Methods for increasing lymphocyte numbers in tumors using IL-7 fusion proteins

Abstract

1. A method for increasing lymphocyte count in a subject in need thereof, comprising administering to the subject (i) a modified interleukin-7 of the following formula (I): A-IL-7, wherein A is an oligopeptide consisting of 1 to 10 amino acid residues, and IL-7 is a polypeptide capable of binding to an IL-7 receptor; or (ii) an interleukin-7 fusion protein comprising: (a) a modified interleukin-7, (b) a second domain containing an oligopeptide having 1 to 10 amino acid residues consisting of methionine, glycine, or a combination thereof; and (c) a third domain that extends the half-life of the interleukin-7 fusion protein.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 895,787, filed September 4, 2019, and U.S. Provisional Application No. 62 / 935,828, filed November 15, 2019. [Background technology]

[0002] T cells play a central role in the immune response, recognizing cancer antigens, destroying cancer cells, and differentiating and proliferating into memory cells to attack cancer. Therefore, the diversity of T cell recognition, which allows them to recognize a variety of antigens expressed on cancer cells, the expansion of T cell clones that respond to cancer-specific antigens, and their differentiation into memory T cells in cancer tissues, can maximize anticancer treatment. Furthermore, cytotoxic chemotherapy and radiation therapy, which are used as standard anticancer treatments, destroy immune cells in the bone marrow and blood, eliminating T cells essential for anticancer immune function and counteracting the anticancer effects of immunotherapy drugs. Recent reports have shown that T cell deficiency in cancer patients is associated with reduced chemotherapy response rates and reduced patient survival. To date, IL-2 (Aldesleukin®) is the only cytokine therapeutic approved for inducing T cell proliferation and hyperactivity. However, the efficacy of IL-2 administration is limited by the proliferation of regulatory T cells, which induces immunosuppression, and its practical clinical use is limited by serious side effects such as cytokine storm and capillary leak syndrome due to excessive immune responses.

[0003] Interleukin-7 (IL-7), an important growth and activation factor for T cells, is primarily involved in the differentiation, proliferation, and survival of naive and memory T cells, which are involved in antigen recognition and targeting while destroying cancer cells. It does not induce the proliferation of regulatory T cells, which suppress the activation of effector T cells. Furthermore, increased levels of IL-7 in the body lead to T cell proliferation, and IL-7 translocates into cells (transcytosis) through binding to the IL-7 receptor (CD127), which is primarily expressed on expanded T cells. This maintains homeostasis and is known as a homeostatic cytokine. Therefore, serious immune-related side effects, such as cytokine storm caused by an excessive immune response, which has been a major issue in the clinical safety of other cytokine treatments, have not been reported in previous clinical trials using recombinant human IL-7 (rhIL-7) by Cytheris under the code name CYT107.

[0004] IL-7 is encoded by the gene IL7 and binds to the IL-7 receptor (CD127). IL-7 is an immunostimulatory cytokine that can promote immune responses mediated by B cells and T cells. IL-7 plays an important role in the adaptive immune system.

[0005] A co-pending application (U.S. Application No. 15 / 126,313), the entire contents of which are incorporated herein by reference, discloses modified IL-7 proteins with improved expression and half-life, fusion proteins containing modified IL-7, and methods for producing the same. Summary of the Invention

[0006] The present disclosure provides methods for increasing lymphocyte numbers using denatured IL-7 or a fusion protein comprising denatured IL-7.

[0007] One embodiment provides a method of increasing lymphocyte numbers in a subject in need thereof, comprising administering to the subject denatured IL-7 or a fusion protein thereof.

[0008] The subject in need thereof may be a mammal suffering from cancer or a malignant tumor. The cancer may be a solid tumor. In an exemplary embodiment, the solid tumor may be a locally advanced or metastatic solid tumor or glioblastoma.

[0009] In one embodiment, the subject may be a human.

[0010] In one embodiment, the subject may receive prior or concurrent cancer treatment. The modified IL-7 or its fusion protein can be administered separately or simultaneously with other anti-cancer drugs.

[0011] Accordingly, various aspects of the present disclosure include the following embodiments.

[0012] 1. A method for increasing lymphocyte count in a subject in need thereof, comprising: (i) a modified interleukin-7 of the following formula (I): A-IL-7 formula (I) where A is an oligopeptide consisting of 1 to 10 amino acid residues, IL-7 is a polypeptide capable of binding to the IL-7 receptor; or (ii) an interleukin-7 fusion protein comprising: a first domain comprising a denatured interleukin-7 of formula (I); a second domain comprising an oligopeptide having 1 to 10 amino acid residues consisting of methionine, glycine, or a combination thereof; and Third domain extends half-life of interleukin-7 fusion proteins to a subject at a dose greater than 600 μg / kg.

[0013] 2. The method of claim 1, wherein the subject is suffering from cancer, infection, chronic failure of the right ventricle of the heart, Hodgkin's disease and cancer of the lymphatic system, leukemia, leak or rupture of the thoracic duct, side effects of prescription medications including anti-cancer drugs (e.g., chemotherapy), antivirals, and glucocorticoids, malnutrition due to a low-protein diet, radiation therapy, uremia, autoimmune disease, immunodeficiency syndrome, high stress levels, trauma, thymectomy, or a combination thereof; or idiopathic, acute radiation syndrome (ARS), or a combination thereof.

[0014] 3. The method according to 1 or 2 above, wherein the IL-7 has an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 6.

[0015] 4. The method according to any one of 1 to 3, wherein A is linked to the N-terminus of IL-7.

[0016] 5. In any one of the above 1 to 4, A is methionine (M), glycine (G), methionine-methionine, glycine-glycine, methionine-glycine, glycine-methionine, methionine-methionine-methionine, methionine-methionine-glycine, methionine-glycine-methionine, glycine-methionine-methionine, methionine-glycine-glycine, glycine-methionine-glycine, glycine-glycine-methionine, glycine-glycine-glycine, MMMM, MGMM, MGGM, MGGG, MGMG, GMMM, GMGG, GGGG, MMMMM, MMGMM, MMGGM, MMGMMG, MMMMG, GGGGG, GGMMM, GGGMG, MGMGMG, MMMGGG, MMGGMM, GGMMGG, MGMGMGMG, MMMMGGGG, MMGGMMGG, MMMMGGGG, MGMGMGMGMG, and / or MMMMMGGGGG.

[0017] 6. The method of claim 5, wherein the third domain is linked to the N-terminus or C-terminus of the first domain or the second domain. In one embodiment, the third domain is linked to the C-terminus of the second domain.

[0018] 7. The method according to any one of 1 to 6, wherein the third domain is any one selected from the group consisting of an Fc region of an immunoglobulin or a portion thereof, 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 sequence of amino acids (XTEN), hydroxyethyl starch (HES), an albumin-binding small molecule, and a combination thereof.

[0019] 8. The method according to any one of 1 to 7, wherein the third domain comprises the Fc region of a modified immunoglobulin.

[0020] 9. The method according to any one of 1 to 8, wherein the modified immunoglobulin is selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and combinations thereof.

[0021] 10. In any one of 1 to 9 above, the Fc region of the modified immunoglobulin comprises, from the N-terminus to the C-terminus, a hinge region, a CH2 domain, and a CH3 domain; wherein the hinge region comprises a human IgD hinge region; the CH2 domain contains a portion of the amino acid residues of the CH2 domains of human IgD and human IgG4; The method, wherein the CH3 domain comprises a portion of amino acid residues of the CH3 domain of human IgG4.

[0022] 11. The method according to any one of 1 to 10 above, wherein the Fc region of the modified immunoglobulin is represented by the following formula (I): Formula (I) N'-(Z1)pY-Z2-Z3-Z4-C' where N' is the N-terminus of the polypeptide and C' is the C-terminus of the polypeptide; p is an integer of 0 or 1; Z1 is an amino acid sequence having 5 to 9 consecutive amino acid residues from the 98th amino acid residue among the 90th to 98th amino acid residues of SEQ ID NO: 7 toward the N-terminus; Y is an amino acid sequence having 5 to 64 consecutive amino acid residues from the 162nd amino acid residue among the 99th to 162nd amino acid residues of SEQ ID NO: 7 toward the N-terminus; Z2 is an amino acid sequence having 4 to 37 consecutive amino acid residues from the 163rd amino acid residue to the C-terminus among the 163rd to 199th amino acid residues of SEQ ID NO: 7; Z3 is an amino acid sequence having 71 to 106 consecutive amino acid residues from the 220th amino acid residue among the 115th to 220th amino acid residues of SEQ ID NO: 8 toward the N-terminus; and Z4 is an amino acid sequence having 80 to 107 consecutive amino acid residues from the 221st amino acid residue among the 221st to 327th amino acid residues of SEQ ID NO:8 toward the C-terminus.

[0023] 12. The method according to any one of 1 to 11 above, wherein the third domain has an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 to 14.

[0024] 13. The method according to any one of 2 to 12, wherein the cancer is a solid tumor, lymphatic cancer, or leukemia.

[0025] 14. The method according to claim 13, wherein the solid tumor is synovial sarcoma, invasive ductal carcinoma, rectal cancer, colon cancer, ovarian cancer, ascending colon cancer, anal cancer, invasive ductal carcinoma, adenocarcinoma, rectal cancer with para-aortic metastasis, neuroendocrine carcinoma (cervix), sigmoid colon cancer, or glioblastoma.

[0026] 15. Any one of 1 to 14 above, wherein the subject has previously undergone or is concurrently undergoing one or more cancer treatments including surgery, radiation, and / or chemotherapy.

[0027] 16. Any one of 1 to 15 above, wherein (i) denatured interleukin-7 or (ii) interleukin-7 fusion protein is administered at a dose ranging from more than 600 μg / kg to about 2,000 μg / kg.

[0028] 17. Any one of 1 to 16, wherein (i) the denatured interleukin-7 or (ii) the interleukin-7 fusion protein is administered at a dose of about 720 μg / kg or more, about 960 μg / kg or more, about 1,200 μg / kg or more, about 1,700 μg / kg or more, or about 2,000 μg / kg.

[0029] 18. Any one of the methods 1 to 16, wherein (i) denatured interleukin-7 or (ii) interleukin-7 fusion protein is administered at a dose of about 720 μg / kg or more, about 840 μg / kg or more, or about 1,440 μg / kg or more.

[0030] 19. Any one of 1 to 16, wherein (i) denatured interleukin-7 or (ii) interleukin-7 fusion protein is administered at 720 μg / kg or more, 840 μg / kg or more, 960 μg / kg or more, 1,200 μg / kg or more, 1,440 μg / kg or more, 1,700 μg / kg or more, or 2,000 μg / kg.

[0031] 20. Any one of 1 to 19, wherein (i) the denatured interleukin-7 or (ii) the interleukin-7 fusion protein is administered two or more times at intervals of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks.

[0032] 21. Any one of 1 to 19, wherein (i) the denatured interleukin-7 or (ii) the interleukin-7 fusion protein is administered two or more times at intervals of 10 days, 20 days, 30 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, or 100 days.

[0033] 22. Any one of 1 to 21, wherein (i) the denatured interleukin-7 or (ii) the interleukin-7 fusion protein is administered parenterally, intramuscularly, subcutaneously, intraocularly, intravenously, intraperitoneally, intradermally, intraorbitally, intracerebrally, intracranially, intraspinally, intraventricularly, intrathecally, intracapsularly, intraarticularly, or intratumorally.

[0034] 23. Any one of the methods 1 to 22 above, further comprising (ii) administering an interleukin-7 fusion protein.

[0035] 24. The method according to any one of 1 to 23, wherein (i) the denatured interleukin-7 comprises the amino acid sequence of SEQ ID NO: 18, and (ii) the interleukin-7 fusion protein comprises the amino acid sequence of SEQ ID NO: 24.

[0036] 25. Any one of 1 to 24, wherein the subject has a lymphocyte count of 1000 or less lymphocyte cells per μl of blood and / or serum as defined by the Common Terminology Criteria for Adverse Events (CTCAE) version 4.0.

[0037] 26. A method according to any one of 1 to 25, wherein the lymphocytes are T cells.

[0038] 27. Any one of 1 to 26, wherein (i) the denatured interleukin-7 or (ii) the interleukin-7 fusion protein is administered two or more times in an amount of about 720 μg / kg at intervals of about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks.

[0039] 28. Any one of 1 to 26, wherein (i) the denatured interleukin-7 or (ii) the interleukin-7 fusion protein is administered two or more times in an amount of about 840 μg / kg at intervals of about 2 weeks, about 3 weeks, about 4 weeks, or about 5 weeks.

[0040] 29. Any one of 1 to 26, wherein (i) the denatured interleukin-7 or (ii) the interleukin-7 fusion protein is administered two or more times in an amount of about 960 μg / kg at intervals of about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, or about 6 weeks.

[0041] 30. Any one of 1 to 26, wherein (i) the denatured interleukin-7 or (ii) the interleukin-7 fusion protein is administered two or more times in an amount of about 1200 μg / kg at intervals of about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, or about 8 weeks.

[0042] 31. Any one of 1 to 26, wherein (i) the denatured interleukin-7 or (ii) the interleukin-7 fusion protein is administered two or more times in an amount of about 1440 μg / kg at intervals of about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 10 weeks, or about 12 weeks, or about 2 months or 3 months.

[0043] 32. Any one of 1 to 26, wherein (i) the denatured interleukin-7 or (ii) the interleukin-7 fusion 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 about 2,000 μg / kg.

[0044] 33. In any one of 1 to 26 above, (i) denatured interleukin-7 or (ii) interleukin-7 fusion 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, or about 750 μg / kg to about 1,200 μg / kg. g, about 800μg / kg to about 1,200μg / kg, about 850μg / kg to about 1,200μg / kg, about 900μg / kg to about 1,200μg / kg, about 950μg / kg kg ~ approx. 1,200μg / kg, approx. 1,000μg / kg ~ approx. 1,200μg / kg, approx. 1,050μg / kg ~ approx. 1,200μg / kg, approx. 1,100μg / kg Approximately 1,200μg / kg, approximately 1,200μg / kg to approximately 2,000μg / kg, approximately 1,300μg / kg to approximately 2,000μg / kg, approximately 1,500μg / kg to approximately 2 ,000μg / kg, approximately 1,700μg / kg to approximately 2,000μg / kg, approximately 610μg / kg to approximately 1,000μg / kg, approximately 650μg / kg to approximately 1,000μg / kg 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.

[0045] 34. The method according to any one of 1 to 26, wherein (i) denatured interleukin-7 or (ii) interleukin-7 fusion 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.

[0046] 35. In any one of 1 to 26, (i) denatured interleukin-7 or (ii) interleukin-7 fusion 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, or about 920 μg / kg. , about 940μg / kg, about 950μg / kg, about 960μg / kg, about 980μg / kg, about 1,000μg / kg, about 1,020μg / kg, about 1,040μg / kg, about 1,060μg / kg, about 1,080μg / kg, about 1,100μg / k g, approximately 1,120μg / kg, approximately 1,140μg / kg, approximately 1,160μg / kg, approximately 1,180μg / kg, approximately 1,200μg / kg, approximately 1,220μg / kg, approximately 1,240μg / kg, approximately 1,260μg / kg, approximately 1,280μg / kg, approximately 1 ,300μg / kg, approximately 1,320μg / kg, approximately 1,340μg / kg, 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 1,48 0μg / 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, 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.

