Method for treating tumors in a subject

Abstract

A C-X-C motif chemokine 12 (CXCL12) antagonist for use in a method for treating a tumor in a subject, the method comprising administering to the subject a C-X-C motif chemokine 12 (CXCL12) antagonist, radiotherapy, and an anti-angiogenic compound, wherein the tumor is a brain tumor, relates to a C-X-C motif chemokine 12 (CXCL12) antagonist.

Description

Technical Field

[0001] The present invention relates to a method for treating tumors in a subject.

Background Art

[0002] Brain cancer is a disease of the brain in which cancer cells occur in brain tissue. The cancer cells grow to form a tumor, i.e., a mass of cancer tissue, which also contains non-cancerous supportive cells (tumor stroma) that interfere with brain functions such as muscle control, sensation, memory, and other normal body functions. A tumor containing cancer cells that grow in an uncontrolled manner, can invade surrounding tissues, and can spread to other parts of the body through the blood and lymphatic systems is called a malignant tumor, and one that does not have a tendency to invade surrounding tissues or spread to distant body parts is called a benign tumor. Tumors that originate from brain tissue are called primary brain tumors, while tumors that spread to the brain from other body sites are referred to as metastatic or secondary brain tumors. Statistics suggest that the incidence of brain cancer is low (1.4% of all new cancer patients per year), and thus, as estimated by the National Cancer Institute (NCI) and the American Cancer Society, it can develop in approximately 23,770 new people per year with about 16,050 deaths. Only about 5% of brain tumors can be attributed to hereditary genetic diseases such as neurofibromatosis, tuberous sclerosis, and several others.

[0003] Depending on how the tumor cells within a tumor look microscopically, the National Cancer Institute (NCI) has enumerated the following grades for brain tumors. Grade I: The tissue is benign. The cells look almost the same as normal brain cells, and they grow slowly. Grade II: The tissue is malignant. The cells do not look as normal as the cells in a grade I tumor. Grade III: The malignant tissue has cells that look very different from normal cells. The abnormal cells are growing actively and have an obviously abnormal appearance (undifferentiated). Grade IV: The malignant tissue has cells that look extremely abnormal and tend to grow rapidly.

[0004] Most of the symptoms caused by brain cancer are not specific to brain cancer and include difficulty walking and / or dizziness / vertigo, seizures, muscle weakness (e.g., weakness in the arms and legs), and headache (persistent and / or severe). Other common symptoms that may occur are nausea, vomiting, blurred vision, changes in a person's attention, drowsiness, decreased intellectual ability and / or confusion, memory impairment, difficulty speaking, voice disorders, or changes in speech such as inability to speak, personality changes, hallucinations, weakness on one side of the body, impaired coordination of movement, feelings of fatigue, and decreased sensations such as pins and needles and / or tactile perception.

[0005] Treatment of brain tumors typically involves surgery, radiation therapy, and chemotherapy. Other treatment options include hyperthermia therapy, immunotherapy, or steroid therapy. Depending on the individual brain tumor, combinations of two or more of the above options may be implemented in the treatment of a subject suffering from a brain tumor. SUMMARY OF THE INVENTION

[0006] The underlying problem of the present invention is to provide a method for treating a tumor in a subject, preferably, the tumor is a brain tumor.

[0007] Another underlying problem of the present invention is to provide a method for treating a malignant brain tumor in a subject.

[0008] Another underlying problem of the present invention is to provide a method for treating a brain tumor in a subject, where the brain tumor is treatable or can be treated by radiation therapy.

[0009] A further underlying problem of the present invention is to provide a method for treating a brain tumor in a subject, where the brain tumor cannot be treated by surgery, typically, due to the size of the tumor or its location close to important adjacent tissues.

[0010] These and other problems are solved by the subject matter of the appended independent claims; preferred embodiments may be employed from the appended dependent claims.

[0011] Nevertheless, the underlying problems of the present invention are solved by the subject matter of the following embodiments, and Embodiment 1 is also referred to as the first aspect of the present invention.

