Method for treating tumors using a combination of IL-7 protein and VEGF antagonist

Abstract

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

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This PCT application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 295,336, filed December 30, 2021, and Korean Patent Application No. 10-2022-0000051, filed January 3, 2022, both of which are incorporated by reference in their entireties.

[0002] References to sequence listings submitted electronically via EFS-WEB The sequence listing, which was filed and submitted electronically with this application as an .XML text file (Name: 4241_029PC02_SequenceListing_ST26, Size: 122,317 bytes, Created: December 30, 2022), is incorporated herein by reference in its entirety.

[0003] The present disclosure generally relates to a combination treatment regimen useful for treating tumors in subjects, for example, human subjects.Specifically, the treatment regimen provided herein comprises a combination of IL-7 protein and VEGF antagonist.In some embodiments, the treatment regimen further comprises an additional therapeutic agent, such as an immune checkpoint inhibitor.Provided herein is a method for treating tumors, comprising using such a treatment regimen to enhance anti-tumor immune response. [Background technology]

[0004] Human cancers harbor numerous genetic and epigenetic mutations that generate neoantigens potentially recognizable by the immune system (Sjoblom et al., Science 314:268-74(2006)). The adaptive immune system, composed of T and B lymphocytes, has potent anticancer potential, with a broad ability to respond to diverse tumor antigens and exquisite specificity. Furthermore, this immune system exhibits considerable plasticity and memory components. Immunotherapy is unique among all cancer treatment modalities in its exploitation of all these attributes of the adaptive immune system.

[0005] Cancer immunotherapy has become well established in recent years and is now one of the more successful treatment options available to 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 immunological pathways important in cancer immune surveillance. And focused efforts have led to the successful development of several immune checkpoint pathway inhibitors, some of which have been approved by the U.S. Food and Drug Administration, including the anti-CTLA-4 antibody ipilimumab (YERVOY®), the anti-PD-1 antibodies nivolumab (OPDIVO®) and pembrolizumab (KEYTRUDA®), and the anti-PD-L1 antibodies atezolizumab (TECENTRIQ®), durvalumab (IMFINZI®), and avelumab (BAVENCIO®).

[0006] Despite these advances, patients with certain malignancies (e.g., metastatic or refractory solid tumors) still have a very poor prognosis. Only a small proportion of such patients actually experience long-term remission of their cancer, and many patients either do not respond to antibodies or respond initially to antibodies but eventually develop resistance. Sharma, P., et al., Cell 168(4):707-723(2017). Furthermore, many cancer patients are lymphopenic, as many of the available standard-of-care cancer treatments (e.g., chemotherapy and radiation therapy) 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, there remains a need for new treatment options with acceptable safety profiles and high efficacy in cancer patients, including those with lymphopenia. Summary of the Invention

[0007] Provided herein are methods of treating a tumor in a subject in need of such treatment, comprising administering to the subject an IL-7 protein in combination with an antagonist of vascular endothelial growth factor ("VEGF") ("VEGF antagonist").

[0008] In some embodiments, tumor volume is reduced in the subject following administration, hi some embodiments, tumor volume is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% following administration.

[0009] Also provided herein is a method of increasing an anti-tumor immune response in a subject in need thereof, comprising administering to the subject an IL-7 protein in combination with an antagonist of vascular endothelial growth factor ("VEGF") ("VEGF antagonist").

[0010] In some embodiments, the subject's anti-tumor immune response is increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 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 following administration. In some embodiments, the increased anti-tumor immune response comprises: (i) a decrease in tumor volume; (ii) an increase in 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 increase in the survival time of the subject; (viii) a decrease in expression of TOX on CD8+ T cells in the tumor, tumor-draining lymph nodes (TDLNs), or both; (ix) an increase in the number of stem cell-like T cells in the tumor, TDLNs, or both; or (x) a combination thereof.

[0011] In some embodiments, the IL-7 protein and the VEGF antagonist are administered to the subject simultaneously. In some embodiments, the IL-7 protein and the VEGF antagonist are administered to the subject sequentially.

[0012] In some embodiments, an additional therapeutic agent is administered to the subject. In some embodiments, the additional therapeutic agent comprises an immune checkpoint inhibitor, an immune checkpoint activator, a standard of care, or a combination thereof.

[0013] In some embodiments, the immune checkpoint inhibitor comprises a CTLA-4 antagonist (e.g., an anti-CTLA-4 antibody), a PD-1 antagonist (e.g., an anti-PD-1 antibody, an anti-PD-L1 antibody), a TIM-3 antagonist (e.g., an anti-TIM-3 antibody), or a combination thereof. In some embodiments, the immune checkpoint activator comprises an OX40 agonist (e.g., an anti-OX40 antibody), a LAG-3 agonist (e.g., an anti-LAG-3 antibody), a 4-1BB (CD137) agonist (e.g., an anti-CD137 antibody), a GITR agonist (e.g., an anti-GITR antibody), or a combination thereof. In some embodiments, the standard of care comprises chemotherapy, radiation, or both.

[0014] In some embodiments, the IL-7 protein is not wild-type IL-7.

[0015] In some embodiments, the IL-7 protein comprises an oligopeptide consisting of 1-10 amino acid residues. In some embodiments, the oligopeptide is selected from the group consisting of methionine (M), glycine (G), methionine-methionine (MM), glycine-glycine (GG), methionine-glycine (MG), glycine-methionine (GM), methionine-methionine-methionine (MMM), methionine-methionine-glycine (MMG), methionine-glycine-methionine (MGM), glycine-methionine-methionine (GMM), methionine-glycine-glycine (MGG), glycine-methionine-glycine (GMG), glycine- Glycine-methionine (GGM), glycine-glycine-glycine (GGG), methionine-glycine-glycine-methionine (MGGM) (SEQ ID NO: 41), methionine-methionine-glycine-glycine (MMGG) (SEQ ID NO: 42), glycine-glycine-methionine-methionine (GGMM) (SEQ ID NO: 43), methionine-glycine-methionine-glycine (MGMG) (SEQ ID NO: 44), glycine-methionine-methionine-glycine (GMMG) (SEQ ID NO: 45), glycine-glycine-glycine-methionine methionine (GGGM) (SEQ ID NO: 46), methionine-glycine-glycine-glycine (MGGG) (SEQ ID NO: 47), glycine-methionine-glycine-glycine (GMGG) (SEQ ID NO: 48), glycine-glycine-methionine-glycine (GGMG) (SEQ ID NO: 49), glycine-glycine-methionine-methionine-methionine (GGMMM) (SEQ ID NO: 50), glycine-glycine-glycine-methionine-methionine (GGGMM) (SEQ ID NO: 51), glycine-glycine-glycine-glycine-methionine (GGGGM) (SEQ ID NO: 52), methionine-glycine-methionine-methionine-methionine (MGMMM) (SEQ ID NO: 53), methionine-glycine-glycine-methionine-methionine (MGGMM) (SEQ ID NO: 54), methionine-glycine-glycine-glycine-methionine (MGGGM) (SEQ ID NO: 55), methionine-methionine-glycine-methionine-methionine (MMGMM) (SEQ ID NO: 56), methionine-methionine-glycine-glycine-methionine (MMGGM) (SEQ ID NO: 57),Methionine-methionine-glycine-glycine-glycine (MMGGG) (SEQ ID NO: 58), methionine-methionine-methionine-glycine-methionine (MMMGM) (SEQ ID NO: 59), methionine-glycine-methionine-glycine-methionine (MGMGM) (SEQ ID NO: 60), glycine-methionine-glycine-methionine-glycine (GMGMG) (SEQ ID NO: 61), glycine-methionine-methionine-methionine-glycine (GMMMG) (SEQ ID NO: 62), glycine-glycine-methionine-glycine-methionine (GGMGM) (SEQ ID NO: 63), glycine-glycine-methionine-methionine-glycine (GGMMG) (SEQ ID NO: 64), glycine-methionine-methionine-glycine-methionine In some embodiments, the oligopeptide comprises methionine-glycine-methionine-methionine-glycine (GMGMGM) (SEQ ID NO:65), methionine-glycine-methionine-methionine-glycine (MGMMG) (SEQ ID NO:66), glycine-methionine-glycine-glycine-methionine (GMGGM) (SEQ ID NO:67), methionine-methionine-glycine-methionine-glycine (MMGMG) (SEQ ID NO:68), glycine-methionine-methionine-glycine-glycine (GMMGG) (SEQ ID NO:69), glycine-methionine-glycine-glycine-glycine (GMGGG) (SEQ ID NO:70), glycine-glycine-methionine-glycine-glycine (GGMGG) (SEQ ID NO:71), glycine-glycine-glycine-glycine-glycine (GGGGG) (SEQ ID NO:72), or a combination thereof. In some embodiments, the oligopeptide is methionine-glycine-methionine (MGM).

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

[0017] In some embodiments, the Fc is a hybrid Fc comprising a hinge region, a CH2 domain, and a CH3 domain, wherein the hinge region comprises a human IgD hinge region, the CH2 domain comprises a portion of the human IgD CH2 domain and a portion of the human IgG4 CH2 domain, and the CH3 domain comprises a portion of the human IgG4 CH3 domain.

[0018] In some embodiments, the IL-7 protein comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to a sequence set forth in any one of SEQ ID NOs: 1-6, 15-26, and 80-85.

[0019] In some embodiments, 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, an siRNA, shRNA, miRNA, dsRNA, aptamer, PNA, or a vector comprising the same that targets VEGF. In some embodiments, the VEGF antagonist is an anti-VEGF antibody. In some embodiments, the anti-VEGF antibody comprises aflibercept (EYLEA® and ZALTRAP®), bevacizumab (AVASTIN®, ZIRABEV®, and MVASI®), ranibizumab (LUCENTIS®, BYOOVIZ®, SUSVIMO®), or a combination thereof.

[0020] In some embodiments, the tumor is derived from cancer including breast cancer, head and neck cancer, uterine cancer, brain cancer, skin cancer, kidney cancer, lung cancer, colon 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. In some embodiments, the brain tumor comprises glioblastoma. In some embodiments, the glioblastoma comprises recurrent glioblastoma. In some embodiments, the glioblastoma is refractory to standard treatment. In some embodiments, the skin cancer comprises Merkel cell carcinoma (MCC), basal cell carcinoma (BCC), cutaneous squamous cell carcinoma (cSCC), melanoma, or a combination thereof.

[0021] In some embodiments, the IL-7 protein is administered intramuscularly, parenterally, subcutaneously, intraocularly, intravenously, intraperitoneally, intradermally, intraorbitally, intracerebrally, intracranially, intraspinal, intraventricularly, intrathecally, intracisternally, intracapsularly, or intratumorally to the subject. In some embodiments, the VEGF antagonist is administered intravenously, parenterally, intramuscularly, subcutaneously, intraocularly, intraperitoneally, intradermally, intraorbitally, intracerebrally, intracranially, intraspinal, intraventricularly, intrathecally, intracisternally, intracapsularly, or intratumorally to the subject. In some aspects, the additional therapeutic agent is administered to the subject parenterally, intravenously, intramuscularly, subcutaneously, intraocularly, intraperitoneally, intradermally, intraorbitally, intracerebrally, intracranially, intraspinally, intraventricularly, intrathecally, intracisternally, intracapsularly, or intratumorally.

[0022] In some embodiments, 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 embodiments, the IL-7 protein is administered at a concentration of about 610 μg / kg to about 1,200 μg / kg, about 650 μg / kg to about 1,200 μg / kg, about 700 μg / kg to about 1,200 μg / kg, about 750 μg / kg to about 1,200 μg / kg, about 800 μg / kg to about 1,200 μg / kg, about 850 μg / kg to about 1,200 μg / kg, about 900 μg / kg to about 1,200 μg / kg, about 950 μg / kg to about 1,200 μg / kg, about 1,000 μg / kg to about 1,200 μg / kg, about 1,050 μg / kg to about 1,200 μg / kg, about 1,100 μg / kg to about 1,200 μg / kg, or about 1,200 μg / kg. g / kg ~ approx. 2,000 μg / kg, approx. 1,300 μg / kg ~ approx. 2,000 μg / kg, approx. 1,500 μg / kg ~ approx. 2,000 μg / kg, approx. 1,7 00μg / kg ~ approx. 2,000μg / kg, approx. 610μg / kg ~ approx. 1,000μg / kg, approx. 650μg / kg ~ approx. 1,000μg / kg, approx. 700μ The therapeutic agent is administered at a dose of about 750 μg / kg to about 1,000 μg / kg, about 800 μg / kg to about 1,000 μg / kg, about 850 μg / kg to about 1,000 μg / kg, about 900 μg / kg to about 1,000 μg / kg, or about 950 μg / kg to about 1,000 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 700 μg / kg to about 900 μg / kg, about 750 μg / kg to about 950 μg / kg, about 700 μg / kg to about 850 μg / kg, about 750 μg / kg to about 850 μg / kg, about 700 μg / kg to about 800 μg / kg, about 800 μg / kg to about 900 μg / kg, about 750 μg / kg to about 850 μg / kg, or about 850 μg / kg to about 950 μg / kg.In some embodiments, the IL-7 protein is administered at a concentration of 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, Approximately 980μg / kg, approximately 1,000μg / kg, approximately 1,020μg / kg, approximately 1,040μg / kg, approximately 1,060μg / kg, approximately 1,080μg / kg, approximately 1,100μg / kg, approximately 1,120μg / kg, approximately 1,140μg / kg , about 1,160μg / kg, about 1,180μg / kg, about 1200μg / kg, about 1,220μg / kg, about 1,240μg / kg, about 1,260μg / kg, about 1,280μg / kg, about 1,300μg / kg, about 1,320μg / k g, approximately 1,340μg / kg, approximately 1,360μg / kg, approximately 1,380μg / kg, approximately 1,400μg / kg, approximately 1,420μg / kg, approximately 1,440μg / kg, approximately 1,460μg / kg, approximately 1,480μg / kg, approximately 1,500μ g / kg, approximately 1,520μg / kg, approximately 1,540μg / kg, approximately 1,560μg / kg, approximately 1,580μg / kg, approximately 1,600μg / kg, approximately 1,620μg / kg, approximately 1,640μg / kg, approximately 1,660μg / kg, approximately 1,68 The antibody is administered at a dose of about 0 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.

[0023] In some embodiments, the IL-7 protein is administered at a dosing frequency of once per week, once per 2 weeks, once per 3 weeks, once per 4 weeks, once per 5 weeks, once per 6 weeks, once per 7 weeks, once per 8 weeks, once per 9 weeks, once per 10 weeks, once per 11 weeks, or once per 12 weeks. In some embodiments, the IL-7 protein is administered at a dosing frequency of once per 8 weeks.

[0024] In some embodiments, the VEGF antagonist is administered to the subject at a dose of about 0.1 mg / kg to about 20 mg / kg. In some embodiments, the VEGF antagonist is administered at a dosing frequency of once per week, once per 2 weeks, once per 3 weeks, once per 4 weeks, once per 5 weeks, once per 6 weeks, once per 7 weeks, once per 8 weeks, once per 9 weeks, once per 10 weeks, once per 11 weeks, or once per 12 weeks. In some embodiments, the VEGF antagonist is administered at a dosing frequency of once per 2 weeks.

[0025] In some embodiments, the anti-VEGF antibody is bevacizumab. In some embodiments, the bevacizumab is administered at a dose of about 5 mg / kg to about 15 mg / kg. In some embodiments, 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.

[0026] In some embodiments, the anti-VEGF antibody is aflibercept. In some embodiments, the aflibercept is administered at a dose of about 4 mg / kg to about 15 mg / kg. In some embodiments, the aflibercept is administered at a dose of about 4 mg / kg. [Brief description of the drawings]

[0027] [Figure 1]Figure 1 shows the antitumor effect of combined treatment with IL-7 protein and VEGF antagonist in a colon adenocarcinoma animal model. A shows a schematic diagram of the schedule of tumor inoculation and treatment administration. B shows a comparison of tumor volume (mm3) at different time points after tumor inoculation in animals of different treatment groups. Treatment groups were: (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 presented as mean ± SEM. Statistical analysis (one-way ANOVA) was performed using GraphPad Prism 9.1.0 software. "**" = p<0.01, and "****" = p<0.0001. [Diagram 2] FIG. 1 shows a schematic diagram of the dosing schedule for the Phase 2 study described in Examples 3 and 4. [Diagram 3] 1 shows a general outline of the dosing schedule for the Phase 2 trials described in Examples 3 and 4. [Figure 4A] Figure 4A shows interim results of the Phase 2 study described in Examples 3 and 4. Figure 4A shows the current status of the 20 subjects enrolled in the Phase 2 study. Specifically, the following subject statuses are shown: (1) alive (gray bars), (2) dead (dark gray bars), (3) partial response to treatment (filled circles), (4) stable disease (filled triangles), (5) unconfirmed disease progression (open squares), (6) confirmed disease progression (filled squares), and (7) treatment completion (filled diamonds). [Figure 4B] Figure 4B shows interim results of the Phase 2 study described in Examples 3 and 4. Figure 4B shows absolute lymphocyte counts at weeks 0 and 4 after the first dose of treatment. (*) indicates a statistically significant difference between week 4 and week 0 (baseline) by Wilcoxan paired signed rank test. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] The present disclosure provides a combination regimen comprising a combination of a VEGF antagonist and an IL-7 protein that can be used to treat tumors. As demonstrated herein, administration of a combination of a VEGF antagonist and an IL-7 protein can significantly improve anti-tumor immunity compared to either the IL-7 protein or the VEGF antagonist alone. Thus, in some aspects, the combination regimen described herein provides an effective and viable treatment option for many cancers. Further aspects of the present disclosure are provided throughout the application.