[0047] 36. Any one of 32 to 35, wherein (i) the denatured interleukin-7 or (ii) the interleukin-7 fusion protein is administered at a 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.

[0048] 37. In the above 26, the T cells are CD4 + T cells and / or CD8 + The method is a T cell.

[0049] 38. In the above 26, the T cells are CD4 + / CD8 + The method is a T cell.

[0050] 39. The method of any one of 1 to 38, wherein the subject has a lymphocyte count of 800 lymphocyte cells per μl of blood or less.

[0051] 40. The method of any one of 1 to 39, wherein the subject has a lymphocyte count of 500 lymphocyte cells per μl of blood or less.

[0052] 41. The method of any one of 1 to 40, wherein the subject has a lymphocyte count of 200 lymphocyte cells per μl of blood or less.

[0053] 42. The method according to any one of 15 to 41, wherein the subject is administered the same as an anti-cancer drug.

[0054] 43. The method according to claim 42, wherein the anticancer agent is an anticancer compound.

[0055] 44. Any one of 1 to 43, wherein the number of intratumoral tumor-infiltrating lymphocytes (TILs) is increased after administration of (i) denatured interleukin-7 or (ii) an interleukin-7 fusion protein compared to the number of intratumoral TILs before administration.

[0056] 45. In any one of 1 to 44, the TIL is CD4 + TIL is the way.

[0057] 46. ​​In any one of 1 to 44, the TIL is CD8 + TIL is the way.

[0058] 47. The method of any one of 44 to 46, wherein the number of TILs is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 125%, at least about 150%, at least about 200%, at least about 250%, or at least about 300% after administration.

[0059] 48. In any one of 1 to 26 above, (i) denatured interleukin-7 or (ii) interleukin-7 fusion protein is administered at a concentration of about 650 μg / kg to 680 μg / kg, about 680 μg / kg to 700 μg / kg, about 700 μg / kg to 720 μg / kg, about 720 μg / kg to 740 μg / kg, about 740 μg / kg to 750 μg / kg, about 750 μg / kg to 760 μg / kg, about 760 μg / kg to 780 μg / kg, about 780 μg / kg to 800 μg / kg, about 800 μg / kg to 820 μg / kg, or about 820 μg / kg to 840 μg / kg, approximately 840μg / kg~850μg / kg, approximately 850μg / kg~860μg / kg, approximately 860μg / kg~880μg / kg, approximately 880μg / kg~900μg / kg, approximately 900μg / kg~920μg / kg, approximately 920μg / kg~940μg / kg, approximately 940μg / kg kg~950μg / kg, approx. 950μg / kg~960μg / kg, approx. 960μg / kg~980μg / kg, approx. 980μg / kg~1000μg / kg, approx. 1,000μg / kg~1,020μg / kg, approx. 1,020μg / kg~1,040μg / kg, approx. 1,040μg / kg kg~1,060μg / kg, approx. 1,060μg / kg~1,080μg / kg, approx. 1,080μg / kg~1,100μg / kg, approx. 1,100μg / kg~1,120μg / kg, approx. 1,120μg / kg~1,140μg / kg, approx. 1,140μg / kg~1,160μg / kg, approximately 1,160μg / kg~1,180μg / kg, approximately 1,180μg / kg~1,200μg / kg, approximately 1,200μg / kg~1,220μg / kg, approximately 1,220μg / kg~1,240μg / kg, approximately 1,240μg / kg~1,260μg / kg, approximately 1,260 μg / kg~1,280μg / kg, approx. 1,280μg / kg~1,300μg / kg, approx. 1,300μg / kg~1,320μg / kg, approx. 1,320μg / kg~1,340μg / kg, approx. 1,340μg / kg~1,360μg / kg, approx. 1,360μg / kg~1,38 0μg / kg, approximately 1,380μg / kg~1,400μg / kg, approximately 1,400μg / kg~1,420μg / kg, approximately 1,420μg / kg~1,440μg / kg, approximately 1,440μg / kg~1,460μg / kg, approximately 1,480μg / kg, approximately 1,480μg / kg~1,500μg / kg, approximately 1,500μg / kg~1,520μg / kg, approximately 1,520μg / kg~1,540μg / kg, approximately 1,540μg / kg~1,560μg / kg, approximately 1,560μg / kg~1,580μg / kg, approximately 1,580μg / kg~1,600μg / kg, approximately 1,600μg / kg~1,620μg / kg, approximately 1 ,620μg / kg~1,640μg / kg, approx. 1,640μg / kg~1,660μg / kg, approx. 1,660μg / kg~1,680μg / kg, approx. 1,680μg / kg~1,700μg / kg, approx. 1,700μg / kg~1,720μg / kg, approx. 1,720μg / kg~1,740μg / kg, approx. 1,740μg / kg~1, 760μg / kg, approximately 1,760μg / kg~1,780μg / kg, approximately 1,780μg / kg~1,800μg / kg, approximately 1,800μg / kg~1,820μg / kg, approximately 1,820μg / kg~1,840μg / kg, approximately 1,840μg / kg~1,860μg / kg, approximately 1,860μg / kg~1,880μg / kg, approximately 1, The method is administered at a dose of 880 μg / kg to 1,900 μg / kg, about 1,900 μg / kg to 1,920 μg / kg, about 1,920 μg / kg to 1,940 μg / kg, about 1,940 μg / kg to 1,960 μg / kg, about 1,960 μg / kg to 1,980 μg / kg, or about 1,980 μg / kg to 2,000 μg / kg.

[0060] 49. The method of 48, wherein (i) the denatured interleukin-7 or (ii) the interleukin-7 fusion protein is administered two, three, four, or five times at said doses, at intervals of 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, about 12 weeks, about 13 weeks, about 14 weeks, or about 15 weeks. In certain embodiments, for example, the following are provided: (Item 1) 1. A method of increasing lymphocyte count in a subject in need thereof, comprising: (i) a modified interleukin-7 of formula (I): A-IL-7 formula (I) where A is an oligopeptide consisting of 1 to 10 amino acid residues, IL-7 is a polypeptide capable of binding to the IL-7 receptor; or (ii) an interleukin-7 fusion protein comprising: (a) Denatured interleukin-7, (b) a second domain comprising an oligopeptide having 1 to 10 amino acid residues consisting of methionine, glycine, or a combination thereof; and (c) A third domain that extends the half-life of interleukin-7 fusion proteins. to a subject at a dose greater than about 600 μg / kg. (Item 2) The method of item 1, wherein the subject is suffering from cancer; an infection; chronic failure of the right ventricle of the heart; Hodgkin's disease; a leak or rupture of the thoracic duct; a side effect of prescription medications including anti-cancer drugs (e.g., chemotherapy), antivirals, or glucocorticoids; malnutrition due to a low-protein diet, radiation therapy, uremia, an autoimmune disease, an immunodeficiency syndrome, thymectomy, or a combination thereof; or idiopathic, acute radiation syndrome (ARS), or a combination thereof. (Item 3) 3. The method according to item 1 or 2, wherein the IL-7 has an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 6. (Item 4) 4. The method of item 3, wherein A is linked to the N-terminus of IL-7. (Item 5) 4. The method of item 3, wherein A is methionine, glycine, methionine-methionine, glycine-glycine, methionine-glycine, glycine-methionine, methionine-methionine-methionine, methionine-methionine-glycine, methionine-glycine-methionine, glycine-methionine-glycine, glycine-methionine-glycine, glycine-glycine-methionine, or glycine-glycine-glycine. (Item 6) 6. The method of item 5, wherein the third domain is linked to the N-terminus or C-terminus of the first domain or the second domain. (Item 7) 7. The method of any one of items 4 to 6, wherein the third domain is any one selected from the group consisting of an Fc region of an immunoglobulin or a portion thereof, albumin, an albumin-binding polypeptide, Pro / Ala / Ser (PAS), the C-terminal peptide of the β subunit of human chorionic gonadotropin (CTP), polyethylene glycol (PEG), a long unstructured hydrophilic sequence of amino acids (XTEN), hydroxyethyl starch (HES), an albumin-binding small molecule, and a combination thereof. (Item 8) 8. The method of claim 7, wherein the third domain comprises an Fc region of a modified immunoglobulin. (Item 9) 9. The method of claim 8, wherein the modified immunoglobulin is selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and combinations thereof. (Item 10) The Fc region of a modified immunoglobulin comprises, from the N-terminus to the C-terminus, a hinge region, a CH2 domain, and a CH3 domain; wherein the hinge region comprises a human IgD hinge region; the CH2 domain contains a portion of the amino acid residues of the CH2 domains of human IgD and human IgG4; 10. The method according to item 9, wherein the CH3 domain comprises a portion of amino acid residues of the CH3 domain of human IgG4. (Item 11) The method according to item 10, wherein the Fc region of the modified immunoglobulin is represented by the following formula (I): Formula (I) N'-(Z1)pY-Z2-Z3-Z4-C' where N' is the N-terminus of the polypeptide and C' is the C-terminus of the polypeptide; p is an integer of 0 or 1; Z1 is an amino acid sequence having 5 to 9 consecutive amino acid residues from the 98th amino acid residue among the 90th to 98th amino acid residues of SEQ ID NO: 7 toward the N-terminus; Y is an amino acid sequence having 5 to 64 consecutive amino acid residues from the 162nd amino acid residue among the 99th to 162nd amino acid residues of SEQ ID NO: 7 toward the N-terminus; Z2 is an amino acid sequence having 4 to 37 consecutive amino acid residues from the 163rd amino acid residue to the C-terminus among the 163rd to 199th amino acid residues of SEQ ID NO: 7; Z3 is an amino acid sequence having 71 to 106 consecutive amino acid residues from the 220th amino acid residue among the 115th to 220th amino acid residues of SEQ ID NO: 8 toward the N-terminus; and Z4 is an amino acid sequence having 80 to 107 consecutive amino acid residues from the 221st amino acid residue among the 221st to 327th amino acid residues of SEQ ID NO:8 toward the C-terminus. (Item 12) Item 2. The method according to Item 1, wherein the third domain has an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 to 14. (Item 13) 3. The method according to item 2, wherein the cancer is a solid tumor, lymphatic cancer or leukemia. (Item 14) Item 14. The method of item 13, wherein the solid tumor is synovial sarcoma, invasive ductal carcinoma, rectal cancer, colon cancer, ovarian cancer, ascending colon cancer, anal cancer, invasive ductal carcinoma, adenocarcinoma, rectal cancer with para-aortic metastasis, neuroendocrine carcinoma (cervix), sigmoid colon cancer, or glioblastoma. (Item 15) 15. The method of any one of paragraphs 1, 2, 13 or 14, wherein the subject has previously undergone, is concurrently undergoing, or will undergo one or more of the following cancer treatments including surgery, radiation and / or chemotherapy. (Item 16) 3. The method of claim 1 or 2, wherein (i) the denatured interleukin-7 or (ii) the interleukin-7 fusion protein is administered at a dose ranging from greater than about 600 μg / kg to about 2,000 μg / kg. (Item 17) 3. The method of claim 1 or 2, wherein (i) the denatured interleukin-7 or (ii) the interleukin-7 fusion protein is administered at a dose of about 720 μg / kg or more, about 960 μg / kg or more, about μg / kg or more, 1,200 μg / kg or more, about 1,700 μg / kg or more, or about 2,000 μg / kg. (Item 18) 3. The method of claim 1 or 2, wherein (i) the denatured interleukin-7 or (ii) the interleukin-7 fusion protein is administered at a dose of about 720 μg / kg or more, about 840 μg / kg or more, or about 1,440 μg / kg or more. (Item 19) 3. The method of item 1 or 2, wherein (i) the denatured interleukin-7 or (ii) the interleukin-7 fusion protein is administered at a dose of about 720 μg / kg or more, about 840 μg / kg or more, about 960 μg / kg or more, about 1,200 μg / kg or more, about 1,440 μg / kg or more, about 1,700 μg / kg or more, or about 2,000 μg / kg. (Item 20) 3. The method of claim 1 or 2, wherein (i) the denatured interleukin-7 or (ii) the interleukin-7 fusion protein is administered two or more times at intervals of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, or 15 weeks. (Item 21) 3. The method of claim 1 or 2, wherein (i) the denatured interleukin-7 or (ii) the interleukin-7 fusion protein is administered two or more times at intervals of 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 days. (Item 22) 3. The method of claim 1 or 2, wherein (i) the denatured interleukin-7 or (ii) the interleukin-7 fusion protein is administered parenterally, intramuscularly, subcutaneously, intraocularly, intravenously, intraperitoneally, intradermally, intraorbitally, intracerebrally, intracranially, intraspinally, intraventricularly, intrathecally, intracapsularly, intraarticularly, or intratumorally. (Item 23) (ii) The method of item 1 or 2, comprising administering an interleukin-7 fusion protein. (Item 24) 24. The method of claim 23, wherein the (ii) interleukin-7 fusion protein comprises the amino acid sequence of SEQ ID NO: 24. (Item 25) 3. The method of item 1 or 2, wherein the subject has a lymphocyte count of about 1000 lymphocyte cells per μl of blood or less as defined by the Common Terminology Criteria for Adverse Events (CTCAE) version 4.0. (Item 26) 26. The method of item 25, wherein the lymphocytes are T cells. (Item 27) 3. The method of claim 1 or 2, wherein (i) the denatured interleukin-7 or (ii) the interleukin-7 fusion protein is administered two or more times in an amount of about 720 μg / kg at intervals of about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, or about 6 weeks. (Item 28) 3. The method of claim 1 or 2, wherein (i) the denatured interleukin-7 or (ii) the interleukin-7 fusion protein is administered two or more times in an amount of about 840 μg / kg at intervals of about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, or about 6 weeks. (Item 29) 3. The method of claim 1 or 2, wherein (i) the denatured interleukin-7 or (ii) the interleukin-7 fusion protein is administered two or more times in an amount of about 960 μg / kg at intervals of about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, or about 9 weeks. (Item 30) 3. The method of claim 1 or 2, wherein (i) the denatured interleukin-7 or (ii) the interleukin-7 fusion protein is administered two or more times in an amount of about 1,200 μg / kg at intervals of about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, or about 10 weeks. (Item 31) 3. The method of claim 1 or 2, wherein (i) the denatured interleukin-7 or (ii) the interleukin-7 fusion protein is administered two or more times in an amount of about 1,440 μg / kg at intervals of 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, about 12 weeks, about 13 weeks, about 14 weeks, about 15 weeks, about 2 months, or about 3 months. (Item 32) 3. The method of item 1 or 2, wherein (i) the denatured interleukin-7 or (ii) the interleukin-7 fusion 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. (Item 33) (i) Denatured interleukin-7 or (ii) interleukin-7 fusion protein: 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, approximately 850μg / kg to approximately 1,200μg / kg, approximately 900μg / kg to approximately 1,200μg / kg, approximately 950μg / kg to approximately 1,200μg / kg, approximately 1, 1,20 0μg / kg ~ approx. 2,000μg / kg, approx. 1,300μg / kg ~ approx. 2,000μg / kg, approx. 1,500μg / kg ~ approx. 2,000μg / kg, approx. 1,700 μg / kg ~ approx. 2,000 μg / kg, approx. 610 μg / kg ~ approx. 1,000 μg / kg, approx. 650 μg / kg ~ approx. 1,000 μg / kg, approx. 700 μg / kg ~ approx. 1 3. The method of claim 1 or 2, wherein the patient is administered at a dose of 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. (Item 34) 3. The method of claim 1 or 2, wherein (i) the denatured interleukin-7 or (ii) the interleukin-7 fusion 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. (Item 35) (i) Denatured interleukin-7 or (ii) interleukin-7 fusion protein, at concentrations 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 g, approximately 960μg / kg, approximately 980μg / kg, approximately 1,000μg / kg, approximately 1,020μg / kg, approximately 1,040μg / kg, approximately 1,060μg / kg, approximately 1,080μg / kg, approximately 1,100μg / kg, approximately 1,120μg / kg, approximately 1,1 40μg / kg, approximately 1,160μg / kg, approximately 1,180μg / kg, approximately 1,200μg / kg, approximately 1,220μg / kg, approximately 1,240μg / kg, approximately 1,260μg / kg, approximately 1,280μg / kg, approximately 1,300μg / kg, approximately 1,320 μg / kg, approximately 1,340μg / kg, approximately 1,360μg / kg, approximately 1,380μg / kg, approximately 1,400μg / kg, approximately 1,420μg / kg, approximately 1,440μg / kg, approximately 1,460μg / kg, approximately 1,480μg / kg, approximately 1,500μg / kg, approximately 1,520μg / kg, approximately 1,540μg / kg, approximately 1,560μg / kg, approximately 1,580μg / kg, approximately 1,600μg / kg, approximately 1,620μg / kg, approximately 1,640μg / kg, approximately 1,660μg / kg, approximately 1,680μg / k 3. The method of item 1 or 2, wherein the medicament is administered at a dose of 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. (Item 36) 3. The method of item 1 or 2, wherein (i) the denatured interleukin-7 or (ii) the interleukin-7 fusion 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, once per 12 weeks, once per 13 weeks, once per 14 weeks, or once per 15 weeks. (Item 37) T cells are CD4 + and / or CD8 + 27. The method of item 26, wherein the cell is a T cell. (Item 38) T cells are CD4 + / CD8 + 27. The method of item 26, wherein the cell is a T cell. (Item 39) 26. The method of claim 25, wherein the subject has a lymphocyte count of about 800 lymphocyte cells per μl of blood or less. (Item 40) 26. The method of claim 25, wherein the subject has a lymphocyte count of about 500 lymphocyte cells per μl of blood or less. (Item 41) 26. The method of claim 25, wherein the subject has a lymphocyte count of about 200 lymphocyte cells per μl of blood or less. (Item 42) 3. The method of item 1 or 2, wherein the subject has been administered, is concurrently administered, or will be administered an anti-cancer agent. (Item 43) 43. The method of item 42, wherein the anticancer agent is an anticancer compound. (Item 44) 43. The method of item 25 or 42, wherein the number of intratumoral tumor-infiltrating lymphocytes (TILs) is increased after administration of (i) denatured interleukin-7 or (ii) an interleukin-7 fusion protein compared to the number of intratumoral TILs before administration. (Item 45) TILs are CD4 + 45. The method of item 44, wherein the TIL. (Item 46) TILs are CD8 + 45. The method of item 44, wherein the TIL. (Item 47) 45. The method of item 44, wherein the number of TILs is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 125%, at least about 150%, at least about 200%, at least about 250%, or at least about 300% after administration. (Item 48) 1. Use of (i) and / or (ii) of the following for increasing lymphocyte count in a subject in need thereof: (i) a modified interleukin-7 of formula (I): A-IL-7 formula (I) where A is an oligopeptide consisting of 1 to 10 amino acid residues, IL-7 is a polypeptide capable of binding to the IL-7 receptor; and / or (ii) an interleukin-7 fusion protein comprising: (a) Denatured interleukin-7; (b) a second domain comprising an oligopeptide having 1 to 10 amino acid residues consisting of methionine, glycine, or a combination thereof; and (c) A third domain that extends the half-life of interleukin-7 fusion proteins. to a subject at a dose greater than about 600 μg / kg. (Item 49) 1. The use of (i) and / or (ii) of the following in the manufacture of a medicament for use in increasing lymphocyte count in a subject in need thereof, wherein the medicament is administered to the patient at a dose in excess of about 600 μg / kg: (i) a modified interleukin-7 of formula (I): A-IL-7 formula (I) where A is an oligopeptide consisting of 1 to 10 amino acid residues, IL-7 is a polypeptide capable of binding to the IL-7 receptor; and / or (ii) an interleukin-7 fusion protein comprising: (a) Denatured interleukin-7; (b) a second domain comprising an oligopeptide having 1 to 10 amino acid residues consisting of methionine, glycine, or a combination thereof; and (c) A third domain that extends the half-life of interleukin-7 fusion proteins. (Item 50) A pharmaceutical composition for increasing lymphocyte count in a subject in need thereof, comprising as an active ingredient: (i) a modified interleukin-7 of formula (I): A-IL-7 formula (I) where A is an oligopeptide consisting of 1 to 10 amino acid residues, IL-7 is a polypeptide capable of binding to the IL-7 receptor; and / or (ii) an interleukin-7 fusion protein comprising: (a) Denatured interleukin-7; (b) a second domain comprising an oligopeptide having 1 to 10 amino acid residues consisting of methionine, glycine, or a combination thereof; and (c) A third domain that extends the half-life of interleukin-7 fusion proteins. and administered to a subject at a dose greater than about 600 μg / kg. [Brief explanation of the drawings]