[0012] Embodiment 1. A method for treating a tumor in a subject, the method comprising the step of administering to the subject: - a C-X-C motif chemokine 12 (CXCL12) antagonist, - radiotherapy, and - an anti-angiogenic compound wherein the tumor is a brain tumor. Embodiment 2. The method according to Embodiment 1, wherein the tumor is selected from the group consisting of glioblastoma, glioma, acoustic neuroma, astrocytoma, brain metastasis, choroid plexus carcinoma, craniopharyngioma, germinoma, medulloblastoma, meningioma, pediatric brain tumor, pineoblastoma, and pituitary tumor. Embodiment 3. The method according to Embodiment 1 or 2, wherein the tumor is selected from the group consisting of glioblastoma and glioma. Embodiment 4. The method according to any one of Embodiments 1 to 3, wherein the tumor is a grade IV astrocytoma or glioblastoma. Embodiment 5. The method according to Embodiment 4, wherein the tumor is a supratentorial glioblastoma. Embodiment 6. The glioblastoma is a glioblastoma having a non-methylated O 6 -methylguanine DNA methyltransferase (MGMT) promoter status, according to the method of Embodiment 4 or 5. Embodiment 7. The method according to any one of Embodiments 1 to 6, preferably any one of Embodiments 4 to 6, wherein the tumor is non-responsive to temozolomide. Embodiment 8. The method according to any one of Embodiments 1 to 7, preferably any one of Embodiments 4 to 7, wherein the tumor expresses CXCL12 and VEGF. Embodiment 9. The method according to embodiment 8, wherein the tumor expresses CXCL12 and VEGF in various compartments of the tumor. Embodiment 10. The method according to embodiment 8 or 9, wherein CXCL12 is expressed at the tip of the tumor, in the hyperplastic blood vessels of the tumor, and / or in the microvascular proliferation zone of the tumor. Embodiment 11. The method according to any one of embodiments 8 to 10, preferably embodiment 10, wherein VEGF is expressed in the cellular tumor compartment of the tumor, by the region having pseudopalisading cells of the tumor, and / or in the necrotic peripheral zone of the tumor, and preferably the tumor is glioblastoma. Embodiment 12. The method according to any one of embodiments 1 to 11, wherein the tumor is selected from the group including astrocytoma, ependymoma, and oligodendroglioma. Embodiment 13. The method according to any one of embodiments 1 to 12, preferably any one of embodiments 4 to 11, wherein the tumor is an incompletely resected tumor. Embodiment 14. The method according to embodiment 13, wherein the tumor is an incompletely resected tumor detectable by postoperative T1-weighted contrast-enhanced MRI scan. Embodiment 15. The method according to any one of embodiments 1 to 14, wherein the CXCL12 antagonist inhibits the signal transduction of CXCL12 via one or both of the CXCL12 receptors C-X-C chemokine receptor type 4 (CXCR4) and C-X-C chemokine receptor type 7 (CXCR7). Embodiment 16. The method according to embodiment 15, wherein the CXCL12 antagonist inhibits the binding of CXCL12 to the CXCL12 receptor C-X-C chemokine receptor type 4 (CXCR4) and / or to the CXCL12 receptor C-X-C chemokine receptor type 7 (CXCR7). Embodiment 17. The method according to embodiment 16, wherein the CXCL12 antagonist binds to CXCL12. Embodiment 18. The CXCL12 antagonist is a nucleic acid molecule that binds to CXCL12, and the nucleic acid molecule is preferably selected from the group consisting of a type B SDF-1 binding nucleic acid molecule, a type C SDF-1 binding nucleic acid molecule, a type A SDF-1 binding nucleic acid molecule, and a type D SDF-1 binding nucleic acid molecule, according to the method of Embodiment 17. Embodiment 19. The type B SDF-1 binding nucleic acid molecule contains a central stretch of nucleotides, and the central stretch of nucleotides has the following nucleotide sequence: 5’GUGUGAUCUAGAUGUADWGGCUGWUCCUAGUYAGG 3’ (SEQ ID NO: 52) according to the method of Embodiment 18. Embodiment 20. The central stretch of nucleotides has the following nucleotide sequence: 5’GUGUGAUCUAGAUGUADUGGCUGAUCCUAGUCAGG 3’ (SEQ ID NO: 53) according to the method of Embodiment 19. Embodiment 21. The type B SDF-1 binding nucleic acid molecule contains, in the 5’→3’ direction, a first terminal stretch of nucleotides, the central stretch of nucleotides, and a second terminal stretch of nucleotides, according to the method of Embodiment 19 or 20. Embodiment 22. The type B SDF-1 binding nucleic acid molecule contains, in the 5’→3’ direction, a second terminal stretch of nucleotides, the central stretch of nucleotides, and a first terminal stretch of nucleotides, according to the method of Embodiment 19 or 20. Embodiment 23. The first terminal stretch of nucleotides contains a nucleotide sequence of 5’X1X2SVNS 3’, and the second terminal stretch of nucleotides contains a nucleotide sequence of 5’BVBSX3X43’, where X1 is absent or A, X2 is G, X3 is C, X4 is absent or U; or, X1 is absent, X2 is absent or G, X3 is absent or C, X4 is absent. The method according to Embodiment 21 or 22. Embodiment 24. The first terminal stretch of the nucleotide comprises a nucleotide sequence of 5’X1X2CRWG 3’, and the second terminal stretch of the nucleotide comprises a nucleotide sequence of 5’KRYSX3X4 3’, wherein X1 is absent or A, X2 is G, X3 is C, and X4 is absent or U, Any one of Embodiments 21 to 23, preferably the method according to Embodiment 50. Embodiment 25. The first terminal stretch of the nucleotide comprises a nucleotide sequence of 5’X1X2CGUG 3’, and the second terminal stretch of the nucleotide comprises a nucleotide sequence of 5’UACGX3X43’, wherein X1 is absent or A, X2 is G, X3 is C, and X4 is absent or U, Preferably, the first terminal stretch of the nucleotide comprises a nucleotide sequence of 5’AGCGUG 3’, and the second terminal stretch of the nucleotide comprises a nucleotide sequence of 5’UACGCU 3’. Any one of Embodiments 21 to 24, preferably the method according to Embodiment 23 or 24. Embodiment 26. The first terminal stretch of the nucleotide comprises a nucleotide sequence of 5’X1X2SSBS 3’, and the second terminal stretch of the nucleotide comprises a nucleotide sequence of 5’BVSSX3X4 3’, wherein X1 is absent, X2 is absent or G, X3 is absent or C, and X4 is absent, Preferably, the first terminal stretch of the nucleotide comprises a nucleotide sequence of 5’GCGUG 3’, and the second terminal stretch of the nucleotide comprises a nucleotide sequence of 5’UACGC 3’. Any one of Embodiments 21 to 23, preferably the method according to Embodiment 23. Embodiment 27. The type B SDF-1 binding nucleic acid molecule is any nucleotide sequence among SEQ ID NO: 5 to SEQ ID NO: 20 and SEQ ID NO: 22 to SEQ ID NO: 28, or a nucleotide sequence that is at least 80% identical thereto. Preferably, it is any one of SEQ ID NO: 5 to SEQ ID NO: 7, SEQ ID NO: 16, SEQ ID NO: 22, and SEQ ID NO: 28, or a nucleotide sequence that is at least 80% identical thereto. More preferably, the method according to any one of Embodiments 18 to 26, which comprises any one of SEQ ID NO: 22 and SEQ ID NO: 28, or a nucleotide sequence that is at least 80% identical thereto. Embodiment 28. The type C SDF-1 binding nucleic acid molecule contains a central stretch of nucleotides, and the central stretch of nucleotides is GGUYAGGGCUHRX A and contains the nucleotide sequence of AGUCGG (SEQ ID NO: 108). wherein X A is absent or is A. The method according to Embodiment 18. Embodiment 29. The central stretch of nucleotides is 5’GGUYAGGGCUHRAAGUCGG 3’ (SEQ ID NO: 109), 5’GGUYAGGGCUHRAGUCGG 3’ (SEQ ID NO: 110), or 5’GGUUAGGGCUHGAAGUCGG 3’ (SEQ ID NO: 111), preferably 5’GGUUAGGGCUHGAAGUCGG 3’ (SEQ ID NO: 111). The method according to Embodiment 28. Embodiment 30. The type C SDF-1 binding nucleic acid molecule contains, in the 5’→3’ direction, a first terminal stretch of nucleotides, the central stretch of nucleotides, and a second terminal stretch of nucleotides. The method according to Embodiment 28 or 29. Embodiment 31. The type C SDF-1 binding nucleic acid molecule contains, in the 5’→3’ direction, a second terminal stretch of nucleotides, the central stretch of nucleotides, and a first terminal stretch of nucleotides. The method according to Embodiment 28 or 29. Embodiment 32. The first terminal stretch of the nucleotide comprises the nucleotide sequence of 5’RKSBUSNVGR 3’ (SEQ ID NO: 138), and the second terminal stretch of the nucleotide comprises the nucleotide sequence of 5’YYNRCASSMY 3’ (SEQ ID NO: 139). Preferably, the first terminal stretch of the nucleotide comprises the nucleotide sequence of 5’RKSBUGSVGR 3’ (SEQ ID NO: 140), and the second terminal stretch of the nucleotide comprises the nucleotide sequence of 5’YCNRCASSMY 3’ (SEQ ID NO: 141), the method according to Embodiment 30 or 31. Embodiment 33. The first terminal stretch of the nucleotide comprises the nucleotide sequence of 5’XSSSSV 3’, and the second terminal stretch of the nucleotide comprises the nucleotide sequence of 5’BSSSXS 3’, wherein Xs is absent or is S. Preferably, the first terminal stretch of the nucleotide comprises the nucleotide sequence of 5’SGGSR 3’, and the second terminal stretch of the nucleotide comprises the nucleotide sequence of 5’YSCCS 3’, the method according to Embodiment 30 or 31. Embodiment 34. a) The first terminal stretch of the nucleotide comprises the nucleotide sequence of 5’GCCGG 3’, and the second terminal stretch of the nucleotide comprises the nucleotide sequence of 5’CCGGC 3’; or b) The first terminal stretch of the nucleotide comprises the nucleotide sequence of 5’CGUGCGCUUGAGAUAGG 3’ (SEQ ID NO: 220), and the second terminal stretch of the nucleotide comprises the nucleotide sequence of 5’CUGAUUCUCACG 3’ (SEQ ID NO: 221); or c) The first terminal stretch of the nucleotide comprises the nucleotide sequence of 5’UGAGAUAGG 3’, and the second terminal stretch of the nucleotide comprises the nucleotide sequence of 5’CUGAUUCUCA 3’ (SEQ ID NO: 222); or d) The first terminal stretch of nucleotides comprises the nucleotide sequence 5’GAGAUAGG 3’, and the second terminal stretch of nucleotides comprises the nucleotide sequence 5’CUGAUUCUC 3’. The method according to embodiment 30 or 31. Embodiment 35. The type C SDF-1 binding nucleic acid molecule is any one of SEQ ID NO: 95 to SEQ ID NO: 107, SEQ ID NO: 112 to SEQ ID NO: 137, SEQ ID NO: 223, and SEQ ID NO: 224. Preferably, the method according to any one of embodiments 28 to 34, comprising the nucleotide sequence of any one of SEQ ID NO: 120, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 223, and SEQ ID NO: 224. Embodiment 36. The type A SDF-1 binding nucleic acid molecule comprises a central stretch of nucleotides, and the central stretch of nucleotides comprises 5’AAAGYRACAHGUMAAX A UGAAAGGUARC 3’ (SEQ ID NO: 74). wherein X A is absent or is A. The method according to embodiment 18. Embodiment 37. The central stretch of nucleotides is 5’AAAGYRACAHGUMAAUGAAAGGUARC 3’ (SEQ ID NO: 75), or 5’AAAGYRACAHGUMAAAUGAAAGGUARC 3’ (SEQ ID NO: 76), or 5’AAAGYAACAHGUCAAUGAAAGGUARC 3’ (SEQ ID NO: 77), preferably, the central stretch of nucleotides comprises the nucleotide sequence 5’AAAGYAACAHGUCAAUGAAAGGUARC 3’ (SEQ ID NO: 77). The method according to embodiment 36. Embodiment 38. The type A SDF-1 binding nucleic acid molecule, in the 5’→3’ direction, comprises a first terminal stretch of nucleotides, the central stretch of nucleotides, and a second terminal stretch of nucleotides. The method according to embodiment 36 or 37. Embodiment 39. The method according to embodiment 36 or 37, wherein the type A SDF-1 binding nucleic acid molecule comprises, in the 5'→3' direction, a second terminal stretch of nucleotides, said central stretch of nucleotides, and a first terminal stretch of nucleotides. Embodiment 40. Said first terminal stretch of nucleotides comprises a nucleotide sequence of 5’X1X2NNB V 3’, and said second terminal stretch of nucleotides comprises a nucleotide sequence of 5’BNBNX3X43’, wherein X1 is absent or R, X2 is S, X3 is S, and X4 is absent or Y; or, X1 is absent, X2 is absent or S, X3 is absent or S, and X4 is absent, The method according to embodiment 38 or 39. Embodiment 41. Said first terminal stretch of nucleotides comprises a nucleotide sequence of 5’RSHRYR 3’, and said second terminal stretch of nucleotides comprises a nucleotide sequence of 5’YRYDSY 3’, Preferably, said first terminal stretch of nucleotides comprises a nucleotide sequence of 5’GCUGUG 3’, and said second terminal stretch of nucleotides comprises a nucleotide sequence of 5’CGCAGC 3’, the method according to any one of embodiments 38 to 40, preferably 40. Embodiment 42. Said first terminal stretch of nucleotides comprises a nucleotide sequence of 5’X2BBBS 3’, and said second terminal stretch of nucleotides comprises a nucleotide sequence of 5’SBBVX33’, wherein X2 is absent or S, and X3 is absent or S; Preferably, said first terminal stretch of nucleotides comprises a nucleotide sequence of 5’CUGUG 3’, and said second terminal stretch of nucleotides comprises a nucleotide sequence of 5’CGCAG 3’; Or the first terminal stretch of the nucleotide contains a nucleotide sequence of 5’GCGUG 3’, and the second terminal stretch of the nucleotide contains a nucleotide sequence of 5’CGCGC 3’, any one of Embodiments 38 to 40, preferably the method according to 40. Embodiment 43. The type A SDF-1 binding nucleic acid molecule has a nucleotide sequence of any one of SEQ ID NO: 60 to SEQ ID NO: 73, SEQ ID NO: 78 to SEQ ID NO: 82, SEQ ID NO: 84 to SEQ ID NO: 87, SEQ ID NO: 89 to SEQ ID NO: 94, and SEQ ID NO: 145, or a nucleotide sequence that is at least 80% identical thereto. Preferably, any one of SEQ ID NO: 60, SEQ ID NO: 63, SEQ ID NO: 66, SEQ ID NO: 78, SEQ ID NO: 84, and SEQ ID NO: 146, or a nucleotide sequence that is at least 80% identical thereto. More preferably, any one of SEQ ID NO: 84 and SEQ ID NO: 146, or a method according to any one of Embodiments 36 to 42, comprising a nucleotide sequence that is at least 80% identical thereto. Embodiment 44. The type D SDF-1 binding nucleic acid molecule has a nucleotide sequence of any one of SEQ ID NO: 142 to SEQ ID NO: 144, or a nucleotide sequence that is at least 80% identical thereto, according to the method of Embodiment 18. Embodiment 45. The nucleic acid molecule contains a modification, the modification is preferably a high molecular weight moiety, and / or the modification preferably modifies the characteristics of the nucleic acid molecule from the perspective of residence time in an animal or human body, preferably in a human body, according to any one of Embodiments 18 to 44. Embodiment 46. The modification is selected from the group consisting of an HES moiety, a PEG moiety, a biodegradable modification, and combinations thereof, according to the method of Embodiment 45. Embodiment 47. The modification is a PEG moiety consisting of linear PEG or branched PEG, preferably the molecular weight of the linear or branched PEG is about 20,000 to 120,000 Da, more preferably about 30,000 to 80,000 Da, and most preferably about 40,000 Da, according to the method of Embodiment 46. Embodiment 48. The modification is an HES moiety, preferably, the molecular weight of the HES moiety is about 10,000 to 200,000 Da, more preferably about 30,000 to 170,000 Da, and most preferably about 150,000 Da, the method according to Embodiment 46. Embodiment 49. The modification is attached to the nucleic acid molecule via a linker, preferably, the linker is a biostable or biodegradable linker, the method according to any one of Embodiments 45 to 48. Embodiment 50. The modification is added to the 5'-terminal nucleotide and / or the 3'-terminal nucleotide of the nucleic acid molecule, and / or to the nucleotides of the nucleic acid molecule between the 5'-terminal nucleotide and the 3'-terminal nucleotide of the nucleic acid molecule, the method according to any one of Embodiments 45 to 49. Embodiment 51. The nucleotides of the nucleic acid molecule, or the nucleotides forming the nucleic acid molecule are L-nucleotides, the method according to any one of Embodiments 18 to 50. Embodiment 52. The nucleic acid molecule is an L-nucleic acid molecule, the method according to any one of Embodiments 18 to 51. Embodiment 53. The nucleic acid molecule is GCGUGGUGUGAUCUAGAUGUAUUGGCUGAUCCUAGUCAGGUACGC (SEQ ID NO: 22) an L-ribonucleic acid molecule comprising the nucleotide sequence of, the method according to Embodiment 18. Embodiment 54. The L-ribonucleic acid molecule comprises a 40 kDa polyethylene glycol moiety, and the 40 kDa polyethylene glycol moiety is added to the 5'-end of the nucleotide sequence, the method according to Embodiment 53. Embodiment 55. N-[ω-methoxypoly(oxy-1,2-ethanediyl]] N'-[ω-methoxypoly(oxy-1,2-ethanediyl]-acetyl-9-amino-8-oxo-7-aza-nonyloxy phosphate is interspersed between the 5'-terminal nucleotide and the 40 kDa polyethylene glycol moiety, the method according to Embodiment 54. Embodiment 56. The method according to embodiment 55, wherein the L-ribonucleic acid molecule is present as a sodium salt, and preferably, the L-nucleic acid molecule is NOX-A12. Embodiment 57. The method according to any one of embodiments 1 to 16, wherein the CXCL12 antagonist is an aptamer that binds to CXCL12. Embodiment 58. The method according to any one of embodiments 1 to 16, wherein the CXCL12 antagonist is an antibody that binds to CXCL12. Embodiment 59. The method according to embodiment 58, wherein the antibody that binds to CXCL12 is mAB-30D8, 1131-H12, or 1143-H1. Embodiment 60. The method according to any one of embodiments 1 to 16, wherein the CXCL12 antagonist is a CXCL12 binding protein. Embodiment 61. The method according to embodiment 60, wherein the protein is a TRAP protein or an anticalin. Embodiment 62. The method according to any one of embodiments 1 to 16, wherein the CXCL12 antagonist targets CXCR4. Embodiment 63. The method according to embodiment 62, wherein the CXCL12 antagonist binds to CXCR4. Embodiment 64. The method according to embodiment 62 or 63, wherein the CXCR4-binding CXCL12 antagonist is selected from the group consisting of small molecules, peptides, synthetic peptides, recombinant proteins, fusion proteins, and antibodies. Embodiment 65. The method according to embodiment 64, wherein the CXCR4-binding CXCL12 antagonist is a small molecule. Embodiment 66. The method according to embodiment 65, wherein the CXCR4-binding CXCL12 antagonist is a small molecule selected from the group consisting of prelixafor, mobixafor, Q-122, HPH-112, BKT-300, X4P-002, X4P-003, X4-136, GP-01CR01, GP-01CR11, GP-01CR21, pentixather, BKT-170, BMS-585248, CS-3955, CTCE-0012, CTCE-9908, GBV-4086, HPH-112, HPH-211, NB-325, SP-10, and USL-311. Embodiment 67. The method according to embodiment 66, wherein the small molecule is selected from the group consisting of prelixafor, mobixafor, Q-122, HPH-112, BKT-300, X4P-002, X4P-003, X4-136, GP-01CR01, GP-01CR11, GP-01CR21, and pentixather. Embodiment 68. The method according to embodiment 64, wherein the CXCR4-binding CXCL12 antagonist is a peptide. Embodiment 69. The method according to embodiment 68, wherein the CXCR4-binding CXCL12 antagonist is motixafortide. Embodiment 70. The method according to embodiment 64, wherein the CXCR4-binding CXCL12 antagonist is a synthetic peptide. Embodiment 71. The method according to embodiment 70, wherein the CXCR4-binding CXCL12 antagonist is a synthetic peptide selected from the group consisting of valixafortide, LY-2510924, ALB-408, and POL-5551. Embodiment 72. The method according to embodiment 71, wherein the CXCR4-binding CXCL12 antagonist is selected from the group consisting of valixafortide and LY-2510924. Embodiment 73. The method according to embodiment 64, wherein the CXCR4-binding CXCL12 antagonist is a recombinant protein. Embodiment 74. The method according to embodiment 73, wherein the recombinant protein is selected from the group consisting of PTX-9098 and NNL-121. Embodiment 75. The method according to embodiment 64, wherein the CXCR4-binding CXCL12 antagonist is a fusion protein. Embodiment 76. The method according to embodiment 75, wherein the fusion protein is selected from the group consisting of AD-214 and AM-3114. Embodiment 77. The method according to embodiment 64, wherein the CXCR4-binding CXCL12 antagonist is an antibody or an antigen-binding fragment of the antibody. Embodiment 78. The method according to embodiment 77, wherein the antibody is a monoclonal antibody. Embodiment 79. The method according to embodiment 77 or 78, wherein the CXCR4-binding CXCL12 antagonist is an antibody. Embodiment 80. The method according to embodiment 79, wherein the antibody is a monoclonal antibody selected from the group consisting of ulocuplumab, hz-515H7, KY-1051, PF-06747143, ALX-0651, AT-009, LY-2624587, and STIA-220X. Embodiment 81. The method according to embodiment 80, wherein the antibody is a monoclonal antibody selected from the group consisting of ulocuplumab, hz-515H7, and KY-1051. Embodiment 82. The method according to any one of embodiments 1 to 16, wherein the CXCL12 antagonist binds to CXCR7. Embodiment 83. The method according to embodiment 82, wherein the CXCR7-binding CXCL12 antagonist is selected from the group consisting of small molecules, synthetic peptides, and antibodies. Embodiment 84. The method according to embodiment 83, wherein the CXCR7-binding CXCL12 antagonist is a small molecule. Embodiment 85. The method according to embodiment 84, wherein the CXCR7-binding CXCL12 antagonist is a small molecule selected from the group consisting of ACT-10041239, CCX-650, CCX-662, and CCX-771. Embodiment 86. The method according to embodiment 85, wherein the CXCR7-binding CXCL12 antagonist is ACT-10041239. Method according to Embodiment 83, wherein the CXCR7-binding CXCL12 antagonist is a synthetic peptide. Method according to Embodiment 87, wherein the synthetic peptide is selected from the group consisting of LIH-383 and POL-6926. Method according to Embodiment 88, wherein the synthetic peptide is LIH-383. Method according to Embodiment 83, wherein the CXCR7-binding CXCL12 antagonist is an antibody or an antigen-binding fragment of the antibody. Method according to Embodiment 90, wherein the antibody is a monoclonal antibody. Method according to Embodiment 90 or 91, wherein the antibody is a monoclonal antibody selected from the group consisting of JT-07, X-7Ab, and STIA-230X. Method according to Embodiment 92, wherein the antibody is a monoclonal antibody selected from the group consisting of JT-07 and X-7Ab. Method according to any one of Embodiments 17 to 93, preferably any one of Embodiments 17 to 56, wherein an amount of the CXCL12 antagonist is administered to the subject, and the amount is effective to inhibit angiogenesis of the tumor. Method according to Embodiment 94, wherein angiogenesis is caused by CXCL12-mediated mobilization of endothelial cells or bone marrow-derived angiogenesis-promoting cells. Method according to Embodiment 94 or 95, wherein the CXCL12-mediated mobilization is CXCR4- and / or CXCR7-dependent. Method according to any one of Embodiments 1 to 96, preferably any one of Embodiments 17 to 57, wherein the radiotherapy uses ionizing electromagnetic radiation or a particle beam. Method according to Embodiment 97, wherein the ionizing electromagnetic radiation is selected from the group consisting of X-rays and gamma rays. Method according to Embodiment 98, wherein the X-rays have a wavelength of about 10 picometers to about 10 nanometers. Embodiment 100. The method according to embodiment 98 or 99, wherein the energy of the X-ray is from about 145 eV to about 124 keV. Embodiment 101. The method according to embodiment 98, wherein the gamma-ray has a wavelength of less than 10 picometers. Embodiment 102. The method according to embodiment 101, wherein the energy of the gamma-ray is 100 keV or more. Embodiment 103. The method according to embodiment 97, wherein the radiation therapy uses a particle beam. Embodiment 104. The method according to embodiment 103, wherein the particle beam is a particle beam selected from the group consisting of a proton beam, a photon beam, and an electron beam. Embodiment 105. The method according to any one of embodiments 97 to 104, wherein the radiation therapy uses intensity-modulated radiation therapy (IMRT), tomography also referred to as image-guided radiation therapy (IGRT), and / or stereotactic radiosurgery. Embodiment 106. The method according to any one of embodiments 97 to 105, wherein the radiation therapy is administered by external radiation therapy, preferably external beam radiation therapy. Embodiment 107. The method according to any one of embodiments 97 to 106, wherein the radiation therapy is administered by brachytherapy. Embodiment 108. The method according to embodiment 107, wherein the brachytherapy is provided by a low-dose rate (LDR) implant, a high-dose rate (HDR) implant, a permanent implant, a formulation containing a therapeutic radioactive nuclide, preferably a liquid formulation, or a combination thereof. Embodiment 109. The method according to any one of embodiments 97 to 108, wherein the radiation therapy targets or is directed towards the cells of the tumor. Embodiment 110. The method according to any one of embodiments 97 to 109, wherein the radiation therapy targets or is directed towards the microvasculature of the tumor. Embodiment 111. The method according to embodiment 110, wherein the radiation therapy depletes the microvasculature of the tumor. Embodiment 112. The method according to any one of Embodiments 97 to 111, wherein radiotherapy creates a hypoxic state within the tumor. Embodiment 113. The method according to any one of Embodiments 97 to 112, preferably Embodiment 112, wherein the tumor expresses CXCL12 in the presence of radiotherapy. Embodiment 114. The method according to any one of Embodiments 97 to 113, preferably Embodiment 112 or 113, wherein the tumor is revascularized in the presence of radiotherapy. Embodiment 115. The method according to any one of Embodiments 97 to 114, preferably any one of Embodiments 112 to 114, wherein radiotherapy results in immunosuppression. Embodiment 116. The method according to any one of Embodiments 97 to 115, wherein the total absorbed radiation dose of the radiotherapy is about 10 Gy to about 100 Gy, preferably about 40 Gy to about 60 Gy, more preferably about 40 Gy or about 60 Gy. Embodiment 117. The method according to Embodiment 116, wherein the radiotherapy is normofractionated radiotherapy. Embodiment 118. The method according to Embodiment 116, wherein the radiotherapy is administered as hypofractionated radiotherapy. Embodiment 119. The method according to any one of Embodiments 116 to 118, wherein the individual absorbed radiation of the fractionated radiotherapy is about 0.5 Gy to about 5 Gy, preferably about 2 Gy to about 2.7 Gy, more preferably about 2 Gy or about 2.7 Gy. Embodiment 120. The method according to any one of Embodiments 97 to 119, wherein the radiotherapy includes intraoperative radiotherapy (IORT). Embodiment 121. The method according to any one of Embodiments 1 to 120, wherein the anti-angiogenic compound inhibits angiogenesis. Embodiment 122. The method according to Embodiment 121, wherein angiogenesis is the formation of new blood vessels from existing blood vessels. Embodiment 123. The method according to Embodiment 121 or 122, wherein the anti-angiogenic compound inhibits angiogenesis to and / or within the tumor. Embodiment 124. The anti-angiogenic compound inhibits the growth of the tumor, and the method according to any one of Embodiments 121 to 123. Embodiment 125. The anti-angiogenic compound inhibits the interaction between vascular endothelial growth factor (VEGF) and the VEGF receptor, and the method according to any one of Embodiments 121 to 124. Embodiment 126. The VEGF is human VEGF including any of its isoforms and any form thereof cleaved by proteolysis, and the method according to Embodiment 125. Embodiment 127. The isoform is selected from the group consisting of VEGF121, VEGF165, VEGF189, VEGF206, VEGF145, VEGF183, and VEGF165b, and preferably, the isoform is selected from the group consisting of VEGF121, VEGF165, VEGF189, VEGF206, and VEGF145, and the method according to Embodiment 126. Embodiment 128. The form cleaved by proteolysis of VEGF is VEGF110, and the method according to Embodiment 126. Embodiment 129. The anti-angiogenic compound inhibits the interaction between the VEGF receptor and at least one of human vascular endothelial growth factor (VEGF), an isoform of VEGF, and a form cleaved by proteolysis of VEGF or an isoform of VEGF, and the method according to any one of Embodiments 125 to 128. Embodiment 130. The VEGF receptor is selected from the group consisting of VEGFR1 and VEGFR2, and the method according to any one of Embodiments 125 to 129. Embodiment 131. The VEGF receptor is VEGFR1, and the method according to Embodiment 130. Embodiment 132. The anti-angiogenic compound binds to VEGF, and the method according to any one of Embodiments 125 to 131. Embodiment 133. The anti-angiogenic compound that binds to VEGF is selected from the group consisting of an anti-VEGF antibody, a fragment of an anti-VEGF antibody, a VEGF-binding protein, and a VEGF-binding nucleic acid molecule, and the method according to Embodiment 132. Embodiment 134. The method according to embodiment 133, wherein the anti-angiogenic compound is an anti-VEGF antibody. Embodiment 135. The method according to embodiment 134, wherein the anti-VEGF antibody is a human anti-VEGF antibody, a humanized anti-VEGF antibody, or a human anti-VEGF antibody. Embodiment 136. The method according to embodiment 134 or 135, wherein the anti-VEGF antibody comprises the following CDRs in the light chain variable region: CDR1: QDISNY (SEQ ID NO: 226), CDR2: FTS, and CDR3: QQYSTVPWT (SEQ ID NO: 227). Embodiment 137. The method according to any one of embodiments 134 to 136, wherein the anti-VEGF antibody comprises the following CDRs in the heavy chain variable region: CDR1: GYTFTNYG (SEQ ID NO: 228), CDR2: INTYTGEP (SEQ ID NO: 229), and CDR3: AKYPHYYGSSHWYFDV (SEQ ID NO: 230). Embodiment 138. The method according to embodiment 136 or 137, wherein the antibody comprises the following CDRs in the light chain variable region: CDR1: QDISNY (SEQ ID NO: 226), CDR2: FTS, and CDR3: QQYSTVPWT (SEQ ID NO: 227), and the following CDRs in the heavy chain variable region: CDR1: GYTFTNYG (SEQ ID NO: 228), CDR2: INTYTGEP (SEQ ID NO: 229), and CDR3: AKYPHYYGSSHWYFDV (SEQ ID NO: 230). Embodiment 139. The antibody has the following amino acid sequence: The method according to any one of embodiments 134 to 138, wherein the antibody comprises a light chain comprising the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 231). Embodiment 140. The antibody has the following amino acid sequence: The method according to any one of embodiments 134 to 139, comprising a heavy chain comprising EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 232). Embodiment 141. The antibody has the following amino acid sequence: A light chain comprising DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 231), and the following amino acid sequence: The method according to any one of embodiments 134 to 140, comprising a heavy chain comprising EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 232). Embodiment 142. The method according to any one of embodiments 134 to 141, wherein the anti-VEGF antibody is an IgG1k isotype antibody. Embodiment 143. The method according to any one of embodiments 134 to 142, wherein the anti-VEGF antibody is bevacizumab. Embodiment 144. The method according to embodiment 143, wherein the anti-VEGF antibody is a Fab fragment of bevacizumab. Embodiment 145. The method according to embodiment 144, wherein the Fab fragment of bevacizumab is ranibizumab. Embodiment 146. The method according to embodiment 133, wherein the anti-angiogenic compound is a VEGF-binding protein. Embodiment 147. The method according to embodiment 146, wherein the VEGF-binding protein is a functional derivative of a VEGF receptor. Embodiment 148. The method according to embodiment 147, wherein the VEGF receptor is selected from the group consisting of VEGFR1 and VEGFR2. Embodiment 149. The method according to Embodiment 147 or 148, wherein the functional derivative of the VEGF receptor comprises at least one VEGF binding domain of VEGFR1 and / or VEGFR2. Embodiment 150. The method according to any one of Embodiments 147 to 149, wherein the VEGF binding protein is ziv aflibercept. Embodiment 151. The method according to Embodiment 146, wherein the VEGF binding protein is a VEGF binding anticalin. Embodiment 152. The method according to Embodiment 133, wherein the anti-angiogenic compound that binds to VEGF is a VEGF binding nucleic acid molecule. Embodiment 153. The method according to Embodiment 152, wherein the VEGF binding nucleic acid molecule is a VEGF binding D-nucleic acid molecule. Embodiment 154. The method according to Embodiment 153, wherein the VEGF binding D-nucleic acid molecule is a VEGF binding aptamer. Embodiment 155. The method according to Embodiment 154, wherein the VEGF binding D-nucleic acid molecule is pegaptanib (Macugen), which preferably binds to the VEGF isoform VEGF165. Embodiment 156. The method according to Embodiment 152, wherein the VEGF binding nucleic acid molecule is a VEGF binding L-nucleic acid molecule. Embodiment 157. The method according to Embodiment 156, wherein the VEGF binding nucleic acid molecule is a VEGF binding Spiegelmer. Embodiment 158. The method according to any one of Embodiments 125 to 131, wherein the anti-angiogenic compound binds to VEGFR. Embodiment 159. The method according to Embodiment 158, wherein the VEGFR is VEGFR1, VEGFR2, or both VEGFR1 and VEGFR2, and preferably, the VEGFR is VEGFR2. Embodiment 160. The method according to Embodiment 158 or 159, wherein the anti-angiogenic compound that binds to VEGF is selected from the group consisting of an anti-VEGFR antibody, a fragment of an anti-VEGFR antibody, a VEGFR binding protein, and a VEGFR binding nucleic acid molecule. The method according to Embodiment 160, wherein the anti-angiogenic compound is an anti-VEGFR antibody. The method according to Embodiment 161, wherein the anti-VEGFR antibody is a human anti-VEGFR antibody, a humanized anti-VEGFR antibody, or a human anti-VEGFR antibody. The method according to Embodiment 161 or 162, wherein the anti-VEGFR antibody comprises the following CDRs in the light chain variable region: CDR1: RASQGIDNWLG (SEQ ID NO: 233), CDR2: DASNLDT (SEQ ID NO: 234), CDR3: QQAKAFPPT (SEQ ID NO: 235). The method according to any one of Embodiments 161 to 163, wherein the anti-VEGFR antibody comprises the following CDRs in the heavy chain variable region: CDR1: GFTFSSYSMN (SEQ ID NO: 236), CDR2: SISSSSSYIYYADSVKG (SEQ ID NO: 237), and CDR3: VTDAFDI (SEQ ID NO: 238). The method according to any one of Embodiments 161 to 164, wherein the anti-VEGFR antibody comprises the following CDRs in the light chain variable region: CDR1: RASQGIDNWLG (SEQ ID NO: 233), CDR2: DASNLD (SEQ ID NO: 234), CDR3: QQAKAFPPT (SEQ ID NO: 2345); and the following CDRs in the heavy chain variable region: CDR1: GFTFSSYSMN (SEQ ID NO: 236), CDR2: SISSSSSYIYYADSVKG (SEQ ID NO: 237), and CDR3: VTDAFDI (SEQ ID NO: 238). Embodiment 166. The method according to any one of Embodiments 161 to 165, wherein the anti-VEGFR antibody comprises a light chain having the following amino acid sequence: DIQMTQSPSSVSASIGDRVTITCRASQGIDNWLGWYQQKPGKAPKLLIYDASNLDTGVPS RFSGSGSGTYFTLTISSLQAEDFAVYFCQQAKAFPPTFGGGTKVDIKRTVAAPSVFIFPP SDEQLKSGTA SVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC (SEQ ID NO: 239). Embodiment 167. The method according to any one of Embodiments 161 to 166, wherein the anti-VEGFR antibody comprises a heavy chain having the following amino acid sequence: EVQLVQSGGG LVKPGGSLRL SCAASGFTFS SYSMNWVRQA PGKGLEWVSS ISSSSSYIYY ADSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCARVT DAFDIWGQGT MVTVSSASTK GPSVLPLAPS SKSTSGGTAA LGCLVKDYFP EPVTVSWNSG ALTSGVHTFP AVLQSSGLYS LSSVVTVPSS SLGTQTYICN VNHKPSNTKV DKRVEPKSCD KTHTCPPCPA PELLGGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTL PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK (SEQ ID NO: 240). Embodiment 168. The anti-VEGFR antibody has the following amino acid sequence: A light chain comprising DIQMTQSPSSVSASIGDRVTITCRASQGIDNWLGWYQQKPGKAPKLLIYDASNLDTGVPS RFSGSGSGTYFTLTISSLQAEDFAVYFCQQAKAFPPTFGGGTKVDIKRTVAAPSVFIFPP SDEQLKSGTA SVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFN RGEC (SEQ ID NO: 239), and the following amino acid sequence: The method according to any one of Embodiments 161 to 167, comprising a heavy chain comprising EVQLVQSGGG LVKPGGSLRLSCAASGFTFS SYSMNWVRQA PGKGLEWVSS ISSSSSYIYY ADSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCARVT DAFDIWGQGT MVTVSSASTK GPSVLPLAPS SKSTSGGTAA LGCLVKDYFP EPVTVSWNSG ALTSGVHTFP AVLQSSGLYS LSSVVTVPSS SLGTQTYICN VNHKPSNTKV DKRVEPKSCD KTHTCPPCPA PELLGGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTL PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK (SEQ ID NO: 240). Embodiment 169. The method according to any one of Embodiments 161 to 168, wherein the anti-VEGFR antibody is an IgG isotype antibody. Embodiment 170. The method according to any one of Embodiments 161 to 169, wherein the anti-VEGFR antibody is ramucirumab (Cyramza). The method according to Embodiment 160, wherein the anti-angiogenic compound is a VEGFR-binding protein. The method according to Embodiment 171, wherein the VEGFR-binding protein is a VEGFR-binding anti-kallikrein. The method according to Embodiment 160, wherein the anti-angiogenic compound that binds to VEGFR is a VEGFR-binding nucleic acid molecule. The method according to Embodiment 173, wherein the VEGFR-binding nucleic acid molecule is a VEGFR-binding D-nucleic acid molecule. The method according to Embodiment 174, wherein the VEGFR-binding D-nucleic acid molecule is a VEGFR-binding aptamer. The method according to Embodiment 152, wherein the VEGFR-binding nucleic acid molecule is a VEGFR-binding L-nucleic acid molecule. The method according to Embodiment 176, wherein the VEGF-binding nucleic acid molecule is a VEGFR-binding Spiegelmer. The method according to any one of Embodiments 121 to 124, wherein the anti-angiogenic compound inhibits the signal transduction of VEGFR. The method according to Embodiment 178, wherein the VEGFR is VEGFR1, VEGFR2, VEGFR3, and any combination thereof. The method according to Embodiment 178 or 179, wherein the anti-angiogenic compound inhibits the intracellular signal transduction of the VEGFR. The method according to any one of Embodiments 178 to 180, wherein the anti-angiogenic compound inhibits the signal cascade induced by the binding of VEGF to the VEGFR. The method according to Embodiment 180 or 181, wherein the anti-angiogenic compound is a tyrosine kinase inhibitor. The method according to Embodiment 182, wherein the tyrosine kinase inhibitor is a multi-tyrosine kinase inhibitor. Embodiment 184. The method according to Embodiment 182 or 183, wherein the tyrosine kinase inhibitor is selected from the group consisting of pazopanib (Votrient), sunitinib (Sutent), sorafenib (Nexavar), axitinib (Inlyta), ponatinib (Iclusig), cabozantinib (Cometriq / Cabometyx), regorafenib (Stivarga), vandetanib (Caprelsa), and lenvatinib (Lenvima). Embodiment 185. The method according to any one of Embodiments 1 to 184, further comprising the step of administering chemotherapy to the subject. Embodiment 186. The method according to Embodiment 185, wherein the chemotherapy is chemotherapy for the treatment of the cancer. Embodiment 187. The method according to Embodiment 185 or 186, wherein the tumor is glioblastoma and the chemotherapy is chemotherapy for the treatment of glioblastoma. Embodiment 188. The method according to any one of Embodiments 185 to 187, wherein the chemotherapy is selected from the group consisting of temozolomide, lomustine, irinotecan, dianhydrogalactitol, a combination of irinotecan and bevacizumab, and a combination of procarbazine, lomustine, and vincristine (PCV). Embodiment 189. The method according to Embodiment 188, comprising the step of administering temozolomide to the subject. Embodiment 190. The method according to any one of Embodiments 1 to 189, wherein when the CXCL12 antagonist is preferably a Spiegelmer or an aptamer, more preferably a Spiegelmer, the CXCL12 antagonist is administered intravenously or subcutaneously to the subject. Embodiment 191. The method according to any one of Embodiments 1 to 190, preferably Embodiment 190, wherein the CXCL12 antagonist is continuously administered to the subject over a period of time. Embodiment 192. The method according to Embodiment 191, wherein the period is 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, or a period longer than that. Embodiment 193. The method according to any one of Embodiments 1 to 192, preferably any one of Embodiments 190 to 192, wherein the CXCL12 antagonist is a CXCL12-binding nucleic acid molecule containing a 40 kDa PEG moiety. Embodiment 194. The method according to Embodiment 193, wherein the CXCL12 antagonist is NOX-A12. Embodiment 195. The method according to Embodiment 193 or 194, wherein the weekly dose of the CXCL12 antagonist administered to the subject is about 100 mg to about 1,000 mg, preferably about 200 mg to about 600 mg, more preferably about 400 to about 600 mg. Embodiment 196. The method according to any one of Embodiments 1 to 195, preferably any one of Embodiments 190 to 195, wherein the anti-angiogenic compound is administered to the subject intravenously, subcutaneously, or orally, preferably the anti-angiogenic compound is administered intravenously, and more preferably, when the anti-angiogenic compound is bevacizumab, it is administered intravenously. Embodiment 197. The method according to any one of Embodiments 1 to 196, preferably any one of Embodiments 190 to 196, wherein the anti-angiogenic compound is administered weekly, biweekly, or every three weeks over a period of time. Embodiment 198. The method according to Embodiment 197, wherein the period is 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks. Embodiment 199. (a) The anti-angiogenic compound is administered weekly, and the period is 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks. (b) The anti-angiogenic compound is administered biweekly, and the period is 2 weeks, 4 weeks, or 6 weeks, or (c) The anti-angiogenic compound is administered every three weeks, and the period is 3 weeks or 6 weeks. The method according to Embodiment 197 or 198. Embodiment 200. The anti-angiogenic compound is an anti-VEGF antibody, preferably an IgG antibody, more preferably bevacizumab, and the method according to any one of Embodiments 1 to 199, preferably any one of Embodiments 190 to 199. Embodiment 201. (a) When the anti-angiogenic compound is administered weekly, the dosage is 5 mg / kg per kg of the subject's body weight. (b) When the anti-angiogenic compound is administered every other week, the dosage is 10 mg / kg per kg of the subject's body weight, or (c) When the anti-angiogenic compound is administered every three weeks, the dosage is 15 mg / kg per kg of the subject's body weight. The method according to any one of Embodiments 1 to 200, preferably any one of Embodiments 190 to 200. Embodiment 202. The chemotherapy is administered to the subject, and the method according to any one of Embodiments 1 to 201, preferably any one of Embodiments 190 to 201. Embodiment 203. The chemotherapy is administered to the subject daily, and the method according to any one of Embodiments 1 to 202, preferably any one of Embodiments 190 to 202. Embodiment 204. The chemotherapy is administered to the subject over a period, preferably the period is 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks, and more preferably the chemotherapy is administered to the subject daily over the period, and the method according to any one of Embodiments 1 to 203, preferably any one of Embodiments 190 to 203. Embodiment 205. The chemotherapy includes the step of administering temozolomide to the subject, and the method according to any one of Embodiments 1 to 204, preferably any one of Embodiments 190 to 203, more preferably any one of Embodiments 202 to 204. Embodiment 206. The period during which the chemotherapy is administered to the subject is the same as the period during which the radiotherapy is administered to the subject, and the method according to any one of Embodiments 1 to 205, preferably any one of Embodiments 190 to 205. Embodiment 207. The daily dose of temozolomide administered to the subject is about 75 mg / m per body surface area 2 The method according to any one of Embodiments 190 to 206, preferably any one of Embodiments 202 to 206. Embodiment 208. The radiation therapy is administered to the subject for several days of the week over a period of time, the method according to any one of Embodiments 1 to 207, preferably any one of Embodiments 190 to 207. Embodiment 209. The radiation therapy is administered to the subject for 5 consecutive days of the week over a period of time, the method according to Embodiment 208. Embodiment 210. The period is 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks, the method according to Embodiment 208 or 209. Embodiment 211. The radiation therapy is fractionated radiation therapy, the method according to any one of Embodiments 208 to 210. Embodiment 212. The absorbed radiation dose is about 2 Gy or about 2.7 Gy per session, preferably there are daily sessions for 5 consecutive days in a week, the method according to any one of Embodiments 208 to 211. Embodiment 213. The period constitutes the first stage of treatment, the method according to any one of Embodiments 191 to 212. Embodiment 214. In the said first stage of treatment, - The CXCL12 antagonist is NOX-A12, which is continuously intravenously administered to the subject over a period of 6 weeks, and the weekly dose administered to the subject is about 200 mg / kg per body weight of the subject, about 400 mg / kg per body weight of the subject, about 600 mg / kg per body weight of the subject, or about 800 mg / kg per body weight of the subject. - The anti-angiogenic compound is bevacizumab, which is intravenously administered to the subject every other week over a period of 6 weeks, and the bi-weekly dose administered to the subject is 10 mg / kg per body weight of the subject. - The radiotherapy is standard fractionated radiotherapy, which is administered to the subject for 5 consecutive days out of a week over a period of 6 weeks, with a daily absorbed radiation dose of about 2 Gy, or The radiotherapy is hypofractionated radiotherapy, which is administered to the subject for 5 consecutive days out of a week over a period of 3 weeks, with a daily absorbed radiation dose of about 2.7 Gy. The method according to embodiment 213. In the first stage of the treatment according to embodiment 215, chemotherapy is administered to the subject, and the chemotherapy is temozolomide, which is continuously administered orally or intravenously to the subject over a period of 6 weeks. The daily dose of temozolomide administered to the subject is 75 mg / m per body surface area of the subject 2 of the method according to embodiment 214. Embodiment 216. The second stage of the treatment follows the first stage of the treatment and is the method according to any one of embodiments 213 to 215. Embodiment 217. In the second stage of the treatment, the CXCL12 antagonist is intravenously administered to the subject, and the method is as described in embodiment 216. Embodiment 218. The CXCL12 antagonist is continuously administered to the subject over a second period, and the method is as described in embodiment 217. Embodiment 219. The second period is an arbitrary period from 1 week to 20 weeks, or a period longer than 20 weeks, and the method is as described in embodiment 218. Embodiment 220. The CXCL12 antagonist is a CXCL12-binding nucleic acid molecule containing a 40 kDa PEG moiety, and the method is as described in any one of embodiments 216 to 219. Embodiment 221. The CXCL12 antagonist is NOX-A12, and the method is as described in embodiment 220. Embodiment 222. The weekly dose of the CXCL12 antagonist administered to the subject is about 200 mg, about 400 mg, about 600 mg, or about 800 mg, and the method is as described in embodiment 220 or 221. The method according to any one of embodiments 216 to 222, wherein the anti-angiogenic compound is administered intravenously to the subject. The method according to any one of embodiments 216 to 223, wherein the anti-angiogenic compound is administered weekly, bi-weekly, or every three weeks over a second period. The method according to embodiment 224, wherein the second period is any period from 1 week to 20 weeks, or a period longer than 20 weeks. Embodiment 226. (a) The anti-angiogenic compound is administered weekly and the second period is a multiple of 1 week. (b) The anti-angiogenic compound is administered bi-weekly and the second period is a multiple of 2 weeks, or (c) The anti-angiogenic compound is administered every three weeks and the second period is a multiple of 3 weeks. The method according to embodiment 224 or 225. The method according to any one of embodiments 216 to 226, wherein the anti-angiogenic compound is an anti-VEGF antibody, preferably an IgG antibody, more preferably bevacizumab. Embodiment 228. (a) When the anti-angiogenic compound is administered weekly, the dosage is about 5 mg / kg per kg of the subject's body weight. (b) When the anti-angiogenic compound is administered bi-weekly, the dosage is about 10 mg / kg per kg of the subject's body weight, or (c) When the anti-angiogenic compound is administered every three weeks, the dosage is about 15 mg / kg per kg of the subject's body weight. The method according to any one of embodiments 216 to 227. The method according to any one of embodiments 216 to 228, wherein in the second stage of treatment, the chemotherapy is administered to the subject in cycles of 28 days. Embodiment 230. In the second stage, the chemotherapy is administered for 1, 2, 3, 4, 5, 6 cycles, or more than 6 cycles, preferably 6 cycles, and is any one of Embodiments 216 to 229, preferably the method according to Embodiment 229. Embodiment 231. The method according to Embodiment 230, wherein the cycle is 28 days. Embodiment 232. The method according to Embodiment 231, wherein temozolomide is administered to the subject for the first 5 days of the cycle, followed by a 23-day drug holiday. Embodiment 233. The method according to any one of Embodiments 229 to 232, wherein the chemotherapy comprises temozolomide. Embodiment 234. The daily dose of temozolomide is about 150 to about 200 mg / m 2 per body surface area of the subject, and is the method according to Embodiment 233. Embodiment 235. In the second stage of treatment, - The CXCL12 antagonist is NOX-A12, which is continuously intravenously administered to the subject for a period of about 1 to about 20 weeks. The weekly dose administered to the subject is about 200 mg / kg per body weight of the subject, about 400 mg / kg per body weight of the subject, about 600 mg / kg per body weight of the subject, or about 800 mg / kg per body weight of the subject. - The anti-angiogenic compound is bevacizumab, which is intravenously administered to the subject every other week for a period of about 1 to about 20 weeks. The bi-weekly dose administered to the subject is 10 mg / kg per body weight of the subject. The method according to any one of Embodiments 216 to 234. Embodiment 236. In the second stage of treatment, - The CXCL12 antagonist is NOX-A12, which is continuously intravenously administered to the subject for a period of about 1 to about 20 weeks. The weekly dose administered to the subject is about 200 mg / kg per body weight of the subject, about 400 mg / kg per body weight of the subject, about 600 mg / kg per body weight of the subject, or about 800 mg / kg per body weight of the subject. - The anti-angiogenic compound is bevacizumab, which is intravenously administered to the subject every other week over a period of about one week to about twenty weeks, and the bi-weekly dose administered to the subject is about 10 mg / kg per body weight of the subject. - The chemotherapy is temozolomide, which is administered to the subject orally or intravenously during 6 cycles, each cycle consisting of 28 days, and temozolomide is administered to the subject on each of the 1st, 2nd, 3rd, 4th, and 5th days of each cycle, and the daily dose of temozolomide administered to the subject is about 150 to about 200 mg / m 2 of the body surface area of the subject. The method according to any one of embodiments 216 to 234. Embodiment 237. In the second stage of treatment, radiotherapy is not administered to the subject, the method according to any one of embodiments 226 to 236, preferably embodiment 235 or 236.