[0029] 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 may of course vary. As will be apparent to one skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has separate components and features that can be easily separated from or combined with the features of any of the other embodiments without departing from the scope or spirit of the present disclosure. Any method described can be carried out in the order of events described or in any other order that is logically possible.

[0030] The headings provided herein are not intended to be limitations of the various aspects of the disclosure, which aspects may be defined by reference to the entire specification. It should also be understood that the terminology used herein is for the purpose of describing particular aspects only, and not for the purpose of limitation, since the scope of the disclosure is limited only by the appended claims.

[0031] I. Definition In order that this disclosure may be more readily understood, certain terms are first defined. As used in this application, unless otherwise stated herein, each of the following terms shall have the meaning set forth below. Further definitions are set forth throughout this application.

[0032] Reference to the term "a" or "an" entity refers to one or more of that entity; for example, "a nucleotide sequence" is understood to refer to one or more nucleotide sequences. Thus, the terms "a" (or "an"), "one or more," and "at least one" may be used interchangeably herein.

[0033] Furthermore, as used herein, "and / or" should be understood as a specific disclosure of each of two particular features or elements with or without the other. Thus, the term "and / or" used herein in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to include each of the following embodiments: A, B, and C, A, B, or C, A or C, A or B, B or C, A and C, A and B, B and C, A (only), B (only), and C (only).

[0034] Where an embodiment is described herein using the language "comprising," it is understood that similar embodiments separately described in terms of "consisting of" and / or "consisting essentially of" are also provided.

[0035] Unless otherwise defined, all technical and scientific terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press, The Dictionary of Cell and Molecular Biology, 5th ed., 2013, Academic Press, and Oxford Dictionary Of Biochemistry and Molecular Biology, 2nd ed., 2008, Oxford University Press provide those of ordinary skill in the art with a general dictionary of many of the terms used in this disclosure.

[0036] Units, prefixes, and symbols are written in the format accepted by the International System of Units (SI). Numerical ranges are inclusive of the numbers that define the range. When a range of values ​​is listed, it is understood that each intervening integer value between the upper and lower limit values ​​listed for that range, and each fractional part thereof, is also expressly disclosed, along with each subrange between such values. The upper and lower limits of any range may be independently included or excluded from the range, and each range that includes either the upper or lower limit, each range that does not include either, or each range that includes both, is also encompassed by the present disclosure. Thus, ranges listed herein are understood to be shorthand for all values ​​within the range, including the recited endpoints. For example, a range of 1 to 10 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0037] When a value is explicitly stated, it is understood that values ​​that are about the same number or amount as the stated value (e.g., 10) (e.g., ±10%) are also included within the scope of the disclosure. When a combination is disclosed, each subcombination of the elements of the combination is also specifically disclosed and is included within the scope of the disclosure. Conversely, when different elements or groups of elements are disclosed separately, their combinations are also disclosed. When any element of a disclosure is disclosed as having multiple alternatives, the disclosed examples in which each alternative is excluded, alone or in any combination with other alternatives, are also disclosed herein, and two or more elements of a disclosure may have such an exclusion, and all combinations of elements with such exclusions are disclosed herein.

[0038] The term "about" is used herein to mean approximately, roughly, roughly, or within a range. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values ​​set forth. In general, the term "about" can modify a numerical value to be above or below the set forth value, for example, by a variance of up to 10 percent (higher or lower).

[0039] As used herein, the term "interleukin-7" or "IL-7" refers to IL-7 polypeptides and derivatives and analogs thereof that have substantial amino acid sequence identity to wild-type adult mammalian IL-7 and have substantially equivalent biological activity, e.g., in standard bioassays or assays of IL-7 receptor binding affinity. For example, IL-7 refers to the amino acid sequence of a recombinant or non-recombinant polypeptide having the amino acid sequence of i) a naturally occurring 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., human, bovine, or ovine. The nucleic acid and amino acid sequences of IL-7 are well known in the art. For example, the human IL-7 amino acid sequence has a Genbank accession number of P13232 (SEQ ID NO:1), the mouse IL-7 amino acid sequence has a Genbank accession number of P10168 (SEQ ID NO:3), the rat IL-7 amino acid sequence has a Genbank accession number of P56478 (SEQ ID NO:2), the monkey IL-7 amino acid sequence has a Genbank accession number of NP_001279008 (SEQ ID NO:4), the bovine IL-7 amino acid sequence has a Genbank accession number of P26895 (SEQ ID NO:5), and the ovine IL-7 amino acid sequence has a Genbank accession number of Q28540 (SEQ ID NO:6). In some embodiments, the IL-7 polypeptides of the present disclosure are variants of the IL-7 protein. As is evident from the present disclosure, in some embodiments, the IL-7 polypeptides useful in the present invention do not include a signal peptide (see, e.g., Table 1A). [Table 1-1] [Table 1-2]

[0040] As used herein, the term "vascular endothelial growth factor" or "VEGF" refers to a protein that is produced by many cells and stimulates the formation of blood vessels (i.e., angiogenesis). VEGF is part of a protein family that includes five major members: VEGF-A (also simply referred to as "VEGF"), placenta growth factor (PGF), VEGF-B, VEGF-C, and VEGF-D. Unless otherwise indicated, the term "VEGF" refers to the VEGF-A member of the family, including all isoforms (see, e.g., Table 1B) and variants thereof. There are at least 17 known isoforms of human VEGF (i.e., VEGF-A) due to alternative promoter usage, alternative splicing, and / or alternative initiation. The sequences of the different VEGF isoforms are shown in Table 1B (below). [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6]

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

[0042] The terms "antibody" and "antibodies" are terms of art and may be used interchangeably herein to refer to a molecule that comprises an antigen-binding site that specifically binds to an antigen. As used herein, these terms include whole antibodies, and any antigen-binding fragments (i.e., "antigen-binding portions") or single chains thereof. An "antibody" refers, in one aspect, to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains, or antigen-binding portions thereof, interconnected by disulfide bonds. In some aspects, an "antibody" refers to a single-chain antibody comprising a single variable domain, e.g., a VHH domain. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. In certain naturally occurring antibodies, the heavy chain constant region consists of three domains: CH1, CH2, and CH3. In certain naturally occurring antibodies, each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL.

[0043] The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). Each VH and VL contains three CDRs and four FRs arranged from amino-terminus to carboxy-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 the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0044] Antibodies are typically -5 ~10 -11 The dissociation constant (K D ) specifically binds to its cognate antigen with high affinity, reflected by approximately 10 -4 K exceeds M D As used herein, an antibody that "specifically binds" to an antigen is one that binds to the antigen and to a substantially identical antigen with high affinity, i.e., has a KD of 10 or greater. -7 M or less, 10 -8 M or less, 5×10 -9 M or less, or 10 -8 M~10 -10 By "substantially identical" to a given antigen is meant an antibody that binds with a high affinity of M or less, but does not bind with high affinity to unrelated antigens. An antigen is "substantially identical" to a given antigen if it exhibits a high degree of sequence identity to the given antigen, for example, if it exhibits at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the sequence of the given antigen. By way of example, an antibody that specifically binds to VEGF may, in certain embodiments, have cross-reactivity with VEGF antigens from a particular primate species (e.g., cynomolgus anti-VEGF antibodies), but cannot cross-react with VEGF molecules from other species or with molecules other than VEGF.

[0045] Immunoglobulins may be derived from any of the commonly known isotypes, including, but not limited to, IgA, secretory IgA, IgG, and IgM. IgG subclasses are also well known to those of skill 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) that is encoded by the heavy chain constant region genes. In some embodiments, one or more amino acids of an isotype can be mutated to alter effector functions. The term "antibody" includes, by way of example, both naturally occurring (Ab) and non-naturally occurring antibodies, monoclonal and polyclonal antibodies, chimeric and humanized antibodies, human or non-human antibodies, fully synthetic antibodies, and single chain antibodies. Non-human antibodies can be humanized by recombinant methods to reduce their immunogenicity in humans. Unless expressly stated, and unless the context dictates otherwise, the term "antibody" also includes antigen-binding fragments or portions of any of the aforementioned immunoglobulins, including monovalent and divalent fragments or portions, as well as single-chain antibodies.

[0046] By "isolated antibody" is meant 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 of different species. Moreover, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0047] The term "monoclonal antibody" ("mAb") refers to a non-naturally occurring preparation of antibody molecules of single molecular composition, i.e., essentially identical in primary sequence and displaying a single binding specificity and affinity for a particular epitope. A mAb is an example of an isolated antibody. MAbs can be produced by hybridoma, recombinant, transgenic, or other techniques known to those of skill in the art.

[0048] A "human" antibody (HuMAb) refers to an antibody having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Additionally, if the antibody contains a constant region, the constant region is also derived from a human germline immunoglobulin sequence. A human antibody of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" as used herein 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.

[0049] "Humanized antibody" refers to an antibody in which some, most, or all of the amino acids outside the CDR domains of a non-human antibody are replaced with the corresponding amino acids from a human immunoglobulin. In one embodiment of a humanized form of an antibody, some, most, or all of the amino acids outside the CDR domains are replaced with amino acids from a human immunoglobulin, while some, most, or all of the amino acids within one or more CDR regions remain unchanged. Minor additions, deletions, insertions, substitutions, or modifications of amino acids are permissible as long as they do not abrogate the ability of the antibody to bind to a specific antigen. A "humanized" antibody retains the same antigen specificity as the original antibody.

[0050] By "chimeric antibody" is meant an antibody whose variable region is derived from one species and whose constant region is derived from another species, such as an antibody whose variable region is derived from a mouse antibody and whose constant region is derived from a human antibody.

[0051] 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.

[0052] An "antigen-binding portion" of an antibody (also called an "antigen-binding fragment") means one or more fragments of an antibody that retain the ability to specifically bind to the antigen bound by the whole antibody.

[0053] As used herein, the terms "specific binding," "selective binding," "selectively binds," and "specifically binds" mean that an antibody binds to an epitope on a given antigen. Typically, an antibody binds to an epitope on a given antigen at a binding affinity of approximately 10% or more, as determined, for example, by surface plasmon resonance (SPR) techniques on a BIACORE™ 2000 instrument using a given antigen as the analyte and the antibody as the ligand, or by Scatchard analysis of antibody binding to antigen-positive cells. -7 Less than M, e.g., about 10 -8 M, 10 -9 M or 10 -10 The equilibrium dissociation constant (K D ) and (ii) binds to a given antigen with an affinity that is at least two-fold higher than the affinity with which it binds to a nonspecific antigen other than the given antigen or a closely related antigen (e.g., BSA, casein).

[0054] As used herein, the term "naturally occurring" refers to the fact that the subject can be found in nature when applied to an object.For example, a polypeptide or polynucleotide sequence that can be isolated from a natural source and exists in an organism (including viruses) that has not been intentionally modified by man in a laboratory is naturally occurring.

[0055] A "polypeptide" refers to a chain comprising at least two contiguously linked amino acid residues, with no upper limit on the length of the chain. One or more amino acid residues of a protein may contain modifications, such as, but not limited to, glycosylation, phosphorylation, or disulfide bond formation. A "protein" may include one or more polypeptides. Unless otherwise specified, the terms "protein" and "polypeptide" may be used interchangeably.

[0056] As used herein, the term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. A nucleic acid molecule may be single-stranded or double-stranded and may be cDNA.

[0057] "Conservative amino acid substitution" means that an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In some aspects, a predicted non-essential amino acid residue in an antibody is replaced with another amino acid residue from the same side chain family. Methods for identifying conservative nucleotide and amino acid substitutions that do not abolish antigen binding are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

[0058] In the case of nucleic acids, the term "substantial homology" indicates that two nucleic acids, or their designated sequences, when optimally aligned and compared, are identical in at least about 80%, at least about 90%-95%, or at least about 98%-99.5% of the nucleotides, with appropriate nucleotide insertions or deletions. Alternatively, substantial homology exists when the segments will hybridize under selective hybridization conditions, to the complement of the strand.

[0059] In the case of polypeptides, the term "substantial homology" indicates that two polypeptides, or their designated sequences, when optimally aligned and compared, are identical in at least about 80% of the amino acids, at least about 90%-95%, or at least about 98%-99.5% of the amino acids, with appropriate amino acid insertions or deletions.

[0060] The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = number of identical positions / total number of positions x 100), taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap. The comparison of sequences and determination of the percent identity between two sequences can be accomplished using a mathematical algorithm, for example, as described in the non-limiting examples below.

[0061] The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at worldwideweb.gcg.com) using a NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70, or 80 and length weights of 1, 2, 3, 4, 5, or 6. The percent identity between two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) as incorporated into the ALIGN program (version 2.0) using a PAM120 weighted residue table, a gap length penalty of 12, and a gap penalty of 4. Additionally, percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm incorporated into the GAP program of the GCG software package (available at worldwideweb.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0062] The nucleic acid and protein sequences described herein can further be used as "query sequences" to perform searches against public databases, for example to identify related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=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, wordlength=3 to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See worldwideweb.ncbi.nlm.nih.gov.

[0063] Nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is "isolated" or "substantially pure" when it has been purified from other cellular components or other contaminants, such as other cellular nucleic acids (e.g., other parts of chromosomes) or proteins, by standard techniques, including alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other techniques well known in the art. See F. Ausubel, et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).

[0064] Nucleic acids, e.g., cDNA, can be mutated according to standard techniques to produce gene sequences. In the case of coding sequences, these mutations can affect the amino acid sequence as desired. In particular, DNA sequences that are substantially homologous to or derived from natural V, D, J, constant sequences, switches, and other such sequences described herein are contemplated (where "derived" indicates that the sequence is identical to or modified from another sequence).

[0065] The term "vector" as used herein is intended to mean a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. Since plasmids are the most commonly used form of vector, "plasmid" and "vector" can be used interchangeably herein. However, also included are other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0066] The term "recombinant host cell" (or simply "host cell") as used herein is intended to mean 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 are intended to refer not only to the particular subject cell, but also to the progeny of such a cell. Since certain modifications may occur in subsequent generations, either by mutation or environmental influences, such progeny may not in fact be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0067] As used herein, the term "linked" refers to the attachment of two or more molecules. The attachment may be covalent or non-covalent. The attachment may be genetic (i.e., recombinantly fused). Such attachment may be achieved using a wide range of art-recognized techniques, such as chemical conjugation and recombinant protein production.

[0068] "Cancer" refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division and growth can result in the formation of malignant tumors that invade adjacent tissues and may metastasize to distant parts of the body via the lymphatic system or bloodstream. As used herein, "cancer" refers to primary cancers, metastatic cancers, and recurrent cancers. Non-limiting examples of suitable cancers that can be treated according to the present disclosure are provided elsewhere in this disclosure.

[0069] The term "fusion protein" refers to a protein created by the joining of two or more genes that originally encoded separate proteins. Translation of the fusion gene results in a single polypeptide or multiple polypeptides with functional properties derived from each of the original proteins. In some embodiments, the two or more genes may contain substitutions, deletions, and / or additions in their nucleotide sequences.

[0070] An "Fc receptor" or "FcR" is a receptor that binds to the Fc region of an immunoglobulin. FcRs that bind IgG antibodies include receptors of the FcγR family, including allelic variants and alternatively spliced ​​forms of these receptors. The FcγR family consists of three activating receptors (FcγRI, FcγRIII, and FcγRIV in mice; FcγRIA, FcγRIIA, and FcγRIIIA in humans) and one inhibitory receptor (FcγRIIB). The 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, whereas natural killer (NK) cells selectively express one activating Fc receptor (FcγRIII in mice and FcγRIIIA in humans) but do not selectively express inhibitory FcγRIIB in mice and humans. Human IgG1 binds to most human Fc receptors and is considered to be equivalent to mouse IgG2a with respect to the type of activating Fc receptor it binds.

[0071] "Fc region" (fragment crystallizable region) or "Fc domain" or "Fc" refers to the C-terminal region of an antibody heavy chain that mediates 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 includes the constant region of an antibody except for the first constant region immunoglobulin domain (e.g., CH1 or CL). In IgG, IgA and IgD antibody isotypes, the Fc region includes two identical protein fragments derived from the second (CH2) and third (CH3) constant domains of the antibody's two heavy chains, while the Fc region of IgM and IgE includes three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. In the case of IgG, the Fc region includes the immunoglobulin domains CH2 and CH3, as well as the hinge between the CH1 and CH2 domains. Although the definition of the boundaries of the Fc region of an immunoglobulin heavy chain may vary as defined herein, the human IgG heavy chain Fc region is defined to extend from amino acid residues D221 for IgG1, V222 for IgG2, L221 for IgG3, and P224 for IgG4 to the carboxy-terminus of the heavy chain, where the numbering is according to the EU index as per Kabat. The CH2 domain of the human IgG Fc region extends from amino acid 237 to amino acid 340, and the CH3 domain is located C-terminal to the CH2 domain in the Fc region, i.e., from amino acid 341 to amino acid 447 or 446 (if the C-terminal lysine residue is absent) or 445 (if the C-terminal glycine and lysine residues are absent) of IgG. As used herein, an Fc region may be a native sequence Fc, including any allotypic variants, or a variant Fc (e.g., a non-naturally occurring Fc). Fc may refer to this region alone or in the context of a protein polypeptide that contains Fc, such as a "binding protein comprising an Fc region," also referred to as an "Fc fusion protein" (e.g., an antibody or immunoadhesin).