[0061] [Figure 1a] Figures 1a, 1b and 1c show the pharmacokinetic profiles by IV, SC and IM routes in rats, respectively. [Figure 1b]Figures 1a, 1b and 1c show the pharmacokinetic profiles by IV, SC and IM routes in rats, respectively. [Figure 1c] Figures 1a, 1b and 1c show the pharmacokinetic profiles by IV, SC and IM routes in rats, respectively. [Figure 2a] Figures 2a and 2b show the dose-dependent GX-I7 concentration-time profiles on linear and log scales. [Figure 2b] Figures 2a and 2b show the dose-dependent GX-I7 concentration-time profiles on linear and log scales. [Figure 3a] Figures 3a and 3b show the correlation of serum exposure (Cmax and AUClast) versus dose, respectively. [Figure 3b] Figures 3a and 3b show the correlation of serum exposure (Cmax and AUClast) versus dose, respectively. [Figure 4a] Figure 4a and Figure 4b show the changes in absolute lymphocyte counts (ALC) compared to baseline in the low-dose (60-120 μg / kg), medium-dose (240-480 μg / kg), and high-dose (720-1,200 μg / kg) groups. Figure 4a shows the results for the solid tumor patient group, and Figure 4b shows the results for the glioblastoma patient group. [Figure 4b] Figure 4a and Figure 4b show the changes in absolute lymphocyte counts (ALC) compared to baseline in the low-dose (60-120 μg / kg), medium-dose (240-480 μg / kg), and high-dose (720-1,200 μg / kg) groups. Figure 4a shows the results for the solid tumor patient group, and Figure 4b shows the results for the glioblastoma patient group. [Figure 5a] Figures 5a and 5b show the change in CD3+ counts compared to baseline in the low-dose (60-120 μg / kg), medium-dose (240-480 μg / kg), and high-dose (720-1,200 μg / kg) groups. Figure 5a shows the results for the solid tumor patient group, and Figure 5b shows the results for the glioblastoma patient group. [Figure 5b]Figures 5a and 5b show the change in CD3+ counts compared to baseline in the low-dose (60-120 μg / kg), medium-dose (240-480 μg / kg), and high-dose (720-1,200 μg / kg) groups. Figure 5a shows the results for the solid tumor patient group, and Figure 5b shows the results for the glioblastoma patient group. [Figure 6a] Figures 6a and 6b show the change in CD4+ counts compared to baseline in the low-dose (60-120 μg / kg), medium-dose (240-480 μg / kg), and high-dose (720-1,200 μg / kg) groups. Figure 6a shows the results for the solid tumor patient group, and Figure 6b shows the results for the glioblastoma patient group. [Figure 6b] Figures 6a and 6b show the change in CD4+ counts compared to baseline in the low-dose (60-120 μg / kg), medium-dose (240-480 μg / kg), and high-dose (720-1,200 μg / kg) groups. Figure 6a shows the results for the solid tumor patient group, and Figure 6b shows the results for the glioblastoma patient group. [Figure 7a] Figures 7a and 7b show the change in CD8+ counts compared to baseline in the low-dose (60-120 μg / kg), medium-dose (240-480 μg / kg), and high-dose (720-1,200 μg / kg) groups. Figure 7a shows the results for the solid tumor patient group, and Figure 7b shows the results for the glioblastoma patient group. [Figure 7b] Figures 7a and 7b show the change in CD8+ counts compared to baseline in the low-dose (60-120 μg / kg), medium-dose (240-480 μg / kg), and high-dose (720-1,200 μg / kg) groups. Figure 7a shows the results for the solid tumor patient group, and Figure 7b shows the results for the glioblastoma patient group. [Figure 8a]Figures 8a and 8b show the changes in absolute lymphocyte count (ALC) in non-lymphopenic (baseline ALC ≥ 1,000 cells / mm3) and lymphopenic (baseline ALC < 1,000 cells / mm3) solid tumor patients, respectively, divided into low-dose (60-120 μg / kg), medium-dose (240-480 μg / kg), and high-dose (720-1,200 μg / kg) groups. *p < 0.05, **p < 0.01, ***p < 0.001 vs. baseline (week 0) group (Wilcoxon matched-pairs signed-rank test). [Figure 8b] Figures 8a and 8b show the changes in absolute lymphocyte count (ALC) in non-lymphopenic (baseline ALC ≥ 1,000 cells / mm3) and lymphopenic (baseline ALC < 1,000 cells / mm3) solid tumor patients, respectively, divided into low-dose (60-120 μg / kg), medium-dose (240-480 μg / kg), and high-dose (720-1,200 μg / kg) groups. *p < 0.05, **p < 0.01, ***p < 0.001 vs. baseline (week 0) group (Wilcoxon matched-pairs signed-rank test). [Figure 9a] Figures 9a and 9b show the change in absolute lymphocyte count (ALC) in non-lymphopenic glioblastoma patients (baseline ALC ≥ 1,000 cells / mm3) and lymphopenic (baseline ALC < 1,000 cells / mm3) solid tumor patients, respectively, divided into low-dose (60-120 μg / kg), medium-dose (240-480 μg / kg), and high-dose (720-1,200 μg / kg) groups. *p<0.05, **p<0.01, ***p<0.001 vs. baseline (week 0) group (Wilcoxon matched-pairs signed-rank test). [Figure 9b]Figures 9a and 9b show the change in absolute lymphocyte count (ALC) in non-lymphopenic glioblastoma patients (baseline ALC ≥ 1,000 cells / mm3) and lymphopenic (baseline ALC < 1,000 cells / mm3) solid tumor patients, respectively, divided into low-dose (60-120 μg / kg), medium-dose (240-480 μg / kg), and high-dose (720-1,200 μg / kg) groups. *p<0.05, **p<0.01, ***p<0.001 vs. baseline (week 0) group (Wilcoxon matched-pairs signed-rank test). [Figure 10a] Figures 10a, 10b, and 10c show the changes in Ki67, CD127, and Treg ratios in CD4+ and CD8+ cells, respectively, after administration of IL-7 fusion protein to the low-dose (60-120 μg / kg), medium-dose (240-480 μg / kg), and high-dose (720-1,200 μg / kg) groups. NS: not significant; *p<0.05, **p<0.01, ***p<0.001 vs. baseline (week 0) group (by Wilcoxon matched-pairs signed-rank test). [Figure 10b] Figures 10a, 10b, and 10c show the changes in Ki67, CD127, and Treg ratios in CD4+ and CD8+ cells, respectively, after administration of IL-7 fusion protein to the low-dose (60-120 μg / kg), medium-dose (240-480 μg / kg), and high-dose (720-1,200 μg / kg) groups. NS: not significant; *p<0.05, **p<0.01, ***p<0.001 vs. baseline (week 0) group (by Wilcoxon matched-pairs signed-rank test). [Figure 10c] Figures 10a, 10b, and 10c show the changes in Ki67, CD127, and Treg ratios in CD4+ and CD8+ cells, respectively, after administration of IL-7 fusion protein to the low-dose (60-120 μg / kg), medium-dose (240-480 μg / kg), and high-dose (720-1,200 μg / kg) groups. NS: not significant; *p<0.05, **p<0.01, ***p<0.001 vs. baseline (week 0) group (by Wilcoxon matched-pairs signed-rank test). [Figure 11]Figure 11 is an illustration of the timeline of blood sampling in the monkey model. [Figure 12a] Figures 12a and 12b show the expression patterns of Ki67 in CD8+ and CD4+ cells, respectively, in the blood samples of the monkeys in which the study was performed. [Figure 12b] Figures 12a and 12b show the expression patterns of Ki67 in CD8+ and CD4+ cells, respectively, in the blood samples of the monkeys in which the study was performed. [Figure 13a] Figures 13a and 13b show the change in absolute lymphocyte count (ALC) compared to baseline values ​​with repeated dosing from time zero (week 0) to 15 weeks in the low-dose (120 μg / kg) group (Figure 13a), the medium-dose (360-600 μg / kg) group, and the high-dose (840-1,440 μg / kg) group (Figure 13b). [Figure 13b] Figures 13a and 13b show the change in absolute lymphocyte count (ALC) compared to baseline values ​​with repeated dosing from time zero (week 0) to 15 weeks in the low-dose (120 μg / kg) group (Figure 13a), the medium-dose (360-600 μg / kg) group, and the high-dose (840-1,440 μg / kg) group (Figure 13b). [Figure 14a] Figures 14a, 14b, and 14c show the change in absolute lymphocyte count (ALC) compared to baseline (week 0) with repeated administration of IL-7 fusion protein at intervals of 8 weeks or more in patients with glioblastoma receiving chemotherapy with TMZ (temozolomide 150 mg / m2), Avastin / irinotecan (A; Avastin 10 mg / kg, I; irinotecan 100 mg / m2), and PCV (PCV; CCNU 240 mg, vincristine 2 mg, procarbazine 150 mg & 100 mg, vincristine 2 mg), respectively. [Figure 14b]Figures 14a, 14b, and 14c show the change in absolute lymphocyte count (ALC) compared to baseline (week 0) with repeated administration of IL-7 fusion protein at intervals of 8 weeks or more in patients with glioblastoma receiving chemotherapy with TMZ (temozolomide 150 mg / m2), Avastin / irinotecan (A; Avastin 10 mg / kg, I; irinotecan 100 mg / m2), and PCV (PCV; CCNU 240 mg, vincristine 2 mg, procarbazine 150 mg & 100 mg, vincristine 2 mg), respectively. [Figure 14c] Figures 14a, 14b, and 14c show the change in absolute lymphocyte count (ALC) compared to baseline (week 0) with repeated administration of IL-7 fusion protein at intervals of 8 weeks or more in patients with glioblastoma receiving chemotherapy with TMZ (temozolomide 150 mg / m2), Avastin / irinotecan (A; Avastin 10 mg / kg, I; irinotecan 100 mg / m2), and PCV (PCV; CCNU 240 mg, vincristine 2 mg, procarbazine 150 mg & 100 mg, vincristine 2 mg), respectively. [Figure 15a] Figures 15a and 15b show the changes in CD4+ and CD8+ T cell subsets, and changes in chemokine receptor CCR and other immune cells (B cells and NK cells) after administration of IL-7 fusion protein. NS: not significant, p>0.05, *p<0.05, **p<0.01, ***p<0.001 vs. baseline (week 0) group (by Wilcoxon matched-pairs signed-rank test). [Figure 15b] Figures 15a and 15b show the changes in CD4+ and CD8+ T cell subsets, and changes in chemokine receptor CCR and other immune cells (B cells and NK cells) after administration of IL-7 fusion protein. NS: not significant, p>0.05, *p<0.05, **p<0.01, ***p<0.001 vs. baseline (week 0) group (by Wilcoxon matched-pairs signed-rank test). DETAILED DESCRIPTION OF THE INVENTION

[0062] definition Unless otherwise stated or implied from context, the following terms and phrases include the meanings provided below. Unless otherwise stated or apparent from context, the following terms and phrases do not exclude the meaning that the term or phrase has acquired in the art. Definitions are provided to aid in the description of particular embodiments and are not intended to limit the aspects provided herein, as the scope of the aspects provided herein is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the relevant art to which the aspects provided herein belong.