[0013] More specifically, the problem underlying the present invention is solved in a first aspect by a method for treating a tumor in a subject, the method comprising administering to the subject - a C-X-C motif chemokine 12 (CXCL12) antagonist, - radiotherapy, and - an anti-angiogenic compound , wherein the tumor is a brain tumor.

[0014] More specifically, the problem underlying the present invention is solved in a second aspect by a C-X-C motif chemokine 12 (CXCL12) antagonist for use in a method for treating a brain tumor in a subject, the method comprising administering to the subject - a C-X-C motif chemokine 12 (CXCL12) antagonist, - radiotherapy, and - an anti-angiogenic compound , wherein the tumor is a brain tumor.

[0015] More specifically, the problem underlying the present invention is solved in a third aspect by radiotherapy for use in a method for treating a brain tumor in a subject, the method comprising administering to the subject - a C-X-C motif chemokine 12 (CXCL12) antagonist, - radiotherapy, and - an anti-angiogenic compound wherein the tumor is a brain tumor.

[0016] More specifically, the problem underlying the present invention is solved in a fourth aspect by an anti-angiogenic compound for use in a method for treating a brain tumor in a subject, the method comprising administering to the subject - a C-X-C motif chemokine 12 (CXCL12) antagonist, - radiotherapy, and - an anti-angiogenic compound wherein the tumor is a brain tumor.

[0017] More specifically, the problem underlying the present invention is solved in a fifth aspect by chemotherapy for use in a method for treating a brain tumor in a subject, the method comprising administering to the subject - a C-X-C motif chemokine 12 (CXCL12) antagonist, - radiotherapy, - an anti-angiogenic compound, and - chemotherapy wherein the tumor is a brain tumor.

Mode for Carrying Out the Invention

[0018] Any embodiment of the first aspect is also an embodiment of each and any one of the second, third, fourth, and fifth aspects, and vice versa, as will be understood by those skilled in the art. Embodiments of the invention are also understood by those skilled in the art to be embodiments of the first, second, third, fourth, and fifth aspects of the invention, unless explicitly stated otherwise. The same applies when reference is made to embodiments of the invention.

[0019] to a subject - a C-X-C motif chemokine 12 (CXCL12) antagonist, - radiotherapy, - an anti-angiogenic compound, and - chemotherapy The method of the invention for treating a brain tumor, which comprises the step of administering, can in principle be applied to any brain tumor.

[0020] Brain tumors can be classified based on the cells that form such tumors or based on the microscopic appearance of the tumor cells. The most common brain tumors are gliomas, meningiomas, pituitary adenomas, vestibular schwannomas, and medulloblastomas. Gliomas include several subtypes such as astrocytomas, oligodendrogliomas, ependymomas, and choroid plexus papillomas. The classification based on the microscopic appearance of the cells forming the brain tumor is based on grades I-IV as defined in the introduction incorporated herein by reference.

[0021] In one embodiment of the invention, the brain tumor is a grade IV brain tumor. A particularly preferred brain tumor is glioblastoma, which is a grade IV astrocytoma. Among glioblastomas, a further distinction can be made between supratentorial glioblastoma and infratentorial glioblastoma. Both forms can be treated by the method of the invention, and supratentorial glioblastoma occurs more frequently.

[0022] Brain tumors treatable by the method of the invention can be further or alternatively characterized from the perspective of treatment options available to or already applied to a subject suffering from the tumor.

[0023] In one embodiment of the present invention, the tumors that can be treated by the method of the present invention are those that are sensitive to radiotherapy, those that can be treated by radiotherapy, or those that have been treated by radiotherapy before administering the method of the present invention to a subject suffering from a brain tumor. This type of radiotherapy is known to those skilled in the art. It will be understood by those skilled in the art that this type of radiotherapy can be the same type of radiotherapy administered to a subject in accordance with the present invention, the respective disclosures of which are incorporated herein by reference.

[0024] In one embodiment of the present invention, the tumors that can be treated by the method of the present invention are those that remain after surgery, typically incompletely resected tumors. As shown above, surgery is still one of the most frequently used treatments for the treatment of brain tumors, and it will be recognized that some tumors cannot be treated by surgery because the associated tissue damage caused by surgery can result in even more severe overall damage or even death than the brain tumor itself. However, when a brain tumor is treated by surgery, some residual tumor tissue can escape surgical removal, typically due to its small size or its location within the subject's brain. The location within the subject's brain can be inaccessible to surgery for mechanical reasons, such as the inability to access the tumor tissue due to non-tumor brain tissue, or for medical reasons, such as the tumor tissue being too close to an important structure that should not be damaged for the health of the subject. This type of residual tumor tissue can be subjected to the method of the present invention in one embodiment of the present invention.

[0025] Means for typically detecting postoperatively a tumor that is an incompletely resected tumor are magnetic resonance imaging (MRI) scans. Preferably, the MRI scan is a contrast-enhanced MRI scan, more preferably a T1-weighted contrast-enhanced MRI scan. The basis of T1-weighted imaging is longitudinal relaxation, i.e., the return of the longitudinal magnetization after excitation to its equilibrium value. T1-weighted magnetic resonance images are typically created by using short echo times (TE) and repetition times (TR). The final image is a reflection of more than one of these pulse sequence parameters weighted according to the type of sequence and its timing. The T1 signal mainly determines the contrast and brightness in this type of image, although the proton density will always contribute to the image intensity. The T1 dependence is mainly determined by the repetition time or any prepulse (such as in an inversion recovery pulse sequence). Accordingly, in one embodiment, the tumor is a tumor that provides a signal in a T1-weighted MRI scan; in a preferred embodiment, the tumor is an incompletely resected tumor that provides a signal in a T1-weighted MRI scan. In both embodiments, the T1-weighted MRI scan is a contrast-enhanced T1-weighted MRI scan.

[0026] Due to larger longitudinal and transverse magnetization, fat will have a higher signal and will appear bright on T1-contrast MR images. Conversely, water has low longitudinal magnetization before the RF pulse, and thus the low transverse magnetization after the RF pulse produces a low signal that appears dark on T1-contrast images. Often, a gadolinium compound, a paramagnetic contrast agent, is administered and both pre-contrast and post-contrast T1-weighted images are acquired.

[0027] In one embodiment of the present invention, a tumor to be treated can be characterized by certain biochemical features. Such biochemical features include, but are not limited to, resistance to pharmaceuticals, and the secretion of factors such as chemokines and / or angiogenic factors. It will be understood by those skilled in the art that tumors can also exhibit combinations of such biochemical features.

[0028] In one embodiment of the present invention, resistance to one or more pharmaceuticals is resistance to temozolomide (TMZ). Temozolomide is an alkylating agent and is sold under the trade name Temodar among others. It is used as a standard therapy in the treatment of brain tumors such as glioblastoma (primary treatment) and anaplastic astrocytoma (secondary treatment). It is taken orally or via intravenous infusion. The most commonly seen side effects associated with temozolomide are nausea, vomiting, constipation, loss of appetite, alopecia (hair loss), headache, fatigue, seizures, rash, neutropenia or lymphopenia (low white blood cell count), and thrombocytopenia (low platelet count).

[0029] Resistance to temozolomide therapy is a major cause of treatment failure in gliomas and glioblastomas, and therefore it is extremely important to provide means for treating gliomas and glioblastomas resistant to temozolomide therapy. The demethylase O6-methylguanine-DNA methyltransferase (MGMT) is involved in intrinsic TMZ resistance (TMZ-R) and recurrence by directly removing the alkyl group from the O6 position of guanine. However, epigenetic silencing of MGMT is frequently seen in gliomas, and even gliomas with low MGMT levels (deficiency and low expression) are sufficient to confer TMZ-R, suggesting the existence of an MGMT-independent mechanism of acquired TMZ-R. Gliomas with low MGMT levels exhibited genetic mutations. For example, recurrent gliomas exhibited transcriptional silencing of the MGMT gene, followed by dysfunction of the mismatch repair (MMR) system and hyperfunction of the DNA repair system. Having a deficiency in MMR activated the DNA damage repair (DDR) system, leading to TMZ-R. Therefore, the functional availability of the DDR system probably regulates the response of recurrent gliomas to TMZ. Thus, the hypothesis is put forward that DDR signaling is enhanced in TMZ-R glioma cells and that it contributes to their phenotypic resistance. Rho-associated kinase 2 (ROCK2) is hyperactivated in the TMZ-R model, and inhibition of ROCK2 has been shown to reverse TMZ-R with increased TMZ sensitivity, suggesting an association between ROCK2 and TMZ-R, but the mechanism remains unclear (Zhang X et al., Cell Death & Disease 13, Article number 138 (2022)).

[0030] Accordingly, in one embodiment of the present invention, the tumor is a tumor that is non-responsive or resistant to temozolomide. Also, in one embodiment of the present invention, the tumor is a glioblastoma having a non-methylated O 6 -methylguanine DNA methyltransferase (MGMT) promoter status.

[0031] In one embodiment of the present invention, the tumor is a tumor that secretes factors, and the factors are chemokines and / or angiogenic factors. It is particularly preferred that the tumor expresses both chemokines and angiogenic factors. In a preferred embodiment, the chemokine is CXC motif chemokine 12 (CXCL12), and the angiogenic factor is vascular endothelial growth factor (VEGF).

[0032] CXC motif chemokine 12 (CXCL12), also known as stromal cell-derived factor 1 (SDF-1), is a major chemokine involved in the attraction or repulsion of many types of leukocytes by signaling through the receptor CXC motif chemokine receptor 4 (CXCR4) and potentially through its alternative receptor CXCR7.

[0033] CXCL12 is an angiogenic CXC motif chemokine that does not contain the ELR motif typical of IL-8-like chemokines (Salcedo, Wasserman et al. 1999; Salcedo and Oppenheim 2003), but binds to and activates the G protein-coupled receptor CXCR4. As a result of alternative splicing, there are additional forms of CXCL12, such as CXCL12α (68 amino acids, SEQ ID NO: 1), and CXCL12β (SEQ ID NO: 2), which has five additional amino acids at the C-terminus compared to CXCL12α (Shirozu, Nakano et al. 1995).

[0034] The amino acid sequence conservation between CXCL12 from different species is remarkable: human CXCL12α (SEQ ID NO: 1) and murine CXCL12α (SEQ ID NO: 3) are virtually identical. There is only a single conservative change from V to I at position 18 (Shirozu, Nakano et al. 1995).

[0035] Since the CXCL12 receptor CXCR4 is widely expressed on leukocytes, mature dendritic cells, endothelial cells, brain cells, and megakaryocytes, the activity of CXCL12 is multifaceted. This chemokine exhibits the broadest range of biological functions of any that have been identified to date. The most significant functional effects of CXCL12 are - Homing and attachment of epithelial cells to sites of neovascularization in the choroid portion of the retina; - CXCL12 is required to maintain stem and progenitor cells, such as hematopoietic progenitor (usually CD34+) cells, in the adult bone marrow; - SDF1 supports the proliferation of pre-B cells and increases the growth of bone marrow B cell precursors, which induces the specific migration of pre- and pro-B cells, but does not act as a significant chemoattractant for mature B cells; - CXCL12 is one of the most potent T cell chemoattractants; and - CXCL12 and its receptor CXCR4 are essential for embryonic development.

[0036] Alterations in the expression levels of CXCL12 or its receptor CXCR4, or alterations in the responses to these molecules, are associated with retinopathy (Brooks, Caballero et al. 2004; Butler, Guthrie et al. 2005; Meleth, Agron et al. 2005); breast cancer (Muller, Homey et al. 2001; Cabioglu, Sahin et al. 2005), ovarian cancer (Scotton, Wilson et al. 2002), pancreatic cancer (Koshiba, Hosotani et al. 2000), thyroid cancer (Hwang, Chung et al. 2003), and nasopharyngeal cancer (Wang, Wu et al. 2005); glioma (Zhou, Larsen et al. 2002); neuroblastoma (Geminder, Sagi-Assif et al. 2001); B-cell chronic lymphocytic leukemia (Burger, Tsukada et al. 2000); WHIM syndrome (WHIM is an abbreviation for Warts, Hypogammaglobulinemia, Infections, Myelokathexis syndrome) (Gulino, Moratto et al. 2004; Balabanian, Lagane et al. 2005b; Kawai, Choi et al. 2005); immunodeficiency syndromes (Arya, Ginsberg et al. 1999; Marechal, Arenzana-Seisdedos et al. 1999; Soriano, Martinez et al. 2002); pathological angiogenesis (Salvucci, Yao et al. 2002; Yamaguchi, Kusano et al. 2003; Grunewald, Avraham et al. 2006); inflammation (Murdoch 2000; Fedyk, Jones et al. 2001; Wang, Guan et al. 2001); multiple sclerosis (Krumbholz, Theil et al. 2006); rheumatoid arthritis / osteoarthritis (Buckley, Amft et al. 2000; Kanbe, Takagishi et al. 2002; Grassi, Cristino et al.It is said to be associated with many human diseases such as (2004).

[0037] Tumors, including solid and hematological neoplasms and malignancies, are not just a mass of cancer cells: infiltration of tumors by immune cells is a characteristic of cancer. Many human cancers have a complex chemokine network that affects the degree and phenotype of this infiltrate, as well as tumor growth, survival, migration, and angiogenesis. Most solid tumors contain many non-malignant stromal cells. In fact, the number of stromal cells can sometimes be greater than that of cancer cells. The main stromal cells found in cancer are macrophages, lymphocytes, endothelial cells, and fibroblasts.

[0038] Cells from different cancer types have different profiles of chemokine-receptor expression, but the CXCL12 receptor CXCR4 is most commonly found on mouse and human tumor cells: tumor cells from at least 23 different types of human cancers of epithelial, mesenchymal, and hematopoietic origin express CXCR4 (Balkwill 2004), and CXCL12 is the only known ligand for CXCR4. In addition to the bone marrow and secondary lymphoid tissues where it is constitutively expressed, CXCL12 is found at the primary tumor site in lymphoma (Corcione, Ottonello et al. 2000), as well as in brain tumors of both neuronal and astrocytic lineages. Furthermore, it is present at high levels in ovarian cancer (Scotton, Wilson et al. 2002) and pancreatic cancer (Koshiba, Hosotani et al. 2000), as well as at the sites of metastasis in breast cancer (Muller, Homey et al. 2001) and thyroid cancer (Hwang, Chung et al. 2003), neuroblastoma, and hematological malignancies (Geminder, Sagi-Assif et al. 2001).

[0039] In addition to CXCR4, another CXCL12 receptor has been identified: RDC1 / CXCR7 (Balabanian, Lagane et al. 2005a, Burns, Summers et al. 2006). In vitro and in vivo studies using prostate cancer cell lines suggest that alterations in CXCR7 / RDC1 expression are associated with enhanced adhesion and invasion activities in addition to a survival advantage. In vitro and in vivo studies have shown that both receptors for CXCL12, namely CXCR4 and CXCR7, promote tumor growth, metastatic potential, and resistance to (chemotherapy-induced) apoptosis in a number of tumors, such as breast cancer, glioblastoma, ovarian cancer, neuroblastoma, lung cancer, colorectal, and prostate cancer (Burns et al, 2006; Li et al, 2008; Scotton et al, 2002; Yang et al, 2008; Zagzag et al, 2008).

[0040] Therefore, CXCR4 and CXCR7 expression appears to be a common feature of several tumors.

[0041] Vascular endothelial growth factor (VEGF) is a basic heparin-binding homodimeric glycoprotein of 45,000 daltons. These properties are consistent with those of VEGF165, the major VEGF isoform. The human VEGF gene is located on chromosome 6p21.3 and is organized into eight exons separated by seven introns. Alternative splicing has been shown to result in four major VEGF isoforms (VEGF121, VEGF165, VEGF189, and VEGF206) consisting of 121, 165, 189, and 206 amino acids, respectively, after cleavage of the signal sequence (Ferrara & Henzel 1989, Biochem Biophys Res Comm 161:851; Houck et al. 1991, Mol Endocrinol 5:1806; Leung et al. 1989, Science 246:1306). Low-frequency splice variants have also been reported, including VEGF145 (Poltorak et al. 1997, J Biol Chem 272:7151), VEGF183 (Jingjing et al. 1999, Invest Ophthalmol Vis Sci 40:752), and VEGF165b (Bates et al. 2002, Cancer Res 62:4123). Additional levels of regulation of VEGF biological activity are provided by proteolytic cleavage mechanisms that include all VEGF isoforms, resulting in the VEGF110 form (Houck et al. 1992, J Biol Chem 267:26031).