[0072] A "native sequence Fc region" or "native sequence Fc" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include native sequence human IgG1 Fc region, native sequence human IgG2 Fc region, native sequence human IgG3 Fc region, and native sequence human IgG4 Fc region, as well as naturally occurring variants thereof. Native sequence Fc includes the various allotypes of Fc (see, e.g., Jefferis et al. (2009) mAbs 1:1).

[0073] Furthermore, the Fc (native or variant) of the present disclosure may be in a form having native, more or less glycosylation compared to the native form, or may be in a deglycosylated form. The Fc glycosylation of an immunoglobulin can be modified by conventional methods, such as chemical, enzymatic, and genetic engineering methods using microorganisms. Removal of the glycosylation from the Fc fragment significantly reduces the binding affinity to the C1q portion of the first component of complement C1, and reduces or eliminates ADCC or CDC, so that no unwanted immune response is induced in vivo. In this regard, a deglycosylated or nonglycosylated 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 have been enzymatically removed from the Fc fragment. Furthermore, the term "nonglycosylated" refers to an Fc fragment produced in a nonglycosylated form by a prokaryote, preferably in E. coli.

[0074] As used herein, the term "immune response" refers to a biological response in a vertebrate to foreign agents that protects the organism against these agents and the diseases caused by them. As used herein, the term "anti-tumor immune response" refers to an immune response to tumor antigens. Immune responses are mediated by the action of cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and soluble macromolecules (including antibodies, cytokines, and complement) produced by any of these cells or the liver that selectively target, bind to, damage, destroy, and / or eliminate from the vertebrate body invading pathogens, pathogen-infected cells or tissues, cancer or other abnormal cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues. Immune responses include, for example, T cells, e.g., effector T cells or Th cells, e.g., CD4 + Or CD8 + Examples of such effects include activation or inhibition of T cells, or inhibition of Treg cells.

[0075] The term "immunotherapy" refers to the treatment of a subject suffering from a disease or at risk of suffering from or having a recurrence of a disease by methods that involve inducing, enhancing, suppressing, or otherwise modifying the immune response. "Treatment" or "therapy" of a subject refers to any type of intervention or process performed on a subject, or the administration of an active agent to a subject, for the purpose of reversing, alleviating, ameliorating, inhibiting, slowing, or preventing the onset, progression, development, severity, or recurrence of a symptom, complication, or pathology, or biochemical manifestation associated with a disease.

[0076] The term "effector T cells" (Teff) refers to T cells with cytolytic activity (e.g., CD4 + and CD8 +The term "IL-7" refers to T cells, which secrete cytokines to activate and induce other immune cells, and T helper (Th) cells, but does not include regulatory T cells (Treg cells). The combination of IL-7 protein and a VEGF antagonist (e.g., an anti-VEGF antibody) can stimulate Teff cells, e.g., CD4 + and CD8 + T cells become activated and / or their frequency increases.

[0077] As used herein, the term "regulatory T cells" (Tregs) refers to a population of T cells that have the ability to reduce or suppress the induction and proliferation of effector T cells, thereby regulating the immune response. In some embodiments, Tregs can suppress immune responses by secreting anti-inflammatory cytokines such as IL-10, TGF-β, and IL-35, which can prevent the activation and differentiation of naive T cells into effector T cells. In some embodiments, Tregs can also produce cytolytic molecules such as granzyme B, which can induce apoptosis of effector T cells. In some embodiments, regulatory T cells are endogenous regulatory T cells (nTregs) (i.e., develop in the thymus). In some embodiments, regulatory T cells are induced regulatory T cells (iTregs) (i.e., naive T cells that differentiate into Tregs in peripheral tissues upon exposure to a specific stimulus). Methods for identifying Tregs are well known in the art. For example, Tregs express certain phenotypic markers (e.g., CD25, Foxp3, or CD39) that can be measured using flow cytometry. See, e.g., International Publication No. WO2017 / 062035 A1; Gu J., et al., Cell Mol Immunol 14(6):521-528(2017). In some embodiments, Tregs express CD45RA - CD39 + T cells.

[0078] As used herein, the term "tumor infiltrating lymphocytes" or "TILs" refers to lymphocytes (e.g., effector T cells) that have migrated from the periphery (e.g., from the blood) into a tumor. In some embodiments, the tumor infiltrating lymphocytes are CD4+ TILs. In some embodiments, the tumor infiltrating lymphocytes are CD8+ TILs.

[0079] The increased ability to stimulate an immune response or immune system may be due to improved agonist activity of T cell costimulatory receptors and / or improved antagonist activity of inhibitory receptors. The increased ability to stimulate an immune response or immune system may be reflected in the fold increase in EC50 or maximum activity level in assays that measure immune responses, such as assays that measure cytokine or chemokine release, cytolytic activity (directly determined on target cells or indirectly by detecting CD107a or granzymes), and changes in proliferation. The ability to stimulate an immune response or immune system activity may be increased by at least 10%, 30%, 50%, 75%, 2-fold, 3-fold, 5-fold, or more.

[0080] For example, a "variant" of an IL-7 protein is defined as an amino acid sequence in which one or more amino acids have been changed. A variant may have "conservative" changes, such as the replacement of a leucine with an isoleucine, where the substituted amino acid has similar structural or chemical properties. Less commonly, a variant may have "non-conservative" changes, such as the replacement of a glycine with a tryptophan. Similar minor changes may also include amino acid deletions or insertions, or both. Guidance on determining the type and number of amino acid residues that can be substituted, inserted, or deleted without impairing biological activity can be found using computer programs well known in the art, such as software for molecular modeling or alignment generation. For example, variant IL-7 proteins included in the present disclosure include IL-7 proteins that retain IL-7 activity. Similarly, variants of the VEGF antagonists described herein refer to those that retain antagonist activity against VEGF. Variants, including additions, substitutions, or deletions, are also included within the disclosure so long as they retain substantially the same biological activity as the corresponding non-variant (e.g., wild-type) form. For example, truncated forms of IL-7 that retain the same biological activity as the full-length form of the IL-7 protein are included in the disclosure. The activity of the therapeutic agents described herein (e.g., IL-7 protein and / or VEGF antagonists) can be measured using any suitable method known in the art.

[0081] To determine the percent identity of two amino acid sequences or two nucleic acids, the sequences are aligned for optimal comparison (e.g., a first amino acid sequence or a nucleic acid sequence can be introduced with gaps for optimal alignment with a second amino acid or nucleic acid sequence). The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology=number of identical positions / total number of positions×100). The determination of the percent homology between two sequences can be achieved using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul, et al., (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=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.

[0082] A "subject" includes any human or non-human animal. The term "non-human animal" includes, but is not limited to, vertebrates, such as non-human primates, sheep, dogs, and rodents, such as mice, rats, and guinea pigs. In some aspects, the subject is a human. The terms "subject" and "patient" are used interchangeably herein.

[0083] The term "therapeutically effective amount" or "therapeutically effective dosage" refers to an amount of an agent that produces a desired biological, therapeutic, and / or prophylactic result. The result can be reduction, amelioration, alleviation, attenuation, delay, and / or relief of one or more of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. With respect to solid tumors, an effective amount includes an amount sufficient to shrink the tumor and / or reduce the rate of growth of the tumor (e.g., inhibit tumor growth), or prevent or slow other undesirable cell proliferation. In some embodiments, an effective amount is an amount sufficient to slow the progression of the tumor. In some embodiments, an effective amount is an amount sufficient to prevent or slow the recurrence of the tumor. An effective amount can be administered in one or more administrations. An effective amount of a drug or composition can (i) reduce the number of cancer cells, (ii) reduce tumor size, (iii) inhibit, delay, slow to some extent, or stop the invasion of cancer cells into peripheral organs, (iv) inhibit (i.e., slow to some extent, or stop) tumor metastasis, (v) inhibit tumor growth, (vi) prevent or delay the onset and / or recurrence of tumors, and / or (vii) alleviate to some extent one or more symptoms associated with cancer. In some embodiments, 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 proven to affect a significant reduction in cancer, such as an advanced solid tumor, or a slowing (regression) of the progression of cancer. The ability of a therapeutic agent to promote disease regression can be assessed using a variety of methods known to the skilled practitioner, for example, by assaying the activity of the agent in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or in in vitro assays.

[0084] The term "dosing frequency" refers to the number of times that a therapeutic agent (e.g., an IL-7 protein and / or a VEGF antagonist) is administered to a subject within a particular period of time. Dosing frequency can be indicated as the number of doses per given time, such as, for example, once a day, once a week, or once every two weeks. As used herein, "dosing frequency" is applicable when a subject receives multiple (or repeated) doses of a therapeutic agent. Suitable dosing frequencies for any of the IL-7 proteins and VEGF antagonists described herein are provided elsewhere in this disclosure.

[0085] As used herein, the term "standard of care" refers to a treatment that is accepted by medical professionals as an appropriate treatment for a particular type of disease and is widely used by medical practitioners. The term may be used interchangeably with any of the following terms: "best practice," "standard of care," and "standard of care." As further described elsewhere in this disclosure, in some aspects, the standard of care includes chemotherapy (e.g., temozolomide). In some aspects, the standard of care includes radiation therapy. In some aspects, the standard of care includes both radiation therapy and chemotherapy (e.g., temozolomide).

[0086] As used herein, the term "drug" refers to any bioactive agent (e.g., the IL-7 protein and / or VEGF antagonists disclosed herein) 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 embodiments, the drugs disclosed herein are anti-cancer drugs. Unless otherwise specified, the term "drug" may be used interchangeably with the term "therapeutic agent."

[0087] By way of example, an "anti-cancer drug" promotes the regression of a cancer in a subject or prevents further tumor growth. In some embodiments, a therapeutically effective amount of a drug promotes the regression of a cancer to the point that the cancer disappears. "Promoting the regression of a cancer" means that administration of an effective amount of the drug, alone or in combination with an anti-tumor drug, reduces the growth or size of a tumor, necroses the tumor, reduces the severity of at least one disease symptom, increases the frequency and duration of disease-free periods, or prevents functional impairment or disability due to disease affliction. Furthermore, the terms "effective" and "effectiveness" in relation to treatment include both pharmacological effectiveness and physiological safety. Pharmacological effectiveness refers to the ability of a drug to promote the regression of a cancer in a patient. Physiological safety refers to the level of toxicity or other adverse physiological effects (adverse effects) at the cell, organ, and / or organism level resulting from administration of the drug.

[0088] For example, in the treatment of tumors, a therapeutically effective amount of an anti-cancer drug can inhibit cell proliferation or tumor growth by at least about 10%, at least about 20%, at least about 40%, at least about 60%, or at least about 80% compared to untreated subjects, or in some embodiments, compared to patients treated with standard treatment therapy. In some embodiments, tumor regression can be observed and continues for a period of at least about 20 days, at least about 40 days, or at least about 60 days. Regardless of these ultimate measurements of therapeutic efficacy, the evaluation of immunotherapeutic agents must also take into account "immune-related" response patterns.

[0089] As used herein, the term "immune checkpoint inhibitor" refers to a molecule that completely or partially reduces, inhibits, prevents, or modulates one or more immune checkpoint proteins. Checkpoint proteins regulate T cell activation or function. A number of checkpoint proteins are known, such as CTLA-4 and its ligands CD80 and CD86, and PD-1 and its ligands PD-L1 and PD-L2. Pardoll, DM, Nat Rev Cancer 12(4):252-64(2012). These proteins are responsible for costimulatory or inhibitory interactions of T cell responses. Immune checkpoint proteins regulate and maintain self-tolerance, as well as the duration and extent of physiological immune responses. Immune checkpoint inhibitors include or are derived from antibodies. Suitable immune checkpoint inhibitors useful in the present disclosure are set forth elsewhere in this disclosure.

[0090] As used herein, the term "reference subject" refers to a corresponding subject (e.g., a cancer subject) who was not administered a combination treatment described herein (e.g., a combination of a VEGF antagonist and an IL-7 protein). In some embodiments, the reference subject refers to a corresponding subject who was administered an IL-7 protein alone or a VEGF antagonist alone. In some embodiments, the reference subject was not administered an IL-7 protein or 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 prior to administration of a combination of an IL-7 protein and a VEGF antagonist. In certain embodiments, the term "reference subject" refers to the average of a population of subjects (e.g., cancer subjects).

[0091] As used herein, the terms "ug" and "uM" are used interchangeably with "μg" and "μM," respectively.

[0092] The various aspects described herein are described in further detail in the following subsections.

[0093] II. Method of Disclosure Provided herein is a method of treating a tumor in a subject in need of tumor (or cancer) treatment, comprising administering to the subject a combination of an interleukin-7 (IL-7) protein and an antagonist of vascular endothelial growth factor ("VEGF") ("VEGF antagonist"). Non-limiting examples of IL-7 proteins and VEGF antagonists useful in the methods are provided elsewhere in this disclosure. As demonstrated herein, treatment regimens including an IL-7 protein and a VEGF antagonist (such as those described herein) can be associated with much improved anti-tumor immunity compared to either therapeutic agent alone.

[0094] Thus, in some embodiments, administering the combination treatment described herein (e.g., a combination of a VEGF antagonist and an IL-7 protein) can increase the anti-tumor immune response in a subject. In some embodiments, after administration, the anti-tumor immune response 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 in a reference subject. For example, in some embodiments, the anti-tumor immune response in a subject is increased compared to the anti-tumor immune response in a subject before administration of the combination treatment. In some embodiments, the anti-tumor immune response in a subject is increased compared to the corresponding subject administered with an IL-7 protein alone or a VEGF antagonist alone. In some embodiments, the anti-tumor immune response in the subject is increased as compared to a corresponding subject that is not administered either the IL-7 protein or the VEGF antagonist alone.

[0095] In some embodiments, an increase in anti-tumor immune response may be associated with one or more of the following therapeutic effects: (i) a decrease in tumor volume and / or growth, (ii) an increase in effector activity (e.g., ability to kill tumor cells) of tumor-specific T cells, (iii) an increase in the number of tumor-specific effector T cells, e.g., in the tumor, (iv) an increase in the number of tumor-infiltrating lymphocytes (TIL), e.g., in the tumor, (v) a decrease in the number of myeloid-derived suppressor cells (MDSC), e.g., in the tumor, (vi) a decrease in the number of regulatory T cells (Treg), e.g., in the tumor, (vii) an increase in the survival time of the subject, (viii) a decrease in the expression of TOX on CD8+ T cells, e.g., in the tumor, tumor-draining lymph nodes (TDLN), or both, (ix) an increase in the number of stem cell-like T cells, e.g., in the tumor, TDLN, or both, or (x) any combination thereof. Unless otherwise specified, the term "number" refers to both absolute numbers and percentages. Any of the therapeutic effects described above or elsewhere in this disclosure can be determined using any suitable method provided herein and / or known in the art.

[0096] In some embodiments, administering the combination treatment described herein (e.g., a combination of a VEGF antagonist and an IL-7 protein) reduces tumor volume in a subject. In some embodiments, the tumor volume is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% after administration compared to the corresponding value in a reference subject. In some embodiments, administering the combination treatment described herein (e.g., a combination of a VEGF antagonist and an IL-7 protein) can reduce tumor growth in a subject. In some embodiments, the 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% compared to the corresponding value in a reference subject.

[0097] In some embodiments, administration of a combination treatment described herein (e.g., a combination of a VEGF antagonist and an IL-7 protein) increases tumor-specific T cells (e.g., CD8 + T cells and / or CD4 + The effector activity of T cells (e.g., CD8 T cells) that may be useful in the treatment of cancer may be increased. In some embodiments, the effector activity 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. In some embodiments, the effector activity is increased by at least about 0.5-fold, at least about 1-fold, at least about 2-fold, at least about 3 ... + T cells and / or CD4 +Effector activities include any cytolytic activity of T cells. Non-limiting examples of such effector activities include the ability to produce effector molecules (e.g., cytokines, perforin, and / or granzymes) upon antigenic stimulation, the ability to recognize and kill tumor cells, or both.

[0098] In some embodiments, administration of a combination treatment described herein (e.g., a combination of a VEGF antagonist and an IL-7 protein) increases tumor-specific effector T cells (e.g., CD8 + T cells and / or CD4 + In some embodiments, 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.

[0099] Without being bound to any one theory, in some embodiments, the increase in number is due to an increase in the proliferation of tumor-specific effector T cells. Thus, in some embodiments, the proliferation of tumor-specific effector T cells increases 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 after administration of a combination treatment described herein (e.g., a combination of an IL-7 protein and a VEGF antagonist) compared to the corresponding value in a reference subject. In some embodiments, the increase in the number of tumor-specific effector T cells is due to the ability of the T cells to survive and / or persist. In some embodiments, the ability of tumor-specific effector T cells to survive and / or persist in a subject 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. In some embodiments, the combination treatments described herein (e.g., a combination of a VEGF antagonist and an IL-7 protein), when administered to a subject, can increase both the proliferation and survival / persistence of tumor-specific effector T cells.

[0100] In some embodiments, the tumor-specific effector T cell is a tumor-infiltrating lymphocyte (TIL). Thus, in some embodiments, the number of TILs in the tumor of a subject can be increased by administering the combination treatment described herein (e.g., a combination of a VEGF antagonist and an IL-7 protein). In some embodiments, the number of TILs in the tumor of a subject 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 in a reference subject. In some embodiments, the increase in the number of TILs in the tumor of a subject can be associated with an increase in the recruitment of TILs from the periphery to the tumor. In some embodiments, recruitment of TILs into 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 following administration of a combination treatment described herein (e.g., a combination of an IL-7 protein and a VEGF antagonist) compared to corresponding values ​​in reference subjects.