[0063] The term "pharmaceutical composition" is defined herein to refer to a mixture or solution containing at least one therapeutic agent that is administered to a mammal to prevent or treat a particular disease or condition affecting the mammal. In embodiments, the mammal may be a human.

[0064] The term "pharmaceutically acceptable" is defined herein to refer to compounds, substances, compositions and / or dosage forms that are suitable for use in contact with the tissues of human patients without undue toxicity, irritation, allergic response and other problems and complications commensurate with a reasonable benefit / risk ratio within the scope of sound medical judgment.

[0065] As used herein, the term "treat" or "treatment" includes treatment that relieves, reduces, reduces, alleviates, or delays the progression of at least one symptom in a human patient. For example, treatment can be the reduction of one or several symptoms of a disorder or the complete eradication of a disorder, such as cancer. Within the meaning of this disclosure, the term "treat" also refers to halting, delaying the onset (i.e., the period before clinical signs of disease), and / or reducing the risk of disease onset or worsening. The term "protect" is used herein to mean preventing, delaying, treating, or all of the above, as appropriate, the development or persistence or worsening of a disease in a subject.

[0066] As used herein, the terms "prevent," "prophylactic," or "prevention" include the prevention of at least one symptom associated with or caused by the condition, disease, or disorder being prevented.

[0067] The term "pharmacologically effective amount" or "clinically effective amount" of a therapeutic combination is an amount sufficient to provide an observable improvement over clinically observable baseline signs and symptoms of the disorder being treated with the combination.

[0068] As used herein, the phrase "a human patient in need of such treatment" refers to a human patient diagnosed with or suffering from a confirmed proliferative disorder.

[0069] The term "about" or "approximately" means within 10%, 9%, 8%, 7%, 6%, or 5% of a given value or range.

[0070] The term "reduced" or "reducing" is generally used herein to mean a statistically significant decrease. In some embodiments, "reduced" or "reducing" refers to a decrease of at least 10% compared to a reference level, e.g., at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or a decrease of up to 100% (e.g., an absent or undetectable level compared to a reference level), or any decrease between 10% and 100% compared to a reference level. In the context of a marker or symptom, such terms refer to a statistically significant decrease in such a level. The decrease may be, for example, at least 10%, at least 20%, at least 30%, at least 40%, or more, preferably to a level that is within the normal range for individuals without a given disease.

[0071] Denatured IL-7 Modified IL-7 that can be used in embodiments can have the following structure: A-IL-7 formula (I) Here, A is an oligopeptide consisting of 1 to 10 amino acid residues, and IL-7 is interleukin 7, a polypeptide capable of binding to the IL-7 receptor (also known as CD127), or a polypeptide having the activity of IL-7 or an activity similar thereto.

[0072] As used herein, the term "polypeptide having the activity of IL-7 or an activity similar thereto" refers to a polypeptide or protein having the same or similar sequence and activity as IL-7. Unless otherwise specified in the embodiments, the term can be used interchangeably with the first domain of an IL-7 fusion protein or a modified IL-7 fusion protein, which are used interchangeably herein.

[0073] IL-7 includes polypeptides consisting of the amino acid sequences represented by SEQ ID NOs: 1-6. Furthermore, IL-7 can have approximately 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with the sequences of SEQ ID NOs: 1-6. Sequence identity of peptide sequences can be determined using known sequence alignment or comparison software. For example, sequence identity can be determined using the BLASTP program (blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE=Proteins) using default settings.

[0074] IL-7 may include the IL-7 protein or a fragment thereof, wherein the fragment is capable of binding to the IL-7 receptor. As used herein, the term "IL-7 protein" can be used as a concept that includes "IL-7 protein and its fragment (capable of binding to the IL-7 receptor)." IL-7 may be obtained from human, rat, mouse, monkey, cow, or sheep.

[0075] Unless otherwise specified, the terms "protein," "polypeptide," and "peptide" can be used interchangeably.

[0076] Specifically, human IL-7 can have the amino acid sequence represented by SEQ ID NO: 1 (Genbank accession number P13232); rat IL-7 can have the amino acid sequence represented by SEQ ID NO: 2 (Genbank accession number P56478); mouse IL-7 can have the amino acid sequence represented by SEQ ID NO: 3 (Genbank accession number P10168); monkey IL-7 can have the amino acid sequence represented by SEQ ID NO: 4 (Genbank accession number NP_001279008); bovine IL-7 can have the amino acid sequence represented by SEQ ID NO: 5 (Genbank accession number P26895), and ovine IL-7 can have the amino acid sequence represented by SEQ ID NO: 6 (Genbank accession number Q28540).

[0077] The IL-7 protein or fragment thereof can also include variously modified proteins or peptides, i.e., mutants. The modifications can be made by substituting, deleting, or adding at least one protein to wild-type IL-7 without altering the function of IL-7. These variant proteins or peptides can have at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the wild-type protein.

[0078] Typically, the wild-type amino acid residue is substituted with alanine, although substitutions can be made with conservative amino acid substitutions that have no or weak effect on the overall protein charge, ie, polarity or hydrophobicity.

[0079] Conservative amino acid substitutions can be found in Table 1 below.

[0080] [Table 1]

[0081] For each amino acid, further conservative substitutions include "homologs" of the amino acid. In particular, a "homolog" refers to an amino acid having a methylene group (CH2) inserted into the side chain at the beta position of the amino acid side chain. Examples of "homologs" may include, but are not limited to, homophenylalanine, homoarginine, homoserine, etc.

[0082] In the structure of denatured IL-7, moiety A may be linked directly to the N-terminus of IL-7 or via a linker, and unless otherwise specified, the term can be used interchangeably with the second domain of an IL-7 fusion protein.

[0083] In one embodiment, A may be linked to the N-terminus of IL-7. A may contain 1 to 10 amino acids, and the amino acids may be selected from the group consisting of methionine, glycine, and combinations thereof. In one embodiment, when A is a single amino acid residue, it is glycine.

[0084] Methionine and glycine do not induce immune responses in the human body. Protein therapeutics produced from E. coli always contain methionine at the N-terminus, but no adverse reactions have been reported. Glycine is also widely used as a GS linker, and it has been reported that commercial products, like dulaglutide, do not induce immune responses (Cell Biophys. 1993 Jan-Jun;22(103):189-224).

[0085] In an exemplary embodiment, A may be an oligopeptide containing 1 to 10 amino acids selected from the group consisting of methionine (Met, M), glycine (Gly, G), and combinations thereof. In one embodiment, A may be an oligopeptide consisting of 1 to 5 amino acids. For example, A may be methionine (M), glycine (G), methionine-methionine, glycine-glycine, methionine-glycine, glycine-methionine, methionine-methionine-methionine, methionine-glycine-methionine, glycine-methionine-glycine, glycine-methionine-glycine, glycine-glycine-methionine, glycine-glycine-methionine, glycine-glycine-glycine, MMMM, or MG. It may have any one N-terminal sequence selected from the group consisting of MM, MGGM, MGGG, MGMG, GMMM, GMGG, GGGG, MMMMM, MMGMM, MMGGM, MMGMMG, MMMMG, GGGGG, GGMMM, GGGMG, MGMGMG, MMMGGG, MMGGMM, GGMMGG, MGMGMGMG, MMMMGGGG, MMGGMMGG, MMMMGGGG, MGMGMGMGMG, or / and MMMMMGGGGGG. In embodiments, A can be methionine, glycine, methionine-methionine, glycine-glycine, methionine-glycine, glycine-methionine, methionine-methionine-methionine, methionine-methionine-glycine, methionine-glycine-methionine, glycine-methionine-methionine, methionine-glycine-glycine, glycine-methionine-glycine, glycine-glycine-methionine, or glycine-glycine-glycine.

[0086] Fusion protein of denatured IL-7 or denatured IL-7 fusion protein Yet another embodiment provides an IL-7 fusion protein comprising: 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 that extends the half-life of the IL-7 fusion protein.

[0087] The third domain may be linked to the N-terminus or C-terminus of the first domain or the second domain, or a denatured IL-7 comprising the first domain and the second domain may be linked to both termini of the third domain.

[0088] The third domain may be a fusion partner for increasing in vivo half-life, and may preferably comprise any one selected from the group consisting of the Fc region of an immunoglobulin or a portion thereof, albumin, albumin-binding polypeptide, Pro / Ala / Ser (PAS), the C-terminal peptide of the β subunit of human chorionic gonadotropin (CTP), polyethylene glycol (PEG), a long unstructured hydrophilic sequence of amino acids (XTEN), hydroxyethyl starch (HES), an albumin-binding small molecule, and a combination thereof.

[0089] When the third domain is an Fc region of an immunoglobulin, it may be the Fc region of a modified immunoglobulin. In particular, the Fc region of the modified immunoglobulin may have attenuated antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) due to alteration of Fc receptor and / or complement binding affinity. The modified immunoglobulin may be selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and combinations thereof. Specifically, the Fc region of the modified immunoglobulin may comprise, from the N-terminus to the C-terminus, a hinge region, a CH2 domain, and a CH3 domain. In particular, the hinge region may comprise a human IgD hinge region; the CH2 domain may comprise some amino acid residues of human IgD and some amino acid residues of the CH2 domain of human IgG4; and the CH3 domain may comprise some amino acid residues of the CH3 domain of human IgG4.

[0090] Furthermore, two fusion proteins can form a dimer. For example, when the third domain is an Fc region, the Fc regions can bind to each other to form a dimer.

[0091] As used herein, the terms "Fc region," "Fc fragment," or "Fc" refer to a protein that comprises immunoglobulin heavy chain constant region 2 (CH2) and heavy chain constant region 3 (CH3), but does not comprise the variable regions of the heavy and light chains and the light chain constant region (CL1), and may further comprise the hinge region of the heavy chain constant region. In one embodiment, a hybrid Fc or hybrid Fc fragment thereof may be designated "hFc" or "hyFc."

[0092] The term "Fc region variant" as used herein refers to a variant produced by substituting some amino acids in the Fc region or by combining different types of Fc regions. The Fc region variant can prevent cutoff at the hinge region. Specifically, the amino acid at position 144 and / or the amino acid at position 145 of SEQ ID NO: 9 may be modified. Preferably, the variants include a variant in which the amino acid K at position 144 is substituted with G or S, and a variant in which the amino acid E at position 145 is substituted with G or S.

[0093] The Fc region or Fc region variant of the modified immunoglobulin may also be represented by the following formula (I): Formula (I) N'-(Z1)pY-Z2-Z3-Z4-C'

[0094] In the above formula (I), N' is the N-terminus of the polypeptide and C' is the C-terminus of the polypeptide; p is an integer of 0 or 1; Z1 is an amino acid sequence having 5 to 9 consecutive amino acid residues from the 98th amino acid residue among the 90th to 98th amino acid residues of SEQ ID NO: 7 toward the N-terminus; Y is an amino acid sequence having 5 to 64 consecutive amino acid residues from the 162nd amino acid residue among the 99th to 162nd amino acid residues of SEQ ID NO: 7 toward the N-terminus; Z2 is an amino acid sequence having 4 to 37 consecutive amino acid residues from the 163rd amino acid residue to the C-terminus among the 163rd to 199th amino acid residues of SEQ ID NO: 7; Z3 is an amino acid sequence having 71 to 106 consecutive amino acid residues from the 220th amino acid residue among the 115th to 220th amino acid residues of SEQ ID NO: 8 toward the N-terminus; Z4 is an amino acid sequence having 80 to 107 consecutive amino acid residues from the 221st amino acid residue among the 221st to 327th amino acid residues of SEQ ID NO:8 toward the C-terminus.

[0095] Furthermore, the Fc fragment may have native, increased, or decreased glycosylation compared to the native form, or may be deglycosylated. The glycosylation of immunoglobulin Fc can be modified by conventional methods, such as chemical, enzymatic, or microbial genetic engineering. Removal of glycosylation from the Fc fragment drastically reduces the binding affinity for the C1q portion of the first complement component C1, reducing or eliminating ADCC or CDC, thereby preventing the induction of unwanted immune responses in vivo. In this regard, a deglycosylated or nonglycosylated immunoglobulin Fc region may be more suitable as a drug carrier in some embodiments. As used herein, the term "deglycosylated" refers to an Fc region from which glycosylation has been enzymatically removed. The term "nonglycosylated" refers to an Fc fragment produced in a nonglycosylated form by a prokaryote, preferably E. coli.

[0096] Additionally, the Fc region of the modified immunoglobulin 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). Additionally, the Fc region of the modified immunoglobulin may comprise the amino acid sequence of SEQ ID NO: 14 (nonlytic mouse Fc).

[0097] The Fc region of the modified immunoglobulin may be that described in U.S. Pat. No. 7,867,491, and the production of the Fc region of the modified immunoglobulin may be carried out with reference to the disclosure of U.S. Pat. No. 7,867,491, the entire contents of which are incorporated herein by reference.

[0098] The second domain may be linked directly to the N-terminus of the first domain or via a linker, and the result may be in the form of second domain-first domain or second domain-linker-first domain.

[0099] The third domain may be linked directly to the first or second domain, or may be linked via a linker. Specifically, the result may be in the form of second domain-first domain-third domain, third domain-second domain-first domain, second domain-first domain-linker-third domain, third domain-linker-second domain-first domain, second domain-linker-first domain-linker-third domain, or third domain-linker-second domain-first domain.

[0100] When the linker is a peptide linker, linkage can occur at any linkage region. They can be linked using cross-linking agents known in the art. Examples of cross-linking agents 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(succinimidyl propionate), and bifunctional maleimides, such as bis-N-maleimide-1,8-octane.

[0101] Furthermore, the linker can be an albumin linker or a peptide linker, which can be a peptide of 10 to 20 amino acid residues consisting of Gly and Ser residues.

[0102] When the linker is formed by any one selected from the group consisting of chemical bonds, the chemical bond may be a disulfide bond, a diamine bond, a sulfide-amine bond, a carboxy-amine bond, an ester bond, or a covalent bond.