[0042] VEGF is the main regulator of angiogenesis during normal and pathological processes, including those associated with tumor growth (reviewed in Ferrara & Gerber 2001, Acta Haematol 106:148, or Ferrara et al. 2003, Nat Med 9:669). VEGF has a major regulatory function during developmental angiogenesis (Ferrara et al. 1996, Nature 380:439; Carmeliet et al. 1996, Nature 380:14756). The well-documented in vitro activities of VEGF are its ability to promote the growth of vascular endothelial cells (ECs) derived from arteries, veins, and lymphatics (reviewed in Ferrara & Davis-Smyth 1997, Endocr Rev 18:4), as well as certain non-endothelial cells (reviewed in Matsumoto & Claesson-Welsh 2001, Sci STKE 2001(112):p.RE21). VEGF has been shown to be a survival factor for ECs both in vitro and in vivo (Yuan et al. 1996, Proc Natl Acad Sci USA 93:14765; Gerber et al. 1998, J Biol Chem 273:13313; Gerber et al. 1998, J Biol Chem 273:30336; Benjamin et al. 1999, J Clin Invest 103:159). VEGF stimulated the production of surfactant protein by cultured type II alveolar epithelial cells (Compernolle et al. 2002, Nat Med 8:702). VEGF induces vasodilation in vitro (Ku et al. 1993, Am J Physiol 265:H586) and is thought to play a role in inflammation due to its ability to induce vascular leakage (Dvorak et al. 1995, Am J Pathol 146:1029; Senger et al. 1983, Science 219:983). VEGF exhibits chemotactic effects on endothelial cells and increases the expression of proteolytic enzymes in endothelial cells involved in matrix degradation.VEGF also has an effect on bone marrow-derived cells and promotes monocyte activation and chemotaxis (Clauss et al. 1990, J Exp Med 172:1535). VEGF enhanced colony formation by a mature subset of granulocyte macrophages and erythroid progenitor cells stimulated with colony-stimulating factors (Bates et al. 2002, Cancer Res 62:4123). VEGF also exhibits an immune effect through inhibition of the maturation of antigen-presenting dendritic cells (Gabrilovitch et al. 1996, Nat Med 2:1096).

[0043] VEGF binds to two related receptor tyrosine kinases (RTKs) named Flt-1 (VEGFR-1) (de Vries et al. 1992, Science 255:989; Shibuya et al. 1990, Oncogene 5:519) and KDR / Flk-1 (VEGFR-2) (Terman et al. 1992, Biochem Biophys Res Commun 187:1579). The fms-like tyrosine kinase Flt-4 (VEGFR-3) is a member of the same family of RTKs but is not a receptor for VEGF; instead, it binds to VEGFC and VEGFD8. In addition to these RTKs, VEGF interacts with a family of co-receptors called neuropilins. Binding of VEGF to VEGFR-1 and VEGFR-2 induces homodimerization of the two receptor subunits, which in turn induces autophosphorylation of their tyrosine kinase domains located in the cytoplasm. Autophosphorylation of the tyrosine kinase domain then participates in a series of specific signaling events and ultimately regulates the various biological activities of VEGF on endothelial cells. Most of the mitogenic and survival activities of VEGF appear to be mediated by VEGFR-2, including the expression of the anti-apoptotic proteins Bcl-2 and A1. Survival signaling by VEGFR-2 is mediated by the PI3 kinase / Akt pathway (Gerber et al. 1998, J Biol Chem 273:30336). VEGFR-2 has also been shown to induce other signaling pathways, including phospholipase C gamma and the mitogen-activated protein kinase MAPK p44 / 42 (Gille et al. 2001, J Biol Chem 276:3222).

[0044] In one embodiment of the present invention, CXCL12 is expressed at the tip of the tumor (the outermost tumor section where the tumor spreads towards the surrounding tissue), in the compartment having hyperplastic blood vessels (blood vessels having a thickened wall due to the proliferation of endothelial cells), and / or in the compartment of tumor microvascular proliferation (see, for example, Comba A et al., Front. Oncol., 05 August 2021; https: / / doi.org / 10.3389 / fonc.2021.703764).

[0045] In one embodiment of the present invention, VEGF is expressed in the cellular tumor compartment of the tumor where the proliferation of tumor cells is most prominent, in the compartment where pseudopalisading cells (characterized by the rosette-like organization of tumor cells at the edge of the tumor necrosis area) are present, and / or in the necrotic peripheral zone of the dead malignant cells of the tumor.

[0046] In a particularly preferred embodiment of the present invention, CXCL12 is expressed at the tip of the tumor, in the hyperplastic blood vessels of the tumor, and / or in the tumor microvascular proliferation zone, and VEGF is expressed in the cellular tumor compartment of the tumor, by the tumor pseudopalisading cells and / or the necrotic peripheral zone of the tumor.

[0047] According to the method of the present invention, a CXCL12 antagonist is administered to a subject suffering from a tumor. The CXCL12 antagonist used according to the present invention can use different modes of action. Such modes of action include the CXCL12 antagonist inhibiting the signaling of CXCL12 via one or both of the CXCL12 receptors C-X-C chemokine receptor type 4 (CXCR4) and C-X-C chemokine receptor type 7 (CXCR7). In a preferred embodiment, the CXCL12 antagonist directly binds to CXCL12 by physical interaction or the like, and as a result, preferably, one or several effects otherwise caused by CXCL12 will be reduced or eliminated. Preferably, such reduced or eliminated effects are effective or useful in the treatment of the disease. In a more preferred embodiment, the binding of the CXCL12 antagonist to CXCL12 interferes with the binding of CXCL12 to CXCL12 receptors such as CXCR4 and / or CXCR7. In an alternative preferred embodiment, the CXCL12 antagonist directly binds to a CXCL12 receptor such as CXCR4 and / or CXCR7 by physical interaction or the like, and as a result, CXCL12 no longer binds to such a CXCL12 receptor, or binds at a reduced rate, with a reduced affinity, or with a reduced shorter binding on-time. Preferably, due to such interference with the binding of CXCL12 to the CXCL12 receptor, one or several effects otherwise caused by CXCL12 will be reduced or eliminated. Preferably, such reduced or eliminated effects are effective or useful in the treatment of the disease.

[0048] Such inhibition of CXCL12 signaling via one or both of the CXCL12 receptors CXCR4 and CXCR7 can be achieved, as will be appreciated by those skilled in the art, by (a) providing a molecule that binds to CXCL12 and inhibits the binding of CXCL12 to CXCR4 and / or CXCR7, (b) providing a molecule that binds to CXCR4 and inhibits the binding of CXCL12 to CXCR4, and (c) providing a molecule that binds to CXCR7 and inhibits the binding of CXCL12 to CXCR7. Such molecules that bind to CXCL12 are preferably selected from the group consisting of aptamers, preferably CXCL12-binding aptamers, Spiegelmers, preferably CXCL12-binding Spiegelmers, antibodies, preferably CXCL12-binding antibodies, proteins that bind to CXCL12, CXCL12-binding anticalins, CXCL12-binding peptides, fusion proteins containing a CXCL12-binding moiety, and CXCL12-binding small molecules.

[0049] Such molecules that bind to CXCR4 and inhibit CXCL12 signaling are preferably selected from the group consisting of aptamers, preferably CXCR4-binding aptamers, Spiegelmers, preferably CXCR4-binding Spiegelmers, antibodies, preferably CXCR4-binding antibodies, proteins that bind to CXCR4, CXCR4-binding anticalins, CXCR4-binding peptides, fusion proteins containing a CXCR4-binding moiety, and CXCR4-binding small molecules.

[0050] Such molecules that bind to CXCR7 are preferably selected from the group consisting of aptamers, preferably CXCR7-binding aptamers, Spiegelmers, preferably CXCR7-binding Spiegelmers, antibodies, preferably CXCR7-binding antibodies, proteins that bind to CXCR4, CXCR7-binding anticalins, CXCR7-binding peptides, fusion proteins containing a CXCR7-binding moiety, and CXCR7-binding small molecules.

[0051] Such inhibition of CXCL12 signaling via one or both of the CXCL12 receptors CXCR4 and CXCR7 can be achieved by a compound that binds to CXCR4 and / or CXCR7, and such binding of the compound disrupts signaling, preferably by disrupting the signaling characteristics of CXCR4 and / or CXCR7 rather than by interfering with the binding of CXCL12 to CXCR4 and / or CXCR7, as will also be understood by those skilled in the art.

[0052] Molecules that bind to CXCR4 and, by virtue of their binding to CXCR4, interfere with the signaling characteristics of CXCR4, preferably the signaling induced by the binding of CXCL12 to CXCR4, are preferably selected from the group consisting of aptamers, preferably CXCR4-binding aptamers, Spiegelmers, preferably CXCR4-binding Spiegelmers, proteins that bind to CXCR4, antibodies or antigen-binding portions of antibodies, preferably CXCR4-binding antibodies or CXCR4-binding fragments of CXCR4-binding antibodies, anticalins, preferably CXCR4-binding anticalins, CXCR4-binding peptides, fusion proteins containing a CXCR4-binding portion, and CXCR4-binding small molecules.

[0053] The generation and identification of each of such aptamers, Spiegelmers, antibodies, binding proteins, anticalins, peptides, fusion proteins, and small molecules are known to those skilled in the art and are also disclosed herein.

[0054] In a preferred embodiment of the invention, CXCL12 is human CXCL12, CXCR4 is human CXCR4, and CXCR7 is human CXCR7.

[0055] Regarding this part of the method of the present invention, namely administering a CXCL12 antagonist to a subject suffering from a tumor, it should be recognized that animal studies have shown that radiation-induced devascularization is efficiently restored by angiogenesis after mobilization of bone marrow-derived cells, which leads to rapid tumor recurrence (Kioi et al. 2010, J Clin Invest 120:694). CXCL12 is presumed to play an important role in angiogenesis by mobilizing endothelial cells and other bone marrow-derived angiogenesis-promoting cells through CXCR4- and CXCR7-dependent mechanisms. Notably, irradiation further increases CXCL12 expression (Kioi et al. 2010, J Clin Invest 120:694; Kozin et al. 2010, Cancer Res 70:5679; Liu et al. 2014, Neuro Oncol 16:21).

[0056] In one embodiment of the present invention, the CXCL12 antagonist inhibits the signal transduction of CXCL12 via one or both of the CXCL12 receptors C-X-C chemokine receptor type 4 (CXCR4) and C-X-C chemokine receptor type 7 (CXCR7). In a preferred embodiment thereof, the CXCL12 antagonist inhibits the binding of CXCL12 to the CXCL12 receptor C-X-C chemokine receptor type 4 (CXCR4) and / or to the CXCL12 receptor C-X-C chemokine receptor type 7 (CXCR7). The interaction between CXCL12 and its receptor CXCR4 is also referred to as the CXCL12-CXCR4 axis. Such a CXCL12-CXCR4 axis can be targeted by a CXCL12 antagonist. Similarly, the interaction between CXCL12 and its receptor CXCR7 is also referred to as the CXCL12-CXCR7 axis. Such a CXCL12-CXCR7 axis can be targeted by a CXCL12 antagonist. In an even more preferred embodiment, such binding of CXCL12 to the receptor is brought about by a CXCL12 antagonist that binds to CXCL12.

[0057] Among several CXCL12 antagonists that bind to CXCL12, there are nucleic acid molecules that bind to CXCL12. Such CXCL12-binding nucleic acid molecules can be CXCL12-binding aptamers or CXCL12-binding Spiegelmers.

[0058] An aptamer is a nucleic acid molecule made from D-nucleotides as a constituent unit that specifically binds to a target molecule by a mechanism different from Watson-Crick base pairing. A Spiegelmer is a nucleic acid molecule made from L-nucleotides as a constituent unit that specifically binds to a target molecule by a mechanism different from Watson-Crick base pairing. Both aptamers and Spiegelmers can be made from ribonucleotides, deoxyribonucleotides, or a combination of both ribonucleotides and deoxyribonucleotides. Aptamers and Spiegelmers themselves are known to those skilled in the art. Among several, they are described in "The Aptamer Handbook" (eds. Klussmann, 2006), and Spiegelmers containing both ribonucleotides and deoxyribonucleotides are disclosed in International Patent Application WO2012 / 095303.

[0059] In one embodiment of the present invention, the CXCL12 antagonist is an L-nucleic acid molecule that binds to CXCL12, and the L-nucleic acid molecule is preferably selected from the group consisting of type B CXCL12-binding nucleic acid molecules, type C CXCL12-binding nucleic acid molecules, type A CXCL12-binding nucleic acid molecules, and type D CXCL12-binding nucleic acid molecules, which are defined in more detail herein.

[0060] CXCL12-binding L-nucleic acid molecules can be characterized from the perspective of stretches of nucleotides, which are also referred to herein as boxes (see Example 1).

[0061] The various types of CXCL12-binding nucleic acid molecules comprise three different stretches of nucleotides: a first terminal stretch of nucleotides, a central stretch of nucleotides, and a second terminal stretch of nucleotides. Generally, the CXCL12-binding nucleic acid molecules of the present invention comprise, at their 5' and 3' termini, a first terminal stretch of nucleotides and a second terminal stretch of nucleotides (also referred to as the 5'-terminal stretch of nucleotides and the 3'-terminal stretch of nucleotides), which are terminal stretches of nucleotides. The first terminal stretch of nucleotides and the second terminal stretch of nucleotides can hybridize to each other due to their base complementarity in principle, and when hybridization occurs, a double-stranded structure is formed. However, such hybridization is not necessarily realized intramolecularly under physiological and / or non-physiological conditions. The three stretches of nucleotides of the CXCL12-binding nucleic acid molecule, namely the first terminal stretch of nucleotides, the central stretch of nucleotides, and the second terminal stretch of nucleotides, are arranged relative to each other in the 5'→3' direction: the first terminal stretch of nucleotides - the central stretch of nucleotides - the second terminal stretch of nucleotides. However, alternatively, the second terminal stretch of nucleotides, the central stretch of nucleotides, and the first terminal stretch of nucleotides are arranged relative to each other in the 5'→3' direction.

[0062] Differences in the sequences of defined boxes or stretches between the various CXCL12-binding nucleic acid molecules affect the binding affinity for CXCL12. Based on the binding assays for the various CXCL12-binding nucleic acid molecules used in accordance with the present invention, the central stretch and the nucleotides forming it are, individually and more preferably as a whole, essential for binding to human CXCL12.

[0063] The terms "stretch" and "stretch of nucleotides" are used interchangeably herein unless the contrary is indicated.

[0064] In a preferred embodiment of the present invention, the nucleic acid used in accordance with the present invention is a single nucleic acid molecule. In a further embodiment, the single nucleic acid molecule exists as a number of single nucleic acid molecules or as a number of single nucleic acid molecule species.

[0065] It will be appreciated by those skilled in the art that the nucleic acid molecules used in accordance with the present invention preferably consist of nucleotides that are covalently linked to each other through phosphodiester bonds or linkages.

[0066] It is within the scope of the present invention that the nucleic acid used in accordance with the present invention contains two or more stretches or portions thereof that can in principle hybridize to each other. Such hybridization results in the formation of a double-stranded structure. It will be appreciated by those skilled in the art that such hybridization may or may not occur, particularly under in vitro and / or in vivo conditions. Also, in the case of such hybridization, it is not necessarily the case that hybridization occurs over the entire length of the two stretches such that such hybridization and hence the formation of a double-stranded structure can in principle occur, at least based on the base pairing rules. As preferably used herein, a double-stranded structure is a structure formed by a portion of a nucleic acid molecule or by two or more separate strands or two spatially separated stretches of a single strand of a nucleic acid molecule, and there is at least one, preferably two or more, base pairings that are preferably base pairings according to the Watson-Crick base pairing rules. It will also be appreciated by those skilled in the art that other base pairings such as Hoogsteen base pairing can be present in or form such a double-stranded structure. It should also be recognized that the property of two stretches hybridizing indicates that such hybridization is presumed to occur, preferably due to the base complementarity of the two stretches.

[0067] In a preferred embodiment of the present invention, the term "arrangement" as used herein means the order or sequence of structural or functional traits or elements described herein in relation to the nucleic acids disclosed herein.

[0068] It will be appreciated by those skilled in the art that the nucleic acids used in accordance with the present invention may bind to CXCL12. Without wishing to be bound by any theory, CXCL12 binding results from a combination of three-dimensional structural traits or elements of the nucleic acid molecule caused by the orientation and folding pattern of the primary sequence of nucleotides that form such traits or elements, preferably such traits or elements being the first terminal stretch of nucleotides, the central stretch of nucleotides, and the second terminal stretch of nucleotides of the CXCL12-binding nucleic acid molecule. It is clear that the individual traits or elements can be formed by a variety of different individual sequences, the degree of variation of which can vary depending on the three-dimensional structure that such elements or traits must form. The overall binding characteristics of the claimed nucleic acid result from the interrelationship of the various elements and traits that ultimately result in the interaction of the claimed nucleic acid with its target, namely CXCL12. Again, without wishing to be bound by any theory, the central stretch of nucleotides characteristic of the CXCL12-binding nucleic acid appears to be important for mediating the binding of the claimed nucleic acid molecule to CXCL12. Thus, the nucleic acids according to the present invention are suitable for interaction with CXCL12. Also, it will be appreciated by those skilled in the art that the nucleic acids used in accordance with the present invention are antagonists to CXCL12. For this reason, the nucleic acids according to the present invention are suitable for the treatment and prevention of brain cancer according to the method of the present invention.

[0069] The nucleic acid molecules used in accordance with the present invention shall also include nucleic acids that are essentially homologous to the specific sequences disclosed herein. The term "substantially homologous" is understood to mean that the homology exceeds at least 75%, preferably 85%, more preferably 90%, most preferably 95%, 96%, 97%, 98%, or 99%, etc.

[0070] The actual percentage of homologous nucleotides present in the nucleic acids used according to the present invention will depend on the total number of nucleotides present in the nucleic acid. The percentage of modification can be based on the total number of nucleotides present in the nucleic acid.

[0071] The homology between two nucleic acid molecules can be determined as is known to those skilled in the art. More specifically, a sequence comparison algorithm can be used to calculate the percent sequence homology of a test sequence compared to a reference sequence based on specified program parameters. The test sequence is preferably a sequence or nucleic acid molecule that is said to be homologous, or a sequence or nucleic acid molecule that is being tested to determine whether it is homologous to a different nucleic acid molecule and, if so, to what extent. Such a different nucleic acid molecule is also referred to as a reference sequence. In one embodiment, the reference sequence is a nucleic acid molecule described herein, preferably a nucleic acid molecule having a sequence according to any one of SEQ ID NO:5 to SEQ ID NO:225, more preferably a nucleic acid molecule having a sequence according to any one of SEQ ID NO:22, SEQ ID NO:28, SEQ ID NO:120, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:223, SEQ ID NO:224, SEQ ID NO:84, SEQ ID NO:146, SEQ ID NO:142, SEQ ID NO:143, and SEQ ID NO:144. The optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman (Smith & Waterman, 1981), by the homology alignment algorithm of Needleman & Wunsch (Needleman & Wunsch, 1970), by the similarity search method of Pearson & Lipman (Pearson & Lipman, 1988), by computerized execution of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics software package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection.

[0072] One example of an algorithm suitable for determining percent sequence identity is the algorithm used in the Basic Local Alignment Search Tool (hereinafter “BLAST”), see, e.g., Altschul et al. (Altschul et al., 1990 and Altschul et al., 1997). Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (hereinafter “NCBI”). The default parameters used in determining sequence identity using available software from NCBI, such as BLASTN (for nucleotide sequences) and BLASTP (for amino acid sequences), are described in McGinnis et al. (McGinnis et al., 2004).

[0073] The nucleic acid molecules used in accordance with the present invention are also intended to include nucleic acid molecules having a certain degree of identity compared to the nucleic acid molecules disclosed herein and defined by their nucleotide sequences. More specifically, the present invention provides at least 75%, preferably 85%, more preferably 90%, most preferably greater than 95%, 96%, 97%, 98%, or 99% identity compared to the nucleic acid molecules disclosed herein and defined by their nucleotide sequences or a portion thereof. Such nucleic acid molecules having identity are also included.

[0074] The terms nucleic acid and nucleic acid molecule are used interchangeably herein. Further, the nucleic acid molecules used in accordance with the present invention preferably include such nucleic acid molecules in which the nucleic acid molecule or the portion thereof includes a nucleic acid sequence or a part thereof disclosed herein, to the extent that the nucleic acid molecule or the portion is involved in or can bind to CXCL12, such as metabolites or derivatives of the nucleic acid molecules used in accordance with the present invention. Such nucleic acid molecules can be derived, for example, by truncation, from those disclosed herein. The truncation can relate to either or both ends of the nucleic acid molecules disclosed herein. Also, the truncation can relate to the internal sequence of nucleotides, i.e., it can relate to the nucleotides between the 5' and 3' terminal nucleotides, respectively. Further, the truncation is intended to include deletions of as few as a single nucleotide from the sequences of the nucleic acids disclosed herein. The truncation can also relate to stretches of more than one of the nucleic acid molecules used in accordance with the present invention, and the stretches can be as short as 1 nucleotide in length. The binding of the nucleic acid molecules used in accordance with the present invention can be determined by those skilled in the art using routine experimentation or by using or adopting the methods described herein, preferably in the Examples section.

[0075] In addition, it is within the scope of the present invention that one or some parts of the nucleic acid molecules used according to the present invention are present as D-nucleic acids, or at least one or some parts of the nucleic acid molecules are L-nucleic acids. The term "part" of a nucleic acid molecule shall mean just 1 nucleotide. Such nucleic acid molecules are generally referred to herein as D- and L-nucleic acids, respectively. Therefore, in a particularly preferred embodiment, the nucleic acid molecule used according to the present invention consists of L-nucleotides and contains at least 1 D-nucleotide. Such D-nucleotides are preferably attached to a part different from the stretch defining the nucleic acid molecule used according to the present invention, preferably to such a part where the interaction with other parts of the nucleic acid molecule is involved. Preferably, such D-nucleotides are attached to either end of any stretch used according to the present invention and to the end of any nucleic acid molecule, respectively. In a more preferred embodiment, such D-nucleotides can act as spacers or linkers, preferably attaching modifications such as PEG and HES to the nucleic acids according to the present invention.

[0076] The nucleic acid molecules used in accordance with the present invention are part of a longer nucleic acid molecule, which contains several parts, and at least one such part being the nucleic acid molecule or a part thereof used in accordance with the present invention is also within the scope of the present invention. Other parts of these longer nucleic acid molecules can be either one or several D-nucleic acids or L-nucleic acids. In connection with the present invention, any combination can be used. These other parts of the longer nucleic acid molecule can exhibit functions different from binding, preferably binding to CXCL12. One possible function is to enable interaction with other molecules, such other molecules being preferably different from CXCL12, for example for immobilization, cross-linking, detection, or amplification. In a further embodiment of the present invention, the nucleic acid molecule used in accordance with the present invention contains some of the nucleic acid molecules used in accordance with the present invention as individual parts or combined parts. Such nucleic acid molecules containing some of the nucleic acid molecules used in accordance with the present invention are also encompassed by the term longer nucleic acid.

[0077] As used herein, an L-nucleic acid molecule is a nucleic acid consisting of L-nucleotides, preferably consisting entirely of L-nucleotides.

[0078] As used herein, a D-nucleic acid molecule is a nucleic acid molecule consisting of D-nucleotides, preferably consisting entirely of D-nucleotides.