[0101] In some embodiments, administering a combination treatment described herein (e.g., a combination of a VEGF antagonist and an IL-7 protein) can reduce the number of regulatory T cells (Tregs) in a subject's tumor. In some embodiments, regulatory T cells are CD4 + In some embodiments, the regulatory T cells are Foxp3 +In some embodiments, the number and / or percentage of regulatory T cells is decreased 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.

[0102] In some embodiments, administering the combination treatments described herein (e.g., a combination of a VEGF antagonist and an IL-7 protein) can reduce the number of myeloid-derived suppressor cells (MDSCs) in a subject's tumor. As used herein, the term "myeloid-derived suppressor cells" (MDSCs) refers to a heterogeneous population of immune cells defined by their myeloid origin, immature state, and ability to potently suppress T cell responses. They are known to proliferate in certain pathological conditions, such as chronic infection and cancer. In some embodiments, the MDSCs are monocytic MDSCs (M-MDSCs). In some embodiments, the MDSCs are polymorphonuclear MDSCs (PMN-MDSCs). In some embodiments, following administration, the number of MDSCs in the tumor is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% compared to the corresponding value in a reference subject.

[0103] As will be apparent to one of skill in the art, in some embodiments, one or more of the therapeutic effects described above may result in (i) an increase in the subject's survival time (e.g., progression-free survival time), (ii) an increase in the response rate, (iii) a decrease in disease progression, or (iv) a combination thereof. Thus, in some embodiments, administering a combination treatment described herein (e.g., a combination of a VEGF antagonist and an IL-7 protein) can increase the survival time of a subject (e.g., a tumor-bearing subject). In some embodiments, the survival time 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. In some embodiments, after administration, the subject's survival time is increased by at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 1 year or more compared to the reference subject. In some embodiments, after administration, the subject's survival time is increased by a level higher (about 1 month higher, about 2 months higher, about 3 months higher, about 4 months higher, about 5 months higher, about 6 months higher, about 7 months higher, about 8 months higher, about 9 months higher, about 10 months higher, about 11 months higher, or about 1 year higher) than the survival time of the reference subject.

[0104] In some embodiments, administering a combination treatment (e.g., a combination of a VEGF antagonist and an IL-7 protein) described herein can increase progression-free survival of a subject (e.g., a cancer patient). For example, in some embodiments, the progression-free survival of a subject is increased by at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 1 year compared to a reference subject.

[0105] In some embodiments, administering a combination treatment described herein (e.g., a combination of a VEGF antagonist and an IL-7 protein) can increase the response rate in a subject population. For example, the response rate in a subject population is 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 higher than the corresponding value in a reference subject.

[0106] In some embodiments, administration of a combination therapy described herein (e.g., a combination of a VEGF antagonist and an IL-7 protein) can reduce cancer progression. In some embodiments, following administration, disease progression 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 corresponding values ​​in reference subjects.

[0107] In some embodiments, administration of the combination treatments described herein (e.g., a combination of a VEGF antagonist and an IL-7 protein) can reduce, for example, expression of TOX in CD8+ T cells present in a tumor, a tumor-draining lymph node, or both. In some embodiments, following administration, 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% compared to the corresponding value in a reference subject. Without being bound by any theory, it is believed that, at least in CD8+ T cells, TOX plays an important role in inducing T cell exhaustion (see, e.g., Khan et al., Nature 571: 211-218 (2019)). Thus, in some embodiments, the combination treatments described herein (e.g., a combination of a VEGF antagonist and an IL-7 protein) can help reduce T cell exhaustion when administered to a subject.

[0108] In some embodiments, administering the combination treatments described herein can 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" refers to T cells that are less differentiated while 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 embodiments, stem cell-like T cells useful in the present disclosure are TCF1+ and TOX-. In some embodiments, stem cell-like T cells are further TIM3-. In some embodiments, following administration, the number of stem cell-like 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.

[0109] In some embodiments, the subject treated with the methods is a non-human animal, such as a rat or a mouse. In some embodiments, the subject treated with the methods is a human.

[0110] As will be apparent from the present disclosure, the combination treatments described herein (e.g., a combination of a VEGF antagonist and an IL-7 protein) can be used to treat any suitable cancer known in the art. Non-limiting examples of cancers (or tumors) that can be treated by the disclosure set forth 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), colon cancer, endometrial cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), prostate cancer (e.g., hormone refractory prostate cancer), thyroid cancer, pancreatic cancer, foetal cancer, and ovarian cancer. cervical cancer, cancer of the stomach, cancer of the bladder, hepatocellular carcinoma, breast cancer, colon cancer, and head and neck cancer (or carcinoma), stomach cancer, germ cell tumors, childhood sarcoma, sinonasal natural killer, melanoma (e.g., metastatic malignant melanoma, e.g., cutaneous or intraocular malignant melanoma), bone cancer, skin cancer, uterine cancer, cancer of the anal region, testicular cancer, fallopian tube cancer, endometrial carcinoma, 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 of childhood, ureteral cancer, renal pelvic cancer, tumor angiogenesis, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, virus-associated cancers or cancers of viral origin (e.g., tumors associated with or derived from human papillomavirus (HPV)), and the two major blood cell lineages, i.e., myeloid (producing granulocytes, erythrocytes, platelets, macrophages, and mast cells) or lymphoma. Hematological malignancies originating from any of the cytoplasmic cell lineages (producing B cells, T cells, NK cells, and plasma cells), e.g., all types of leukemias, lymphomas, and myelomas, e.g., acute, chronic, lymphocytic, and / or myeloid leukemias, e.g., acute leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML), anaplastic AML (MO), myeloblastic leukemia (Ml), myeloblastic leukemia (M2;cytoplasmic leukemia (M3 or M3 variant [M3V]), myelomonocytic leukemia (M4 or M4 variant [M4E] with eosinophilia), monocytic leukemia (M5), erythroleukemia (M6), megakaryoblastic leukemia (M7), isolated granulocytic sarcoma, and chloroma; lymphomas, e.g., Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), B-cell hematological malignancies, e.g., B-cell lymphoma, T-cell lymphoma, lymphoplasmacytic lymphoma, monocytoid 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, hemangiocentric 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 somatic Liquid lymphoma, B-cell lymphoma, lymphoblastic lymphoma (LBL), hematopoietic neoplasms of the lymphatic system, acute lymphoblastic leukemia, diffuse large B-cell 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 Sézary syndrome), and Waldenström-Stöckel syndrome. Lymphoplasmacytic lymphoma (LPL) with hematopoietic macroglobulinemia; myelomas, e.g., IgG myeloma, light chain myeloma, nonsecretory myeloma, smoldering myeloma (also called asymptomatic myeloma), solitary plasmacytoma, and multiple myeloma, chronic lymphocytic leukemia (CLL), hairy cell lymphoma; hematopoietic tumors of myeloid lineage, tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; seminoma, teratoma, fibrosarcoma, rhabdomyosarcoma, and rhabdomyosarcoma; tumors of mesenchymal origin, including rhabdomyosarcoma, and osteosarcoma; as well as other tumors, such as melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, follicular thyroid carcinoma and teratoma, hematopoietic tumors of the lymphatic system, such as T-cell tumors and B-cell tumors, including, but not limited to, T-prolymphocytic leukemia (T-PLL) including small cell and cerebriform cell types, T-cell types of large granular lymphocyte leukemia (LGL); a / d T-NHL hepatosplenic lymphoma; peripheral / post-thymic T-cell lymphoma (pleomorphic and immunoblastic subtypes); angiocentric (nasal) T-cell lymphoma; head and neck cancer, renal cancer, rectal cancer, thyroid cancer; acute myeloid lymphoma, and any combination thereof.

[0111] In some embodiments, 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, colon cancer, prostate cancer, liver cancer, bladder cancer, kidney cancer, pancreatic cancer, thyroid cancer, esophageal cancer, eye cancer, stomach cancer (gastric cancer), gastrointestinal cancer, carcinoma, sarcoma, leukemia, lymphoma, myeloma, or a combination thereof. In some embodiments, the cancer (tumor) that can be treated with the present disclosure is pancreatic cancer. In some embodiments, the cancer (or tumor) that can be treated is brain tumor. In some embodiments, the brain tumor includes a glioma. In some embodiments, the brain tumor includes a glioblastoma. In some embodiments, the glioblastoma includes a primary glioblastoma. In some embodiments, the glioblastoma includes a secondary glioblastoma. In some embodiments, the glioblastoma that can be treated with the present disclosure is refractory (e.g., to standard of care including previous cancer treatments, e.g., radiation therapy and / or chemotherapy, such as temozolomide). In some embodiments, the cancer (or tumor) that can be treated is 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.

[0112] In some embodiments, the disclosures provided herein (e.g., combinations of IL-7 proteins and VEGF antagonists) can also be used to treat metastatic cancer, unresectable refractory cancer (e.g., cancer refractory to previous cancer therapy, e.g., immunotherapy and / or standard of care), and / or recurrent cancer. In some embodiments, the previous cancer therapy comprises chemotherapy. In some embodiments, the chemotherapy comprises a platinum-based therapy. In some embodiments, the platinum-based therapy comprises a platinum-based anti-neoplastic agent selected from the group consisting of cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, satraplatin, and any combination thereof. In some embodiments, the platinum-based therapy comprises cisplatin. In some embodiments, the platinum-based therapy comprises carboplatin.

[0113] In some embodiments, the subject treated by the disclosure provided herein has undergone one, two, three, four, five, or more previous cancer treatments. In some embodiments, the subject is treatment naive (i.e., has never undergone a previous cancer treatment). In some embodiments, the subject has progressed on other cancer treatments. In some embodiments, the previous cancer treatment included immunotherapy. In some embodiments, the previous cancer treatment included chemotherapy. In other embodiments, the previous cancer treatment included radiation therapy. In some embodiments, the previous cancer treatment included standard of care, e.g., chemotherapy (e.g., temozolomide) and / or radiation therapy. In some embodiments, the tumor has recurred. In some embodiments, the tumor is metastatic. In some embodiments, the tumor is not metastatic. Thus, as further described elsewhere in this disclosure, in some embodiments, the subject treated by the present disclosure is afflicted with a cancer that is resistant to a previous cancer treatment (e.g., standard of care). In some aspects, subjects that can be treated with the present disclosure suffer from glioblastoma that is resistant to standard treatments (e.g., radiation therapy and / or temozolomide).

[0114] In some embodiments, a unit dose (e.g., for use in humans) of an IL-7 protein disclosed herein can range from about 0.001 mg / kg to about 10 mg / kg. In some embodiments, the unit dose of an IL-7 protein ranges from about 0.01 mg / kg to about 2 mg / kg. In some embodiments, the unit dose ranges from about 0.02 mg / kg to about 1 mg / kg.

[0115] In some embodiments, the IL-7 protein disclosed herein can be administered to a subject at a dose based on body weight. In some embodiments, the IL-7 protein can be administered at a dose based on body weight of about 20 μg / kg to about 600 μg / kg. In some embodiments, the IL-7 protein of the present disclosure can be administered at a dose based on body 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 embodiments, the IL-7 protein is administered to a subject at a dose of 60 μg / kg. In some embodiments, the IL-7 protein is administered to a subject at a dose of 120 μg / kg. In some embodiments, the IL-7 protein is administered to a subject at a dose of 240 μg / kg.

[0116] In some embodiments, the IL-7 protein disclosed herein can be administered to a subject at a dose of greater than about 600 μg / kg. In some embodiments, 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.

[0117] In some embodiments, the IL-7 protein of the present disclosure is administered at a concentration of 610 μg / kg to about 1,200 μg / kg, 650 μg / kg to about 1,200 μg / kg, about 700 μg / kg to about 1,200 μg / kg, about 750 μg / kg to about 1,200 μg / kg, about 800 μg / kg to about 1,200 μg / kg, about 850 μg / kg to about 1,200 μg / kg, about 900 μg / kg to about 1,200 μg / kg, about 950 μg / kg to about 1,200 μg / kg, about 1,000 μg / kg to about 1,200 μg / kg, about 1,050 μg / kg to about 1,200 μg / kg, about 1,100 μg / kg to about 1,200 μg / kg, about 1,200 μg / kg, about 1,200 μg / kg, about 1,200 μg / kg, about 1,300 μg / kg, about 1,400 μg / kg, about 1,500 μg / kg, about 1,600 μg / kg, about 1,700 μg / kg, about 1,800 μg / kg, about 1,900 μg / kg, about 1,100 μg / kg, about 1,200 μg / kg, about 1,200 μg / kg, about 1,300 μg / kg, about 1,400 μg / kg, about 1,500 μg / kg, about 1,600 μg / kg, about 1,700 μg / kg, about 1,800 μg / kg, about 1,900 μg / kg, about 1,2 μg / kg ~ approx. 2,000 μg / kg, approx. 1,300 μg / kg ~ approx. 2,000 μg / kg, approx. 1,500 μg / kg ~ approx. 2,000 μg / kg, approx. 1, 700μg / kg ~ approx. 2,000μg / kg, approx. 610μg / kg ~ approx. 1,000μg / kg, approx. 650μg / kg ~ approx. 1,000μg / kg, approx. 700μ The therapeutic agent is administered at a dose of about 750 μg / kg to about 1,000 μg / kg, about 800 μg / kg to about 1,000 μg / kg, about 850 μg / kg to about 1,000 μg / kg, about 900 μg / kg to about 1,000 μg / kg, or about 950 μg / kg to about 1,000 μg / kg.

[0118] In some embodiments, the IL-7 protein of the present disclosure is administered at a dose of 610 μg / kg to about 1,200 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of 650 μg / kg to about 1,200 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 700 μg / kg to about 1,200 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 750 μg / kg to about 1,200 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 800 μg / kg to about 1,200 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 850 μg / kg to about 1,200 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 900 μg / kg to about 1,200 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 950 μg / kg to about 1,200 μg / kg. In some embodiments, the IL-7 protein disclosed herein is administered at a dose of about 1,000 μg / kg to about 1,200 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,050 μg / kg to about 1,200 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,100 μg / kg to about 1,200 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,200 μg / kg to about 2,000 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,300 μg / kg to about 2,000 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,500 μg / kg to about 2,000 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,700 μg / kg to about 2,000 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 610 μg / kg to about 1,000 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 650 μg / kg to about 1,000 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 700 μg / kg to about 1,000 μg / kg.In some embodiments, the IL-7 protein is administered at a dose of about 750 μg / kg to about 1,000 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 800 μg / kg to about 1,000 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 850 μg / kg to about 1,000 μg / kg. In some embodiments, the IL-7 protein of the present disclosure is administered at a dose of about 900 μg / kg to about 1,000 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 950 μg / kg to about 1,000 μg / kg.

[0119] In some embodiments, the IL-7 protein is administered at a dose of about 700 μg / kg to about 900 μg / kg, about 750 μg / kg to about 950 μg / kg, about 700 μg / kg to about 850 μg / kg, about 750 μg / kg to about 850 μg / kg, about 700 μg / kg to about 800 μg / kg, about 800 μg / kg to about 900 μg / kg, about 750 μg / kg to about 850 μg / kg, or about 850 μg / kg to about 950 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 700 μg / kg to about 900 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 750 μg / kg to about 950 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 700 μg / kg to about 850 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 750 μg / kg to about 850 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 700 μg / kg to about 800 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 800 μg / kg to about 900 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 750 μg / kg to about 850 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 850 μg / kg to about 950 μg / kg.

[0120] In some embodiments, the IL-7 protein is administered at a concentration of 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 98 0μg / kg, approximately 1,000μg / kg, approximately 1,020μg / kg, approximately 1,020μg / kg, approximately 1,040μg / kg, approximately 1,060μg / kg, approximately 1,080μg / kg, approximately 1,100μg / kg, approximately 1,120μg / kg, approximately 1,1 1,3 20μg / kg, approximately 1,340μg / kg, approximately 1,360μg / kg, approximately 1,380μg / kg, approximately 1,400μg / kg, approximately 1,420μg / kg, approximately 1,440μg / kg, approximately 1,460μg / kg, approximately 1,480μg / kg, approximately 1, 500μg / kg, approximately 1,520μg / kg, approximately 1,540μg / kg, approximately 1,560μg / kg, approximately 1,580μg / kg, approximately 1,600μg / kg, approximately 1,620μg / kg, approximately 1,640μg / kg, approximately 1,660μg / kg, approximately 1 and 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.

[0121] In some embodiments, the IL-7 protein is administered at a dose of about 650 μg / kg. In some embodiments, the IL-7 protein disclosed herein is administered at a dose of about 680 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 700 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 720 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 740 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 750 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 760 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 780 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 800 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 820 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 840 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 850 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 860 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 880 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 900 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 920 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 940 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 950 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 960 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 980 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,000 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,020 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,040 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,060 μg / kg.In some embodiments, the IL-7 protein is administered at a dose of about 1,080 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,100 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,120 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,140 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,160 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,180 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,200 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,220 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,240 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,260 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,280 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,300 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,320 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,340 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,360 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,380 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,400 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,420 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,440 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,460 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,480 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,500 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,520 μg / kg.In some embodiments, the IL-7 protein is administered at a dose of about 1,540 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,560 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,580 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,600 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,620 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,640 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,660 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,680 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,700 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,720 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,740 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,760 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,780 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,800 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,820 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,840 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,860 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,880 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,900 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,920 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,940 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,960 μg / kg. In some embodiments, the IL-7 protein is administered at a dose of about 1,980 μg / kg.In some embodiments, the IL-7 protein is administered at a dose of about 2,000 μg / kg.

