGM) (SEQ ID NO:52), methionine-methionine-glycine-methionine-glycine-methionine (MMGGM) (SEQ ID NO:53), methionine-methionine-glycine-glycine-methionine (MMGGM) (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-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-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-methionine-glycine-glycine (GMMGG) (SEQ ID NO:70), 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.

17. The method of claim 18, wherein the oligopeptide is methionine-glycine-methionine (MGM).

18. The method of any one of claims 1 to 17, wherein the IL-7 protein comprises a half-life extending moiety.

19. The method of claim 18, wherein the half-life extending moiety comprises Fc, albumin, albumin binding polypeptide, Pro / Ala / Ser (PAS), C-terminal peptide (CTP) of the beta subunit of human chorionic gonadotropin, polyethylene glycol (PEG), long unstructured hydrophilic amino acid sequence (XTEN), hydroxyethyl starch (HES), albumin binding small molecules, or a combination thereof.

20. The method of claim 19, wherein the half-life extending moiety is Fc.

21. The method of claim 18 or 20, wherein 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, wherein the CH2 domain comprises a portion of a human IgD CH2 domain and a portion of a human IgG4 CH2 domain, and wherein the CH3 domain comprises a portion of a human IgG4 CH3 domain.

22. The method of any one of claims 1 to 21, 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 the sequence shown in any one of SEQ ID NOs: 1-6, 15-26, and 80-85.

23. The method of any one of claims 1 to 22, wherein the VEGF antagonist comprises an antibody or antigen-binding fragment thereof that specifically binds to the VEGF ("anti-VEGF antibody"), a polynucleotide encoding the anti-VEGF antibody, an antisense oligonucleotide, siRNA, shRNA, miRNA, a dsRNA targeting VEGF, an aptamer, a PNA, or a vector comprising the same.

24. The method of claim 23, wherein the VEGF antagonist is an anti-VEGF antibody.

25. The method of claim 24, wherein the anti-VEGF antibody comprises aflibercept ( and ), bevacizumab ( and ), Ranibizumab or a combination thereof.

26. The method of any one of claims 1 to 25, wherein the tumor is derived from a cancer selected from the group consisting of breast cancer, head and neck cancer, uterine cancer, brain cancer, skin cancer, kidney cancer, lung cancer, colorectal cancer, prostate cancer, liver cancer, bladder cancer, kidney cancer, pancreatic cancer, thyroid cancer, esophageal cancer, eye cancer, stomach (gastric) cancer, gastrointestinal cancer, ovarian cancer, carcinoma, sarcoma, leukemia, lymphoma, myeloma, or a combination thereof.

27. The method of claim 26, wherein the brain cancer comprises glioblastoma.

28. The method of claim 27, wherein the glioblastoma comprises recurrent glioblastoma.

29. The method of claim 27 or 28, wherein the glioblastoma is refractory to standard of care therapy.

30. The method of any one of claims 26 to 29, wherein the skin cancer comprises Merkel cell carcinoma (MCC), basal cell carcinoma (BCC), cutaneous squamous cell carcinoma (cSCC), melanoma, or a combination thereof.

31. The method of any one of claims 1 to 30, wherein the IL-7 protein is administered to the subject intramuscularly, parenterally, subcutaneously, ocularly, intravenously, intraperitoneally, intradermally, intraorbitally, intracerebrally, intracranially, intraspinally, intraventricularly, intrathecally, intracisternalally, intracapsularly, or intratumorally.

32. The method of any one of claims 1 to 31, wherein the VEGF antagonist is administered to the subject intravenously, parenterally, intramuscularly, subcutaneously, ocularly, intraperitoneally, intradermally, intraorbitally, intracerebrally, intracranially, intraspinally, intraventricularly, intrathecally, intracisternallly, intracapsularly, or intratumorally.

33. The method of any one of claims 9 to 32, wherein the additional therapeutic agent is administered to the subject parenterally, intravenously, intramuscularly, subcutaneously, ocularly, intraperitoneally, intradermally, intraorbitally, intracerebrally, intracranially, intraspinally, intraventricularly, intrathecally, intracisternallly, intracapsularly, or intratumorally.

34. The method of any one of claims 1 to 33, wherein the IL-7 protein is administered 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.