[0079] The terms nucleic acid and nucleic acid molecule are used interchangeably herein unless expressly indicated to the contrary.

[0080] Also, unless indicated to the contrary, any nucleotide sequence is specified herein in the 5'→3' direction.

[0081] As preferably used herein, any position of a nucleotide is determined or referenced relative to the 5' end of an array, stretch, or sub-stretch. Thus, the second nucleotide is the second nucleotide counted from the 5' end of each of the array, stretch, and sub-stretch, respectively. Accordingly, the second nucleotide from the last is the second nucleotide counted from the 3' end of each of the array, stretch, and sub-stretch, respectively.

[0082] The nucleic acid molecules used according to the present invention can exist as single-stranded or double-stranded nucleic acids, regardless of whether they exist as D-nucleic acids, L-nucleic acids, or D,L-nucleic acids, or whether they are DNA or RNA, which is also within the scope of the present invention. Typically, the nucleic acid molecules used according to the present invention are single-stranded nucleic acids that exhibit a secondary structure defined by the primary sequence and can thus form a tertiary structure. However, the nucleic acid molecules used according to the present invention can also be double-stranded in the sense that two strands that are complementary or partially complementary to each other hybridize to each other, or can hybridize to each other at least based on their base complementarity.

[0083] The nucleic acid molecules used according to the present invention can be modified. Such modifications can relate to a single nucleotide of the nucleic acid and are well known in the art. Examples of such modifications are described, among others, by Venkatesan et al. (Venkatesan, Kim et al., 2003) and Kusser (Kusser, 2000). Such modifications can be an H atom, an F atom, or an O-CH3 group or an NH2-group at the 2'-position of the individual nucleotides that make up the nucleic acid. Also, the nucleic acid molecules used according to the present invention can contain at least one LNA nucleotide. In one embodiment, the nucleic acid according to the present invention consists of LNA nucleotides.

[0084] In one embodiment of the present invention, the nucleic acid molecule used in accordance with the present invention can be a nucleic acid molecule divided into a number of parts. As used herein, a nucleic acid molecule divided into a number of parts refers to a nucleic acid molecule consisting of at least two separate nucleic acid strands. These at least two nucleic acid strands form a functional unit, and the functional unit is a ligand for the target molecule. The at least two nucleic acid strands can be derived from any of the nucleic acid molecules used in accordance with the present invention by cleaving the nucleic acid molecule to produce two strands or by synthesizing one nucleic acid corresponding to the first part of the entire nucleic acid molecule and the other nucleic acid molecule corresponding to the second part of the entire nucleic acid molecule. It should be recognized that both cleavage and synthesis can be applied to produce nucleic acid molecules divided into a number of parts having more than two strands as exemplified above. In other words, the at least two separate nucleic acid strands are typically different from two strands that are complementary to each other and hybridize, although to some extent there can be a certain degree of complementarity between the at least two separate nucleic acid strands, whereby such complementarity can result in hybridization of the separate strands.

[0085] Finally, it is also within the scope of the present invention that a completely closed, i.e., circular, structure is achieved for the nucleic acid molecule used in accordance with the present invention, i.e., the nucleic acid molecule is closed in one embodiment, preferably by covalent linkage, and more preferably such covalent linkage is made between the 5' and 3' ends of the nucleic acid sequence disclosed herein or any derivative thereof.

[0086] An option for determining the binding constant of the nucleic acid molecule used in accordance with the present invention is the use of the methods described in Examples 3 and 4, which confirm the above finding that the nucleic acid molecule used in accordance with the present invention exhibits a preferred K D value range. A suitable measure for expressing the strength of the binding between an individual nucleic acid molecule and a target, which in this case is CXCL12, is the so-called K D value, which itself and the method for its determination are known to those skilled in the art.

[0087] Preferably, the K value indicated by the nucleic acid molecule used according to the present invention is less than 1 μM. A K value of about 1 μM is said to be characteristic of non-specific binding of the nucleic acid to the target. As will be appreciated by those skilled in the art, the K values of a group of compounds such as the nucleic acid molecules used according to the present invention are within a certain range. The above-mentioned K value of about 1 μM is the preferred upper limit of the K value. The lower limit of the K value of the target-binding nucleic acid may be as low as about 10 picomoles or higher. That the K value of an individual nucleic acid molecule that binds to CXCL12 is preferably within this range is within the scope of the present invention. The preferred range can be defined by selecting any first numerical value within this range and any second numerical value within this range. The preferred upper limit K value is 250 nM and 100 nM, and the preferred lower limit K value is 50 nM, 10 nM, 1 nM, 100 pM, and 10 pM. A more preferred upper limit K value is 2.5 nM, and a more preferred lower limit K value is 100 pM. D value is less than 1 μM. The K D value is said to be characteristic of non-specific binding of the nucleic acid to the target. As will be appreciated by those skilled in the art, the K D values of a group of compounds such as the nucleic acid molecules used according to the present invention are within a certain range. The above-mentioned K D value is the preferred upper limit of the K D value. The lower limit of the K value of the target-binding nucleic acid D may be as low as about 10 picomoles or higher. The K D values of individual nucleic acid molecules that bind to CXCL12 are preferably within this range, which is within the scope of the present invention. The preferred range can be defined by selecting any first numerical value within this range and any second numerical value within this range. The preferred upper limit K D value is 250 nM and 100 nM, and the preferred lower limit K D value is 50 nM, 10 nM, 1 nM, 100 pM, and 10 pM. A more preferred upper limit K D value is 2.5 nM, and a more preferred lower limit K D value is 100 pM.

[0088] In addition to the binding characteristics of the nucleic acid molecules used according to the present invention, the nucleic acid molecules used according to the present invention inhibit the function of each target molecule, which in this case is CXCL12. Inhibition of the function of CXCL12, such as inhibition of the stimulation of each receptor described above, is achieved by binding of the nucleic acid molecule used according to the present invention to CXCL12 and forming a complex between the nucleic acid molecule and CXCL12. Such a complex of the nucleic acid molecule and CXCL12 cannot stimulate receptors that are normally stimulated by CXCL12, such as CXCR4 and CXCR7. Thus, inhibition of receptor function by the nucleic acid molecule is independent of each receptor that can be stimulated by CXCL12, but results from preventing the stimulation of the receptor by CXCL12 by the nucleic acid molecule.

[0089] The possibility of determining the inhibition constant of a nucleic acid molecule used according to the present invention is the use of the methods described in Examples 5 and 6 (for CXCR4 and CXCR7 respectively), which confirms the above finding that the nucleic acid molecule used according to the present invention exhibits a preferred inhibition constant that enables the use of the nucleic acid molecule in the claimed method of treatment and in any treatment scheme including such method of treatment. In this case, a suitable measure for expressing the strength of the inhibitory effect of an individual nucleic acid molecule on the interaction between a target, which is CXCL12 in this case, and its respective receptor is the so-called half-maximal inhibitory concentration (abbreviation IC 50 ), which is known to those skilled in the art per se as well as the method for its determination.

[0090] Preferably, the IC 50 value shown by the nucleic acid molecule used according to the present invention is below 1 μM. An IC 50 value of about 1 μM is said to be characteristic of non-specific inhibition of target function by a nucleic acid molecule. As will be recognized by those skilled in the art, the IC 50 values of a group of compounds such as the nucleic acid molecule used according to the present invention are within a certain range. The above IC 50 value of about 1 μM is the preferred upper limit of the IC 50 value. The lower limit of the IC 50 of a target-binding nucleic acid molecule may be as low as about 10 picomoles or higher. That the IC 50 values of individual nucleic acid molecules that bind to CXCL12 are preferably within this range is within the scope of the present invention. A preferred range can be defined by selecting any first numerical value within this range and any second numerical value within this range. The preferred upper limit IC 50 values are 250 nM and 100 nM, and the preferred lower limit IC 50 values are 50 nM, 10 nM, 1 nM, 100 pM, and 10 pM. A more preferred upper limit IC 50 value is 2.5 nM, and a more preferred lower limit IC 50 value is 100 pM.

[0091] The nucleic acid molecules used in accordance with the present invention can have any length, provided that they can still bind to the target molecule. It will be recognized in the art that there are preferred lengths for the nucleic acid molecules used in accordance with the present invention. Typically, the length is 15 to 120 nucleotides. It will be recognized by those skilled in the art that any integer between 15 and 120 can be a possible length for the nucleic acid molecules used in accordance with the present invention. More preferred ranges for the length of the nucleic acid molecules used in accordance with the present invention are lengths of about 20 to 100 nucleotides, about 20 to 80 nucleotides, about 20 to 60 nucleotides, about 20 to 50 nucleotides, and about 29 to 45 nucleotides.

[0092] It is within the scope of the present invention that the nucleic acids used in accordance with the present invention preferably are a high molecular weight moiety and / or preferably include a moiety that enables modification of the characteristics of the nucleic acid, particularly from the viewpoint of residence time in a living body, preferably in a human body. Particularly preferred embodiments of such modification are PEGylation and HESylation of the nucleic acids used in accordance with the present invention. As used herein, PEG represents poly(ethylene glycol) and HES represents hydroxyethly starch. The PEGylation preferably used herein is a modification of the nucleic acids used in accordance with the present invention, such modification consisting of a PEG moiety, which is attached to the nucleic acids used in accordance with the present invention. The HESylation preferably used herein is a modification of the nucleic acids used in accordance with the present invention, such modification consisting of a HES moiety, which is attached to the nucleic acids used in accordance with the present invention. These modifications, as well as the process of modifying nucleic acids using such modifications, are described in European Patent Application EP1306382, the disclosure of which is incorporated herein by reference in its entirety.

[0093] When PEG is such a high molecular weight moiety, the molecular weight is preferably from about 20,000 to about 120,000 Da, more preferably from about 30,000 to about 80,000 Da, and most preferably about 40,000 Da. When HES is such a high molecular weight moiety, the molecular weight is preferably from about 50 to about 1000 kDa, more preferably from about 100 to about 700 kDa, and most preferably from about 200 to about 500 kDa. HES exhibits a molar substitution of 0.1 to 1.5, more preferably 1 to 1.5, and exhibits a substitution pattern expressed as a C2 / C6 ratio of approximately 0.1 to 15, preferably approximately 3 to 10. The process of HES modification is described, for example, in German patent application DE12004006249.8, the disclosure of which is incorporated herein by reference in its entirety.

[0094] Modifications can, in principle, be made at any position in the nucleic acid molecule used according to the invention. Preferably, such modifications are made to the 5'-terminal nucleotide, the 3'-terminal nucleotide, and / or any nucleotide between the 5'- and 3'-nucleotides of the nucleic acid molecule.

[0095] The modifications, and preferably the PEG and / or HES moieties, can be attached directly or indirectly, preferably through a linker, to the nucleic acid molecule used according to the invention. It is also within the scope of the invention for the nucleic acid molecule used according to the invention to comprise one or more modifications, preferably one or more PEG and / or HES moieties. In one embodiment, an individual linker molecule attaches more than one PEG moiety or HES moiety to the nucleic acid molecule used according to the invention. The linker used in connection with the present invention can itself be linear or branched. This type of linker is known to those skilled in the art and is further described in patent applications WO2005 / 074993 and WO2003 / 035665.

[0096] In a preferred embodiment of the present invention, the linker is a biodegradable linker. The biodegradable linker can modify the characteristics of the nucleic acid used according to the present invention, particularly from the perspective of residence time, especially in the body of an animal, preferably in the human body, due to the release of the modification from the nucleic acid according to the present invention. The use of a biodegradable linker may enable better control of the residence time of the nucleic acid molecule used according to the present invention. Preferred embodiments of such biodegradable linkers include, but are not limited to, the biodegradable linkers described in International Patent Applications WO2006 / 052790, WO2008 / 034122, WO2004 / 092191, and WO2005 / 099768.

[0097] It is within the scope of the present invention that the modification or the modifying group is a biodegradable modification, and the biodegradable modification can be attached to the nucleic acid molecule used according to the present invention directly or indirectly, preferably through a linker. The biodegradable modification can modify the characteristics of the nucleic acid used according to the present invention, particularly from the perspective of residence time, especially in the body of an animal, preferably in the human body, due to the release or decomposition of the modification from the nucleic acid molecule used according to the present invention. The use of a biodegradable modification may enable better control of the residence time of the nucleic acid according to the present invention. Preferred embodiments of such biodegradable modifications include, but are not limited to, those described in International Patent Applications WO2002 / 065963, WO2003 / 070823, WO2004 / 113394, and WO2000 / 41647, preferably those described in WO2000 / 41647, page 18, lines 4 to 24, which are biodegradable.

[0098] In addition to the modifications described above, other modifications can be used to modify the characteristics of the nucleic acid used according to the present invention, and such other modifications can be selected from the group of chains of proteins, lipids such as cholesterol, and saccharides such as amylase and dextran.

[0099] Without wishing to be bound by any theory, it is believed that the pharmacokinetics will change by modifying the nucleic acid molecules used according to the invention, preferably using a physiologically acceptable polymer, more particularly one or more high molecular weight moieties of the polymers disclosed herein. More particularly, due to the increase in the molecular weight of such modified nucleic acids and due to the fact that the nucleic acid molecules used according to the invention are not metabolized, especially when in the L-form, excretion from the animal body, preferably from the mammalian body, more preferably from the human body, is believed to decrease. Since excretion typically occurs via the kidneys, the glomerular filtration rate of the nucleic acid molecules thus modified is significantly reduced compared to nucleic acid molecules without such high molecular weight modification, which results in an increased residence time in the animal body. In this context, it is particularly noteworthy that the specificity of the nucleic acid molecules used according to the invention is not affected in a detrimental manner, notwithstanding such high molecular weight modification. To that extent, the nucleic acid molecules used according to the invention have, inter alia, surprising features not normally expected from pharmaceutically active compounds, whereby pharmaceutical formulations providing sustained release are not necessarily required to provide sustained release of the nucleic acid molecules used according to the invention. Rather, the nucleic acid molecules used according to the invention in their modified forms containing high molecular weight moieties can themselves already be used as sustained release formulations, since they act, due to their modification, as if they had already been released from a sustained release formulation. To that extent, the modification of the nucleic acid molecules used according to the invention disclosed herein, and the nucleic acid molecules thus modified used according to the invention, and any composition containing the same, can provide a defined, preferably controlled, pharmacokinetics and biodistribution. This includes the residence time in the circulation and the distribution to tissues. Such modifications are further described in patent application WO2003 / 035665.

[0100] However, it is within the scope of the present invention that the nucleic acid molecules used in accordance with the present invention do not contain any modifications, particularly high molecular weight modifications such as PEGylation or HESylation. Such embodiments are particularly preferred when the nucleic acid molecules used in accordance with the present invention exhibit preferential distribution to any target organ or tissue in the body, or when rapid clearance of the nucleic acid molecules used in accordance with the present invention from the body after administration is desired. Nucleic acid molecules used in accordance with the present invention having a preferential distribution profile to any target organ or tissue in the body will be able to establish an effective local concentration in the target tissue while keeping the systemic concentration of the nucleic acid molecule low. This is thought to enable the use of low doses, which is not only beneficial from an economic perspective, but also reduces the unnecessary exposure of other tissues to the nucleic acid agent, and thus reduces the potential risk of side effects. In particular, rapid clearance of the nucleic acid molecules used in accordance with the present invention from the body after administration may be desired in the case of in vivo imaging or specific therapeutic dosing requirements using the nucleic acid molecules used in accordance with the present invention or a medicament containing the same.

[0101] In one embodiment of the present invention, a pharmaceutical composition containing a nucleic acid molecule used in accordance with the present invention comprises at least one of the nucleic acid molecules and a pharmaceutically acceptable excipient. Such a pharmaceutical composition may additionally contain one or more further pharmaceutically active compounds. Such pharmaceutically acceptable excipients can be, for example, water, buffer solutions, PBS, glucose solutions, preferably 5% glucose salt balanced solutions, starch, saccharides, gelatin, or any other acceptable carrier substance. Such excipients are generally known to those skilled in the art. It will be recognized by those skilled in the art that any embodiment, use, and aspect of or related to the medicament of the present invention is also applicable to the pharmaceutical composition of the present invention, and vice versa.

[0102] The indications, diseases, and disorders for treatment and / or prevention for which the nucleic acid, pharmaceutical composition, and medicament are used or prepared in accordance with the present invention are caused by the direct or indirect involvement of CXCL12 in their respective pathogenesis mechanisms.

[0103] In a preferred embodiment of the present invention, the CXCL12 antagonist is NOX-A12. NOX-A12 (CAS-RN 1322069-19-1) is an (L)-oligonucleotide of 45 (L)-ribonucleotides (molecular weight 14.5 kDa) terminating at the 5'-end in a hexylamino linker, to which a branched 40 kDa monomethoxypolyethylene glycol (PEG) moiety is covalently attached. The PEG moiety increases the plasma half-life of the molecule. NOX-A12 is suitable for parenteral administration and has good subcutaneous bioavailability. NOX-A12 is the nucleic acid molecule of SEQ ID NO: 28. It will be appreciated by those skilled in the art that a preferred nucleic acid molecule that binds to CXCL12 is an L-nucleic acid molecule having at least 80% identity with the L-nucleic acid molecule of SEQ ID NO: 28 or SEQ ID NO: 22.

[0104] The chemical structure of NOX-A12 can also be depicted as the 5'-ester with N'-[ω-methoxypoly(oxy-1,2-ethanediyl)]-acetyl-9-amino-8-oxo-7-aza-nonyloxy phosphate, sodium salt, which is N-[ω-methoxypoly(oxy-1,2-ethanediyl)] in the form of L-guanyl-(3’→5’)-L-cytidyl-(3’→5’)-L-guanyl-(3’→5’)-L-uridyl-(3’→5’)-L-guanyl-(3’→5’)-L-guanyl-(3’→5’)-L-uridyl-(3’→5’)-L-guanyl-(3’→5’)-L-uridyl-(3’→5’)-L-guanyl-(3’→5’)-L-adenyl-(3’→5’)-L-uridyl-(3’→5’)-L-cytidyl-(3’→5’)-L-uridyl-(3’→5’)-L-adenyl-(3’→5’)-L-guanyl-(3’→5’)-L-adenyl-(3’→5’)-L-uridyl-(3’→5’)-L-guanyl-(3’→5’)-L-uridyl-(3’→5’)-L-adenyl-(3’→5’)-L-uridyl-(3’→5’)-L-uridyl-(3’→5’)-L-guanyl-(3’→5’)-L-guanyl-(3’→5’)-L-cytidyl-(3’→5’)-L-uridyl-(3’→5’)-L-guanyl-(3’→5’)-L-adenyl-(3’→5’)-L-uridyl-(3’→5’)-L-cytidyl-(3’→5’)-L-cytidyl-(3’→5’)-L-uridyl-(3’→5’)-L-adenyl-(3’→5’)-L-guanyl-(3’→5’)-L-uridyl-(3’→5’)-L-cytidyl-(3’→5’)-L-adenyl-(3’→5’)-L-guanyl-(3’→5’)-L-guanyl-(3’→5’)-L-uridyl-(3’→5’)-L-adenyl-(3’→5’)-L-cytidyl-(3’→5’)-L-guanyl-(3’→5’)-L-cytidyl-(3’→5’), and additionally contains a 40 kDa PEG moiety attached to the 5'-end of the nucleotide sequence by the linker shown above. In the case of NOX-A12, the 40 kDa PEG moiety increases the plasma half-life in humans from a few minutes (for non-PEGylated L-nucleic acid molecules) to almost two days.

[0105] In one embodiment of the present invention, the CXCL12 antagonist is olaptesed pegol. Preferably, the terms NOX-A12 and olaptesed pegol are used synonymously.

[0106] In one embodiment of the present invention, the CXCL12 antagonist is a CXCL12-binding aptamer provided by Aptitude Medical Systems, which was originally provided for the treatment of exudative AMD.

[0107] In another embodiment of the present invention, a CXCL12 antagonist that binds to CXCL12 and inhibits the binding of CXCL12 to the CXCL12 receptors CXCR4 and / or CXCR7 is an antibody that binds to CXCL12, preferably an antibody that binds to CXCL12 and inhibits the binding of CXCL12 to the CXCL12 receptors CXCR4 and / or CXCR7. In this embodiment, CXCL12 is the target or antigen of the antibody. In a preferred embodiment, the antibody is a monoclonal antibody.

[0108] Monoclonal antibodies directed against CXCL12 are known in the art and include, among others, mAB-30D8 (provided by Genentech), 1131-H12 or 1143-H1 (both provided by iOnctura). The antibody sequence of antibody mAB-30D8, including its humanized form, is disclosed in Zhong C et al. (Cancer Therapy Res (2013) 19(16):4433-4445; see Supplementary Figure 4).

[0109] In a further embodiment of the invention, CXCL12 is bound to a protein, such a protein can be a CXCL12-binding TRAP protein (provided by OncoTrap, Inc.) or a CXCL12-binding anticalin. In one embodiment, the TRAP protein is provided through TRAP protein-encoding mRNA delivered to the tumor. The expression product of the mRNA is secreted by the transfected cells, diffuses throughout the tumor environment, captures CXCL12 with nanomolar binding affinity, and as a result, CXCL12 is trapped and its function in the tumor microenvironment is lost. In one embodiment and as preferably used herein, the anticalin is a target-binding polypeptide. This class of target-binding polypeptides is described, among others, in German patent application DE19742706.

[0110] In addition to the above classes of compounds, it will be appreciated by those skilled in the art that small molecules can also be used in principle as antagonists of CXCL12. The latter is even more due to the direct or indirect interaction, preferably the direct physical interaction, of such small molecules, and the binding of CXCL12 to its receptors CXCR4 and / or CXCR7 is inhibited. As preferably used herein, a small molecule is a molecule having a molecular weight of less than 900 Da. Alternatively, a small molecule complies with Lipinski's rule of five, i.e., the compound violates no more than one of the following criteria: a total of no more than 5 hydrogen bond donors (the total number of nitrogen-hydrogen and oxygen-hydrogen bonds); no more than 10 hydrogen bond acceptors (all nitrogen or oxygen atoms); a molecular weight of less than 500 daltons; an octanol-water partition coefficient (log P) of no more than 5.

[0111] In another embodiment of the present invention, the CXCL12 antagonist binds to CXCR4 and inhibits the binding of CXCL12 to CXCR4. In other words, CXCR4 is the target and antigen, respectively, to which this embodiment of the CXCL12 antagonist binds. Similar to the case of a CXCL12 antagonist that binds to CXCL12 and inhibits the binding of CXCL12 to CXCR4 and / or CXCR7, a CXCL12 antagonist that binds to CXCR4 can be a compound selected from the group consisting of the following classes of compounds: aptamers, Spiegelmers, antibodies, target-binding proteins, target-binding peptides, synthetic peptides, peptides, anticalins, small molecules, and fusion proteins.

[0112] In the method of the present invention, various small molecules that can be used as CXCL12 antagonists due to their binding to CXCR4 are disclosed in the prior art. Such small molecules include, but are not limited to, plerixafor (Sanofi), mobilerixafor (X4 Pharmaceuticals), Q-122 (Que Oncology), HPH-112 (Harmonic Pharma), BKT-300 (Biokine Therapeutics), X4P-002 (X4 Pharmaceuticals), X4P-003 (X4 Pharmaceuticals), X4-136 (X4 Pharmaceuticals), GP-01CR01 (GPCR Therapeutics), GP-01CR11 (GPCR Therapeutics), GP-01CR21 (GPCR Therapeutics), pentixather (Pentixapharm), BKT-170 (Biokine Therapeutics), BMS-585248 (Bristol-Myers Squibb), CS-3955 (Kureha), CTCE-0012 (British Canadian Biosciences), CTCE-9908 (British Canadian Biosciences), GBV-4086 (Globavir Biosciences), HPH-112 (Harmonic Pharma), HPH-211 (Harmonic Pharma), NB-325 (Novaflux Biosciences), SP-10 (Samaritan Pharmaceuticals), and USL-311 (Upsher-Smith Laboratories).