[0122] In some embodiments, the IL-7 protein can be administered at a fixed dose. In some embodiments, the IL-7 protein can be administered at a fixed dose of about 0.25 mg to about 9 mg. In some embodiments, the IL-7 protein can be administered at a fixed dose of about 0.25 mg, about 1 mg, about 3 mg, about 6 mg, or about 9 mg.

[0123] In some embodiments, a subject disclosed herein is administered a single dose of IL-7 protein at any of the doses described above. In some embodiments, an IL-7 protein disclosed herein is administered to a subject in multiple doses (i.e., repeated administrations). In some embodiments, an IL-7 protein is administered to a subject at least two times, 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.

[0124] When IL-7 protein is administered multiple times to a subject (e.g., having a tumor), in some embodiments, the IL-7 protein is administered at a dosing frequency of about once per week, about once per 2 weeks, about once per 3 weeks, about once per 4 weeks, about once per 5 weeks, about once per 6 weeks, about once per 7 weeks, about once per 8 weeks, about once per 9 weeks, about once per 10 weeks, about once per 11 weeks, or about once per 12 weeks, e.g., at one of the doses described above. In some embodiments, the IL-7 protein is administered at a dosing frequency of about once per 10 days, about once per 20 days, about once per 30 days, about once per 40 days, about once per 50 days, about once per 60 days, about once per 70 days, about once per 80 days, about once per 90 days, or about once per 100 days. In some embodiments, the IL-7 protein is administered once every 3 weeks. In some embodiments, the IL-7 protein is administered once per week. In some embodiments, the IL-7 protein is administered once per two weeks. In some embodiments, the IL-7 protein is administered once per four weeks. In some embodiments, the IL-7 protein is administered once per six weeks. In some embodiments, the IL-7 protein is administered once per eight weeks. In some embodiments, the IL-7 protein is administered once per nine weeks. In some embodiments, the IL-7 protein is administered once per twelve weeks. In some embodiments, the IL-7 protein is administered once per ten days. In some embodiments, the IL-7 protein is administered once per twenty days. In some embodiments, the IL-7 protein is administered once per thirty days. In some embodiments, the IL-7 protein is administered once per forty days. In some embodiments, the IL-7 protein is administered once per fifty days. In some embodiments, the IL-7 protein is administered once per sixty days. In some embodiments, the IL-7 protein is administered once every 70 days. In some embodiments, the IL-7 protein is administered once every 80 days. In some embodiments, the IL-7 protein is administered once every 90 days. In some embodiments, the IL-7 protein is administered once every 100 days. As demonstrated herein, in some embodiments, the IL-7 protein is administered at a dose of about 1,200 μg / kg and with a dosing interval of about 8 weeks.

[0125] In some embodiments, the VEGF antagonist can be administered to the subject (e.g., in combination with an IL-7 protein as described herein) at any suitable dose and / or frequency of administration to treat the tumors described herein. In some embodiments, the VEGF antagonist is administered to the subject at a dose of about 0.001 mg / kg to about 20 mg / kg. In some embodiments, the VEGF antagonist is administered at a dose of about 1 mg / kg to about 20 mg / kg. In some embodiments, the VEGF antagonist (e.g., bevacizumab) is administered at a dose of about 5 mg / kg to about 15 mg / kg. In some embodiments, the 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 embodiments, the VEGF antagonist is administered to the subject at a dose of about 5 mg / kg. In some embodiments, the VEGF antagonist is administered to the subject at a dose of about 7.5 mg / kg. In some embodiments, the VEGF antagonist is administered to the subject at a dose of about 10 mg / kg. In some embodiments, the VEGF antagonist is administered to the subject at a dose of about 12.5 mg / kg. In some embodiments, the VEGF antagonist is administered to the subject at a dose of about 15 mg / kg. In some embodiments, 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 embodiments, the VEGF antagonist (e.g., aflibercept) is administered at a dose of about 4 mg / kg.

[0126] In some embodiments, a subject disclosed herein is administered a single dose of a VEGF antagonist (e.g., in combination with an IL-7 protein) at any of the doses described above. In some embodiments, a VEGF antagonist disclosed herein is administered to a subject (e.g., alone or in combination with an IL-7 protein) in multiple doses (i.e., repeated administrations). In some embodiments, a VEGF antagonist is administered to a subject 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, or at least about 10 times or more.

[0127] When a VEGF antagonist is administered multiple times to a subject (e.g., having a tumor) (e.g., alone or in combination with an IL-7 protein), in some embodiments, the VEGF antagonist is administered, for example, at one of the doses described herein, at a dosing frequency of about once a day, about once every 2 days, about once every 3 days, about once every 4 days, about once every 5 days, about once every 6 days, about once a week, about once every 2 weeks, about once every 3 weeks, about once every 4 weeks, about once every 5 weeks, about once every 6 weeks, about once every 7 weeks, about once every 8 weeks, about once every 9 weeks, about once every 10 weeks, about once every 11 weeks, or about once every 12 weeks. As demonstrated herein, in some embodiments, the VEGF antagonist is administered at a dose of about 10 mg / kg and with a dosing interval of about 2 weeks.

[0128] Thus, in some embodiments, a subject described herein (e.g., having a tumor) is administered a single dose (e.g., one of the doses described herein) of an IL-7 protein and a single dose (e.g., one of the doses described herein) of a VEGF antagonist. In such embodiments, the IL-7 protein and the VEGF antagonist can be administered to the subject simultaneously. In some embodiments, the IL-7 protein and the VEGF antagonist can be administered simultaneously as a single composition in a pharma- ceutically acceptable carrier. In some embodiments, the IL-7 protein and the VEGF antagonist can be administered simultaneously as separate compositions. In such embodiments, the IL-7 protein and the VEGF antagonist are administered sequentially to the subject. When the IL-7 protein and the VEGF antagonist are administered sequentially to the subject, in some embodiments, the period between administration of the IL-7 protein and administration of the VEGF antagonist is at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 11 weeks, at least about 12 weeks, or more. In some embodiments, the IL-7 protein is administered to the subject prior to administration of the VEGF antagonist. In some embodiments, the IL-7 protein is administered to the subject after administration of the VEGF antagonist.

[0129] In some embodiments, a subject described herein (e.g., having a tumor) is administered a single dose (e.g., one of the doses described herein) of an IL-7 protein and multiple doses (e.g., at least about two, at least about three, or at least about four) of a VEGF antagonist. In some embodiments, a subject described herein (e.g., having a tumor) is administered multiple doses (e.g., one of the doses described herein) of an IL-7 protein and a single dose (e.g., one of the doses described herein) of a VEGF antagonist. In some embodiments, a subject described herein (e.g., having a tumor) is administered multiple doses (e.g., one of the doses described herein) of an IL-7 protein and multiple doses (e.g., one of the doses described herein) of a VEGF antagonist.

[0130] When multiple doses of an IL-7 protein and / or a VEGF antagonist are administered to a subject, in some embodiments, at least one of the doses of the IL-7 protein and at least one of the doses of the VEGF antagonist are administered to the subject simultaneously (e.g., as a single composition or two separate compositions). In some embodiments, each of the multiple doses of the IL-7 protein and each of the multiple doses of the VEGF antagonist are administered to the subject simultaneously (e.g., as a single composition or two separate compositions). In some embodiments, one or more of the multiple doses of the IL-7 protein and one or more of the multiple doses of the VEGF antagonist are administered to the subject sequentially. In some embodiments, (i) at least one of the multiple doses of the IL-7 protein and at least one of the multiple doses of the 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 the IL-7 protein and at least one of the multiple doses of the VEGF antagonist are administered sequentially (e.g., on separate days).

[0131] As is evident from the present disclosure, in some embodiments, the subject receives multiple cycles of the therapeutic regimen described herein (combination of VEGF antagonist and IL-7 protein). In some embodiments, the subject receives at least about 2 cycles, at least about 3 cycles, at least about 4 cycles, at least about 5 cycles, at least about 6 cycles, at least about 7 cycles, at least about 8 cycles, at least about 9 cycles, at least about 10 cycles of the therapeutic regimen. In some embodiments, the subject receives 2 cycles of the therapeutic regimen. In some embodiments, the subject receives 3 cycles of the therapeutic regimen. In some embodiments, the subject receives 4 cycles of the therapeutic regimen. In some embodiments, the subject receives 5 cycles of the therapeutic regimen. In some embodiments, the subject receives 6 cycles of the therapeutic regimen. In some embodiments, the subject receives 7 cycles of the therapeutic regimen. In some embodiments, the subject receives 8 cycles of the therapeutic regimen. In some embodiments, the subject receives 9 cycles of the therapeutic regimen. In some embodiments, the subject receives 10 cycles of the therapeutic regimen. When multiple cycles of a treatment regimen are administered to a subject, in some embodiments, each of the treatment cycles is the same, hi some embodiments, one or more of the treatment cycles are different (e.g., different doses and / or dosing intervals).

[0132] In some embodiments, the length of each treatment cycle is about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, or about 12 weeks. As demonstrated herein, in some embodiments, the length of each treatment cycle is about 8 weeks. Unless otherwise specified, each treatment cycle includes at least one dose of IL-7 protein and at least one dose of VEGF antagonist. In some embodiments, each treatment cycle includes one dose of IL-7 protein and one dose of VEGF antagonist. In some embodiments, each treatment cycle includes one dose of IL-7 protein and two doses of VEGF antagonist. In some embodiments, each treatment cycle includes one dose of IL-7 protein and three doses of VEGF antagonist. In some embodiments, each treatment cycle includes one dose of IL-7 protein and four doses of VEGF antagonist. In some embodiments, each treatment cycle comprises one dose of IL-7 protein and five doses of a VEGF antagonist. As demonstrated herein (see, e.g., Example 3), in some embodiments, each treatment cycle comprises one dose of an IL-7 protein and four doses of a VEGF antagonist, which are administered at two-week dosing intervals. For example, in some embodiments, an IL-7 protein and a VEGF antagonist are administered to a subject on day 1 of a treatment cycle, and then an additional dose of a VEGF antagonist is administered on days 15, 29, and / or 43 of the treatment cycle.

[0133] In some embodiments, the combination treatment of IL-7 protein and VEGF antagonist described herein can be used with one or more additional therapeutic agents. Non-limiting examples of such additional therapeutic agents include, for example, chemotherapeutic agents, small molecule drugs, radiation, antibodies that stimulate immune response to certain cancers, or combinations thereof. Thus, in some embodiments, the present invention provides a method for treating a tumor in a subject in need of tumor treatment, comprising administering to the subject a combination of IL-7 protein and VEGF antagonist and an additional therapeutic agent. Non-limiting examples of such combinations include therapies that enhance tumor antigen presentation (e.g., dendritic cell vaccines, GM-CSF-secreting cellular vaccines, CpG oligonucleotides, imiquimod); therapies that inhibit negative immune regulation, for example, by inhibition of the CTLA-4 and / or PD1 / PD-L1 / PD-L2 pathways and / or depletion or blockage of Tregs or other immune suppressive cells (e.g., myeloid-derived suppressor cells); therapies that stimulate positive immune regulation, for example, by agonists that stimulate the CD-137, OX-40, and / or CD40 or GITR pathways and / or stimulate T cell effector function; therapies that systemically increase the frequency of anti-tumor T cells; for example, by using antagonists of CD25 (e.g., daclizumab) or ex vivo. These include therapies that deplete or inhibit Tregs, such as Tregs in tumors by in vivo anti-CD25 bead depletion; therapies that affect the function of suppressor myeloid cells in tumors; therapies that improve the immunogenicity of tumor cells (e.g., anthracyclines); adoptive transfer of T cells or NK cells, including genetically modified cells, e.g., cells modified with chimeric antigen receptors (CAR-T therapy); therapies that inhibit metabolic enzymes such as indoleamine dioxygenase (IDO), dioxygenase, arginase, or nitric oxide synthase; therapies that reverse / prevent T cell anergy or exhaustion; therapies that induce innate immune activation and / or inflammation at the tumor site; administration of immune stimulatory cytokines; or blockade of immune suppressive cytokines.

[0134] In some embodiments, additional therapeutic agents that can be used with the combination treatment of IL-7 protein and VEGF antagonist include immune checkpoint inhibitors (i.e., block signaling by a particular immune checkpoint pathway). Thus, in some embodiments, provided herein is a method of treating a tumor in a subject in need of tumor treatment, comprising administering to the subject a combination of IL-7 protein and both a VEGF antagonist and an immune checkpoint inhibitor. Non-limiting examples of immune checkpoint inhibitors that can be used in the methods of the present invention include CTLA-4 antagonists (e.g., anti-CTLA-4 antibodies), PD-1 antagonists (e.g., anti-PD-1 antibodies, anti-PD-L1 antibodies), TIM-3 antagonists (e.g., anti-TIM-3 antibodies), or combinations thereof. Non-limiting examples of such immune checkpoint inhibitors are well known in the art. For example, suitable anti-PD-1 antagonists are described in, e.g., U.S. Patent Nos. 6,808,710, 7,488,802, 8,008,449, 8,168,757, 8,354,509, 8,609,089, 8,779,105, and 9,217,034, 7,635,757, 7,943,743, 8,217,149, 8,981,063, and 9,624,298, each of which is incorporated by reference in its entirety. Suitable anti-CTLA-4 antagonists are described, for example, in U.S. Patent Nos. 5,977,318, 6,051,227; 6,682,736, 6,984,720, 7,034,121, 7,605,238, 10,479,833, and International Publication No. WO2007 / 113648, each of which is incorporated herein by reference in its entirety. Suitable TIM-3 antagonists are described, for example, in U.S. Patent Nos. 10,508,149, 10,533,052, and 10,894,830, each of which is incorporated herein by reference in its entirety.

[0135] In some embodiments, additional therapeutic agents that can be used include immune checkpoint activators (i.e., promote signaling through a particular immune checkpoint pathway). In some embodiments, immune checkpoint activators include OX40 agonists (e.g., anti-OX40 antibodies), LAG-3 agonists (e.g., anti-LAG-3 antibodies), 4-1BB (CD137) agonists (e.g., anti-CD137 antibodies), GITR agonists (e.g., anti-GITR antibodies), or any combination thereof.

[0136] In some embodiments, the therapeutic regimen provided herein (VEGF antagonist and IL-7 protein combination) is not used in combination with one or more other anti-cancer treatments. Thus, in some embodiments, the subject being treated with a VEGF antagonist and IL-7 protein combination does not receive one or more of the following anti-cancer treatments: (1) any combination treatment approved or experimental for the treatment of cancer, including chemotherapy, hormonal therapy, immunotherapy, radiation therapy, investigational drugs or herbal therapy, (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).

[0137] II.A. IL-7 Proteins Useful in the Disclosure Disclosed herein are IL-7 proteins that can be used in combination with VEGF antagonists to treat cancer (or tumors). In some embodiments, the IL-7 proteins useful for use herein may be wild-type IL-7 or modified IL-7 (i.e., not wild-type IL-7 protein) (e.g., IL-7 variants, functional fragments of IL-7, IL-7 derivatives, or any combination thereof, e.g., fusion proteins, chimeric proteins, etc.), so long as they have one or more biological activities of IL-7, e.g., can bind to IL-7R and can induce early T cell development and promote T cell homeostasis (see ElKassar and Gress. J Immunotoxicol. 2010 Mar;7(1):1-7). In some embodiments, the IL-7 proteins of the present disclosure are not wild-type IL-7 proteins (i.e., include one or more modifications). Non-limiting examples of such modifications may include oligopeptides and / or half-life extending moieties (see, e.g., WO2016 / 200219, which is incorporated by reference in its entirety).

[0138] IL-7 binds to its receptor, which consists of two chains: IL-7Rα (CD127) (Ziegler and Liu, 2006), which is shared with thymic stromal lymphopoietin (TSLP), and γ (CD132), which is shared with IL-2, IL-15, IL-9, and IL-21. IL-7Rα is expressed almost exclusively on lymphoid cells, whereas γc is expressed on most hematopoietic cells. After binding to its receptor, IL-7 signals through two distinct pathways: Jak-Stat (Janus kinase-signal transducer and activator of transcription) and PI3K / Akt, which are responsible for differentiation and survival, respectively. The absence of IL-7 signaling is responsible for the reduced thymic cellularity observed in mice treated with anti-IL-7 neutralizing monoclonal antibodies (MAbs; Grabstein et al., 1993), IL-7- / - mice (von Freeden-Jeffry et al., 1995), IL-7Rα- / - mice (Peschon et al., 1994; Maki et al., 1996), γc- / - mice (Malissen et al., 1997), and Jak3- / - mice (Park et al., 1995). In the absence of IL-7 signaling, mice lack T cells, B cells, and NK-T cells. IL-7α- / - mice (Peschon et al., 1994) have a similar but more severe phenotype to IL-7- / - mice (von Freeden-Jeffry et al., 1995), likely because TSLP signaling is also abrogated in IL-7α- / - mice. IL-7 is required for the development of γδ cells (Maki et al., 1996) and NK-T cells (Boesteanu et al., 1997).