35. The method of any one of claims 1 to 34, wherein the IL-7 protein is administered at a concentration of between about 610 μg / kg and about 1,200 μg / kg, between about 650 μg / kg and about 1,200 μg / kg, between about 700 μg / kg and about 1,200 μg / kg, between about 750 μg / kg and about 1,200 μg / kg, between about 800 μg / kg and about 1,200 μg / kg, or between about 1000 μg / kg and about 1000 μg / kg. between about 850 μg / kg and about 1,200 μg / kg, between about 900 μg / kg and about 1,200 μg / kg, between about 950 μg / kg and about 1,200 μg / kg, between about 1,000 μg / kg and about 1,200 μg / kg, between about 1,050 μg / kg and about 1,200 μg / kg, between about 1,100 μg / kg and about 1,200 μg / kg , between about 1,200 μg / kg and about 2,000 μg / kg, between about 1,300 μg / kg and about 2,000 μg / kg, between about 1,500 μg / kg and about 2,000 μg / kg, between about 1,700 μg / kg and about 2,000 μg / kg, between about 610 μg / kg and about 1,000 μg / kg, between about 650 μg / kg and about 1,000 μg / kg, The dosage may be between about 700 μg / kg and about 1,000 μg / kg, between about 750 μg / kg and about 1,000 μg / kg, between about 800 μg / kg and about 1,000 μg / kg, between about 850 μg / kg and about 1,000 μg / kg, between about 900 μg / kg and about 1,000 μg / kg, or between about 950 μg / kg and about 1,000 μg / kg.

36. The method of any one of claims 1 to 35, wherein the IL-7 protein is administered at a dose of between about 700 μg / kg and about 900 μg / kg, between about 750 μg / kg and about 950 μg / kg, between about 700 μg / kg and about 850 μg / kg, between about 750 μg / kg and about 850 μg / kg, between about 700 μg / kg and about 800 μg / kg, between about 800 μg / kg and about 900 μg / kg, between about 750 μg / kg and about 850 μg / kg, or between about 850 μg / kg and about 950 μg / kg.

37. The method of any one of claims 1 to 36, wherein the IL-7 protein is administered at about 650 μg / kg, about 680 μg / kg, 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 820 μg / kg, about 840 μg / kg, about 850 μg / kg, about 860 μg / kg, about 880 μg / kg, about 900 μg / kg, about 920 μg / kg, about 940 μg / kg, about 950 μg / kg , about 960μg / kg, about 980μg / kg, about 1,000μg / kg, about 1,020μg / kg, about 1,040μg / kg, about 1,060μg / kg, about 1,080μg / kg, about 1,100μg / kg, about 1,120μg / kg, about 1,140μg / kg, about 1,160μg / kg, about 1,180μg / kg, about 1,200μg / kg, about 1,220μg / kg, about 1,240μg / kg, about 1,260μg / kg, about 1,280μg / kg, about 1,300μg / kg, about 1,320μg / kg, about 1,340μg / kg, about 1,360μg / kg, about 1,380μg / kg, about 1,400μg / kg, about 1,420μg / kg, about 1,440μg / kg, about 1,460μg / kg, about 1,480μg / kg, about 1,500μg / kg, about 1,520μg / kg, about 1,540μg / kg, about 1,560μg / kg, about 1,580μg / kg, about 1,600μg / kg, about 1,620μg / kg, about 1,640μg / kg, about 1,660μg / kg, about 1,680 μg / kg, about 1,700 μg / 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.

38. The method of any one of claims 1 to 37, wherein the IL-7 protein is administered at a dosing frequency of once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every 10 weeks, once every 11 weeks, or once every 12 weeks.

39. The method of claim 38, wherein the IL-7 protein is administered at a dosing frequency of once every eight weeks.

40. The method of any one of claims 1 to 39, wherein the VEGF antagonist is administered to the subject at a dose of about 0.1 mg / kg to about 20 mg / kg.

41. The method of any one of claims 1 to 40, wherein the VEGF antagonist is administered at a dosing frequency of once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every 10 weeks, once every 11 weeks, or once every 12 weeks.

42. The method of claim 41, wherein the VEGF antagonist is administered at a dosing frequency of once every two weeks.

43. The method of any one of claims 25 to 42, wherein the anti-VEGF antibody is bevacizumab.

44. The method of claim 43, wherein the bevacizumab is administered at a dose of about 5 mg / kg to about 15 mg / kg.

45. The method of claim 44, wherein the bevacizumab is administered at a dose of about 5 mg / kg, about 7.5 mg / kg, about 10 mg / kg, about 12.5 mg / kg, or about 15 mg / kg.

46. ​​The method of any one of claims 25 to 42, wherein the anti-VEGF antibody is aflibercept.

47. The method of claim 46, wherein the aflibercept is administered at a dose of about 4 mg / kg to about 15 mg / kg.

48. The method of claim 47, wherein the aflibercept is administered at a dose of about 4 mg / kg.

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