[0103] The modified IL-7 can have the structure of A-IL-7, which includes a polypeptide having the activity of IL-7 or an activity similar thereto, and an oligopeptide consisting of 1 to 10 amino acids.

[0104] In one embodiment, the modified IL-7 can have an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 to 20. Furthermore, the modified IL-7 can have a sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 15, 16, 17, 18, 19, or 20.

[0105] In another exemplary embodiment, the modified IL-7 or 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 the Fc region of a modified immunoglobulin linked to the C-terminus of the first domain.

[0106] The IL-7 fusion protein can have an amino acid sequence selected from the group consisting of SEQ ID NOs: 21 to 25. Furthermore, the IL-7 fusion protein can have a sequence having at least about 70%, at least about 75%, at least about 80%, 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 21, 22, 23, 24, or 25.

[0107] The IL-7 fusion proteins of SEQ ID NOs: 21-25 can be encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 29-39.

[0108] The nucleic acid molecule may further comprise a signal or leader sequence.

[0109] As used herein, the term "signal sequence" refers to a segment that directs the secretion of biologically active molecules, drugs, and fusion proteins, and which is cleaved after translation in the host cell. In one embodiment, the signal sequence is a polynucleotide encoding an amino acid sequence that initiates translocation of a protein across the endoplasmic reticulum (ER) membrane. In one embodiment, useful signal sequences include antibody light chain signal sequences, such as antibody 14.18 (Gillies et al., J. Immunol. Meth. 1989.125:191-202), antibody heavy chain signal sequences, such as MOPC141 and antibody heavy chain signal sequences (Sakano et al., Nature. 1980.286:676-683), and other signal sequences known in the art (see, e.g., Watson et al., Nucleic Acid Research. 1984.12:5145-5164).

[0110] The characteristics of signal peptides are well known in the art, and signal peptides typically contain 16-30 amino acids, but may contain more or fewer amino acid residues. Existing signal peptides consist of three regions: a basic N-terminal region, a central hydrophobic region, and a more polar C-terminal region.

[0111] The central hydrophobic region contains 4 to 12 hydrophobic residues that anchor the signal sequence through the membrane lipid bilayer during translocation of the immature polypeptide. After initiation, the signal sequence is frequently cleaved within the lumen of the ER by cellular enzymes known as signal peptidases. In particular, the signal sequence may be a secretory signal sequence for tissue plasminogen activator (tPa), a signal sequence for herpes simplex virus glycoprotein D (HSV gDs), or growth hormone. Preferably, a secretory signal sequence used in higher eukaryotic cells, including mammals, can be used. The secretory signal sequence may be the signal sequence contained in wild-type IL-7 or may be substituted with a codon frequently expressed in the host cell.

[0112] An isolated nucleic acid molecule encoding a modified IL-7 or IL-7 fusion protein may be contained in an expression vector.

[0113] The term "vector" as used herein refers to a nucleic acid vector containing a nucleotide sequence that can be introduced into a host cell and recombined to be inserted into the host cell's genome or replicate autonomously as an episome. Vectors may include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors, and analogs thereof. Examples of viral vectors include, but are not limited to, retroviruses, adenoviruses, and adeno-associated viruses.

[0114] As used herein, the term "gene expression" or "expression" of a target protein is understood to refer to the transcription of a DNA sequence, translation of an mRNA transcript, and secretion of the fusion protein product or fragment thereof.

[0115] As used herein, the term "host cell" refers to prokaryotic and eukaryotic cells into which a recombinant expression vector can be introduced. As used herein, the terms "transduced," "transformed," and "transfected" refer to the introduction of a nucleic acid (e.g., a vector) into a cell using techniques known in the art.

[0116] As used herein, the term "gene expression" or "expression" of a target protein is understood to refer to the transcription of a DNA sequence, translation of an mRNA transcript, and secretion of an Fc fusion protein product or antibody or antibody fragment thereof.

[0117] A useful expression vector may be RcCMV (Invitrogen, Carlsbad) or a variant thereof. The expression vector may also contain the polyadenylation signal sequence of human cytomegalovirus (CMV) to promote sustained transcription of the target gene in mammalian cells and bovine growth hormone to increase the stability of the post-transcriptional RNA. In an exemplary embodiment, the expression vector is pAD15, a modified form of RcCMV.

[0118] The expression vector may be contained in a suitable host cell adapted for expression and / or secretion of the target protein upon transduction or transfection of the DNA sequence of the embodiment.

[0119] Examples of suitable host cells include, but are not limited to, immortal hybridoma cells, NS / 0 myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, human amniotic fluid-derived cells (CapT cells), or COS cells.

[0120] Modified IL-7 proteins and fusion proteins thereof can be produced by the methods described in co-pending application (U.S. Application No. 15 / 126,313), the entire contents of which are incorporated herein by reference.

[0121] composition U.S. Application No. 15 / 773,273, the entire contents of which are incorporated herein by reference, discloses a dosage form containing a denatured IL-7 fusion protein. In some embodiments, the pharmaceutical dosage form may comprise (a) a denatured IL-7 fusion protein; (b) a basic buffer solution at a concentration of 10-50 mM; (c) a sugar solution at a concentration of 2.5-5 w / v%; and (d) a surfactant solution at a concentration of 0.05-6 w / v%. The dosage form may further comprise an amino acid, a sugar alcohol (e.g., sorbitol, xylitol, maltitol, mannitol, or a mixture thereof). The dosage form may have a pH of about 5.

[0122] Pharmaceutical compositions containing denatured IL-7 or IL-7 fusion proteins can be administered to a subject by direct administration (e.g., local administration via injection, implantation, or topical administration into a tissue region) or by appropriate means systemically (e.g., parenterally or orally). When the composition is administered parenterally, intravenously, subcutaneously, intraocularly, intraperitoneally, intramuscularly, orally, intrarectally, intraorbitally, intracerebrally, intracranially, intraspinally, intrathecally, intracapsularly, intraarticularly, intranasally, or by aerosol administration, the composition preferably contains an aqueous or physiologically compatible suspension of body fluids or a portion of such a solution. Thus, a physiologically acceptable carrier or transporter can be added to the composition and delivered to the patient without negatively affecting the patient's electrolyte and / or volume balance. Thus, the physiologically acceptable carrier or transporter can be physiological saline.

[0123] Additionally, DNA constructs (or genomic constructs) containing nucleic acids containing modified IL-7 or IL-7 fusion proteins can be used as part of a gene therapy protocol.

[0124] To reconstitute or complement the function of a desired protein, an expression vector capable of expressing the fusion protein in a specific cell can be administered in conjunction with a biologically effective carrier, which can be any formulation or composition capable of efficiently delivering the gene encoding the desired protein or IL-7 fusion protein into cells in vivo.

[0125] For gene therapy using nucleic acids encoding modified IL-7 or IL-7 fusion proteins, the gene of interest may be inserted into a viral vector, a recombinant bacterial plasmid, or a recombinant eukaryotic plasmid. The viral vector may include recombinant retrovirus, adenovirus, adeno-associated virus, herpes simplex virus-1, or other vectors, including transplanted T cells or proliferative in vitro isolated T cell aggregates.

[0126] Uses and treatment regimens of modified IL-7 or its fusion proteins 1. Use of modified IL-7 or a fusion protein thereof in the manufacture of a medicament for use in treating a proliferative disorder, said medicament comprising a therapeutically effective dose in the range of greater than about 600 μg / kg to about 2,000 μg / kg administered two or more times at intervals of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, or 15 weeks to a subject in need of treatment.

[0127] In embodiments, there is provided use of modified IL-7 or a fusion protein thereof in the manufacture of a medicament for use in treating a proliferative disorder, said medicament comprising a therapeutically effective dose in the range of greater than about 600 μg / kg to about 2,000 μg / kg administered two or more times at intervals of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, or 15 weeks to a subject in need of treatment.

[0128] 1. Use of modified IL-7 or a fusion protein thereof for use in the treatment of a proliferative disorder, comprising administering to a subject in need thereof two or more doses of a compound of Formula (I) or a pharmaceutically acceptable salt thereof at a therapeutically effective dose of about 720 μg / kg or more, about 960 μg / kg or more, about 1,200 μg / kg or more, about 1,700 μg / kg or more, or about 2,000 μg / kg of modified IL-7 or a fusion protein thereof at intervals of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, or 15 weeks, or at intervals of 10 days, 20 days, 30 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, or 100 days.

[0129] 1. Use of modified IL-7 or a fusion protein thereof for use in the manufacture of a medicament for use in the treatment of a proliferative disorder, comprising administering to a subject in need thereof two or more doses of a compound of Formula (I) or a pharmaceutically acceptable salt thereof at a therapeutically effective dose of about 720 μg / kg or more, about 960 μg / kg or more, about 1,200 μg / kg or more, about 1,700 μg / kg or more, or about 2,000 μg / kg of modified IL-7 or a fusion protein thereof at intervals of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, or 15 weeks, or at intervals of 10 days, 20 days, 30 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, or 100 days.

[0130] 1. Use of modified IL-7 or a fusion protein thereof for use in the treatment of a proliferative disorder, comprising administering to a subject in need thereof two or more doses of a compound of Formula (I) or a pharmaceutically acceptable salt thereof at a therapeutically effective dose of about 720 μg / kg or more, about 840 μg / kg or more, about 960 μg / kg or more, about 1,200 μg / kg or more, about 1,440 μg / kg or more, about 1,700 μg / kg or more, or about 2,000 μg / kg of modified IL-7 or a fusion protein thereof at intervals of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, or 15 weeks, or at intervals of 10 days, 20 days, 30 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, or 100 days.

[0131] 1. Use of modified IL-7 or a fusion protein thereof for use in the manufacture of a medicament for use in the treatment of a proliferative disorder, comprising administering to a subject in need thereof two or more doses of a compound of Formula (I) or a pharmaceutically acceptable salt thereof at a therapeutically effective dose of about 720 μg / kg or more, about 840 μg / kg or more, about 960 μg / kg or more, about 1,200 μg / kg or more, about 1,440 μg / kg or more, about 1,700 μg / kg or more, or about 2,000 μg / kg of modified IL-7 or a fusion protein thereof at intervals of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, or 15 weeks, or at intervals of 10 days, 20 days, 30 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, or 100 days.

[0132] 1. Use of modified IL-7 or a fusion protein thereof in the manufacture of a medicament for use in increasing lymphocyte count or lymphocyte production in a subject in need thereof, said medicament comprising a therapeutically effective dose in the range of greater than about 600 μg / kg to about 2,000 μg / kg administered two or more times to the subject at intervals of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, or 15 weeks.

[0133] In embodiments, there is provided use of modified IL-7 or a fusion protein thereof in the manufacture of a medicament for use in increasing lymphocyte count or lymphocyte production in a subject in need thereof, said medicament comprising a therapeutically effective dose in the range of greater than about 600 μg / kg to about 2,000 μg / kg administered two or more times to a subject at intervals of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, or 15 weeks.

[0134] 10. Use of modified IL-7 or a fusion protein thereof for use in increasing lymphocyte number or lymphocyte production in a subject in need thereof, comprising administering to the subject two or more doses of a compound of Formula (I) or a pharmaceutically acceptable salt thereof at a therapeutically effective dose of about 720 μg / kg or more, about 960 μg / kg or more, about 1,200 μg / kg or more, about 1,700 μg / kg or more, or about 2,000 μg / kg of modified IL-7 or a fusion protein thereof at intervals of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, or 15 weeks, or at intervals of 10 days, 20 days, 30 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, or 100 days.

[0135] 1. Use of modified IL-7 or a fusion protein thereof for use in the manufacture of a medicament for use in increasing lymphocyte count or lymphocyte production in a subject in need thereof, comprising administering to the subject two or more doses of a compound of Formula (I) or a pharmaceutically acceptable salt thereof at a therapeutically effective dose of about 720 μg / kg or more, about 960 μg / kg or more, 1,200 μg / kg or more, about 1,700 μg / kg or more, or about 2,000 μg / kg of modified IL-7 or a fusion protein thereof at intervals of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, or 15 weeks, or at intervals of 10 days, 20 days, 30 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, or 100 days.

[0136] 10. Use of modified IL-7 or a fusion protein thereof for use in increasing lymphocyte number or lymphocyte production in a subject in need thereof, comprising administering to the subject two or more doses of a compound of Formula (I) or a pharmaceutically acceptable salt thereof at a therapeutically effective dose of about 720 μg / kg or more, about 840 μg / kg or more, about 960 μg / kg or more, about 1,200 μg / kg or more, about 1,440 μg / kg or more, about 1,700 μg / kg or more, or about 2,000 μg / kg of modified IL-7 or a fusion protein thereof at intervals of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, or 15 weeks, or at intervals of 10 days, 20 days, 30 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, or 100 days.

[0137] At intervals of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, or 15 weeks, or at intervals of 10 days, 20 days, 30 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, or 100 days, the subject is administered a dose of denatured IL-7 or a fusion protein thereof at a dose of about 720 μg / kg or more, about 840 μg / kg or more, about 960 μg / kg or more, 1. Use of modified IL-7 or a fusion protein thereof for use in the manufacture of a medicament for use in increasing lymphocyte count or lymphocyte production in a subject in need thereof, comprising administering two or more doses of a compound of formula (I) or a pharmaceutically acceptable salt thereof at a therapeutically effective dose of at least about 1,200 μg / kg, at least about 1,440 μg / kg, at least about 1,700 μg / kg, or at least about 2,000 μg / kg.

[0138] In embodiments, the proliferative disease for said use is a cancer or tumor, which may be, but is not limited to, a solid tumor, a lymphatic cancer, or a leukemia.

[0139] The solid tumor can be synovial sarcoma, invasive ductal carcinoma, rectal cancer, colon cancer, ovarian cancer, ascending colon cancer, anal cancer, invasive ductal carcinoma of the breast, adenocarcinoma, rectal cancer with periaortic metastases, neuroendocrine carcinoma (cervix), sigmoid colon cancer, or glioblastoma.

[0140] In embodiments, the subject may have previously undergone or may be concurrently undergoing one or more of the following cancer treatments, including surgery, radiation, and chemotherapy.

[0141] (i) Modified interleukin-7 or (ii) interleukin-7 fusion protein, or pharmaceutical compositions containing same, can be administered parenterally, intramuscularly, subcutaneously, intraocularly, intravenously, intranasally, intradermally, intraorbitally, intracerebrally, intracranially, intraspinally, intraventricularly, intrathecally, intracapsularly, intraarticularly, or intratumorally.

[0142] In some embodiments, the subject can have a lymphocyte count of about 1000 lymphocytes per μL of blood or less as determined by Common Terminology Criteria for Adverse Events (CTCAE) version 4.0. The lymphocytes can be T cells. The T cells can be CD4 + and / or CD8 + The lymphocyte count may include T cells. Whole blood or serum may be used to measure lymphocytes in a blood sample. Thus, the term "blood" when used in connection with lymphocyte count includes whole blood and / or serum.

[0143] In still other embodiments, the subject has a lymphocyte count of 800 lymphocyte cells / μl of blood or less, a lymphocyte count of about 500 lymphocyte cells / μl of blood or less, or a lymphocyte count of about 200 lymphocyte cells / μl of blood or less.