[0139] In some embodiments, an IL-7 protein useful in the present disclosure comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1-6. In some embodiments, an IL-7 protein comprises an amino acid sequence having about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% or more sequence identity to the sequence of SEQ ID NOs: 1-6. In some embodiments, an IL-7 protein 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% identity to the sequence set forth in SEQ ID NO:1. In some embodiments, 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% identical to the sequence set forth in SEQ ID NO: 2. In some embodiments, 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% identical to the sequence set forth in SEQ ID NO:3. In some embodiments, 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% identical to the sequence set forth in SEQ ID NO: 4. In some embodiments, 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% identical to the sequence set forth in SEQ ID NO:5. In some embodiments, 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% the sequence set forth in SEQ ID NO:6.

[0140] In some embodiments, the IL-7 protein comprises a modified IL-7 or a fragment thereof that retains one or more biological activities of wild-type IL-7. Thus, in some embodiments, an IL-7 protein useful in the present disclosure comprises a fragment of the amino acid sequence set forth in any one of SEQ ID NOs: 1-6 that retains one or more biological activities of the wild-type IL-7 protein. In some embodiments, the IL-7 protein may be derived from a human, rat, mouse, monkey, cow, or sheep.

[0141] In some embodiments, human IL-7 may have the amino acid sequence represented by SEQ ID NO: 1 (Genbank Accession No. P13232), rat IL-7 may have the amino acid sequence represented by SEQ ID NO: 2 (Genbank Accession No. P56478), mouse IL-7 may have the amino acid sequence represented by SEQ ID NO: 3 (Genbank Accession No. P10168), monkey IL-7 may have the amino acid sequence represented by SEQ ID NO: 4 (Genbank Accession No. NP001279008), bovine IL-7 may have the amino acid sequence represented by SEQ ID NO: 5 (Genbank Accession No. P26895), and ovine IL-7 may have the amino acid sequence represented by SEQ ID NO: 6 (Genbank Accession No. Q28540). As will be apparent to one of skill in the art, the IL-7 protein represented by 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. Thus, as will be apparent from the present disclosure, in some embodiments, the IL-7 proteins that can be used in the methods provided herein lack a signal peptide. The amino acid sequences of such exemplary IL-7 proteins are set forth in SEQ ID NOs: 80-85 (see also Table 1A).

[0142] In some embodiments, an IL-7 protein useful in the present disclosure 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 amino acid sequence set forth in any one of SEQ ID NOs: 80-85. In some embodiments, an 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% identity to the sequence set forth in SEQ ID NO: 80. In some embodiments, an IL-7 protein comprises the amino acid sequence set forth in SEQ ID NO: 80. In some embodiments, 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% identical to the sequence set forth in SEQ ID NO: 81. In some embodiments, 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% identical to the sequence set forth in SEQ ID NO: 82. In some embodiments, 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% identical to the sequence set forth in SEQ ID NO: 82. In some embodiments, 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% identical to the sequence set forth in SEQ ID NO: 83. In some embodiments, 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% identical to the sequence set forth in SEQ ID NO: 84. In some embodiments, 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% identical to the sequence set forth in SEQ ID NO: 84.In some embodiments, 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% identity with the sequence set forth in SEQ ID NO: 85. In some embodiments, the IL-7 protein comprises the amino acid sequence set forth in SEQ ID NO:85.

[0143] In some embodiments, an IL-7 protein useful in the present disclosure comprises an IL-7 fusion protein. In some embodiments, the IL-7 fusion protein comprises (i) an oligopeptide and (i) an IL-7 or variant thereof (e.g., an IL-7 protein set forth in any one of SEQ ID NOs: 1-6 and 80-85). In some embodiments, the oligopeptide is linked to the N-terminal region of IL-7 or a variant thereof.

[0144] In some embodiments, the oligopeptides disclosed herein consist of 1-10 amino acids. In some embodiments, the oligopeptides consist of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or 10 amino acids. In some embodiments, one or more amino acids of the oligopeptide are selected from the group consisting of methionine, glycine, methionine-methionine, glycine-glycine, methionine-glycine, glycine-methionine, methionine-methionine-methionine, methionine-methionine-glycine, methionine-glycine-methionine, glycine-methionine-methionine, methionine-glycine-glycine, glycine-methionine-methionine, methionine-glycine-glycine, glycine-methionine-glycine, glycine-methionine-glycine, glycine-glycine-methionine, and glycine-glycine-glycine. In some embodiments, the oligopeptide is methionine-glycine-methionine.

[0145] In some embodiments, the IL-7 fusion protein comprises (i) IL-7 or a variant thereof and (ii) a half-life extending moiety. In some embodiments, the half-life extending moiety extends the half-life of IL-7 or a variant thereof. In some embodiments, the half-life extending moiety is linked to the C-terminal region of IL-7 or a variant thereof.

[0146] In some embodiments, the IL-7 fusion protein comprises (i) IL-7 (a first domain), (ii) a second domain comprising an amino acid sequence having 1-10 amino acid residues consisting of methionine, glycine, or a combination thereof, e.g., MGM, and (iii) a third domain comprising a half-life extending moiety. In some embodiments, the half-life extending moiety may be linked to the N-terminus or C-terminus of the first domain or the second domain. Additionally, the IL-7 comprising the first domain and the second domain may be linked to both termini of the third domain.

[0147] Non-limiting examples of half-life extending moieties include Fc, albumin, albumin binding polypeptide, Pro / Ala / Ser (PAS), the C-terminal peptide of the beta subunit of human chorionic gonadotropin (CTP), polyethylene glycol (PEG), long unstructured hydrophilic amino acid sequences (XTEN), hydroxyethyl starch (HES), albumin binding small molecules, and combinations thereof.

[0148] In some embodiments, the half-life extending moiety is an Fc. In some embodiments, the Fc is derived from an engineered immunoglobulin with attenuated antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) due to engineered binding affinity with the Fc receptor and / or complement. In some embodiments, the engineered immunoglobulin can be selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and combinations thereof. In some embodiments, the Fc is a hybrid Fc ("hFc" or "hyFc") comprising a hinge region, a CH2 domain, and a CH3 domain. In some embodiments, the hinge region of the hybrid Fc disclosed herein comprises a human IgD hinge region. In some embodiments, the CH2 domain of the hybrid Fc comprises a portion of a human IgD CH2 domain and a portion of a human IgG4 CH2 domain. In some embodiments, the CH3 domain of the hybrid Fc comprises a portion of a human IgG4 CH3 domain. Thus, in some aspects, the hybrid Fc disclosed herein comprises a hinge region, a CH2 domain, and a CH3 domain, wherein the hinge region comprises a human IgD hinge region, the CH2 domain comprises a portion of the human IgD CH2 domain and a portion of the human IgG4 CH2 domain, and the CH3 domain comprises a portion of the human IgG4 CH3 domain.

[0149] In some embodiments, the Fc disclosed herein may be an Fc variant. As used herein, the term "Fc variant" refers to an Fc prepared by substituting a portion of amino acids in the Fc region or by combining different types of Fc regions. The Fc region variant can be prevented from being cleaved at the hinge region. Specifically, in some embodiments, the Fc variant comprises a modification of the 144th and / or 145th amino acids of SEQ ID NO:9. In some embodiments, the 144th amino acid (K) and / or the 145th amino acid (K) are substituted with G or S.

[0150] In some embodiments, the Fc or Fc variant disclosed herein can be represented by the formula: N'-(Z1)pY-Z2-Z3-Z4-C, wherein: N' comprises the N-terminus, Z1 comprises an amino acid sequence having 5 to 9 consecutive amino acid residues from the amino acid residue at position 98 toward the N-terminus among the amino acid residues at positions 90 to 98 of SEQ ID NO: 7, Y comprises an amino acid sequence having 5 to 64 consecutive amino acid residues from the amino acid residue at position 162 toward the N-terminus among the amino acid residues at positions 99 to 162 of SEQ ID NO: 7, Z2 comprises an amino acid sequence having 4 to 37 consecutive amino acid residues from the amino acid residue at position 163 toward the C-terminus among the amino acid residues at positions 163 to 199 of SEQ ID NO: 7, Z3 comprises an amino acid sequence having 71 to 106 consecutive amino acid residues from the amino acid residue at position 220 toward the N-terminus among the amino acid residues at positions 115 to 220 of SEQ ID NO:8, Z4 comprises an amino acid sequence having 80 to 107 consecutive amino acid residues from the amino acid residue at position 221 to the C-terminus among the amino acid residues at positions 221 to 327 of SEQ ID NO:8.

[0151] In some embodiments, the Fc region disclosed herein may comprise the amino acid sequence of SEQ ID NO:9 (hyFc), SEQ ID NO:10 (hyFcM1), SEQ ID NO:11 (hyFcM2), SEQ ID NO:12 (hyFcM3), or SEQ ID NO:13 (hyFcM4). In some embodiments, the Fc region may comprise the amino acid sequence of SEQ ID NO:14 (non-lytic mouse Fc).

[0152] Other non-limiting examples of Fc regions that can be used in the present disclosure are described in US Pat. No. 7,867,491, which is incorporated by reference in its entirety.

[0153] In some embodiments, the IL-7 fusion proteins disclosed herein comprise both an oligopeptide and a half-life extending moiety.

[0154] In some embodiments, the IL-7 protein may be fused to albumin, a variant, or a fragment thereof. An example of an IL-7-albumin fusion protein can be found in International Application Publication No. WO2011 / 124718 A1. In some embodiments, the IL-7 protein is fused to pre-pro B cell growth stimulatory factor (PPBSF), optionally via a flexible linker. See US2002 / 0058791 A1. In some embodiments, the IL-7 protein useful in the present disclosure is a conformer of IL-7 having a specific three-dimensional structure. See US2005 / 0249701 A1. In some embodiments, the IL-7 protein may be fused to an Ig chain, where amino acid residues 70 and 91 of the IL-7 protein are glycosylated and amino acid residue 116 of the IL-7 protein is not glycosylated. See US7,323,549 B2. In some embodiments, IL-7 proteins that do not contain potential T cell epitopes (and thus reduce anti-IL-7 T cell responses) can also be used in the present disclosure. See US2006 / 0141581 A1. In some embodiments, IL-7 proteins with one or more amino acid residue mutations in the carboxy-terminal helix D region can be used in the present disclosure. IL-7 mutants can act as IL-7R partial agonists despite having low binding affinity to the receptor. See US2005 / 0054054A1. Any IL-7 proteins described in the above patents or publications are incorporated herein by reference in their entirety.

[0155] Further, non-limiting examples of additional IL-7 proteins useful in the present disclosure include those described in US7708985, US8034327, US8153114, US7589179, US7323549, US7960514, US8338575, US7118754, US7488482, US7670607, US6730512, WO0017362, GB2434578A, WO2010 / 020766A2, WO91 / 01143, Beqet al., Blood, vol.114(4), 816,23 July 2009, Kang et al., J. Virol. Doi:10.1128 / JVI.02768-15, Martin et al. al., Blood, vol. 121(22), 4484, May 30, 2013; McBride et al., Acta Oncologica, 34:3, 447-451, July 8, 2009; and Xu et al., Cancer Science, 109: 279-288, 2018.

[0156] In some embodiments, the oligopeptide disclosed herein is directly linked to the N-terminal region of IL-7 or a variant thereof. In some embodiments, the oligopeptide is linked to the N-terminal region via a linker. In some embodiments, the half-life extending moiety disclosed herein is directly linked to the C-terminal region of IL-7 or a variant thereof. In some embodiments, the half-life extending moiety is linked to the C-terminal region via a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker comprises a peptide of 10-20 amino acid residues consisting of Gly and Ser residues. In some embodiments, the linker is an albumin linker. In some embodiments, the linker is a chemical bond. In some embodiments, the chemical bond comprises a disulfide bond, a diamine bond, a sulfide-amine bond, a carboxy-amine bond, an ester bond, a covalent bond, or a combination thereof. When the linker is a peptide linker, in some embodiments, the connection can occur at any linking region. These can be linked using crosslinking agents well known in the art. In some embodiments, exemplary cross-linking agents can include, but are not limited to, N-hydroxysuccinimide esters such as 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, and 4-azidosalicylic acid; imidoesters including disuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionic acid), and bifunctional maleimides such as bis-N-maleimide-1,8-octane.

[0157] In some embodiments, the IL-7 (or variants thereof) portion of an IL-7 fusion protein disclosed herein 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 set forth in SEQ ID NOs: 15-20. In some embodiments, the IL-7 (or variants thereof) portion of an IL-7 fusion protein disclosed herein comprises the amino acid sequence set forth in SEQ ID NOs: 15-20.

[0158] In some embodiments, the IL-7 fusion protein comprises a first domain comprising a polypeptide having an activity of IL-7 or an activity similar thereto, a second domain comprising an amino acid sequence having 1 to 10 amino acid residues consisting of methionine, glycine, or a combination thereof, and a third domain which is an Fc region of a modified immunoglobulin linked to the C-terminus of the first domain.

[0159] In some embodiments, an IL-7 fusion protein that can be used in the methods 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 set forth in SEQ ID NOs: 21-25. In some embodiments, an IL-7 fusion protein of the present disclosure comprises an amino acid sequence set forth in SEQ ID NOs: 21-25. In some embodiments, an IL-7 fusion protein disclosed herein comprises an amino acid sequence set forth in SEQ ID NOs: 26 and 27.

[0160] Unless otherwise specified, the IL-7 proteins (including fusion proteins) described herein can be administered to a subject (e.g., in combination with a VEGF antagonist) using any suitable route of administration. In some embodiments, the IL-7 protein is administered to a subject intravenously, parenterally, intramuscularly, subcutaneously, intraocularly, intraperitoneally, intradermally, intraorbitally, intracerebrally, intracranially, intraspinally, intraventricularly, intrathecally, intracisternally, intracapsularly, or intratumorally. In some embodiments, the IL-7 protein is administered to a subject intramuscularly.

[0161] II.B. VEGF Antagonists In some aspects, the disclosure provides a method of treating a tumor in a subject in need of tumor treatment, 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, abrogate, reduce, and / or interfere with VEGF activity, including binding to one or more VEGF receptors. VEGF antagonists include molecules that interfere with the interaction of VEGF with a natural VEGF receptor, such as molecules that bind to VEGF or a VEGF receptor and prevent or otherwise impede the interaction of VEGF with the VEGF receptor. Thus, in some aspects, the VEGF antagonists described herein may specifically target VEGF itself. In some aspects, the VEGF antagonists described herein specifically target one or more VEGF receptors.

[0162] Unless otherwise indicated, the VEGF antagonist useful in the present disclosure may be a protein, a peptide, a nucleic acid, a small molecule, or a combination thereof. In some embodiments, the VEGF antagonist useful in the present disclosure includes an antisense oligonucleotide, siRNA, shRNA, miRNA, dsRNA, aptamer, PNA (peptide nucleic acid), or a vector comprising the same, that targets VEGF. In some embodiments, the VEGF antagonist includes an antibody that specifically binds to one or more of VEGF or VEGF receptors (VEGFR), or an antigen-binding fragment thereof ("anti-VEGF antibody" and "anti-VEGFR antibody," respectively), a polynucleotide that encodes an anti-VEGF antibody and / or an anti-VEGFR antibody, or a vector comprising such a polynucleotide.

[0163] Non-limiting examples of suitable VEGF antagonists are well known in the art, such as aflibercept (EYLEA® and ZALTRAP®), bevacizumab (AVASTIN®, ZIRABEV®, and MVASI®), ranibizumab (LUCENTIS®, BYOOVIZ®, SUSVIMO®), ramucirumab (CYRAMZA®), sorafenib (NEXAVAR®), sunitinib (SUTENT®), and serotoninib (SUTENT®). (R)), pazopanib (VOTRIENT®), dasatinib (SPRYCEL®), regorafenib (STIVARGA®), cabozantinib (CABOMETYX®, COMETRIQ®), lenvatinib (LENVIMA®), ponatinib (ICLUSIG®), axitinib (INLYTA®), tivozanib (FOTIVDA®), vandetanib (CAPRELSA®), or combinations thereof.

[0164] In some embodiments, the VEGF antagonist useful in the present disclosure is an anti-VEGF antibody. In some embodiments, the VEGF antagonist is bevacizumab, as described in, for example, US Patent Publication No. 2015 / 0147317, the entire contents of which are incorporated herein by reference. In some embodiments, the VEGF antagonist is a variant of bevacizumab. For example, in some embodiments, such variants cross-compete with bevacizumab. In some embodiments, the bevacizumab variant binds to the same epitope as bevacizumab. In some embodiments, the bevacizumab variant has the same CDR as bevacizumab. Thus, in some embodiments, the method provided herein (e.g., for treating tumor) comprises administering to a subject in need thereof a combination of IL-7 protein and bevacizumab (or a variant thereof). In such embodiments, bevacizumab (or a variant thereof) can be administered to the subject at any suitable dose known in the art (e.g., an FDA approved dose), for example, about 5 mg / kg to about 15 mg / kg. For example, in some embodiments, bevacizumab (or a variant thereof) is administered in combination with an IL-7 protein 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 embodiments, bevacizumab (or a variant thereof) is administered in combination with an IL-7 protein at a dose of about 10 mg / kg. As described herein, in some embodiments, such methods further include administering an additional therapeutic agent (e.g., an immune checkpoint inhibitor) to the subject.

[0165] In some embodiments, the VEGF antagonist useful in the present disclosure is aflibercept, for example, as described in U.S. Patent No. 11,135,266, the entire contents of which are incorporated herein by reference. In some embodiments, the VEGF antagonist is a variant of aflibercept. In some embodiments, the aflibercept variant cross-competes with aflibercept. In some embodiments, the aflibercept variant binds to the same epitope as aflibercept. In some embodiments, the aflibercept variant has the same CDR as aflibercept. Thus, in some embodiments, the methods provided herein (e.g., for treating tumors) include administering to a subject in need thereof a combination of an IL-7 protein and aflibercept (or a variant thereof). In such embodiments, the aflibercept (or a variant thereof) can be administered to the subject at any suitable dose known in the art (e.g., a dose approved by the FDA), for example, about 4 mg / kg to about 15 mg / kg. For example, in some embodiments, 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 embodiments, such methods can further include administering to the subject an additional therapeutic agent (e.g., an immune checkpoint inhibitor).