[0113] In the method of the present invention, peptides that can be used as CXCL12 antagonists due to their binding to CXCR4 are disclosed in the prior art. Such peptides include, but are not limited to, motixafortide (BioLineRx).

[0114] In the method of the present invention, synthetic peptides that can be used as CXCL12 antagonists due to their binding to CXCR4 are disclosed in the prior art. Such synthetic peptides include, but are not limited to, balixafortide (Spexis), LY-2510924 (Eli Lilly), ALB-408 (Pharis Biotec), and POL-5551 (Spexis).

[0115] In the method of the present invention, recombinant proteins that can be used as CXCL12 antagonists due to their binding to CXCR4 are disclosed in the prior art. Such recombinant proteins include, but are not limited to, PTX-9098 (Pertinax Therapeutics) and NNL-121 (Nanoligent).

[0116] In the method of the present invention, fusion proteins that can be used as CXCL12 antagonists due to their binding to CXCR4 are disclosed in the prior art. Such fusion proteins include, but are not limited to, AD-214 (Adalta) and AM-3114 (Adalta).

[0117] In the method of the present invention, antibodies, more specifically monoclonal antibodies, that can be used as CXCL12 antagonists due to their binding to CXCR4 are disclosed in the prior art. Such antibodies include, but are not limited to, ulocuplumab (Bristol-Myers Squibb), hz-515H7 (Centre d’Immunologie Pierre Fabre), KY-1051 (Kymab), PF-06747143 (Pfizer), ALX-0651 (Ablynx), AT-009 (Affitech), LY-2624587 (Eli Lilly), and STIA-220X (Sorrento Therapeutics).

[0118] In another embodiment of the present invention, the CXCL12 antagonist binds to CXCR7 and inhibits the binding of CXCL12 to CXCR7. In other words, CXCR7 is the respective target and antigen to which this embodiment of the CXCL12 antagonist binds. Similar to the case of a CXCL12 antagonist that binds to CXCL12 and inhibits the binding of CXCL12 to CXCR4 and / or CXCR7, a CXCL12 antagonist that binds to CXCR4 can be a compound selected from the group consisting of the following classes of compounds: aptamers, Spiegelmers, antibodies, target-binding proteins, target-binding peptides, synthetic peptides, peptides, anticalins, small molecules, and fusion proteins.

[0119] In the method of the present invention, various small molecules that can be used as CXCL12 antagonists due to their binding to CXCR7 are disclosed in the prior art. Such small molecules include, but are not limited to, ACT-10041239 (Idorsia Pharmaceutical), CCX-650 (ChemoCentryx), CCX-662 (ChemoCentryx), and CCX-771 (ChemoCentryx).

[0120] In the method of the present invention, various synthetic peptides that can be used as CXCL12 antagonists due to their binding to CXCR7 are disclosed in the prior art. Such small synthetic peptides include, but are not limited to, LIH-383 (Luxembourg Institute of Health) and POL-6926 (Spexis).

[0121] In the method of the present invention, various antibodies or antibody fragments that can be used as CXCL12 antagonists due to their binding to CXCR7 are disclosed in the prior art. Such antibodies and antibody fragments include, but are not limited to, JT-07 (Jyant Technologies), X-7Ab (The Palo Alto Research Center), and STIA-230X (Serrento Therapeutics).

[0122] It will be understood by those skilled in the art that the amount and dosage of the CXCL12 antagonist to be administered to a subject will vary depending on factors such as the subject's age, weight, height, gender, general medical condition, and previous medical history, among others. However, such a dosage of the CXCL12 antagonist to be administered to a subject will preferably be effective in inhibiting tumor angiogenesis. Preferably, such angiogenesis is brought about by CXCL12-mediated mobilization of endothelial cells or bone marrow-derived angiogenesis-promoting cells. More preferably, the CXCL12-mediated mobilization is CXCR4- and / or CXCR7-dependent.

[0123] It will be understood by those skilled in the art that the exact dosage for administration to a subject in the practice of the method of the present invention can be determined by routine means.

[0124] The means by which the CXCL12 antagonist is administered (route of administration and / or formulation) are defined by the chemical nature of the anti-CXCL12 antagonist. For example, if the CXCL12 antagonist is a Spiegelmer, an aptamer, or an antibody, it will typically be administered by intravenous administration. According to the present invention, the CXCL12 antagonist will be administered continuously to the subject. Even if the CXCL12 antagonist is administered discontinuously to the subject, it is preferable that the therapeutic activity level of the CXCL12 antagonist be achieved within the patient. Such levels are preferably therapeutic activity levels from the perspective of the anti-angiogenic effect and / or immune-stimulating effect in the tumor environment such as the tumor and / or the tumor vasculature, and / or the vascular compartment of the subject.

[0125] In an embodiment of the present invention in which the CXCL12 antagonist is a CXCL12-binding Spiegelmer or a CXCL12-binding aptamer, the weekly dose of such a CXCL12 antagonist administered to the subject is about 100 to 1,000 mg, preferably about 200 to about 600 mg, more preferably about 400 to about 600 mg. This dose is applicable particularly when the CXCL12 antagonist is NOX-A12, and preferably NOX-A12 is administered continuously by intravenous infusion.

[0126] According to the present invention, radiotherapy is administered to a subject suffering from a tumor.

[0127] Regarding brain tumors such as glioblastoma, radiotherapy is the most commonly prescribed treatment. It can be used as the primary treatment when the surgeon believes the risk of removing the tumor is too high, or to destroy any residual cancerous cells that are invisible or inaccessible by the surgeon after surgery. During radiotherapy for glioblastoma, ionizing magnetic radiation or particle beams are directed at the tumor to destroy cancerous cells and their supportive environment. As the cancerous cells and their supportive environment are destroyed and eliminated by the body's immune system, the tumor shrinks, which helps to relieve the pressure on the brain.

[0128] Radiation therapy not only directly targets tumor cells but also depletes the tumor microvasculature. The resulting intratumoral hypoxia initiates a series of events that ultimately lead to angiogenesis, immunosuppression, and ultimately tumor regrowth. A major component of this cascade is the overexpression of CXC motif chemokine ligand 12 (CXCL12), formerly known as stromal cell-derived factor 1 (SDF-1).

[0129] In one embodiment of the present invention, the radiation therapy administered to a subject suffering from a tumor is standard care radiation therapy. Generally, there are two forms of standard care radiation therapy schedules for brain cancer, each of which can be in accordance with a preferred embodiment of the method of the present invention. One form is standard fractionated radiation therapy (nfRT); the other form is hypofractionated radiation therapy (hfRT).

[0130] In a first embodiment of the standard fractionated radiotherapy (nfRT), a total of 60 Gy is administered to the subject in 30 sessions of 2 Gy each over a period of 6 weeks. In each week, the radiotherapy is administered to the subject for 5 consecutive days, followed by 2 days without any radiotherapy administration to the subject. Preferably, the standard fractionated radiotherapy is administered to the subject by external beam radiotherapy (EBRT). More preferably, such standard fractionated radiotherapy is administered to the subject in accordance with the guidelines provided by the European Organization for Research and Treatment of Cancer (EORTC), the European Society of Therapeutic Radiology and Oncology (ESTRO), and the Advisory Committee in Radiation Oncology Practice (ACROP), respectively. According to the said guidelines, the gross tumor volume (GTV) is defined as the resection cavity on postoperative magnetic resonance imaging (MRI) and planned computed tomography (CT) scans. To create the clinical target volume (CTV), the GTV must be expanded maximally by 20 mm in all directions (ideally covering abnormal fluid-attenuated inversion recovery (FLAIR) signals as well). It may be reduced at anatomical barriers (e.g., ventricles, falx, tentorium: 5 mm; bone: 0 mm) or at at-risk critical organs (OARs, e.g., brainstem, optic chiasm, optic nerve: 0 mm). The planning target volume (PTV) is then defined as CTV + an isotropic margin of 3 - 5 mm.

[0131] In a second embodiment of standard fractionated radiotherapy (nfRT), a total of 60 Gy is also administered to the subject. However, 46 Gy is administered to the first planned target volume 1 (PTV1) in 2 Gy fractions per session over the first 23 sessions, and then 14 Gy is administered to the second planned target volume 2 (PTV2) in 2 Gy fractions per session over the next 7 sessions (also referred to as "boost" or "cone down"). More specifically and according to guidelines provided by the Radiation Therapy Oncology Group (RTOG), the gross tumor volume 1 (GTV1) resembles the edema (if visible) in the T2-FLAIR sequence and must also encompass the complete resection cavity and any contrast-enhanced lesions in the T1-post-contrast sequence. The clinical target volume 1 (CTV1) is then defined as GTV1 + 2 cm margin. CTV1 can be reduced around the natural margins of the tumor (e.g., skull, falx, etc.). The planned target volume 1 (PTV1) then resembles CTV1 + 2 - 5 mm additional margin. All (T1-) contrast-enhanced lesions / anomalies (including the cavity margin) will be defined as the boost volume (GTV2). The boost clinical target volume (CTV2) will be GTV2 + 2 cm margin. CTV2 can be reduced around the natural margins of the tumor (e.g., skull, falx, etc.). The boost planned target volume (PTV2) resembles CTV2 + 2 - 5 mm additional margin. In connection with the above, it will be understood by those skilled in the art that virtually any glioblastoma has edema. Edema is one of the main causes of the increased intracranial pressure leading to typical glioblastoma symptoms. Since tumor cells are more likely to infiltrate this damaged area, the edema region is also irradiated. Furthermore, it will be recognized that T2 FLAIR is particularly suitable for detecting edema as it allows the distinction between free and tissue-bound fluid. T2 refers to the transverse relaxation. The FLAIR (fluid-attenuated inversion recovery) sequence is a specific MRI sequence very similar to the T2 sequence, but it is normalized against the signal of the fluid in the brain set to zero. Therefore, the fluid will appear black.

[0132] In hypofractionated radiotherapy (hfRT), a subject suffering from a tumor will receive a total dose of approximately 40 Gy, typically 40.05 Gy, administered in 15 daily fractions over a 3-week period. Typically, this means that in each and any of the said 3 weeks, 2.67 Gy is administered to the subject for 5 consecutive days each, followed by 2 days during which radiotherapy is not administered to the subject. More specifically and according to the guidelines provided by the Canadian Cancer Society Research Institute, the gross tumor volume (GTV) is defined as the contrast-enhanced volume in a postoperative planned magnetic resonance imaging (MRI) scan and includes the surgical bed. The clinical target volume (CTV) is a 1.5 cm margin respecting the anatomical boundaries beyond the gross tumor volume contour, to which a 0.5 cm planning target volume (PTV) margin is applied.

[0133] Radiotherapy may use ionizing electromagnetic radiation or ionizing particle beams. Such ionizing electromagnetic radiation can be X-rays or gamma rays. Typically, X-rays have a wavelength of about 10 picometers to about 10 nanometers, and the energy of X-rays is about 145 eV to about 124 keV. For gamma rays, their wavelength is typically less than 10 picometers, and the energy of gamma rays is typically 100 keV or more.

[0134] Such ionizing particle beams can be proton beams, photon beams, or electron beams, and preferably such particle beams are used in external beam radiotherapy (EBRT). In one embodiment of the present invention, EBRT is used in radiotherapy administered to a subject suffering from a tumor according to the present invention, including any of its aspects.

[0135] In external beam radiation therapy (EBRT), tumors are exposed to an external ionizing radiation source, and megavolt X-rays are used to treat deep-seated tumors such as GBM. Such X-rays or photon beams are produced in most radiotherapy devices. Photon beams can reach deep-seated tumors in the body. As they pass through the body, photon beams scatter a small amount of radiation along their paths. These beams do not stop even when they reach the tumor and enter normal tissue beyond it. Megavolt X-rays have an energy of 1 to 25 MeV. Such megavolt X-rays are produced in a linear accelerator (linac). Commercially available medical linacs produce X-rays with an energy range of 4 MeV to a maximum of approximately 25 MeV. On the other hand, in clinical practice, a nominal energy exceeding 15 MeV is rare, and thus the energy range of 4 to 15 MeV is the preferred range. X-rays themselves are produced by the rapid deceleration of electrons in a target material, typically a tungsten alloy, which produces an X-ray spectrum by bremsstrahlung. The shape and intensity of the beam produced by a linac can be modified or narrowed by various means.

[0136] Alternatively, like photon beams, proton beams can also reach deep-seated tumors in the body and are sometimes used, particularly in recurrent brain tumors and recurrent glioblastomas. However, proton beams do not scatter radiation along their paths in the body and they stop when they reach the tumor. Therefore, proton beams are thought to reduce the amount of normal tissue exposed to radiation. Generally, the high cost and size of the equipment limit their use.

[0137] Those skilled in the art will understand that radiation therapy can also use "heavier" particles such as protons or He ions to produce a radiation therapy effect. The use of such heavier particles constitutes another embodiment of the method of the present invention.

[0138] EBRT can be administered to a subject by various means. Some of such means are outlined below and constitute further embodiments of the method of the present invention, namely 3-D conformal radiation therapy, intensity-modulated radiation therapy (IMRT), image-guided radiation therapy (IGRT), and stereotactic radiosurgery.

[0139] 3-D conformal radiation therapy is a common type of EBRT. It uses images from CT, MRI, and PET scans to accurately plan the treatment area. The images are analyzed and the irradiation fields are designed to conform to the shape of the tumor.

[0140] Intensity-modulated radiation therapy (IMRT) is a more advanced form of 3-D conformal radiation therapy. It uses sophisticated software and 3-D images from CT scans to direct radiation directly at the tumor. These pencil-thin beams vary in intensity (to provide a higher dose to certain parts of the tumor) and conform to the specific shape and size of the tumor. This highly focused approach reduces radiation exposure to healthy tissue in the brain. IMRT can also reduce the likelihood of side effects.

[0141] Image-guided radiation therapy (IGRT) or tomography is even more advanced than IMRT by using imaging scans not only for treatment planning before a radiation therapy session but also during the radiation therapy session to guide the location of the radiation beam.

[0142] Stereotactic radiosurgery is the use of focused high-energy beams to treat small tumors with well-defined boundaries in the brain and central nervous system. This precise treatment positions the radiation source closer to the tumor than would be possible with conventional radiotherapy. As such, it can be an option when the risks of surgery are too high due to age or other health problems, or when the tumor cannot be safely reached surgically. Traditional glioblastoma radiotherapy involves multiple sessions scheduled over several months, while stereotactic radiosurgery is delivered in fewer sessions.

[0143] In another embodiment of the invention, the radiation therapy is brachytherapy. Brachytherapy is a type of local internal radiation therapy in which seeds, ribbons, or capsules containing a radiation source, or a liquid formulation containing a radiation source, are placed within or near the tumor, preferably within the resection cavity after surgery. It is often used to treat cancers of the head and neck, breast, cervix, prostate, and eye, and is also applicable in the treatment of brain tumors. Basically, there are three types of brachytherapy: (a) low-dose rate (LDR) implants in which the radiation source remains in place for 1 to 7 days, (b) high-dose rate (HDR) implants in which the radiation source is placed in position for only 10 to 20 minutes per session; and (c) permanent implants in which the radiation source remains in place indefinitely, but the radiation weakens daily. In one embodiment, such a radiation source is a therapeutically active radionuclide. In a preferred embodiment, the therapeutically active radionuclide is 186 Re, 47 Sc, 67 Cu, 89 Sr, 90 Y, 153 Sm, 149 Tb, 161 Tb, 177 Lu, 188 Re, 212 Pb, 213 Bi, 223 Ra, 225 Ac, 226 Th, 227 Th, 131 I, 211Selected from the group containing At, and more preferably, the therapeutic active radionuclide is 186 Re, 47 Sc, 67 Cu, 90 Y, 177 Lu, 188 Re, 212 Pb, 213 Bi, 225 Ac, 227 Th, 131 I, 211 Selected from the group containing At, and most preferably, the therapeutic active radionuclide is 186 Re, 90 Y, 177 Lu, 225 Ac, 227 Th, 131 I, and 211 selected from the group containing At.

[0144] In one embodiment of the present invention, a modified brachytherapy is used. Such modified brachytherapy does not use a solid radiation source to be implanted, but is a beta-emitting radionuclide for therapeutic use having a half-life of 90 hours, a radiation penetration area of 1.8 mm, and a high β / γ energy ratio suitable for cancer brachytherapy 186 uses an injection of Re-containing nanoliposomes. 186 Re nanoliposomes enhance the delivery profile of the long-lived 186 Re energy to achieve long-term tumor retention and a very high absorbed radiation dose to the tumor. Preclinically, such nanoliposomes injected via convection-enhanced delivery achieve very high doses of targeted radiation and a wide therapeutic index. Such modified brachytherapy is described, for example, in Floyd 2021, Int. J. Radiat. Oncol. Biol. Phys. Volume 111, Issue 3, Supplement, 1 November 2021, Page e589.

[0145] In another embodiment of the present invention, the radiation therapy is intraoperative radiation therapy (IORT). Intraoperative radiation therapy (IORT) consists of delivering radiation to the tumor / tumor bed while the area is exposed during surgery. IORT can deliver a precisely high dose of radiation to the tumor bed with minimal exposure to surrounding healthy tissue. It will be appreciated by those skilled in the art that IORT is typically combined with external beam radiation therapy to avoid the regrowth of tumor cells at the site where the tumor has been resected or otherwise treated.

[0146] IORT enables (i) accurate localization of the tumor bed and targeted delivery of a high dose of radiation to the tumor bed; (ii) minimal exposure of dose-limiting normal tissues placed away from the tumor bed and shielded from the radiation; (iii) the opportunity for dose escalation beyond what can be achieved with EBRT; and (iv) the opportunity for re-irradiation, particularly in recurrent cancers where further irradiation with EBRT may not be possible. Thus, IORT can deliver a higher total effective dose to the tumor bed, facilitate dose escalation without significantly increasing complications in normal tissues, and improve the therapeutic ratio compared to EBRT. The methods of IORT are (i) electron IORT / IOERT (electron beam), (ii) kV IORT (X-ray), and (iii) HDR IORT (high dose rate brachytherapy).

[0147] It is within the scope of the present invention that a method for treating a tumor includes anti-angiogenic therapy. Preferably, such anti-angiogenic therapy includes administering an anti-angiogenic compound to a subject suffering from a tumor.

[0148] This part of the method of the present invention, i.e., administering anti-angiogenic therapy to a subject suffering from a tumor, will be appreciated by those skilled in the art to avoid or at least reduce angiogenesis. As preferably used herein, angiogenesis is the formation of new blood vessels within and / or to a tumor. More preferably, angiogenesis starts from existing blood vessels.

[0149] Since malignant tissues also depend on nutrients and oxygen, the formation of new blood vessels is an essential prerequisite for tumor growth. Very small tumors can be supplied by diffusion, but as soon as the tumor becomes larger than 1-2 mm in diameter, a unique vascular system is required for further growth. To generate such a system, tumors secrete various growth factors that stimulate angiogenesis (the angiogenesis switch). The most prominent growth factor in tumor angiogenesis is VEGF (vascular endothelial growth factor). By blocking VEGF signaling through its receptors VEGFR1 (Flt-1) and VEGFR2 (KDR / Flk-1), endothelial cell proliferation within the tumor and subsequent new blood vessel formation are inhibited. In most normal tissues, there is very low or undetectable expression of VEGF receptors, while VEGF is upregulated in most human tumor types. VEGF expression is associated with tumor progression or patient survival in a variety of human cancers.

[0150] Based on this, those skilled in the art will recognize that inhibiting the interaction of VEGF with at least one of its receptors VEGFR1 and VEGFR2, preferably inhibiting the binding of VEGF to at least one of its receptors VEGFR1 and VEGFR2, will result in an anti-angiogenic effect that is therapeutically effective. Such inhibition can be achieved by (a) providing a molecule that binds to VEGF to inhibit the binding of VEGF to VEGFR1 and / or VEGFR2, (b) providing a molecule that binds to VEGFR1 to inhibit the binding of VEGF to VEGFR1, and (c) providing a molecule that binds to VEGFR2 to inhibit the binding of VEGF to VEGFR2, as will be understood by those skilled in the art.

[0151] Such molecules that bind to VEGF are preferably selected from the group consisting of aptamers, preferably VEGF-binding aptamers, Spiegelmers, preferably VEGF-binding antibodies, proteins that bind to VEGF, VEGF-binding anticalins, VEGF-binding peptides, fusion proteins containing a VEGF-binding moiety, and small molecules that bind to VEGF.

[0152] Such molecules that bind to VEGFR1 are preferably selected from the group consisting of aptamers, preferably VEGFR1-binding aptamers, Spiegelmers, preferably VEGFR1-binding antibodies, proteins that bind to VEGFR1, VEGFR1-binding anti-kallikreins, VEGFR1-binding peptides, fusion proteins containing a VEGFR1-binding moiety, and VEGFR1-binding small molecules.

[0153] Such molecules that bind to VEGFR2 are preferably selected from the group consisting of aptamers, preferably VEGFR2-binding aptamers, Spiegelmers, preferably VEGFR2-binding antibodies, proteins that bind to VEGFR2, VEGFR2-binding anti-kallikreins, VEGFR2-binding peptides, fusion proteins containing a VEGFR2-binding moiety, and VEGFR2-binding small molecules.

[0154] Such inhibition of the interaction of VEGF via one or both of the VEGF receptors VEGFR1 and VEGFR2 can be achieved by a compound that binds to VEGFR1 and / or VEGFR2, and it will be understood by those skilled in the art that such binding of the compound interferes with the signal transduction of VEGFR1 and / or VEGFR2 rather than interfering with the binding of VEGF to VEGFR1 and / or VEGFR2.

[0155] Molecules that bind to VEGFR1 and, by virtue of their binding to VEGFR1, interfere with the signal transduction characteristics of VEGFR1, preferably the signal transduction induced by the binding of VEGF to VEGFR1, are preferably selected from the group consisting of aptamers, preferably VEGFR1-binding aptamers, Spiegelmers, preferably VEGFR1-binding antibodies, proteins that bind to VEGFR1, VEGFR1-binding anti-kallikreins, VEGFR1-binding peptides, fusion proteins containing a VEGFR1-binding moiety, and VEGFR1-binding small molecules.

[0156] The generation and identification of each of such aptamers, Spiegelmers, antibodies, binding proteins, anticalins, peptides, fusion proteins, and small molecules are known to those skilled in the art and are also disclosed herein.

[0157] In one embodiment of the invention, VEGF is human VEGF, VEGFR1 is human VEGFR1, and VEGFR2 is human VEGF2.

[0158] VEGF exists as a full-length form, but also as isoforms generated by alternative splicing or proteolytic cleavage by the proteases plasmin, MMP3, and uPA. Such isoforms and proteolytically cleaved forms of VEGF are disclosed, for example, by Vempati et al. (Vempati P et al., Cytokine Growth Factor Rev. 2014 Feb;25(1).1-19) as well as Guyot M and Pages G (Guyot M and Pages G, Methods Mol. Biol 2015;1332:2-23). The isoforms are disclosed in particular by Guyot M and Pages G, and the amino acid sequences of the various forms of VEGF are depicted in FIG. 20. The isoforms of VEGF are preferably those selected from the group consisting of VEGF121, VEGF165, VEGF189, VEGF206, VEGF145, VEGF183, and VEGF165b. The proteolytically cleaved form of VEGF is VEGF110. Whenever reference is made herein to VEGF and to compounds that bind to or interfere with its function, it will be understood by those skilled in the art that such VEGF is, in one embodiment, any of the above-described full-length VEGF, VEGF isoforms, or proteolytically cleaved forms thereof. It will be recognized by those skilled in the art that molecules that bind to VEGF can bind to the form of VEGF used in the generation and identification of each such binding molecule and additionally to one or more of the above-described forms of VEGF, including any isoform or proteolytically cleaved form, within the scope of the present invention.

[0159] In a preferred embodiment of the present invention, the anti-angiogenic compound is an anti-VEGF antibody. The anti-VEGF antibody can be a human, humanized, or chimeric anti-VEGF antibody. The term anti-VEGF antibody as preferably used herein also encompasses any fragment of such an anti-VEGF antibody, and such a fragment still binds or can still bind to VEGF.