[0144] In one embodiment, the number of intratumoral tumor infiltrating lymphocytes (TILs) is increased after administration compared to the number of intratumoral TILs after administration of (i) denatured interleukin-7 or (ii) an interleukin-7 fusion protein. TILs are CD4 + TILs and / or CD8+ It can be a TIL.

[0145] The number of TILs can increase by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 125%, at least about 150%, at least about 200%, at least about 250%, or at least about 300% after administration.

[0146] In one embodiment, (i) denatured interleukin-7 or (ii) interleukin-7 fusion protein can be administered two or more times in an amount of about 720 μg / kg at intervals of about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, or about 8 weeks.

[0147] In one embodiment, (i) denatured interleukin-7 or (ii) interleukin-7 fusion protein can be administered two or more times in an amount of about 840 μg / kg at intervals of about 2 weeks, about 3 weeks, about 4 weeks, or about 5 weeks, about 6 weeks, about 7 weeks, or about 8 weeks.

[0148] In other embodiments, (i) the denatured interleukin-7 or (ii) the interleukin-7 fusion protein can be administered two or more times in an amount of about 960 μg / kg at intervals of about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, or about 10 weeks.

[0149] In yet other embodiments, (i) the denatured interleukin-7 or (ii) the interleukin-7 fusion protein can be administered two or more times in an amount of about 1200 μg / kg at intervals of 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.

[0150] In yet other embodiments, (i) the denatured interleukin-7 or (ii) the interleukin-7 fusion protein can be administered two or more times in an amount of about 1,440 μg / kg at intervals of 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, about 12 weeks, about 13 weeks, about 14 weeks, about 15 weeks, about 1 month, about 2 months, or about 3 months.

[0151] According to one embodiment, (i) denatured interleukin-7 or (ii) interleukin-7 fusion protein is administered at a dose of greater than about 600 μg / kg, greater than about 700 μg / kg, or 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, once per 12 weeks, once per 13 weeks, once per 14 weeks, or once per 15 weeks. It can be administered at a dose of 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.

[0152] According to some embodiments, (i) the denatured interleukin-7 or (ii) the interleukin-7 fusion protein is administered at a dose of about 610 μg / week 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, once per 12 weeks, once per 13 weeks, once per 14 weeks, or once per 15 weeks. kg ~ approx. 1,200μg / kg, approx. 650μg / kg ~ approx. 1,200μg / kg, approx. 700μg / kg ~ approx. 1,200μg / kg, approx. 750μg / kg ~ approx. 1,200μg / kg, approx. 800μg / kg kg ~ approx. 1,200μg / kg, approx. 850μg / kg ~ approx. 1,200μg / kg, approx. 900μg / kg ~ approx. 1,200μg / kg, approx. 950μg / kg ~ approx. 1,200μg / kg, approx. 1,000μg / kg ~ approx. 1,200 μg / kg, approx. 1,050 μg / kg ~ approx. 1,200 μg / kg, approx. 1,100 μg / kg ~ approx. 1,200 μg / kg, approx. 1,200 μg / kg ~ approx. 2,000 μg / kg, Approximately 1,300μg / kg to approximately 2,000μg / kg, approximately 1,500μg / kg to approximately 2,000μg / kg, approximately 1,700μg / kg to approximately 2,000μg / kg, approximately 610μg / kg to approximately 1,000 It can be administered at a dose of 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.

[0153] According to yet another embodiment, (i) denatured interleukin-7 or (ii) interleukin-7 fusion protein is administered at a frequency of about 7 weeks, once every week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks, once every 10 weeks, once every 11 weeks, once every 12 weeks, once every 13 weeks, once every 14 weeks, or once every 15 weeks. The dose can be administered at a range 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.

[0154] According to yet other embodiments, (i) denatured interleukin-7 or (ii) interleukin-7 fusion protein is administered at a dose 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, or about once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, once every eleven weeks, once every 12 weeks, once every thirteen weeks, once every fourteen weeks, or once every fifteen weeks. 780μg / kg, about 800μg / kg, about 820μg / kg, about 840μg / kg, about 850μg / kg, about 860μg / kg, about 880μg / kg, about 900μg / kg, about 920μg / kg, about 940μg / kg, about 950μg / kg, about 960μg / kg, about 98 0μg / kg, approximately 1,000μg / kg, approximately 1,020μg / kg, approximately 1,040μg / kg, approximately 1,060μg / kg, approximately 1,080μg / kg, approximately 1,100μg / kg, approximately 1,120μg / kg, approximately 1,140μg / kg, approximately 1,160μg / kg, approximately 1,180μg / kg, approximately 1,200μg / kg, approximately 1,220μg / kg, approximately 1,240μg / kg, approximately 1,260μg / kg, approximately 1,280μg / kg, approximately 1,300μg / kg, approximately 1,320μg / kg, approximately 1,340μg / kg, approximately 1,360μg / kg, approximately 1,380μg / k g, approx. 1,400 μg / kg, approx. 1,420 μg / kg, approx. 1,440 μg / kg, approx. 1,460 μg / kg, approx. 1,480 μg / kg, approx. 1,500 μg / kg, approx. 1,520 μg / kg, approx. 1,600μg / kg, about 1,620μg / kg, about 1,640μg / kg, about 1,660μg / kg, about 1,680μg / kg, about 1,700μg / kg, about 1,720μg / kg, about 1,740μg / kg, about 1,760μg / kg, about 1,780μg / kg, about 1,800μg / kg, about 1,820μg / kg, about 1,840μg / kg, about 1,860μg / kg, about 1,880μg / kg, about 1,900μg / kg, about 1,920μg / kg, about 1,940μg / kg, about 1,960μg / kg, about 1,980μg / kg, or about 2,It can be administered at a dose of 000 μg / kg.

[0155] In one embodiment, (i) the modified interleukin-7 or (ii) the interleukin-7 fusion protein is administered at a dose of about 650 μg / kg to 680 μg / kg, about 680 μg / kg to 700 μg / kg, at a dosing frequency of once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, once every 11 weeks, once every 12 weeks, once every 13 weeks, or once every 10 days, once every 20 days, once every 30 days, once every 40 days, once every 50 days, once every 60 days, once every 90 days, or once every 100 days. g, about 700μg / kg to 720μg / kg, about 720μg / kg to 740μg / kg, about 740μg / kg to 750μg / kg, about 750μg / kg to 760μg / kg, about 760μg / kg to 780μg / kg, about 780μg / kg to 800μg / kg, about 800μg / kg ~820μg / kg, approximately 820μg / kg~840μg / kg, approximately 840μg / kg~850μg / kg, approximately 850μg / kg~860μg / kg, approximately 860μg / kg~880μg / kg, approximately 880μg / kg~900μg / kg, approximately 900μg / kg~920μg / kg, approx. 920μg / kg~940μg / kg, approx. 940μg / kg~950μg / kg, approx. 950μg / kg~960μg / kg, approx. 960μg / kg~980μg / kg, approx. 980μg / kg~1000μg / kg, approx. 1,000μg / kg~1,020μg / kg, approx. 1,020μ g / kg~1,040μg / kg, approx. 1,040μg / kg~1,060μg / kg, approx. 1,060μg / kg~1,080μg / kg, approx. 1,080μg / kg~1,100μg / kg, approx. 1,100μg / kg~1,120μg / kg, approx. 1,120μg / kg~1,140 μg / kg, approx. 1,140 μg / kg ~ 1,160 μg / kg, approx. 1,160 μg / kg ~ 1,180 μg / kg, approx. 40μg / kg~1,260μg / kg, approx. 1,260μg / kg~1,280μg / kg, approx. 1,280μg / kg~1,300μg / kg, approx. 1,300μg / kg~1,320μg / kg, approx. 1,320μg / kg~1,340μg / kg, approx. 1,340μg / kg~1,360μg / kg, approximately 1,360μg / kg~1,380μg / kg, approximately 1,380μg / kg~1,400μg / kg, approximately 1,400μg / kg~1,420μg / kg, approximately 1,420μg / kg~1,4 40μg / kg, approximately 1,440μg / kg~1,460μg / kg, approximately 1,480μg / kg, approximately 1,480μg / kg~1,500μg / kg, approximately 1,500μg / kg~1,520μg / kg, approximately 1,52 0μg / kg~1,540μg / kg, approx. 1,540μg / kg~1,560μg / kg, approx. 1,560μg / kg~1,580μg / kg, approx. 1,580μg / kg~1,600μg / kg, approx. 1,600μ g / kg~1,620μg / kg, approx. 1,620μg / kg~1,640μg / kg, approx. 1,640μg / kg~1,660μg / kg, approx. 1,660μg / kg~1,680μg / kg, approx. 1,680μg / kg kg~1,700μg / kg, approx. 1,700μg / kg~1,720μg / kg, approx. 1,720μg / kg~1,740μg / kg, approx. 1,740μg / kg~1,760μg / kg, approx. 1,760μg / kg ~1,780μg / kg, approximately 1,780μg / kg~1,800μg / kg, approximately 1,800μg / kg~1,820μg / kg, approximately 1,820μg / kg~1,840μg / kg, approximately 1,840μg / kg~1 It can be administered at a dose of about 1,860 μg / kg, about 1,860 μg / kg to 1,880 μg / kg, about 1,880 μg / kg to 1,900 μg / kg, about 1,900 μg / kg to 1,920 μg / kg, about 1,920 μg / kg to 1,940 μg / kg, about 1,940 μg / kg to 1,960 μg / kg, about 1,960 μg / kg to 1,980 μg / kg, or about 1,980 μg / kg to 1,200 μg / kg.

[0156] In one embodiment, the subject may have received, is receiving, or will receive chemotherapy. The chemotherapeutic agent and the modified IL-7 or fusion protein thereof may be administered simultaneously or sequentially.

[0157] Reference Example 1. Preparation of modified IL-7 protein in which an oligopeptide is bound to IL-7 Modified IL-7 was prepared by conjugating an oligopeptide to the N-terminus of IL-7. The sequence of human IL-7 (SEQ ID NO: 1) was used for IL-7, and the oligopeptides used were methionine (M), glycine (G), MM, GG, MG, GM, MMM, MMG, MGM, GMM, MGG, GMG, GGM, GGG, DDD, or MMMM.

[0158] As shown in Figure 1a, various forms of modified IL-7 were prepared, each having the structure 'A'-IL-7. In this example, methionine (M), glycine (G), MM, GG, MG, GM, MMM, MMG, MGM, GMM, MGG, GMG, GGM, GGG, DDD, or MMMM sequences were used as the secondary domain (oligopeptide, 'A'). Also, MGMM, MGGM, MGGG, MGMG, GMMM, GMGG, GGGG, MMMMM, MMGMM, MMGGM, MMGMMG, MMMMG, GGGGG, GGMMM, GGGGM, MGMGMG, MMMGGG, MMGGMM, GGMMGG, MGMGMGMG, MMMMGGGG, MMGGMMGG, MMMMGGGG, MGMGMGMGMG, and MMMMMGGGGG were generated in 'A'.

[0159] The nucleic acid sequence of SEQ ID NO: 28 was used for the first domain of IL-7 fused to the oligopeptide. The entire nucleic acid sequence of IL-7 fused to the oligopeptide was obtained and inserted into an expression vector. As a negative control, IL-7 protein without oligopeptide modification was produced in the same manner.

[0160] HEK293 cells were transfected with an expression vector containing the A-IL-7 gene. Polyplexes were prepared using 208.3 μg of DNA and 416.6 μg (μL) of polyethyleneimine (PEI) (w / w) based on 300 mL of suspension culture, and then transfected into HEK293F cells. Six days after transfection, the cell cultures were harvested and Western blots were performed to evaluate the expression rate of the target protein. The cultures were then centrifuged at 8,000 rpm for 30 minutes to remove culture debris and filtered using a bottle-top filter with a pore size of 0.22 μm. As a result, liquid cultures containing denatured IL-7, including M-IL-7, G-IL-7, MM-IL-7, GG-IL-7, MG-IL-7, GM-IL-7, MMM-IL-7, MMG-IL-7, MGM-IL-7, GMM-IL-7, MGG-IL-7, GMG-IL-7, GGM-IL-7, GGG-IL-7, DDD-IL-7, and MMMM-IL-7, were obtained. Also, MGMM-IL-7, MGGM-IL-7, MGGG-IL-7, MGMG-IL-7, GMMM-IL-7, GMGG-IL-7, GGGG-IL-7, MMMMM-IL-7, MMGMM-IL-7, MMGGM-IL-7, MGMMG-IL-7, MMMMG-IL-7, GGGGG-IL-7, GGMMM-IL-7, GGG MG-IL-7, MGMGMG-IL-7, MMMGGG-IL-7, MMGGMM-IL-7, GGMMGG-IL-7, MGMGMGMG-IL-7, MMMMGGGG-IL-7, MMGGMMGG-IL-7, MMMMGGGG-IL-7, MGMGMGMGMG-IL-7, and MMMMMGGGGG-IL-7 are produced.

[0161] Reference Example 2: Preparation of IL-7 fusion protein in which an Fc region is linked to the C-terminus of IL-7 We prepared IL-7 fusion proteins (i.e., domain 2-domain 1-domain 3) in which a polypeptide consisting of a heterologous amino acid sequence was further linked to the C-terminus of denatured IL-7. The human IL-7 sequence (SEQ ID NO: 1) was used for the first domain, and the sequence M, G, MM, GG, MG, GM, MMM, MMG, MGM, GMM, MGG, GMG, GGM, GGG, DDD, or MMMM was used for the second domain. The sequence of the Fc region (SEQ ID NO: 9 or 14) was used for the third domain. In other embodiments, an IL-7-Fc fusion protein is produced in which MGMM, MGGM, MGGG, MGMG, GMMM, GMGG, GGGG, MMMMM, MMGMM, MMGGM, MMGMMG, MMMMG, GGGGG, GGMMM, GGGMG, MGMGMG, MMMGGG, MMGGMM, GGMMGG, MGMGMGMG, MMMMGGGG, MMGGMMGG, MMMMGGGG, MGMGMGMGMG, or MMMMMGGGGG is linked to the N-terminus of IL-7.

[0162] Various forms of IL-7 fusion proteins were prepared, each consisting of a second domain, a first domain, and a third domain. In this example, the second domain used was methionine (M), glycine (G), MM, GG, MG, GM, MMM, MMG, MGM, GMM, MGG, GMG, GGM, GGG, DDD, or MMMM; the first domain used was human IL-7; and the third domain used was hybrid Fc (hFc, hyFc) or mouse non-degradable Fc. In further experiments, MGMM, MGGM, MGGG, MGMG, GMMM, GMGG, GGGG, MMMMM, MMGMM, MMGGM, MMGMMG, MMMMG, GGGGG, GGMMM, GGGMG, MGMGMG, MMMGGG, MMGGMM, GGMMGG, MGMGMGMG, MMMMGGGG, MMGGMMGG, MMMMGGGG, MGMGMGMGMG, or MMMMMGGGGG is used as the second domain to generate a modified IL-7 fusion protein.

[0163] In particular, for the hybrid Fc, hFc (hybrid Fc) as disclosed in US Pat. No. 7,867,491 is used, the entire contents of which are incorporated herein by reference. hFc is capable of binding to physiologically active proteins and exhibits superior in vivo half-life compared to the Fc regions of existing denatured immunoglobulins.