[0166] In some embodiments, the VEGF antagonist that can be used in the present disclosure is ranibizumab, for example, as described in U.S. Patent No. 7,169,901, the entire contents of which are incorporated herein by reference. In some embodiments, the VEGF antagonist is a variant of ranibizumab. In some embodiments, the ranibizumab variant cross-competes with ranibizumab. In some embodiments, the ranibizumab variant binds to the same epitope as ranibizumab. In some embodiments, the ranibizumab variant has the same CDR as ranibizumab. Thus, in some embodiments, the method provided herein (e.g., for treating tumor) comprises administering to a subject in need thereof a combination of IL-7 protein and ranibizumab (or a variant thereof). In such embodiments, ranibizumab (or a variant thereof) can be administered to a subject at any suitable dose known in the art (e.g., a dose approved by FDA). In some embodiments, such methods further comprise administering to the subject an additional therapeutic agent (e.g., an immune checkpoint inhibitor).

[0167] In some embodiments, the VEGF antagonist is ramucirumab, as described, for example, in U.S. Patent No. 8,057,791, the entire contents of which are incorporated herein by reference. In some embodiments, the VEGF antagonist is a variant of ramucirumab. In some embodiments, the ramucirumab variant cross-competes with ramucirumab. In some embodiments, the ramucirumab variant binds to the same epitope as ramucirumab. In some embodiments, ramucirumab has the same CDR as ramucirumab. Thus, in some embodiments, the methods provided herein (e.g., for treating a tumor) comprise administering to a subject in need thereof a combination of an IL-7 protein and ramucirumab (or a variant thereof). In such embodiments, ramucirumab (or a variant thereof) can be administered to the subject at any suitable dose known in the art (e.g., a dose approved by the FDA). In some embodiments, such methods further comprise administering to the subject an additional therapeutic agent (e.g., an immune checkpoint inhibitor).

[0168] Unless otherwise specified, the VEGF antagonist described herein can be administered to a subject (e.g., in combination with an IL-7 protein) by any suitable route. In some embodiments, the VEGF antagonist is administered to a subject intravenously, parenterally, intramuscularly, subcutaneously, intraocularly, intraperitoneally, intradermally, intraorbitally, intracerebrally, intracranially, intraspinally, intraventricularly, intrathecally, intracisternally, intracapsularly, or intratumorally. In some embodiments, the VEGF antagonist is administered to a subject intravenously. Thus, in some embodiments, the method of treatment provided herein comprises administering at least one dose of an IL-7 protein intramuscularly and at least one dose of a VEGF antagonist intravenously to a subject.

[0169] III. Nucleic Acids, Vectors, and Host Cells Some embodiments described herein relate to one or more nucleic acid molecules encoding therapeutic agents described herein (e.g., IL-7 protein and / or VEGF antagonists). The nucleic acid may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is "isolated" or "substantially pure" when it has been purified from other cellular components or other contaminants, e.g., other cellular nucleic acids (e.g., other chromosomal DNA, e.g., chromosomal DNA linked to naturally isolated DNA) or proteins by standard techniques, including alkaline / SDS treatment, CsCl banding, column chromatography, restriction enzymes, agarose gel electrophoresis, and others known in the art. See F. Ausubel, et al., ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. The nucleic acids described herein may be, for example, DNA or RNA, and may or may not contain intron sequences. In some embodiments, the nucleic acid is a cDNA molecule. The nucleic acids described herein can be obtained using standard molecular biology techniques well known in the art.

[0170] Some nucleic acid molecules disclosed herein encode IL-7 proteins (e.g., as disclosed herein) that can be used in combination with VEGF antagonists, for example, to treat tumors. Exemplary nucleic acid sequences encoding IL-7 proteins disclosed herein are set forth in SEQ ID NOs: 29-39.

[0171] In some aspects, the disclosure provides vectors comprising an isolated nucleic acid molecule encoding a therapeutic agent (e.g., an IL-7 protein and / or a VEGF antagonist) disclosed herein. In some aspects, the vectors can be used in gene therapy.

[0172] When used as gene therapy (e.g., in humans), nucleic acids encoding therapeutic agents disclosed herein (e.g., IL-7 protein and / or VEGF antagonists) can be administered at dosages ranging from 0.1 mg to 200 mg. In some embodiments, dosages range from 0.6 mg to 100 mg. In some embodiments, dosages range from 1.2 mg to 50 mg.

[0173] Suitable vectors for the present disclosure include expression vectors, viral vectors, and plasmid vectors. In some embodiments, the vector is a viral vector.

[0174] As used herein, an expression vector refers to any nucleic acid construct that, upon introduction into an appropriate host cell, contains the necessary elements for the transcription and translation of an inserted coding sequence, or, in the case of RNA viral vectors, the elements necessary for replication and translation. Expression vectors can include plasmids, phagemids, viruses, and their derivatives.

[0175] As used herein, viral vectors include, but are not limited to, nucleic acid sequences derived from retroviruses, such as Moloney murine leukemia virus, Harvey murine sarcoma virus, mouse mammary tumor virus, and Rous sarcoma virus, lentiviruses, adenoviruses, adeno-associated viruses, SV40 type viruses, polyoma viruses, Epstein-Barr viruses, papilloma viruses, herpes viruses, vaccinia viruses, polio viruses, and RNA viruses, such as retroviruses. One of skill in the art can readily use other vectors known to those of skill in the art. Some viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced with a gene of interest. Non-cytopathic viruses include retroviruses, whose life cycle involves reverse transcription of genomic viral RNA into DNA followed by integration of the provirus into host cell DNA.

[0176] In some embodiments, the vector is derived from an adeno-associated virus. In some embodiments, the vector is derived from a lentivirus. Examples of lentivirus vectors are disclosed in WO9931251, WO9712622, WO9817815, WO9817816, and WO9818934, each of which is incorporated herein by reference in its entirety.

[0177] Other vectors include plasmid vectors. Plasmid vectors have been widely described in the art and are well known to those skilled in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. In the past few years, plasmid vectors have been found to be particularly advantageous for delivering genes to cells in vivo, since they cannot replicate or integrate into the host genome. However, these plasmids have promoters compatible with the host cell and can express peptides from genes functionally encoded within the plasmid. Some commonly used plasmids available from commercial sources include pBR322, pUC18, pUC19, various pcDNA plasmids, pRC / CMV, various pCMV plasmids, pSV40, and pBlueScript. Further examples of specific plasmids include pcDNA3.1 (catalog number V79020), PCDNA3.1 / hygro (catalog number V87020), PCDNA4 / myc-His (catalog number V86320), and pBudCE4.1 (catalog number V53220), all available from Invitrogen (Carlsbad, Calif.). Other plasmids will be known to those of skill in the art. Additionally, plasmids can be custom designed to remove and / or add specific DNA fragments using standard molecular biology techniques.

[0178] The present disclosure also encompasses methods of making the therapeutics (e.g., IL-7 proteins) disclosed herein. In some embodiments, such methods may include expressing the therapeutics (e.g., IL-7 proteins) in cells that contain a nucleic acid molecule (e.g., SEQ ID NOs: 29-39) encoding the therapeutics. Further details regarding methods of making the IL-7 proteins disclosed herein are provided, for example, in U.S. Pat. No. 11,041,007, which is incorporated herein by reference in its entirety. Host cells comprising these nucleotide sequences are encompassed herein. Non-limiting examples of host cells that can be used include immortal hybridoma cells, NS / 0 myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, human amniotic fluid-derived cells (CapT cells), COS cells, or combinations thereof.

[0179] IV. Pharmaceutical Compositions Further provided herein are compositions comprising one or more therapeutic agents (e.g., an IL-7 protein and / or a VEGF antagonist) having a desired degree of purity in a physiologically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA). In some embodiments, the compositions disclosed herein comprise an IL-7 protein or a VEGF antagonist. As disclosed herein, such compositions can be used in combination (e.g., a first composition comprising an IL-7 protein and a second composition comprising a VEGF antagonist). In some embodiments, the compositions disclosed herein comprise both an IL-7 protein and a VEGF antagonist.

[0180] Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include buffers such as phosphate, citric acid, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol alcohol, butyl alcohol, or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; These include proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, such as glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants, such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0181] In some embodiments, the compositions disclosed herein comprise one or more additional components selected from a bulking agent, a stabilizer, a surfactant, a buffer, or a combination thereof.

[0182] Buffers useful in the present disclosure may be weak acids or weak bases used to maintain the acidity (pH) of a solution near a selected value after the addition of another acid or base. A suitable buffer can maximize the stability of the pharmaceutical compositions disclosed herein by maintaining pH control of the composition. A suitable buffer can also ensure physiological compatibility or optimize solubility. Rheology, viscosity, and other properties may also depend on the pH of the composition. Common buffers include Tris buffer, Tris-Cl buffer, histidine buffer, TAE buffer, HEPES buffer, TBE buffer, sodium phosphate buffer, MES buffer, ammonium sulfate buffer, potassium phosphate buffer, potassium thiocyanate buffer, succinate buffer, tartaric acid buffer, DIPSO buffer, HEPPSO buffer, POPSO buffer, PIPES buffer, PBS buffer, MOPS buffer, acetate buffer, phosphate buffer, cacodylate buffer, glycine buffer, sulfate buffer, imidazole buffer, phosphate ... The buffer may include, but is not limited to, ethyl acetate buffer, guanidine hydrochloride buffer, phosphate-citrate buffer, borate buffer, malonate buffer, 3-picoline buffer, 2-picoline buffer, 4-picoline buffer, 3,5-lutidine buffer, 3,4-lutidine buffer, 2,4-lutidine buffer, Aces, diethyl malonate buffer, N-methylimidazole buffer, 1,2-dimethylimidazole buffer, TAPS buffer, bis-Tris buffer, L-arginine buffer, lactate buffer, glycolic acid buffer, or a combination thereof.

[0183] In some embodiments, the compositions disclosed herein further comprise a bulking agent. Bulking agents can be added to pharmaceutical products to add volume and mass to the product, facilitating its accurate measurement and handling. Bulking agents that can be used in the present disclosure include, but are not limited to, sodium chloride (NaCl), mannitol, glycine, alanine, or combinations thereof.

[0184] In some embodiments, the compositions disclosed herein may include a stabilizing agent. Non-limiting examples of stabilizing agents that can be used in the present disclosure include sucrose, trehalose, raffinose, arginine, or combinations thereof.

[0185] In some embodiments, the composition disclosed herein comprises surfactant.In some embodiments, the surfactant can be selected from the following: alkyl ethoxylate, nonylphenol ethoxylate, amine ethoxylate, polyethylene oxide, polypropylene oxide, fatty alcohol such as cetyl alcohol or oleyl alcohol, cocamide MEA, cocamide DEA, polysorbate, dodecyl dimethylamine oxide, or combinations thereof.In some embodiments, the surfactant is polysorbate 20 or polysorbate 80.

[0186] In some embodiments, a composition comprising an IL-7 protein may be formulated using the same formulation as the VEGF antagonist (e.g., used in combination with the IL-7 protein). In some embodiments, the IL-7 protein and the VEGF antagonist are formulated using different formulations.

[0187] The pharmaceutical composition can be formulated for any route of administration to a subject. Specific examples of routes of administration include intramuscular, subcutaneous, intraocular, intravenous, intraperitoneal, intradermal, intraorbital, intracerebral, intracranial, intraspinal, intraventricular, intrathecal, intracisternal, intracapsular, or intratumoral. Parenteral administration, characterized by injection either subcutaneously, intramuscularly, or intravenously, is also contemplated herein. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for dissolving or suspending in liquid prior to injection, or as emulsions. Injectables, solutions, and emulsions also include 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 may also contain minor amounts of nontoxic auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, and other such agents, such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, and cyclodextrins.

[0188] Pharmaceutically acceptable carriers used in parenteral formulations include aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents, and other pharma- ceutically acceptable substances. Examples of aqueous vehicles include sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, dextrose, and lactated Ringer's injection. Non-aqueous parenteral vehicles include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, and peanut oil. Antimicrobial agents in bacteriostatic or fungistatic concentrations can be added to parenteral preparations packaged in multi-dose containers, including phenol or cresol, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride, and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. Buffers include phosphates and citrates. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcellulose, hydroxypropyl methylcellulose, and polyvinylpyrrolidone. Emulsifying agents include Polysorbate 80 (TWEEN® 80). Sequestering or chelating agents of metal ions include EDTA. Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol, and propylene glycol for water-miscible vehicles, and sodium hydroxide, hydrochloric acid, citric acid, or lactic acid for pH adjustment.

[0189] Preparations for parenteral administration include sterile solutions ready for injection, sterile dry soluble products that can be combined with a solvent immediately prior to use, such as lyophilized powders, subcutaneous tablets, sterile suspensions ready for injection, sterile dry insoluble products that can be combined with a vehicle immediately prior to use, and sterile emulsions. Solutions can be either aqueous or non-aqueous.

[0190] If administered intravenously, suitable carriers include saline or phosphate buffered saline (PBS), as well as solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, and polypropylene glycol, and mixtures thereof.

[0191] Topical mixtures containing the antibody are prepared as described for local and systemic administration. The resulting mixture may be a solution, suspension, emulsion, etc., and may be formulated as a cream, gel, ointment, emulsion, solution, elixir, lotion, suspension, tincture, paste, foam, aerosol, irrigate, spray, suppository, bandage, skin patch, or any other formulation suitable for topical administration.

[0192] The antibodies or antigen-binding portions thereof described herein may be formulated as aerosols for local application, such as by inhalation (see, e.g., U.S. Pat. Nos. 4,044,126, 4,414,209, and 4,364,923, which describe aerosols for administering steroids useful in the treatment of inflammatory diseases, particularly asthma). These formulations for administration to the respiratory tract may be in the form of an aerosol or solution for a nebulizer, or as a fine powder for insufflation, either alone or in combination with an inert carrier such as lactose. In such cases, the particles of the formulation have diameters of less than 50 microns in one embodiment, and less than 10 microns in one embodiment.

[0193] The therapeutic agents disclosed herein (e.g., IL-7 proteins and / or VEGF antagonists) can be formulated for topical or local application, e.g., topical application to the skin and mucous membranes, e.g., the eye, in the form of gels, creams, and lotions, and for application to the eye, or for intracapsular or intrathecal application. Local administration is contemplated for transdermal delivery, administration to the eye or mucous membranes, or inhalation therapy. Nasal solutions of the antibodies alone or in combination with other pharma- ceutically acceptable excipients can also be administered.

[0194] Transdermal patches, including iontophoretic and electrophoretic devices, are well known to those of skill in the art and can be used to administer antibodies. For example, such patches are disclosed in U.S. Patent Nos. 6,267,983, 6,261,595, 6,256,533, 6,167,301, 6,024,975, 6,010715, 5,985,317, 5,983,134, 5,948,433, and 5,860,957, each of which is incorporated herein by reference in its entirety.

[0195] In some aspects, pharmaceutical compositions comprising therapeutic agents described herein (e.g., IL-7 proteins and / or VEGF antagonists) are lyophilized powders and can be reconstituted for administration as solutions, emulsions, and other mixtures. They can also be reconstituted and formulated as solids or gels. Lyophilized powders are prepared by dissolving the antibodies or antigen-binding portions thereof described herein, or pharma- ceutically acceptable derivatives thereof, in a suitable solvent. In some aspects, the lyophilized powders are sterilized. The solvent may contain excipients that improve the stability or other pharmacological components of the powder, or a reconstituted solution prepared from the powder. Excipients that can be used include, but are not limited to, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable agents. The solvent may contain a buffer, such as citric acid, sodium or potassium phosphate, or other such buffers known to those of skill in the art, in one embodiment at about neutral pH. The solution is then sterile filtered under standard conditions known to those of skill in the art, followed by lyophilization to provide the desired formulation. In some embodiments, the resulting solution can be distributed into vials for lyophilization. Each vial can contain a single dose or multiple doses of the compound. The lyophilized powder can be stored under appropriate conditions, such as at room temperature at about 4°C.

[0196] Reconstitution of this lyophilized powder with water for injection provides a formulation for use in parenteral administration. When reconstituted, the lyophilized powder is added to sterile water or other suitable carrier. The exact amount depends on the compound selected. Such amounts can be empirically determined.

[0197] The compositions provided herein can also be formulated to target specific tissues, receptors, or other areas of the body of the subject being treated. Many such targeting methods are well known to those skilled in the art. It is envisioned herein that all such targeting methods can be used in the compositions of the present invention. Non-limiting examples of targeting methods can be found in, for example, U.S. Patent Nos. 6,316,652, 6,274,552, 6,271,359, 6,253,872, 6,139,865, 6,131,570, 6,120,751, 6,071,495, 6,071,570 ... See Nos. 6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, 5,900,252, 5,840,674, 5,759,542, and 5,709,874.

[0198] Compositions to be used for in vivo administration can be sterilized, which is readily accomplished, for example, by filtration through sterile filtration membranes.