[0160] In a preferred embodiment of the present invention, the anti-angiogenic antibody comprises the following light chain CDRs: CDR1: QDISNY (SEQ ID NO: 226), CDR2: FTS, and CDR3: QQYSTVPWT (SEQ ID NO: 227); or the following heavy chain CDRs: CDR1: GYTFTNYG (SEQ ID NO: 228), CDR2: INTYTGEP (SEQ ID NO: 229), and CDR3: AKYPHYYGSSHWYFDV (SEQ ID NO: 230), or a combination of the following light chain CDRs: CDR1: QDISNY (SEQ ID NO: 226), CDR2: FTS, and CDR3: QQYSTVPWT (SEQ ID NO: 227); and the following heavy chain CDRs: CDR1: GYTFTNYG (SEQ ID NO: 228), CDR2: INTYTGEP (SEQ ID NO: 229), and CDR3: AKYPHYYGSSHWYFDV (SEQ ID NO: 230).

[0161] In another preferred embodiment of the present invention, the anti-angiogenic antibody comprises a light chain having the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 239) A heavy chain having the following amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 240), or a light chain comprising the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 239 and a heavy chain comprising the following amino acid sequence; It includes a combination with [[SEQ ID NO:240]]: EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0162] In a preferred embodiment of the present invention, the anti-VEGF antibody is bevacizumab. Bevacizumab is a recombinant humanized monoclonal antibody against VEGF; USAN / BAN / JAN name: Bevacizumab; Laboratory code: RO487-6646; CAS registration number: 216974-75-3; Bevacizumab is also referred to as rhuMAb VEGF and anti-VEGF.

[0163] Bevacizumab is an inhibitor of angiogenesis. Therefore, it blocks the formation of new blood vessel systems. Bevacizumab selectively binds with high affinity to all isoforms of human VEGF, which is a major promoter of angiogenesis, thereby inhibiting the binding of VEGF to its receptors (Flt-1, VEGFR1, and KDR, VEGFR2) on the surface of endothelial cells. Neutralizing the biological activity of VEGF by sterically blocking the binding of VEGF to its receptors regresses tumor angiogenesis, normalizes the remaining tumor blood vessel system, inhibits the formation of new tumor blood vessel systems, and thereby inhibits tumor growth. Receptor activation usually induces their tyrosine phosphorylation, and the subsequent series of signal transduction events causes mitogenic activity and survival-promoting activity signals to endothelial cells. In most normal tissues (except for renal glomeruli), there is very low or undetectable expression of VEGF receptors, but in the blood vessel systems of many tumors, there is significant upregulation. Therefore, the neutralization of VEGF by bevacizumab provides a logical basis for the relatively specific inhibition of tumor angiogenesis and thereby the inhibition of tumor growth and metastasis.

[0164] Bevacizumab is a recombinant humanized monoclonal IgG1κ isotype antibody containing human framework regions (93%) and murine complementarity-determining regions (7%). The antibody has a total molecular weight of 149 kDa and is composed of two identical light chains (214 amino acid residues) and two heavy chains (453 residues). The heavy chain exhibits C-terminal heterogeneity (lysine variant) and also contains one N-linked glycosylation site at asparagine 303. The oligosaccharide has a core fucose and a complex biantennary structure with two branches that mainly terminate with 0 (G0), 1 (G1), or 2 (G2) galactose residues. The G0 glycoform predominates with an approximate relative abundance of 80%. Each light chain is covalently linked to the heavy chain at cysteine 226 through a disulfide bond at cysteine 214. The two heavy chains are covalently linked to each other through two interchain disulfide bonds, which is consistent with the structure of human IgG1. Bevacizumab was produced by Genentech through humanization of the murine parental antibody A4.6.1 using hybridomas generated from mice immunized with the 165-residue form of recombinant human vascular endothelial growth factor (rhuVEGF165) conjugated to keyhole limpet hemocyanin.

[0165] Humanization of the A4.6.1 antibody involved the insertion of the six CDRs of A4.6.1 in place of those of the selected human antibody Fab framework (pEMX1), which has a consensus human kappa subgroup I light chain (domains VL-CL) and a shortened human subgroup III immunoglobulin gamma (IgG1) heavy chain (domains VH-CH1). A series of framework residue substitutions were performed to produce the final humanized version, Fab-12, which contains eight substitutions of the human framework outside the CDRs. The VH and VL domains of Fab-12 were combined with the human IgG1 constant domains CH1-CH2-CH3 and CL, respectively, to produce bevacizumab. The expression plasmid pSVID5.ID.LLnspeV.xvegf36HC.LC encoding bevacizumab was introduced into Chinese hamster ovary parental cells CHO DP-12 by lipofection, and the cells were selected in the presence of increasing concentrations of methotrexate (MTX). Isolates were selected for the secretion of active bevacizumab. The isolated subclone 107N was used as a starting point for the production of Phase I and Phase II clinical materials and for the development of the more productive G7 cell line, which was used for the production of Phase III clinical materials, which would be used for the production of the drug substance intended for sale.

[0166] In another embodiment of the invention, the anti-VEGF antibody is ranibizumab (Lucentis; also Roche). The CDRs of bevacizumab and ranibizumab are very similar but have some variations (see the IMGT repertoire (IG and TR available from https: / / www.imgt.org / IMGTrepertoire / GenesClinical / humanized / bevacizumab / bevacizumab_ProteinDisplay.html)). Ranibizumab is the Fab fragment of bevacizumab. In ranibizumab, six amino acids are modified, four of which are in the CDRs. Ranibizumab is not glycosylated. More specifically, ranibizumab has the following amino acid changes compared to bevacizumab: in VH (CDR1-IMGT: T29→D, N36→H, CDR3-IMGT: H109→Y, S112→T), and one amino acid change in V-kappa (M4→L). There is also an additional change in the hinge of bevacizumab (T10>L).

[0167] In yet other embodiments of the present invention, the anti-angiogenic compound that binds to VEGF is a VEGF-binding protein. Preferably, such a VEGF-binding protein is a functional derivative of a VEGF receptor, more preferably VEGFR1 and / or VEGFR2. In a particularly preferred embodiment, the anti-angiogenic compound is ziv-aflibercept (Zaltrap), a "VEGF trap" that inhibits VEGF-A, VEGF-B, and PlGF-2. Zaltrap, also referred to as aflibercept in the scientific literature or known as VEGF trap, is a recombinant fusion protein consisting of the VEGF-binding portions derived from the extracellular domains of human VEGF receptors 1 and 2 fused to the Fc portion of human IgG1. Aflibercept is produced by recombinant DNA technology in a Chinese hamster ovary (CHO) K-1 mammalian expression system. Aflibercept is a dimeric glycoprotein with a protein molecular weight of 97 kilodaltons (kDa), containing glycosylation that constitutes an additional 15% of the total molecular weight, resulting in a total molecular weight of 115 kDa. Aflibercept acts as a soluble decoy receptor that binds to VEGF-A with higher affinity than its native receptor, as well as to the related ligands PlGF and VEGF-B. By acting as a ligand trap, aflibercept prevents the binding of the endogenous ligands to their cognate receptors, thereby blocking receptor-mediated signaling. Aflibercept blocks the activation of VEGF receptors and the proliferation of endothelial cells, thereby inhibiting the growth of new blood vessels that supply oxygen and nutrients to tumors. Aflibercept binds to human VEGF-A (equilibrium dissociation constants of 0.5 pM for VEGF-A165 and 0.36 pM for VEGF-A121), human PlGF (KD of 39 pM for PlGF-2), and VEGF-B (KD of 1.92 pM) to form stable inactive complexes that have no detectable biological activity (see European Medicines Agency, entry on Zaltrap: https: / / www.ema.europa.eu / en / medicines / human / EPAR / zaltrap).

[0168] In one embodiment of the present invention, the anti-angiogenic compound is a compound that binds to VEGFR. VEGFR can be VEGFR1, VEGFR2, or a combination thereof. In a more preferred embodiment, the anti-angiogenic compound that binds to VEGFR is an anti-VEGFR antibody.

[0169] In one embodiment of the present invention, the anti-VEGFR antibody comprises the following light chain CDRs, CDR1: RASQGIDNWLG (SEQ ID NO: 233), CDR2: DASNLDT (SEQ ID NO: 234), and CDR3: QQAKAFPPT (SEQ ID NO: 235), or the following heavy chain CDRs, CDR1: GFTFSSYSMN (SEQ ID NO: 236), CDR2: SISSSSSYIYYADSVKG (SEQ ID NO: 237), and CDR3: VTDAFDI (SEQ ID NO: 238), or the following light chain CDRs, CDR1: RASQGIDNWLG (SEQ ID NO: 233), CDR2: DASNLDT (SEQ ID NO: 234), and CDR3: QQAKAFPPT (SEQ ID NO: 235) and the following heavy chain CDRs, CDR1: GFTFSSYSMN (SEQ ID NO: 236), CDR2: SISSSSSYIYYADSVKG (SEQ ID NO: 237), and CDR3: VTDAFDI (SEQ ID NO: 238).

[0170] In one embodiment of the present invention, the anti-VEGFR antibody has the following light chain: DIQMTQSPSSVSASIGDRVTITCRASQGIDNWLGWYQQKPGKAPKLLIYDASNLDTGVPS RFSGSGSGTYFTLTISSLQAEDFAVYFCQQAKAFPPTFGGGTKVDIKRTVAAPSVFIFPP SDEQLKSGTA SVVCLLNNFYPREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQGLSSPVTKSFN RGEC (SEQ ID NO: 239), or the following heavy chain: EVQLVQSGGG LVKPGGSLRL SCAASGFTFS SYSMNWVRQA PGKGLEWVSS ISSSSSYIYY ADSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCARVT DAFDIWGQGT MVTVSSASTK GPSVLPLAPS SKSTSGGTAA LGCLVKDYFP EPVTVSWNSG ALTSGVHTFP AVLQSSGLYS LSSVVTVPSS SLGTQTYICN VNHKPSNTKV DKRVEPKSCD KTHTCPPCPA PELLGGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTL PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSLSLSPGK (SEQ ID NO: 240), or the following light chain CDRs, CDR1: RASQGIDNWLG (SEQ ID NO: 233), CDR2: DASNLDT (SEQ ID NO: 234), and CDR3: QQAKAFPPT (SEQ ID NO: 235), or the following heavy chain CDRs, CDR1: GFTFSSYSMN (SEQ ID NO: 236), CDR2: SISSSSSYIYYADSVKG (SEQ ID NO: 237), and CDR3: VTDAFDI (SEQ ID NO: 238), or the following light chain CDRs, CDR1: RASQGIDNWLG (SEQ ID NO: 233), CDR2: DASNLDT (SEQ ID NO: 234), and CDR3: QQAKAFPPT (SEQ ID NO: 235), and the following heavy chain CDRs, CDR1: GFTFSSYSMN (SEQ ID NO: 236), CDR2: SISSSSSYIYYADSVKG (SEQ ID NO: 237), and CDR3: VTDAFDI (SEQ ID NO: 238) in combination.

[0171] In one embodiment of the present invention, the anti-VEGFR antibody comprises the following light chain: DIQMTQSPSS VSASIGDRVT ITCRASQGID NWLGWYQQKP GKAPKLLIYD ASNLDTGVPS RFSGSGSGTY FTLTISSLQA EDFAVYFCQQ AKAFPPTFGG GTKVDIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC (SEQ ID NO: 239), and the following heavy chain: EVQLVQSGGG LVKPGGSLRL SCAASGFTFS SYSMNWVRQA PGKGLEWVSS ISSSSSYIYY ADSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCARVT DAFDIWGQGT MVTVSSASTK GPSVLPLAPS SKSTSGGTAA LGCLVKDYFP EPVTVSWNSG ALTSGVHTFP AVLQSSGLYS LSSVVTVPSS SLGTQTYICN VNHKPSNTKV DKRVEPKSCD KTHTCPPCPA PELLGGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTL PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK (SEQ ID NO: 240).

[0172] In another embodiment of the present invention, the anti-angiogenic compound that binds to VEGF is an aptamer, i.e., a D-nucleic acid, and more specifically pegaptanib (Macugen). Pegaptanib (Macugen) is an anti-VEGF aptamer developed as an anti-angiogenic drug for the treatment of neovascular (exudative) age-related macular degeneration. Pegaptanib is a PEGylated aptamer that specifically binds to the 165 isoform of VEGF.

[0173] More preferably, the anti-VEGFR antibody is ramucirumab (Cyramza), a specific VEGFR2 inhibitor. The amino acid sequence of the heavy chain of ramucirumab is EVQLVQSGGG LVKPGGSLRL SCAASGFTFS SYSMNWVRQA PGKGLEWVSS ISSSSSYIYY ADSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCARVT DAFDIWGQGT MVTVSSASTK GPSVLPLAPS SKSTSGGTAA LGCLVKDYFP EPVTVSWNSG ALTSGVHTFP AVLQSSGLYS LSSVVTVPSS SLGTQTYICN VNHKPSNTKV DKRVEPKSCD KTHTCPPCPA PELLGGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTL PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK (SEQ ID NO: 241) and the light chain of ramucirumab is DIQMTQSPSS VSASIGDRVT ITCRASQGID NWLGWYQQKP GKAPKLLIYD ASNLDTGVPS RFSGSGSGTY FTLTISSLQA EDFAVYFCQQ AKAFPPTFGG GTKVDIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC (SEQ ID NO: 242) is as follows.

[0174] These sequences can be obtained from the information provided by the following link: https: / / www.genome.jp / entry / D09371.

[0175] In another embodiment of the present invention, the anti-angiogenic compound is a compound that inhibits the signal transduction of VEGFR, preferably VEGFR1 and VEGFR2. However, inhibition of signal transduction for one or more of the following receptors: platelet-derived growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR), and angiopoietin-1 receptor (Tie-2) is also within the scope of the present invention.

[0176] In a preferred embodiment of the present invention, the anti-angiogenic compound inhibits the signal cascade induced by the binding of VEGF to VEGFR. Assays used to determine whether a compound inhibits the signal cascade are known in the art. More specifically, cell culture assays have been used for such purposes and for the characterization of VEGFR inhibitors. The assays detect the activation of intracellular signal transduction pathways that, in the case of VEGFR, are the Ras / MAPK pathway that regulates cell growth and gene expression; the FAK / paxillin pathway involved in cytoskeletal rearrangement; the PI3K / AKT pathway that regulates cell survival; and the PLCγ pathway that controls vascular permeability.

[0177] More preferably, the anti-angiogenic compound inhibits the tyrosine kinase activity of VEGFR. In such embodiments, the anti-angiogenic compound is a tyrosine kinase inhibitor, more preferably a multi-tyrosine kinase inhibitor.

[0178] In a more preferred embodiment of the present invention, the anti-angiogenic compound that is a tyrosine kinase inhibitor is pazopanib (Votrient, which inhibits VEGFR1-3, PDGFRα and β, FGFR1 and 3, KIT, ITK, LCK, c-FMS), sunitinib (Sutent, which inhibits VEGFR1-3, PDGFRα and β, KIT, FLT-3, CSF-1R, RET), sorafenib (Nexavar, which inhibits intracellular c-RAF, BRAF, and mutant BRAF), and cell surface kinases (KIT, FLT-3, RET, RET / PCT, VEGFR1-3, PDGFR-β), axitinib (Inlyta, which inhibits VEGFR1-3), ponatinib (Iclusig, which inhibits VEGFR, PDGFR, FGFR, EPH receptor, SCR family of kinases, KIT, RET, TIE2, and FLT3), cabozantinib (Cometriq / Cabometyx, which inhibits RET, MET, VEGFR1-3, KIT, TKRB, FLT-3, AXL, ROS1, TYRO3, MER, TIE-2), regorafenib (Stivarga, which inhibits RET, VEGFR1-3, KIT, PDGFRα and β, FGFR1-2, TIE2, DDR2, TRKA, EPH2A, RAF-1, BRAF, BRAF V600E, SAPK2, PTKS, ABL, CSF1R), vandetanib (Caprelsa, which inhibits members of the EGFR and VEGFR families, RET, BRK, TIE-2, EPHR, and Src kinase family), and lenvatinib (Lenvima, which inhibits VEGFR1-3, FGFR1-4, PDGFRα, KIT, and RET), and is selected from the group consisting of.

[0179] One skilled in the art will understand that the amount and dosage of the anti-angiogenic compound to be administered to a subject will vary depending on factors such as the subject's age, weight, height, gender, general medical condition, and previous medical history, among others. However, such a dosage of the anti-angiogenic compound to be administered to a subject will preferably be effective in inhibiting tumor angiogenesis. Preferably, such angiogenesis is brought about by the interaction of VEGF with VEGFR1 and / or VEGFR2.

[0180] One skilled in the art will understand that the exact dosage for the administration of the anti-angiogenic compound to a subject in the practice of the method of the present invention can be determined by routine means.

[0181] The means by which the anti-angiogenic compound is administered (route of administration and / or formulation) is defined by the chemical nature of the anti-angiogenic compound. For example, if the anti-angiogenic compound is a Spiegelmer, aptamer, or antibody, it will typically be administered by intravenous administration. According to the present invention, the anti-angiogenic compound will be administered discontinuously to the subject.

[0182] In embodiments of the present invention where the anti-angiogenic compound is an anti-VEGF antibody and particularly an antibody such as bevacizumab, such an antibody is administered by intravenous infusion weekly, every other week, or every three weeks, preferably every other week or every three weeks. One skilled in the art will understand that the dosing schedule for the anti-angiogenic compound and anti-VEGF antibodies such as bevacizumab is synchronized with the therapy to which the anti-angiogenic compound is combined.

[0183] In one embodiment of the present invention, the method of the present invention includes chemotherapy. In a preferred embodiment, the chemotherapy is chemotherapy used particularly in the treatment of gliomas and glioblastomas, more preferably chemotherapy used as standard care in the treatment of gliomas and glioblastomas.

[0184] In one embodiment of the present invention, chemotherapy includes temozolomide (TMZ). TMZ is preferably administered daily (75 mg / m per day) during radiotherapy for 6 weeks, and then 2 administered in 6 cycles after radiotherapy during the maintenance period. Each cycle of the maintenance period lasts for 28 days, and temozolomide is administered at 150 - 200 mg / m 2 per day for the first 5 days of each cycle, followed by a 23-day drug holiday. In a preferred embodiment, the subject's tumor is sensitive to temozolomide.

[0185] In another embodiment of the present invention, chemotherapy includes lomustine (CCNU). The general scheme for CCNU-based chemotherapy is a dose of 110 mg / m 2 per day on day 1 of a 42-day cycle, according to local standards, and then the treatment continues for a total of up to 6 cycles. In a further embodiment, CCNU is used in the treatment of recurrent glioblastoma.

[0186] In another embodiment of the present invention, chemotherapy includes procarbazine / lomustine / vincristine (PCV) or bevacizumab / irinotecan (BI) (Carvalho 2015, Oncol Res Treat 38:348). In a further embodiment, such chemotherapy is used in the treatment of recurrent glioblastoma.

[0187] In yet another embodiment of the present invention, chemotherapy includes dianhydrogalactitol (VAL-083), an alkylating agent (DNA synthesis inhibitor). Subjects suffering from brain tumors, and particularly glioblastoma, may receive dianhydrogalactitol, and temozolomide (TMZ at 75 mg / m 2 orally daily during radiotherapy), during the maintenance period after radiotherapy. Maintenance period: 30 mg / m 2 of VAL-083 IV on days 1, 2, and 3 of a 21-day cycle.

[0188] In one embodiment of the present invention, the subject is a human subject.

[0189] In one embodiment of the present invention, the antibody can be a human, humanized, or chimeric antibody. Preferably, the antibody is a monoclonal antibody (MAb).

[0190] In one embodiment of the present invention, the antibody can be, but is not limited to, a human, humanized, or chimeric antibody.

[0191] In one embodiment of the present invention and as preferably used herein, the term antibody includes any antigen-binding or any target-binding fragment of an antibody that targets or binds to such an antigen or target. In other words, an antibody fragment of an antibody is a part of the antibody. Exemplary antibody fragments include, but are not limited to, F(ab’)2, Fab’, Fab, Fv, sFv, and the preparation thereof is known to those skilled in the art. Antibody fragments can also include single domain antibodies and IgG4 half molecules as discussed below. Regardless of the structure, antibody fragments bind to the same antigen recognized by the full-length antibody. The term “antibody fragment” also includes isolated fragments consisting of the variable regions of the antibody, such as the “Fv” fragment consisting of the variable regions of the heavy and light chains, and recombinant single-chain polypeptide molecules (the “scFv protein”) in which the variable regions of the light and heavy chains are connected by a peptide linker.

[0192] In one embodiment of the present invention and as preferably used herein, a human antibody is an antibody that can be obtained, for example, from a human or from a transgenic mouse that has been “engineered” to produce specific human antibodies in response to antigen loading. In this technique, elements of the human heavy and light chain loci are introduced into a mouse strain derived from an embryonic stem cell line containing targeted disruption of the endogenous murine heavy and light chain loci. The transgenic mouse can synthesize human antibodies specific for a particular antigen, and the mouse can be used to produce human antibody-secreting hybridomas.

[0193] In one embodiment of the invention and as preferably used herein, a humanized antibody is a recombinant protein in which the CDRs from an antibody of one species, e.g., a rodent antibody, have been transferred from the heavy and light chain variable chains of the rodent antibody to human heavy and light chain variable domains (e.g., framework region sequences). The constant domains of the antibody molecule are derived from those of human antibodies. In certain embodiments, a limited number of framework region amino acid residues from the parent (rodent) antibody may be substituted for human antibody framework region sequences.

[0194] In one embodiment and as preferably used herein, a chimeric antibody is a recombinant protein containing a variable domain that includes the complementarity determining regions (CDRs) of an antibody derived from one species, preferably a rodent antibody, while the constant domains of the antibody molecule are derived from those of human antibodies. For veterinary applications, the constant domains of chimeric antibodies may be derived from those of other species such as cats or dogs.

[0195] It will be understood by those skilled in the art that antibodies, including therapeutic active antibodies, can be produced by routine methods for any target and antigen. Such methods are well known in the art. See, for example, Kohler and Milstein, Nature 256:495 (1975), and Coligan et al. (eds.), CURRENT PROTOCOLS IN IMMUNOLOGY, VOL.1, pages 2.5.1-2.6.7 (John Wiley & Sons 1991). Briefly, a monoclonal antibody can be obtained by injecting a composition containing an antigen into a mouse, removing the spleen to obtain B lymphocytes, fusing the B lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones that produce antibodies against the antigen, culturing the clones that produce antibodies against the antigen, and isolating the antibody from the hybridoma culture.

[0196] MAbs can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography using protein-A or protein-G sepharose, size exclusion chromatography, and ion exchange chromatography. See, e.g., Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. See also Baines et al., “Purification of Immunoglobulin G(IgG),” in METHODS IN MOLECULAR BIOLOGY, VOL.10, pages 79-104 (The Humana Press, Inc. 1992).

[0197] After initial generation of an antibody against an immunogen, the antibody can be sequenced and subsequently prepared by recombinant techniques. Humanization and chimerization of murine antibodies and antibody fragments are well known to those of skill in the art as discussed below.

[0198] Chimeric antibodies are recombinant proteins in which the variable regions of human antibodies are replaced, for example, by the variable regions of murine antibodies, including the complementarity determining regions (CDRs) of the murine antibodies. Chimeric antibodies exhibit reduced immunogenicity and increased stability when administered to a subject. General techniques for cloning murine immunoglobulin variable domains are disclosed, for example, in Orlandi et al., Proc. Nat’l Acad. Sci. USA 6:3833 (1989). Techniques for constructing chimeric antibodies are well known to those of skill in the art. As an example, Leung et al., Hybridoma 13:469 (1994) produced an LL2 chimera by combining the DNA sequences encoding the V κ and V H domains of murine LL2, an anti-CD22 monoclonal antibody, with the respective human κ and IgG1 constant region domains.