[0164] The gene expression vectors were prepared and transfected in the same manner as in Reference Example 1, and the cells were cultured to produce culture media containing various forms of IL-7 fusion proteins. As a result, culture media containing G-IL-7-hyFc, M-IL-7-hyFc, MM-IL-7-hyFc, GG-IL-7-hyFc, MG-IL-7-hyFc, GM-IL-7-hyFc, MMM-IL-7-hyFc, MMG-IL-7-hyFc, MGM-IL-7-hyFc, GMM-IL-7-hyFc, MGG-IL-7-hyFc, GMG-IL-7-hyFc, GGM-IL-7-hyFc, GGG-IL-7-hyFc, DDD-IL-7-hyFc, or MMMM-IL-7-hyFc proteins were obtained. MGMM-IL-7-hyFc, MGGM-IL-7-hyFc, MGGG-IL-7-hyFc, MGMG-IL-7-hyFc, GMMM-IL-7-hyFc, GMGG-IL-7-hyFc, GGGG-IL-7-hyFc, MM MMM-IL-7-hyFc, MMGMM-IL-7-hyFc, MMGGM-IL-7-hyFc, MGMMG-IL-7-hyFc, MMMMG-IL-7-hyFc, GGGGG-IL-7-hyFc, GGMMM-IL-7-hy Fc, GGGMG-IL-7-hyFc, MGMGMG-IL-7-hyFc, MMMGGG-IL-7-hyFc, MMGGMM-IL-7-hyFc, GGMMGG-IL-7-hyFc, MGMGMGMG-IL-7-hyFc, M MMMGGGG-IL-7-hyFc, MMGGMMGG-IL-7-hyFc, MMMMGGGG-IL-7-hyFc, MGMGMGMGG-IL-7-hyFc, or MMMMMGGGGG-IL-7-hyFc is produced.

[0165] In one embodiment, a denatured-IL-7-Fc fusion protein codenamed GX-I7 (SEQ ID NO: 24) can be used. GX-I7 comprises denatured IL-7 of SEQ ID NO: 18 and hyFc fused to the C-terminus of denatured IL-7.

[0166] In one embodiment, the IL-7 fusion protein can be administered at a dose of about 60 μg / kg or more. The dose can range from about 60 μg / kg to about 2,000 μg / kg. The dose can be about 60 μg / kg or more, about 120 μg / kg or more, about 240 μg / kg or more, about 480 μg / kg or more, about 720 μg / kg or more, about 960 μg / kg or more, about μg / kg or more, 1,200 μg / kg or more, about 1,700 μg / kg or more, or about 2,000 μg / kg. In other embodiments, the dose can be about 60 μg / kg or more, about 360 μg / kg or more, about 600 μg / kg or more, about 840 μg / kg or more, or about 1440 μg / kg or more.

[0167] In embodiments, the dose may be about 60 μg / kg or more, about 120 μg / kg or more, about 240 μg / kg or more, about 360 μg / kg or more, about 480 μg / kg or more, about 600 μg / kg or more, about 720 μg / kg or more, about 840 μg / kg or more, about 960 μg / kg or more, about 1,200 μg / kg or more, about 1,440 μg / kg or more, about 1,700 μg / kg or more, or about 2,000 μg / kg.

[0168] In some embodiments, the modified interleukin-7 or interleukin-7 fusion protein is administered two or more times in an amount of about 720 μg / kg, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, or about 8 weeks apart. In some embodiments, the modified interleukin-7 or interleukin-7 fusion protein is administered two or more times in an amount of about 840 μg / kg, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, or about 8 weeks apart. In other embodiments, the modified interleukin-7 or interleukin-7 fusion protein is administered two or more times in an amount of about 960 μg / kg, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, or 9 weeks, or about 10 weeks apart. In some embodiments, the modified interleukin-7 or interleukin-7 fusion protein is administered two or more times in an amount of about 1200 μg / kg, 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 apart. In some other embodiments, the modified interleukin-7 or interleukin-7 fusion protein is administered two or more times in an amount of about 1440 μg / kg, 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, about 12 weeks, about 13 weeks, about 14 weeks, or about 15 weeks apart.

[0169] I. Preclinical research A. Pharmacokinetic study of IL-7 fusion protein after a single dose in rats To observe the pharmacokinetic properties of the IL-7 fusion protein, a pharmacokinetic study was conducted using normal rats. The pharmacokinetic study was conducted by intravenous, subcutaneous, and intramuscular administration. GX-17 (SEQ ID NO: 24) was used as the IL-7 fusion protein.

[0170] The results of a study evaluating the in vivo kinetics and bioavailability of IL-7 fusion protein when administered intravenously, subcutaneously, or intramuscularly once to normal rats.

[0171] When administered intravenously, AUC lastand C max The T values ​​were 349.7 (0.1 mg / kg), 1,440.6 (0.3 mg / kg), and 4,225.9 (1.0 mg / kg) h·ng / mL and 34.3 (0.1 mg / kg), 86.7 (0.3 mg / kg), and 324.5 (1.0 mg / kg) ng / mL, respectively, and were confirmed to increase in a dose-dependent manner. max is 0.083 hours, and the terminal T 1 / 2 were 22.6, 22.6, and 22.0 hours in the 0.1, 0.3, and 1.0 mg / kg dose groups, respectively, and the mean terminal T 1 / 2 The bioavailability was calculated based on the time points at which blood concentrations in the intravenous administration group could be measured: 72 hours (0.1 mg / kg), 120 hours (0.3 mg / kg), and 168 hours (1.0 mg / kg).

[0172] Subcutaneous administration, AUC last and C max The IL-10 values ​​were 363.8 (0.1 mg / kg), 1,675.9 (0.3 mg / kg), and 9,765.4 (1.0 mg / kg) h·ng / mL and 4.1 (0.1 mg / kg), 16.4 (0.3 mg / kg), and 105.9 (1.0 mg / kg) ng / mL, respectively. max The terminal T 1 / 2 The bioavailability was 104 (0.1 mg / kg), 116 (0.3 mg / kg), and 231 (1.0 mg / kg) based on the blood concentration in the intravenous administration group.

[0173] When administered intramuscularly, AUC last and C maxThe values ​​were 732.3 (0.1 mg / kg), 2,898.3 (0.3 mg / kg), and 11,027.8 (1.0 mg / kg) h·ng / mL, and 12.5 (0.1 mg / kg), 51.2 (0.3 mg / kg), and 147.9 (1.0 mg / kg) ng / mL, respectively. These values ​​were confirmed to increase in a dose-dependent manner. max The terminal T 1 / 2 The mean maximal daily intake (MTA) was 48.3, 39.0, and 25.6 hours for the 0.1, 0.3, and 1.0 mg / kg groups, respectively. The bioavailability was 209 (0.1 mg / kg), 201 (0.3 mg / kg), and 261 (1.0 mg / kg) based on the blood concentration in the intravenous administration group.

[0174] Interestingly, in this study, the modified IL-7 fusion protein administered intramuscularly was more bioavailable than intravenously, whereas many existing drug compounds showed lower bioavailability when administered subcutaneously or intramuscularly than when administered intravenously (see Figure 1a-c).

[0175] B. Pharmacokinetic study following administration of IL-7 fusion protein in monkeys The pharmacokinetic profile in cynomolgus monkeys was confirmed through toxicokinetics after a 4-week single-dose toxicity study. After a single administration of 0.6, 2, and 6 mg / kg of IL-7 fusion protein, toxicokinetics was measured for 1 week. As a result, AUC last and C max The T values ​​were 608 (0.6 mg / kg), 2,730 (2 mg / kg), and 15,824 (6 mg / kg) h·ng / mL and 6 (0.6 mg / kg), 29 (2 mg / kg), and 172 (6 mg / kg) ng / mL, respectively, indicating a dose-dependent increase. max is 22-40 hours, and the terminal T 1 / 2 The terminal T was 164, 90, and 69 hours in the 0.6, 2, and 6 mg / kg dose groups, respectively. 1 / 2This is presumably due to a tendency for the blood IL-7 fusion protein concentration to increase slightly at the end of life as a biological variation caused by the low blood IL-7 fusion protein concentration. Therefore, the mean terminal T 1 / 2 was calculated to be approximately 80 hours. The results are shown in Table 2.

[0176] [Table 2]

[0177] C. Safety tests using normal rats and monkeys

[0178] To determine the dose for repeated-dose toxicity of IL-7 fusion protein, a 2-week study was conducted using normal rats and cynomolgus monkeys to measure the (sub)chronic toxicity of repeated administration at the designated dose and duration. A 4-week repeated-dose toxicity study was also conducted to evaluate the exacerbation, delayed onset, and reversibility of toxicities. Safety pharmacology studies were conducted with IL-7 fusion protein (GX-I7) at doses of 0, 1.2, 4, and 12 mg / kg in normal rats and at doses of 0, 0.6, 2, and 6 mg / kg in cynomolgus monkeys. All evaluated doses of IL-7 fusion protein (GX-I7) showed no significant effects on the general behavior, nervous system, respiratory system, or cardiovascular system of the test animals in both normal rats and cynomolgus monkeys.

[0179] When IL-7 fusion protein (GX-I7) was administered subcutaneously to normal rats once a week for a total of five doses over four weeks at a maximum dose of 12 mg / kg / week, no drug-related toxicity findings were observed. Although test drug-related symptoms were observed, no drug exposure was observed in the blood after repeated administration. This was attributed to a masking effect of drug-specific antibodies (anti-drug antibodies). Consequently, a maximum no-observed-adverse-effect dose (NOAEL) could not be determined for IL-7 fusion protein (GX-I7) administered once a week for four weeks in normal rats. Since no drug toxicity was observed at doses up to 6 mg / kg / week in a repeated four-week subcutaneous toxicity study in cynomolgus monkeys, the maximum no-observed-adverse-effect dose (NOAEL) of IL-7 fusion protein (GX-I7) was set at 6 mg / kg / week. This corresponds to a human equivalent dose (HED) of approximately 2 mg / kg.

[0180] II.Clinical research A. Objectives and Demographics This clinical trial was conducted in patients with (a) locally advanced or metastatic solid tumors and (b) glioblastoma patients with severe treatment-related lymphopenia after adjuvant chemotherapy. IL-7 fusion protein was administered every 3, 4, 6, 8, 9, or 12 weeks to evaluate safety, tolerability, and absolute lymphocyte count increasing efficacy.

[0181] (1) All subjects enrolled in the dose escalation phase of metastatic solid tumor patients had either failed existing standard therapies or were histologically diagnosed with locally advanced, recurrent, or metastatic incurable solid tumors. Patients were verified through their cancer diagnosis and related treatment history, and other medical history and screening tests confirmed that they met the inclusion / exclusion criteria for this study. The 21 subjects enrolled consisted of 10 (47.6%) colon cancer, 5 (23.8%) rectal cancer, 2 (9.5%) breast cancer, 1 (4.7%) ovarian cancer, 1 (4.7%) synovial sarcoma, 1 (4.7%) anal cancer, and 1 (4.7%) cervical cancer. The dose and dosing interval to be evaluated in the dose expansion phase (recommended phase 2 dose, RP2D) were selected based on the results of all collected safety, pharmacokinetic / pharmacodynamic, and immunogenicity analyses.

[0182] (2) A clinical study was conducted in patients with glioblastoma to select the recommended phase 2 dose (RP2D) based on the results of safety, pharmacodynamics, and immunogenicity analyses from patients enrolled in Cohorts 1-5 dose escalation phase (15 patients in total). Subjects without serious toxicity and clinically significant disease progression continued to receive the study drug based on the investigator's clinical significance and risk assessment.

[0183] Participating patients ranged in age from 32 to 81 years and included both men and women. Patients had previously been diagnosed with synovial sarcoma, invasive ductal carcinoma, rectal cancer, colon cancer, ovarian cancer, ascending colon cancer, anal cancer, invasive ductal carcinoma of the breast, adenocarcinoma, rectal cancer with para-aortic metastases, neuroendocrine carcinoma (cervix), sigmoid colon cancer, or glioblastoma. Participating patients had previously received one or more of the following cancer treatments: surgery, radiation, and chemotherapy.

[0184] B. Research design Patients with locally advanced or metastatic solid tumors and glioblastoma enrolled in the dose-escalation phase (21, 28, 42, 56, 63, or 84 days per cycle) received a fixed dose of modified IL-7 fusion protein (GX-I7) intramuscularly on the first day of each cycle at intervals of 21, 28, 42, 56, 63, or 84 days.

[0185] The dose escalation phase included intramuscular administration of IL-7 fusion protein at nine (or five) dose levels (60, 120, 240, 480, 720, 960, 1,200, 1,700, and 2,000 μg / kg; or 60, 360, 600, 840, and 1,440 μg / kg) as shown in Table 3 to evaluate safety, tolerability, and pharmacokinetic / pharmacodynamic changes. During intramuscular injection, the injection site was split so that the injection volume did not exceed 2 mL per injection site.

[0186] [Table 3]

[0187] C. Safety 1.Patients with solid tumors Safety was assessed through adverse reactions (abnormal laboratory values, subject-reported clinical symptoms and signs, investigator assessments, etc.) in dose-escalation subjects (a total of 21 patients with locally advanced or metastatic solid tumors). The results are shown in Table 4.

[0188] Of the total adverse reactions, 44 adverse drug reactions (ADRs) judged to be "related to the study drug" occurred in 16 of 21 patients (76.2%). When grades were classified according to the severity of ADRs according to the NCI-CTCAE (version 4.0) system, 29 events were mild (grade 1), 15 events were severe (grade 2), and no grade 3, 4, or 5 ADRs were reported. A total of three serious adverse events (SAEs) were reported, all of which were rated "unrelated to the study drug." Based on the reported ADR frequency, injection site reactions were the most frequent, with 25 events reported in 14 of 21 patients (66.7%), including eight fever events, four rash or rash-papular events, two decreased appetite events, and one each of asthenia, lower back pain, constipation, influenza-like illness, and myalgia. This confirmed the absence of serious adverse drug reactions.

[0189] [Table 4]

[0190] 2. Glioblastoma patients Safety was assessed through adverse reactions (abnormal laboratory test values, clinical symptoms and signs reported by the subjects, investigator assessment, etc.) in 15 glioblastoma patients reported during the dose escalation phase. See Table 5.

[0191] Thirty-two adverse drug reactions (ADRs) judged to be "study drug related" occurred in 13 of 15 subjects (86.7%). The following occurred in each group: 1 patient in the 60 μg / kg group (33.3%, 2 events), 3 patients in the 360 ​​μg / kg group (100.0%, 10 events), 3 patients in the 600 μg / kg group (100.0%, 5 events), 3 patients in the 840 μg / kg group (100.0%, 7 events), and 3 patients in the 1,440 μg / kg group (100.0%, 8 events). When grades were classified for ADR severity according to the NCI-CTCAE (version 4.0) system, 21 events were mild (grade 1), 11 events were severe (grade 2), and no grade 3, 4, or 5 ADRs were reported. According to the reported frequency of ADRs, injection site reactions were the most frequent, with 9 cases reported in 11 of 15 patients (66.0%). Six cases of urticaria, three cases of pruritus, two cases each of heat, fever, and swelling, and one case each of injection site pain, myalgia, peripheral edema, fatigue, and rash were reported. This confirmed that there were no serious adverse reactions to the drug.