[0199] The following examples are illustrative only and should not be construed as limiting the scope of the present disclosure in any way, since many variations and equivalents will become apparent to those of skill in the art upon reading this disclosure. EXAMPLES

[0200] Example 1. Effect of combined treatment with IL-7 protein and VEGF antagonist on tumor volume To evaluate the effect of the combination of VEGF antagonist and IL-7 protein on tumor volume, a colon adenocarcinoma animal model was used. Briefly, MC-38 colon adenocarcinoma tumor cells (5×10 5Cells, subcutaneously) were implanted into C57BL / 6 mice. On the 8th day after tumor inoculation (average tumor size approximately 60 mm 3 ) each animal was treated with IL-7-HyFc protein and antagonistic 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 (intramuscularly, 10 mg / kg per mouse) once (i.e., 8 days after tumor inoculation). Antagonistic anti-VEGF antibody was administered (intravenously, 10 mg / kg per dose per mouse) twice a week for 2 weeks (i.e., 8, 11, 15, and 18 days after tumor inoculation). Tumor volume in these animals was then monitored at different time points. Figure 1A shows a graphical representation of the dosing schedule and Table 2 (below) shows the different treatment groups. [Table 3]

[0201] As shown in FIG. 1B, compared to control animals (i.e., group 1), 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. However, in mice in the combination treatment group (i.e., group 4), the reduction in tumor volume was much more dramatic. The mean tumor volumes from the different treatment groups on day 18 after tumor inoculation were as follows: Group 1: 635.30 mm 3 , 2nd group: 269.46mm 3 , 3rd group: 257.99mm 3 , and the fourth group: 86.33 mm 3 .

[0202] The above results demonstrate that the combination therapy described herein (combining a VEGF antagonist with an IL-7 protein) may be useful in the treatment of certain cancers. Moreover, the data further demonstrate that the IL-7 protein described herein can improve the anti-tumor efficacy of VEGF antagonists, such as those that are commercially available and / or have previously been shown to have anti-tumor efficacy.

[0203] Example 2 Analysis of the antitumor effect of triple combination therapy of IL-7 protein, VEGF antagonist, and immune checkpoint inhibitor To further evaluate the anti-tumor effect of the combination treatment described herein, tumor animal models (e.g., colon adenocarcinoma animal models) are treated with IL-7 protein alone or in combination with a VEGF antagonist (e.g., an anti-VEGF antibody). In some embodiments, some animals are further administered an immune checkpoint inhibitor. Thus, in some embodiments, some animals are treated with a triple combination of IL-7 protein, a VEGF antagonist, and an immune checkpoint inhibitor. After administration, the anti-tumor immune response in each animal is evaluated, for example, by monitoring tumor volume at different time points after treatment.

[0204] Example 3 Effects of combined treatment with IL-7 protein and VEGF antagonist in human glioblastoma subjects A phase 2 study is being conducted in human subjects with recurrent glioblastoma to evaluate both the efficacy and safety of the treatment regimen described herein. As shown in Figure 2 and further described below, eligible subjects (see, e.g., the inclusion and exclusion criteria further described below) were administered the IL-7 protein described herein in combination with bevacizumab (i.e., a VEGF antagonist) (also referred to herein as the "investigational drug"). Eligible subjects are administered the combination treatment for up to six cycles (1 cycle = 8 weeks) or until disease progression or onset of unacceptable toxicity. Tumor assessments are performed every 8 weeks prior to treatment administration. Figure 3 shows an overall summary of the study.

[0205] subject Eligible subjects have been diagnosed with glioblastoma (e.g., recurrent glioblastoma) and have confirmed disease progression following standard of care (concomitant chemotherapy and / or adjuvant chemotherapy with temozolomide).

[0206] IL-7 protein isoforms IL-7 protein will be administered to eligible subjects intramuscularly at a dose of 1,200 μg / kg with an 8-week dosing interval. As above, subjects without severe toxicity and no clinically significant evidence of disease progression will receive up to six cycles of IL-7 protein (based on the investigator's clinical significance and risk assessment). At each dose level, the amount of IL-7 protein administered to a subject can be determined based on the subject's body weight on the first day of each cycle. There are no dose restrictions for overweight subjects.

[0207] Bevacizumab administration Bevacizumab will be administered to eligible subjects intravenously at a dose of 10 mg / kg every 2 weeks until disease progression or unacceptable toxicity (as described elsewhere in this Example).

[0208] Dosage adjustments 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 the subject experiences unacceptable toxicity (CTCAE grade 3 or higher) after IL-7 protein administration, IL-7 protein administration will be discontinued; If toxicity resolves within 28 days, IL-7 protein administration may be resumed; For subjects experiencing clinically manageable toxicity (grade 3 immune-related adverse events) but showing clear signs of clinical benefit (e.g., tumor shrinkage or stable disease), IL-7 protein administration may be continued under close supervision; After the first dose, subjects may achieve a (treatment-relevant) absolute lymphocyte count (ALC) of 20,000 cells / mm 3If ALC exceeds the upper limit of normal (4,000 cells / mm), further treatment should be temporarily discontinued. 3 ), administration can be resumed.

[0209] For VEGF antagonists, dosage adjustments may be made if the subject's body weight changes by more than about 10%. Otherwise, dosage adjustments are generally not recommended. If adverse events occur, administration of the VEGF agonist may be discontinued temporarily or permanently at the discretion of the investigator.

[0210] Injection site reactions Common adverse reactions that may occur at the injection site include allergic reactions (e.g., rash, hives, redness), itching, swelling, and erythema. Appropriate doses of steroids (e.g., prednisolone at a dose of about 5-7.5 mg) may be used as palliative treatment. Antihistamines may be used for prophylactic purposes (e.g., administered to subjects at least 14 days prior to treatment).

[0211] prohibited treatment The following treatments are prohibited during the course of this study: (1) any concomitant treatment, including chemotherapy, hormonal therapy, immunotherapy, radiation therapy, investigational drugs, or herbal therapies approved or investigational for the treatment of cancer; (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) immune stimulants (e.g., IFN-α, IFN-γ, and IL-2).

[0212] Selection Criteria The inclusion criteria for determining subject eligibility included the following: (1) age ≥19 years, (2) patients with histologically diagnosed glioblastoma with confirmed disease progression after attempting standard therapy (RT / CCRT and / or adjuvant chemotherapy (TMZ)), (3) Karnofsky performance status (KPS) ≥60 or ECOG status 0–2, (4) life expectancy >12 weeks, and (5) adequate hematological and end-organ function.

[0213] Exclusion criteria Subjects were excluded from the study if they had one or more of the following: (1) malignancies other than those in the study within 5 years prior to first administration of study drug, (2) prior use of bevacizumab or other VEGF antagonists, or (3) a body mass index (BMI) of ≥ 30 kg / m2. 2 , (4) intracranial hemorrhage confirmed by non-contrast CT or MRI, (5) clinically significant cardiovascular disease, (6) history of arterial or venous thromboembolism within 6 months prior to the study, (7) uncontrolled hypertension (blood pressure ≥ 150 / 90 mmHg with appropriate antihypertensive therapy), (8) history of hypertensive crisis or hypertensive encephalopathy, (9) administration of therapeutic anticoagulants (except low molecular weight heparin or warfarin), (10) pregnancy or breastfeeding, (11) active viral infection, (12) autoimmune disease / syndrome, (13) chronic kidney disease, (14) chronic kidney disease, (15) chronic kidney disease, (16) chronic kidney disease, (17) chronic kidney disease, (18) chronic kidney disease, (19) chronic kidney disease, (20) chronic kidney disease, (21) chronic kidney disease, (22) chronic kidney disease, (23) chronic kidney disease, (24) chronic kidney disease, (25) chronic kidney disease, (26) chronic kidney disease, (27) chronic kidney disease, (28) chronic kidney disease, (29) chronic kidney disease, (30) chronic kidney disease, (31) chronic kidney disease, (32) chronic kidney disease, (33) chronic kidney disease, (34) chronic kidney disease, (35) chronic kidney disease, (36) chronic kidney disease, (37) chronic kidney disease, (38) chronic kidney disease, (39) chronic kidney disease, (40) chronic kidney disease, (41) chronic kidney disease, (42) chronic kidney disease, (43) chronic kidney disease, (44) chronic kidney disease, (45) chronic kidney disease, (46) chronic kidney disease, (47) chronic kidney disease, (48) chronic kidney disease, (49) chronic kidney disease, (50) chronic kidney disease, (51) chronic kidney disease, (52) chronic kidney disease, (53) chronic kidney disease, (54) chronic kidney disease, (55 3) administration of a live attenuated vaccine within 4 weeks prior to Cycle 1, Day 1, or if such a live attenuated vaccine is anticipated to be required during the study; (14) history of severe allergic, anaphylactic, or other hypersensitivity reaction to chimeric or humanized antibodies or fusion proteins; (15) severe infection during the screening period, including but not limited to infectious complications, bacteremia, or severe pneumonia; (16) prior allogeneic bone marrow transplant or prior solid organ transplant.

[0214] Example 4: Interim results of a Phase 2 study involving combination treatment of IL-7 protein and a VEGF antagonist in human glioblastoma subjects In addition to Example 3 above, Figures 4A and 4B show interim results of the study. Twenty subjects were enrolled in the study, but as shown in Figure 4A, 14 subjects are still undergoing treatment (patient numbers 2, 5, 6, 9, and 11-20), and four subjects have progressed (patient numbers 1, 4, 7, and 10). Of the subjects who progressed, two subjects discontinued the study (patient numbers 4 and 7). Also, as shown in Figure 4B, almost all subjects had a significant increase in absolute lymphocyte counts four weeks after the first dose. Further results (e.g., clinical response and survival status) are currently ongoing.

[0215] The above results demonstrate the therapeutic potential of the combination treatments described herein in the treatment of cancers such as glioblastoma.

Claims

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

2. 2. The method of claim 1, wherein tumor volume is reduced in the subject following said administration.

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

5. 5. The method of claim 4, wherein the subject's anti-tumor immune response is increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 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 said administration.

6. 6. The method of claim 4 or 5, wherein the increased anti-tumor immune response comprises: (i) a decrease in tumor volume; (ii) an increase in 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 increase in the survival time of the subject; (viii) a decrease in expression of TOX on CD8+ T cells in the tumor, tumor-draining lymph nodes (TDLNs), or both; (ix) an increase in the number of stem cell-like T cells in the tumor, TDLNs, 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 sequentially to the subject.

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

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

11. 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 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 oligopeptide is selected from the group consisting of 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), and glycine-methionine (GMG). Lysine-glycine-glycine (GGG), methionine-glycine-glycine-methionine (MGGM) (SEQ ID NO: 41), methionine-methionine-glycine-glycine (MMGG) (SEQ ID NO: 42), glycine-glycine-methionine-methionine (GGMM) (SEQ ID NO: 43), methionine-glycine-methionine-glycine (MGMG) (SEQ ID NO: 44), glycine-methionine-methionine-glycine (GMMG) (SEQ ID NO: 45), glycine-glycine-glycine-methionine (GGGM) (SEQ ID NO: 46), methionine-glycine Lysine-glycine-glycine (MGGG) (SEQ ID NO: 47), glycine-methionine-glycine-glycine (GMGG) (SEQ ID NO: 48), glycine-glycine-methionine-glycine (GGMG) (SEQ ID NO: 49), glycine-glycine-methionine-methionine-methionine (GGMMM) (SEQ ID NO: 50), glycine-glycine-glycine-methionine-methionine (GGGMM) (SEQ ID NO: 51), glycine-glycine-glycine-glycine-methionine (GGGGM) (SEQ ID NO: 52), methionine-glycine-methionine- methionine-methionine (MGMMM) (SEQ ID NO: 53), methionine-glycine-glycine-methionine-methionine (MGGMM) (SEQ ID NO: 54), methionine-glycine-glycine-glycine-methionine (MGGGM) (SEQ ID NO: 55), methionine-methionine-glycine-methionine-methionine (MMGMM) (SEQ ID NO: 56), methionine-methionine-glycine-glycine-methionine (MMGGM) (SEQ ID NO: 57), methionine-methionine-glycine-glycine-glycine (MMGGG) (SEQ ID NO: 58),Methionine-methionine-methionine-glycine-methionine (MMMGM) (SEQ ID NO: 59), methionine-glycine-methionine-glycine-methionine (MGMGM) (SEQ ID NO: 60), glycine-methionine-glycine-methionine-glycine (GMGMMG) (SEQ ID NO: 61), glycine-methionine-methionine-methionine-glycine (GMMMG) (SEQ ID NO: 62), glycine-glycine-methionine-glycine-methionine (GGMGM) (SEQ ID NO: 63), glycine-glycine-methionine-methionine-glycine (GGMMG) (SEQ ID NO: 64), glycine-methionine-methionine-glycine-methionine (GMMGM) (SEQ ID NO: 65), methionine-glycine 16. The method of claim 15, comprising the step of: -methionine-methionine-glycine (MGMMG) (SEQ ID NO:66), glycine-methionine-glycine-glycine-methionine (GMGGM) (SEQ ID NO:67), methionine-methionine-glycine-methionine-glycine (MMGMG) (SEQ ID NO:68), glycine-methionine-methionine-glycine-glycine (GMMGG) (SEQ ID NO:69), glycine-methionine-glycine-glycine-glycine (GMGGG) (SEQ ID NO:70), glycine-glycine-methionine-glycine-glycine (GGMGG) (SEQ ID NO:71), glycine-glycine-glycine-glycine-glycine (GGGGGG) (SEQ ID NO:72), or a combination thereof.

17. 19. 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. 19. The method of claim 18, wherein the half-life extending moiety comprises Fc, albumin, an albumin binding polypeptide, Pro / Ala / Ser (PAS), the C-terminal peptide of the beta subunit of human chorionic gonadotropin (CTP), polyethylene glycol (PEG), a long unstructured hydrophilic amino acid sequence (XTEN), hydroxyethyl starch (HES), an albumin binding small molecule, or a combination thereof.

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

21. the Fc is a hybrid Fc comprising a hinge region, a CH2 domain, and a CH3 domain; the hinge region comprises a human IgD hinge region; the CH2 domain comprises a portion of a human IgD CH2 domain and a portion of a human IgG4 CH2 domain; The method of claim 18 or 20, wherein the CH3 domain comprises a portion of a human IgG4 CH3 domain.

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

23. 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 VEGF (an "anti-VEGF antibody"), a polynucleotide encoding an anti-VEGF antibody, an antisense oligonucleotide, an siRNA, shRNA, miRNA, dsRNA, aptamer, PNA targeting VEGF, or a vector comprising them.

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

25. 25. The method of claim 24, wherein the anti-VEGF antibody comprises aflibercept (EYLEA® and ZALTRAP®), bevacizumab (AVASTIN®, ZIRABEV®, and MVASI®), ranibizumab (LUCENTIS®, BYOOVIZ®, SUSVIMO®), or a combination thereof.

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

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

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

29. 29. The method of claim 27 or 28, wherein the glioblastoma is resistant to standard treatment.

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

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

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

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

34. 34. The method of any one of claims 1-33, 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 IL-7 protein is from about 610 μg / kg to about 1,200 μg / kg, from about 650 μg / kg to about 1,200 μg / kg, from about 700 μg / kg to about 1,200 μg / kg, from about 750 μg / kg to about 1,200 μg / kg, from about 800 μg / kg to about 1,200 μg / kg, from about 850 μg / kg to about 1,200 μg / kg, / kg, about 900 μg / kg to about 1,200 μg / kg, about 950 μg / kg to about 1,200 μg / kg, about 1,000 μg / kg to about 1,200 μg / kg , about 1,050 μg / kg to about 1,200 μg / kg, about 1,100 μg / kg to about 1,200 μg / kg, about 1,200 μg / kg to about 2,000 μg / k 35. The method of any one of claims 1-34, wherein the patient is administered at a dose of 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 700 μg / kg to about 1,000 μg / kg, about 750 μg / kg to about 1,000 μg / kg, about 800 μg / kg to about 1,000 μg / kg, about 850 μg / kg to about 1,000 μg / kg, about 900 μg / kg to about 1,000 μg / kg, or about 950 μg / kg to about 1,000 μg / kg.

36. 36. The method of any one of claims 1-35, wherein 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.

37. The IL-7 protein is 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, 1,000 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 1200 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 1,320 μg / kg, about 1,340 μg / kg, Approximately 1,360 μg / kg, approximately 1,380 μg / kg, approximately 1,400 μg / kg, approximately 1,420 μg / kg, approximately 1,440 μg / kg, approximately 1,460 μg / kg, approximately 1,480 μg / kg, approximately 1,500 μg / kg, approximately 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 1,680 μg / kg, about 1,700 μg / k 37. The method of any one of claims 1 to 36, wherein the antibody is administered at a dose of 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. 38. The method of any one of claims 1-37, wherein the IL-7 protein is administered at a dosing frequency of once per week, once per 2 weeks, once per 3 weeks, once per 4 weeks, once per 5 weeks, once per 6 weeks, once per 7 weeks, once per 8 weeks, once per 9 weeks, once per 10 weeks, once per 11 weeks, or once per 12 weeks.

39. 39. The method of claim 38, wherein the IL-7 protein is administered at a dosing frequency of once every 8 weeks.

40. 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. 41. The method of any one of claims 1-40, wherein the VEGF antagonist is administered at a dosing frequency of once per week, once per 2 weeks, once per 3 weeks, once per 4 weeks, once per 5 weeks, once per 6 weeks, once per 7 weeks, once per 8 weeks, once per 9 weeks, once per 10 weeks, once per 11 weeks, or once per 12 weeks.

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

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