[0192] [Table 5-1] [Table 5-2]

[0193] 3. Pharmacokinetics For pharmacokinetic parameters, values ​​were calculated for each subject and the mean, standard deviation, minimum and maximum values ​​were presented in the technical statistics for each dose group.

[0194] Blood was collected before (0 hours) and at 0.5, 6, 12, 24, 48, 72, 168, 336, and 504 hours after administration of the modified IL-7 fusion protein GX-I7. The IL-7 concentration in the blood was analyzed using a commercially available ELISA kit (Human IL-7 Quantikine HS ELISA Kit HS750, manufactured by R&D Systems). The results are shown in tables (PK parameters) and graphs.

[0195] The blood concentration-time curves for each subject in each dose group are shown in Figure 2, and the pharmacokinetic parameters are presented in Table 6. Blood concentration measurements of IL-7 fusion protein before and after intramuscular administration in all dose groups. It was observed that the blood concentration of IL-7 increased in a dose-dependent manner.

[0196] Following the first intramuscular administration, the blood concentration of the denatured IL-7 fusion protein reached its peak within an average of 12 to 48 hours after each dose, after which the blood concentration declined, with the half-life (t1 / 2) varying between 33 and 147 hours.

[0197] Although there are individual differences, C max and AUC last The C tended to increase, and increased more at 1,200 μg / kg than at 960 μg / kg. max and AUC last The correlation between the changes is shown in Figures 3a and 3b.

[0198] [Table 6]

[0199] 4. Pharmacodynamic (PD) properties (a) Clinical trials for patients with solid tumors To evaluate biomarkers that could be used as exploratory pharmacodynamic indicators of IL-7 fusion protein activity in patients with locally advanced or metastatic solid tumors, 21 subjects enrolled in a dose-escalation phase received the study drug. Peripheral blood samples were collected before and after administration to measure various immune cell types and their differentially expressed blood levels.

[0200] (b) Clinical trials for glioblastoma patients Peripheral blood samples were collected before and after study drug administration in 15 subjects enrolled in a dose-escalation phase to evaluate biomarkers that could serve as exploratory pharmacodynamic indicators of IL-7 fusion protein activity in patients with glioblastoma. Changes in various immune cell types and their circulating ratios were measured.

[0201] 4.1 Changes in absolute lymphocyte count (ALC) following administration of IL-7 fusion protein Blood samples were collected from patients in each dose group (cohort) before and 3 weeks after administration of IL-7 fusion protein to measure absolute lymphocyte counts (ALC). Absolute ALC values ​​were measured using an analyzer.

[0202] The changes in absolute ALC values ​​from baseline to 3 weeks in patients with solid tumors and glioblastoma in the low-dose (60-120μg / kg & 60μg / kg), medium-dose (240-480μg / kg & 360-600μg / kg), and high-dose (720-1,200μg / kg & 840-1,440μg / kg) groups are shown in Figure 4a and Figure 4b. As a result, it was confirmed that the absolute change in ALC values ​​increased by 4.4 times compared to the baseline value in the high-dose group (720-1,440μg / kg). This pattern was observed in the CD3 + , CD4 + and CD8 + The same pattern of increase was observed in T cells (see Figures 5a, 5b, 6a, 6b, 7a and 7b).

[0203] 4.2 Changes in ALC in patients with lymphopenia All solid tumor and glioblastoma patients had baseline ALC levels of 1,000 cells / mm before administration of IL-7 fusion proteins. 3 Patients with a lymphopenic phenotype or less were classified as lymphopenic, and IL-7 fusion protein was administered every 3 or 6 weeks (solid tumors) and every 4, 8, 9, or 12 weeks (GBM). Changes in ALC were then observed for lymphopenic and non-lymphopenic patient groups, and the results are shown in Figures 8a, 8b, 9a, and 9b.

[0204] The analysis showed that ALC levels were significantly increased in the non-lymphopenic patient group, with ALC levels exceeding 1,000 cells / mm 3 It was confirmed that ALC values ​​increased to within the normal range 3 weeks after administration in lymphopenic patients with less than 100 mg / kg of levothyroxine. In addition, it was confirmed that the magnitude of change compared to baseline values ​​in non-lymphopenic patients was similar to that in lymphopenic patients, especially in the high-dose group.

[0205] 4.3 Ki67, CD127 (IL-7Rα) and T cell subtype (subset) analysis results In patients with solid tumors, changes in Ki67 expression were a key indicator of increased ALC, and CD4 + T cells and CD8 + The amount of Ki67 expression on both CD4 T cells + T cells and CD8 + The expression levels of CD127, a receptor for IL-7, were significantly decreased at high doses compared to baseline. Furthermore, the expression levels of CD4 T cells, which regulate the immune system, were significantly increased compared to baseline levels. + T cells and CD8 + T cells (CD4 + / Treg ratio and CD8 + / Treg ratio) changes in CD4 + and CD8 + The increase in T cells compared to baseline was greater than the increase in Treg cells in all dose groups, see Figures 10a, 10b and 10c.

[0206] 4.4 Analysis of Ki67 expression and ALC changes for optimal dosing interval setting 4.4.A. Results of Preclinical Studies to Determine Dosing Intervals To confirm the reactivity of IL-7 fusion proteins for determining the administration interval, blood samples were collected in monkeys and Ki67 reactivity was confirmed 7 days after in vitro stimulation with IL-7 fusion proteins. The blood sampling times are shown in Figure 11.

[0207] As a result, we confirmed that Ki67 expression was increased by stimulation with IL-7 fusion protein in PBMCs 14, 10, and 3 days before administration of IL-7 fusion protein, whereas Ki67 expression was decreased by IL-7 fusion protein in blood samples collected on days 4, 11, and 18 after administration of IL-7 fusion protein. This tendency was confirmed by CD4 + and CD8 + This was observed in both T cells and blood samples on days 31, 45, 59, and 73, with increased Ki67 expression.

[0208] The results suggest that after initial T cell expansion with IL-7 fusion proteins, it may take a period of time for regrowth to occur. An inflection point is expected. Furthermore, some individuals (ZC5M03) were observed to regain responsiveness at approximately 5-6 weeks. See Figures 12a and 12b.

[0209] 4.4.B. Clinical Study Results for Dosage Interval Setting

[0210] Measurement of ALC changes following a single administration of IL-7 fusion protein in cancer patients showed that the increase in ALC following the first administration was maintained for up to 12 weeks in the high-dose group. This suggests that the administration interval can be maintained for 8 to 12 weeks or longer (e.g., 15 weeks). See Figures 13a and 13b.

[0211] In a study of glioblastoma patients, IL-7 fusion protein was administered not only alone but also in combination with various anti-cancer drugs, including chemotherapy (TMZ), a standard treatment for glioblastoma patients. ALC significantly increased with repeated administration. The results indicate that the absolute lymphocyte count (ALC), which is typically reduced by existing chemotherapy drugs (which preferentially kill rapidly proliferating cells), can be maintained above a certain level with single or repeated administration of IL-7 fusion protein. Therefore, IL-7 fusion protein is expected to enhance or improve existing anti-cancer treatments. See Figures 14a, 14b, and 14c.

[0212] 4.5 Changes in T cell subsets, NK cells, and B cells after administration of IL-7 fusion proteins CD4 in patients administered GX-I7 as an IL-7 fusion protein + T cells and CD8 + T cell subtype analysis results, CD4 + T cells and CD8 + A dose-dependent increase in T cells was observed. + and CD8 + The increase in T cells was greatest compared to baseline. Their CCR5 expression also increased at each dose compared to baseline, suggesting that administration of IL-7 fusion protein can induce T cell migration to tumor sites. NK cells also showed a dose-dependent increase compared to baseline. However, no increase in B cells compared to baseline was observed. See Figure 15a and Figure 15b.

Claims

1. 1. A composition for increasing lymphocyte numbers in a human subject in need thereof, comprising an interleukin-7 fusion protein comprising: (a) IL-7, (b) a second domain that is an oligopeptide having 1 to 10 amino acid residues consisting of methionine, glycine, or a combination thereof, wherein the oligopeptide is methionine, glycine, methionine-methionine, glycine-glycine, methionine-glycine, glycine-methionine, methionine-methionine-methionine, methionine-methionine-glycine, methionine-glycine-methionine, glycine-methionine-methionine, methionine-glycine-glycine, glycine-methionine-glycine, glycine-glycine-methionine, or glycine-glycine-glycine; and (c) a third domain that extends the half-life of an interleukin-7 fusion protein, the third domain comprising an Fc region of a modified immunoglobulin, the Fc region of the modified immunoglobulin comprising, from the N-terminus to the C-terminus, 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 amino acid residues of the CH2 domains of human IgD and human IgG4, and the CH3 domain comprises a portion of amino acid residues of the CH3 domain of human IgG4. wherein the second domain is linked to the N-terminus of IL-7 and the third domain is linked to the C-terminus of IL-7, and the interleukin-7 fusion protein is administered to a human subject at a dose ranging from 60 μg / kg to 1,440 μg / kg two or more times at intervals of at least three weeks.

2. The composition of claim 1, wherein the subject is suffering from cancer; an infection; chronic failure of the right ventricle of the heart; Hodgkin's disease; a leak or rupture of the thoracic duct; a side effect of prescription medications including anti-cancer drugs (e.g., chemotherapy), antivirals, or glucocorticoids; malnutrition due to a low-protein diet, uremia, an autoimmune disease, an immunodeficiency syndrome, a thymectomy, or a combination thereof; or idiopathic, acute radiation syndrome (ARS), or a combination thereof.

3. The composition of claim 1 or 2, wherein the IL-7 has an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 6.

4. The composition of claim 1, wherein the Fc region of the modified immunoglobulin is represented by the following formula (I): Formula (I) N'-(Z1)p-Y-Z2-Z3-Z4-C' where N' is the N-terminus of the polypeptide and C' is the C-terminus of the polypeptide; p is an integer of 0 or 1; Z1 is an amino acid sequence having 5 to 9 consecutive amino acid residues from the 98th amino acid residue among the 90th to 98th amino acid residues of SEQ ID NO: 7 toward the N-terminus; Y is an amino acid sequence having 5 to 64 consecutive amino acid residues from the 162nd amino acid residue among the 99th to 162nd amino acid residues of SEQ ID NO: 7 toward the N-terminus; Z2 is an amino acid sequence having 4 to 37 consecutive amino acid residues from the 163rd amino acid residue to the C-terminus among the 163rd to 199th amino acid residues of SEQ ID NO: 7; Z3 is an amino acid sequence having 71 to 106 consecutive amino acid residues from the 220th amino acid residue among the 115th to 220th amino acid residues of SEQ ID NO: 8 toward the N-terminus; and Z4 is an amino acid sequence having 80 to 107 consecutive amino acid residues from the 221st amino acid residue among the 221st to 327th amino acid residues of SEQ ID NO:8 toward the C-terminus.

5. The composition of claim 1, wherein the third domain has an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 to 14.

6. The composition of claim 2 , wherein the cancer is a solid tumor, lymphatic cancer, or leukemia.

7. 7. The composition of claim 6, wherein the solid tumor is synovial sarcoma, invasive ductal carcinoma, rectal cancer, colon cancer, ovarian cancer, ascending colon cancer, anal cancer, invasive ductal carcinoma of the breast, adenocarcinoma, rectal cancer with para-aortic metastases, neuroendocrine carcinoma (cervix), sigmoid colon cancer, or glioblastoma.

8. The composition of any one of claims 1 to 7, wherein the subject has previously undergone, is concurrently undergoing, or is scheduled to undergo, one or more of the following cancer treatments: surgery, radiation, and / or chemotherapy.

9. 3. The composition of claim 1 or 2, wherein the interleukin-7 fusion protein is administered two or more times at intervals of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 weeks.

10. 3. The composition of claim 1 or 2, wherein the interleukin-7 fusion protein is administered parenterally, intramuscularly, subcutaneously, intraocularly, intravenously, intraperitoneally, intradermally, intraorbitally, intracerebrally, intracranially, intraspinally, intraventricularly, intrathecally, intracapsularly, intraarticularly, or intratumorally.

11. The composition of claim 1, wherein the interleukin-7 fusion protein comprises the amino acid sequence of SEQ ID NO:

24.

12. 3. The composition of claim 1 or 2, wherein the interleukin-7 fusion protein is administered at a frequency of once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks, once every 10 weeks, once every 11 weeks, once every 12 weeks, once every 13 weeks, once every 14 weeks, or once every 15 weeks.

13. The composition of claim 6, wherein the number of intratumoral tumor-infiltrating lymphocytes (TILs) is increased after administration of the interleukin-7 fusion protein compared to the number of intratumoral TILs before administration.

14. 1. Use of an interleukin-7 fusion protein in the manufacture of a medicament for use in increasing lymphocyte count in a human subject in need thereof, wherein the medicament is administered to the human subject at a dose ranging from 60 μg / kg to 1,440 μg / kg two or more times at intervals of at least three weeks; Interleukin-7 fusion proteins are (a) IL-7; and (b) a second domain that is an oligopeptide having 1 to 10 amino acid residues consisting of methionine, glycine, or a combination thereof, wherein the oligopeptide is methionine, glycine, methionine-methionine, glycine-glycine, methionine-glycine, glycine-methionine, methionine-methionine-methionine, methionine-methionine-glycine, methionine-glycine-methionine, glycine-methionine-methionine, methionine-glycine-glycine, glycine-methionine-glycine, glycine-glycine-methionine, or glycine-glycine-glycine; and (c) a third domain that extends the half-life of an interleukin-7 fusion protein, the third domain comprising an Fc region of a modified immunoglobulin, the Fc region of the modified immunoglobulin comprising, from the N-terminus to the C-terminus, 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 amino acid residues of the CH2 domains of human IgD and human IgG4, and the CH3 domain comprises a portion of amino acid residues of the CH3 domain of human IgG4. wherein the second domain is linked to the N-terminus of IL-7 and the third domain is linked to the C-terminus of IL-7.

15. 1. A pharmaceutical composition for increasing lymphocyte numbers in a human subject in need thereof, comprising, as an active ingredient, an IL-7 fusion protein comprising: (a) IL-7; and (b) a second domain that is an oligopeptide having 1 to 10 amino acid residues consisting of methionine, glycine, or a combination thereof, wherein the oligopeptide is methionine, glycine, methionine-methionine, glycine-glycine, methionine-glycine, glycine-methionine, methionine-methionine-methionine, methionine-methionine-glycine, methionine-glycine-methionine, glycine-methionine-methionine, methionine-glycine-glycine, glycine-methionine-glycine, glycine-glycine-methionine, or glycine-glycine-glycine; and (c) a third domain that extends the half-life of an interleukin-7 fusion protein, the third domain comprising an Fc region of a modified immunoglobulin, the Fc region of the modified immunoglobulin comprising, from the N-terminus to the C-terminus, 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 amino acid residues of the CH2 domains of human IgD and human IgG4, and the CH3 domain comprises a portion of amino acid residues of the CH3 domain of human IgG4. wherein the second domain is linked to the N-terminus of IL-7 and the third domain is linked to the C-terminus of IL-7, and the pharmaceutical composition is administered to a human subject at a dose ranging from 60 μg / kg to 1,440 μg / kg two or more times at intervals of at least three weeks.

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