[0199] Techniques for producing humanized Mabs are well known in the art (see, e.g., Jones et al., Nature 321:522 (1986), Riechmann et al., Nature 332:323 (1988), Verhoeyen et al., Science 239:1534 (1988), Carter et al., Proc. Nat’l Acad. Sci. USA 89:4285 (1992), Sandhu, Crit. Rev. Biotech. 12:437 (1992), and Singer et al., J. Immun. 150:2844 (1993)). Chimeric or murine monoclonal antibodies can be humanized by transferring mouse CDRs from the heavy and light chain variable chains of murine immunoglobulins to the corresponding variable domains of human antibodies. The mouse framework regions (FRs) in chimeric monoclonal antibodies are also replaced with human FR sequences. Merely transferring mouse CDRs to human FRs often results in a decrease or even loss of antibody affinity, and additional modifications may be required to restore the original affinity of the murine antibody. This can be accomplished by replacing one or more human residues in the FR region with their murine counterparts to obtain an antibody that has good binding affinity for its epitope. See, e.g., Tempest et al., Biotechnology 9:266 (1991) and Verhoeyen et al., Science 239:1534 (1988). Preferred residues for substitution include FR residues that are located within 1, 2, or 3 angstroms of the side chain of the CDR residue, adjacent to the CDR sequence, or predicted to interact with the CDR residue.

[0200] Methods for producing fully human antibodies using either combinatorial approaches or transgenic animals transformed with the human immunoglobulin locus are known in the art (e.g., Mancini et al., 2004, New Microbiol. 27:315-28; Conrad and Scheller, 2005, Comb. Chem. High Throughput Screen. 8:117-26; Brekke and Loset, 2003, Curr. Opin. Pharmacol. 3:544-50). Fully human antibodies can also be constructed by gene or chromosomal transfection methods as well as phage display techniques, all of which are known in the art. See, e.g., McCafferty et al., Nature 348:552-553 (1990). When antibodies are intended to be used in vivo, e.g., in tumor therapy after detection of Trop-2 positive cancer, such fully human antibodies are expected to exhibit fewer side effects than chimeric or humanized antibodies and to function in vivo essentially as endogenous human antibodies.

[0201] As an alternative, phage display techniques can be used to generate human antibodies (e.g., Dantas-Barbosa et al., 2005, Genet. Mol. Res. 4:126-40). Human antibodies can be generated from normal humans or from humans presenting a particular disease state such as cancer (Dantas-Barbosa et al., 2005). The advantage of constructing human antibodies from affected individuals is that the circulating antibody repertoire may be biased towards antibodies against disease-related antigens.

[0202] In one non-limiting example of this methodology, Dantas-Barbosa et al. (2005) constructed a phage display library of human Fab antibody fragments from osteosarcoma patients. Generally, total RNA was obtained from circulating blood lymphocytes (ibid.). Recombinant Fabs were cloned from the μ, γ, and κ chain antibody repertoires and inserted into the phage display library (ibid.). The RNA was converted to cDNA and used to generate a Fab cDNA library using specific primers for the heavy and light chain immunoglobulin sequences (Marks et al., 1991, J. Mol. Biol. 222:581-97). Library construction was performed according to Andris-Widhopf et al. (2000, In: Phage Display Laboratory Manual, Barbas et al. (eds), 1 st edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY pp. 9.1 to 9.22). The final Fab fragments were digested with restriction endonucleases and inserted into the bacteriophage genome to generate a phage display library. Such libraries can be screened by standard phage display methods known in the art. Phage display can be performed in a variety of formats; for an overview, see, e.g., Johnson and Chiswell, Current Opinion in Structural Biology 3:5564-571 (1993).

[0203] Human antibodies can also be produced by B cells activated in vitro. See U.S. Pat. Nos. 5,567,610 and 5,229,275. Those skilled in the art will understand that these techniques are exemplary and that any known method for making and screening human antibodies or antibody fragments can be utilized.

[0204] As another alternative, transgenic animals that have been genetically engineered to produce human antibodies can be used to generate antibodies against essentially any immunogenic target using standard immunization protocols. Methods for obtaining human antibodies from transgenic mice are disclosed by Green et al., Nature Genet. 7:13 (1994), Lonberg et al., Nature 368:856 (1994), and Taylor et al., Int. Immun. 6:579 (1994). Non-limiting examples of such systems are the XenoMouse® (registered trademark) manufactured by Abgenix (Fremont, CA) (e.g., Green et al., 1999, J. Immunol. Methods 231:11-23). In XenoMouse® and similar animals, the mouse antibody genes are inactivated and replaced by functional human antibody genes, while the rest of the mouse immune system remains intact.

[0205] The XenoMouse® is transformed with a germline-constructed YAC (yeast artificial chromosome) containing a portion of the human IgH and Ig kappa loci, including most of the variable region sequences, along with accessory genes and regulatory sequences. Antibody-producing B cells can be generated using the human variable region repertoire, which can be processed into hybridomas by known techniques. XenoMouse® immunized with the target antigen is thought to produce human antibodies by a normal immune response, which can be harvested and / or produced by the standard techniques described above. A variety of strains of XenoMouse® are available, each of which can produce different classes of antibodies. Transgenically produced human antibodies have been shown to have therapeutic potential while retaining the pharmacokinetic properties of normal human antibodies (Green et al., 1999). Those skilled in the art will understand that the claimed compositions and methods are not limited to the use of the XenoMouse® system and that any transgenic animal that has been genetically engineered to produce human antibodies can be utilized.

[0206] Antibody fragments are the antigen-binding portions of antibodies, such as F(ab’)2, Fab’, F(ab)2, Fab, Fv, sFv, scFv, etc. Antibody fragments that recognize specific epitopes can be generated by known techniques. For example, F(ab’)2 fragments can be produced by pepsin digestion of antibody molecules. These and other methods are described, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and the references contained therein. See also Nisonoff et al., Arch Biochem. Biophys. 89:230 (1960); Porter, Biochem. J. 73:119 (1959); Edelman et al., in METHODS IN ENZYMOLOGY VOL.1, page 422 (Academic Press 1967); and Coligan at pages 2.8.1-2.8.10 and 2.10.-2.10.4. Alternatively, a Fab’ expression library can be constructed (Huse et al., 1989, Science, 246:1274-1281), which may allow for the rapid and easy identification of monoclonal Fab’ fragments having the desired specificity.

[0207] A single-chain Fv molecule (scFv) comprises a VL domain and a VH domain. The VL and VH domains associate to form a target-binding site. These two domains are further covalently linked by a peptide linker (L). The scFv molecule is designated as VL-L-VH when the VL domain is the N-terminal portion of the scFv molecule, or VH-L-VL when the VH domain is the N-terminal portion of the scFv molecule. Methods for producing scFv molecules and designing suitable peptide linkers are described in U.S. Patent No. 4,704,692, U.S. Patent No. 4,946,778, R. Raag and M. Whitlow, “Single Chain Fvs.” FASEB Vol 9:73-80 (1995), and R.E. Bird and B.W. Walker, Single Chain Antibody Variable Regions, TIBTECH, Vol 9:132-137 (1991).

[0208] Other antibody fragments, such as single domain antibody fragments, are known in the art and can be used in the claimed constructs. Single domain antibodies (VHHs) can be obtained from, for example, camels, alpacas, or llamas by standard immunization techniques. (See, for example, Muyldermans et al., TIBS 26:230-235, 2001; Yau et al., J Immunol Methods 281:161-75, 2003; Maass et al., J Immunol Methods 324:13-25, 2007). VHHs can have strong antigen binding ability and can interact with novel epitopes that are inaccessible with conventional VH-VL pairs. (Muyldermans et al., 2001). Alpaca serum IgG contains approximately 50% IgG antibodies of only the camelid heavy chain (HCAb) (Maass et al., 2007). Alpacas can be immunized with known antigens such as TNF-α, and VHHs that bind to and neutralize the target antigen can be isolated (Maass et al., 2007). PCR primers that amplify virtually all alpaca VHH coding sequences have been identified and can be used to construct an alpaca VHH phage display library, which can be used for antibody fragment isolation by standard biopanning techniques well known in the art (Maass et al., 2007).

[0209] Antibody fragments can be prepared by proteolytic hydrolysis of full-length antibodies or by expression of DNA encoding the fragments in Escherichia coli (E. coli) or another host. Antibody fragments can be obtained by conventional methods, such as pepsin or papain digestion of full-length antibodies. For example, antibody fragments can be produced by enzymatic cleavage of an antibody with pepsin to provide a fragment of approximately 100 kD designated as F(ab’)2. This fragment can be further cleaved using a thiol reducing agent and optionally a blocking group for the sulfhydryl groups resulting from cleavage of the disulfide linkages to produce a Fab’ monovalent fragment of approximately 50 Kd. Alternatively, enzymatic cleavage using papain directly produces two monovalent Fab fragments and one Fc fragment.

[0210] Other methods of cleaving antibodies, such as separation of the heavy chain forming the monovalent light chain-heavy chain fragment, further cleavage of the fragment, or other enzymatic, chemical, or genetic techniques, can be used as long as the fragment binds to the antigen recognized by the intact antibody.

[0211] The terms CXCL12 and SDF-1 are synonyms and are used interchangeably herein.

[0212] In one embodiment of the invention and as preferably used herein, an antagonist of CXCL12.

[0213] It will be understood by those skilled in the art that any reference herein to the present invention and any reference to the present invention is also a reference to each and any aspect disclosed herein, including any embodiment thereof.

[0214] The nucleic acid molecules according to the present invention and the various SEQ ID NOs, chemical properties, actual sequences, and internal reference numbers of the target molecule CXCL12 used herein are summarized in Table 1 below. It should be noted that the nucleic acids are characterized by aptamers, i.e., D-nucleic acid level (D-RNA) using biotinylated human D-CXCL12 (SEQ ID NO: 4), or Spiegelmer level, i.e., L-nucleic acid (L-RNA) using the native structure of CXCL12, L-CXCL12 (human CXCL12α, SEQ ID NO: 1). Although various nucleic acids share one internal reference name, each shares one SEQ ID NO for D-RNA (aptamer) molecules and one SEQ ID NO for L-RNA (Spiegelmer) molecules. This application will be understood by those skilled in the art to contain additional sequences and in particular amino acid sequences.

[0215] JPEG2025522558000001.jpg243170JPEG2025522558000002.jpg241170JPEG2025522558000003.jpg236170JPEG2025522558000004.jpg231170JPEG2025522558000005.jpg233170JPEG2025522558000006.jpg222170JPEG2025522558000007.jpg226170JPEG2025522558000008.jpg225170JPEG2025522558000009.jpg203170JPEG2025522558000010.jpg247170JPEG2025522558000011.jpg244170JPEG2025522558000012.jpg221170JPEG2025522558000013.jpg225170JPEG2025522558000014.jpg209170JPEG2025522558000015.jpg237170JPEG2025522558000016.jpg221170

[0216] The present invention will be further described by and with reference to the following figures and examples, to which additional advantages, features, and embodiments may be adopted.

Brief Description of the Drawings

[0217]

Figure 1

Figure 2A

Figure 2B

Figure 3

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Figure 4B

Figure 5

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Figure 7A

Figure 7B

Figure 8

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Figure 11A

Figure 11B

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Figure 27

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Figure 29

[0218] [Example 1: Nucleic Acids That Bind to Human CXCL12] In the following, the terms "nucleic acid" and "nucleic acid molecule" are used synonymously herein unless the contrary is indicated. Further, the terms "stretch" and "stretch of nucleotides" are used synonymously herein unless the contrary is indicated.

[0219] L-nucleic acid molecules that bind to human CXCL12 and their respective nucleotide sequences are depicted in FIGS. 1-9. Nucleic acids were characterized at the aptamer, i.e., D-nucleic acid level, using competitive or direct pull-down binding assays (see protocol, Example 3) with biotinylated human D-CXCL12. Spiegelmers were tested using surface plasmon resonance measurements (see protocol, Example 5) with a Biacore 2000 instrument and cell culture in vitro chemotaxis assays (see protocol, Example 4) using the native structure of CXCL12 (L-CXCL12).

[0220] CXCL12-binding nucleic acid molecules exhibit different sequence motifs, and three main types are defined in FIGS. 1, 2A and 2B (type A), FIGS. 3, 4A and 4B (type B), FIGS. 5, 4, 7A, 7B and 8 (type C). Nucleic acid molecules exhibit different sequence motifs. For the definition of nucleotide sequence motifs, IUPAC abbreviations for nucleotides with multiple meanings are used. S Strong G or C; W Weak A or U; R Purine G or A; Y Pyrimidine C or U; K Keto G or U; M Imino A or C; B C or U or G not A; D A or G or U not C; H A or C or U not G; V A or C or G not U; N All A or G or C or U

[0221] Unless the contrary is indicated, any nucleic acid sequence or stretch and the sequence of boxes are shown in the 5'→3' direction, respectively.

[0222] <CXCL12-binding nucleic acid molecule of type A> As depicted in Figure 1, all sequences of the CXCL12-binding nucleic acid of type A include one central stretch of nucleotides flanked by a first (5') and a second (3') stretch of nucleotides (also referred to as the first terminal stretch of nucleotides and the second stretch of nucleotides), such that the two stretches can hybridize to each other. However, within the molecule, such hybridization is not necessarily given.

[0223] In the following, the terms "CXCL12-binding nucleic acid of type A" and "type A CXCL12-binding nucleic acid" or type A CXCL12-binding nucleic acid molecule are used synonymously herein unless the contrary is indicated.

[0224] The sequences of the defined boxes or stretches of nucleotides can vary among CXCL12-binding nucleic acids of type A, which affects the binding affinity to CXCL12. Based on the binding assays for various CXCL12-binding nucleic acids summarized as type A CXCL12-binding nucleic acids, the central stretch of nucleotides and its nucleotide sequence described below are individually, and more preferably as a whole, essential for binding to CXCL12.

[0225] The central stretch of nucleotides of all identified sequences of type A CXCL12-binding nucleic acids has the sequence

Chemical formula

Chemical formula

[0226] As described above, type A CXCL12-binding nucleic acids 192-B11 and 192-C10 exhibit an equal binding affinity to CXCL12 as 192-A10-001. However, they show slight differences in the nucleotide sequence of the central stretch of nucleotides. Therefore, the consensus sequence of three molecules that bind to CXCL12 with nearly the same high affinity is the nucleotide sequence [Chemistry] can be summarized by (Type A - 4, SEQ ID NO: 77), the nucleotide sequence of the central stretch of nucleotides of 192 - A10 - 001 [Chemistry] is the nucleotide sequence having the highest binding affinity for Type A CXCL12 - binding nucleic acids.

[0227] Five or six of the six nucleotides of the 5' end stretch (also referred to as the first end stretch) of the type A CXCL12-binding nucleic acid can hybridize to each of five or six of the six nucleotides of the 3' end stretch (also referred to as the second end stretch) to form a terminal helix. Although these nucleotides are variable at several positions, various nucleotides allow hybridization for five or six of the six nucleotides of each of the 5' and 3' end stretches. The 5' and 3' end stretches of the type A CXCL12-binding nucleic acid shown in FIG. 1 can be summarized in a general formula for the 5' end stretch (\"RSHRYR\", type A formula-5-5') and for the 3' end stretch (\"YRYDSY\", type A formula-5-3'). A truncated derivative of the type A CXCL12-binding nucleic acid 192-A10-001 was analyzed in competitive pull-down binding assays against the original molecules 192-A10-001 and 192-A10-008 (FIGS. 2A and 2B). These experiments showed that the reduction of six terminal nucleotides of 192-A10-001 (5' end: GCUGUG; 3' end: CGCAGC) to five nucleotides of the derivative 192-A10-002 (5' end: CUGUG; 3' end: CGCAG) can be done without a reduction in binding affinity. However, shortening to four terminal nucleotides (5' end: UGUG; 3' end: CGCA; 192-A10-003) or less (192-A10-004 / -005 / -006 / -007) led to a decrease in binding affinity to CXCL12 (FIG. 2A). As shown in FIGS. 2A and 2B, the determined 5' and 3' end stretches having lengths of five and four nucleotides of the derivative of the type A CXCL12-binding nucleic acid 192-A10-001 can be described by a general formula for the 5' end stretch (\"X2BBBS\", type A formula-6-5') and for the 3' end stretch (\"SBBVX3\"; type A formula-6-3'), where X2 is absent or \"S\", and X3 is absent or \"S\".

[0228] The nucleotide sequences of the 5’ and 3’ terminal stretches have an impact on the binding affinity of the type A CXCL12-binding nucleic acids. This is shown not only by nucleic acids 192-F10 and 192-E10, but also by derivatives of 192-A10-001 (Figure 2B). The central stretches of 192-F10 and 192-E10 are identical to 192-B11 and 192-C10, but contain slight differences at the 3’ end of the 5’ terminal stretch and at the 5’ end of the 3’ terminal stretch, resulting in a decrease in binding affinity.

[0229] Substitution of the 5' and 3' terminal nucleotides "CUGUG" and "CGCAG" of type A CXCL12-binding nucleic acid 192-A10-002 with "GCGCG" and "CGCGC" (192-A10-015) resulted in a decrease in binding affinity, while substitution with "GCGUG" and "CGCGC" (192-A10-008) resulted in the same binding affinity as shown for 192-A10-002 (Figure 2B). Additionally, nine derivatives (192-A10-014 / -015 / -016 / -017 / -018 / -019 / -020 / -021 / -022 / -023) of type A CXCL12-binding nucleic acid 192-A10-001, each having four 5' and 3' terminal nucleotides, were tested as aptamers for their binding affinity against 192-A10-001 or its derivative 192-A10-008 (both having the same binding affinity to CXCL12). All molecules showed a weaker, very weak, or much weaker binding affinity to CXCL12, respectively, compared to 192-A10-001 (six nucleotides forming the terminal helix) or 192-A10-008 having five terminal nucleotides (Figure 2B). As a result, the sequence and number of nucleotides in the 5' and 3' terminal stretches are essential for effective binding to CXCL12. As shown for type A CXCL12-binding nucleic acids 192-A10-002 and 192-A10-08, the preferred combinations of the 5' and 3' terminal stretches are "CUGUG" and "CGCAG" (the 5' and 3' terminal stretches of type A CXCL12-binding nucleic acid 192-A10-002), and "GCGUG" and "CGCGC" (the 5' and 3' terminal stretches of type A CXCL12-binding nucleic acid 192-A10-008).

[0230] However, when combining the 5' and 3' end stretches of all tested Type A CXCL12-binding nucleic acids, the general formula for the 5' end stretch of the Type A CXCL12-binding nucleic acid is "X1X2NNBV" (Type A formula - 7 - 5'), and the general formula for the 3' end stretch of the Type A CXCL12-binding nucleic acid is "BNBNX3X4" (Type A formula - 7 - 3'), where X1 is "R" or absent, X2 is "S", X3 is "S", and X4 is "Y" or absent; Or, X1 is absent, X2 is "S" or absent, X3 is "S" or absent, and X4 is absent.

[0231] To extend the plasma residence time of the Spiegelmer in vivo, as described in Chapter 2, Spiegelmer 192 - A10 - 008 was covalently linked to a 40 kDa polyethylene glycol (PEG) moiety at the 5' end. The PEG moiety has no effect on the ability of the Spiegelmer to inhibit CXCL12-induced chemotaxis.

[0232] <Type B CXCL12-binding nucleic acid molecule> As depicted in Figure 3, all sequences of the Type B CXCL12-binding nucleic acid contain one central stretch of nucleotides flanked by 5' and 3' end stretches (also referred to as the first and second terminal stretches of nucleotides) that can hybridize to each other. However, within the molecule, such hybridization is not necessarily given.

[0233] Hereinafter, the terms "Type B CXCL12-binding nucleic acid molecule" and "Type B CXCL12-binding nucleic acid" or Type B CXCL12-binding nucleic acid molecule are used synonymously herein unless the contrary is indicated.

[0234] The defined array of boxes or stretches can vary among CXCL12-binding nucleic acids and it affects the binding affinity to CXCL12. Based on the binding assays for various CXCL12-binding nucleic acids, the central stretch of nucleotides and its nucleotide sequence described below are, individually and more preferably as a whole, essential for binding to CXCL12.

[0235] The central stretches of nucleotides of all the specified sequences of CXCL12-binding nucleic acids 193-C2-001, 193-G2-001, 193-F2-001, 193-G1-002, 193-D2-002, 193-A1-002, 193-D3-002, 193-B3-002, 193-H3-002, 193-E3-002, and 193-D1-002 share the sequence

Chem.

Chem.

[0236] Four, five, or six of the six nucleotides of the 5'-end stretch of the CXCL12-binding nucleic acid can hybridize to each of four, five, or six of the six nucleotides of the 3'-end stretch of the CXCL12-binding nucleic acid to form a terminal helix. Although the nucleotides are variable at several positions, various nucleotides enable hybridization to four, five, or six of the six nucleotides of each of the 5'- and 3'-end stretches. The 5'- and 3'-end stretches of the CXCL12-binding nucleic acid shown in Figure 3 can be summarized by general formulas for the 5'-end stretch (「X1X2GCRWG」, where X1 is 「A」 or absent and X2 is 「G」) and for the 3'-end stretch (「KRYSCX3X4」, where X3 is 「G」 and X4 is 「U」 or absent). CXCL12-binding nucleic acids 193-G1-002, 193-D2-002, 193-A1-002, and 193-D3-002 have a weaker binding affinity for CXCL12; however, they share the same nucleotide central stretch as 193-C2-001, 193-G2-001, and 193-F2-001 (Figure 3). The unfavorable binding properties of CXCL12-binding nucleic acids 193-G1-002, 193-D2-002, 193-A1-002, and 193-D3-002 may be due to the number and sequence of the nucleotides in the 5'- and 3'-end stretches.

[0237] Truncated derivatives of CXCL12-binding nucleic acids 193-G2-001 and 193-C2-001 were analyzed in competitive pull-down binding assays against 193-G2-001 and 193-G2-012, respectively (Figures 4A and 4B). These experiments showed that a reduction of the six terminal nucleotides (5'-end: AGCGUG; 3'-end: UACGCU) of CXCL12-binding nucleic acids 193-G2-001 and 193-C2-001 to five nucleotides (5'-end: GCGUG; 3'-end: UACGC) led to molecules (193-C2-002 and 193-G2-012) with comparable binding affinities. The equilibrium dissociation constant K Dwas determined using a pull-down binding assay (K D= 0.3 nM). Shortening to 4 or fewer nucleotides (5'-end: CGUG; 3'-end: UACG; 193-C2-003) or (193-C2-004, 193-C2-005, 193-C2-006, 193-C2-007) resulted in a decrease in binding affinity to CXCL12 as measured by a competitive pull-down binding assay (Figure 4A). The nucleotide sequences of the 5 terminal nucleotides at the 5' and 3' ends each have an impact on the binding affinity of the CXCL12-binding nucleic acid. Substitution of the 5' and 3'-terminal nucleotides "GCGUG" and "UACGC" (193-C2-002, 193-G2-12) with "GCGCG" and "CGCGC" (193-G2-013) resulted in a decrease in binding affinity. Additionally, four different derivatives (193-G2-014 / -015 / -016 / -017) of the CXCL12-binding nucleic acid 193-G2-001 with terminal helices having a length of 4 base pairs were tested. All of them showed a decrease in binding affinity to CXCL12 (Figure 4B). Therefore, the sequences and lengths of the 5' and 3'-terminal nucleotides are essential for effective binding to CXCL12. As shown in Figures 4A and 4B, the 5'- and 3'-terminal stretches with lengths of 5 and 4 nucleotides of the derivatives of the CXCL12-binding nucleic acids 193-C2-003 and 193-G2-012 can be described by general formulas for the 5'-terminal stretch (where "X1X2SSBS", where X1 is absent, X2 is absent or "G") and for the 3'-terminal stretch (where "BVSSX3X4", where X3 is absent or "C" and X4 is absent). As shown for the CXCL12-binding nucleic acids 193-G2-001 and 193-C2-01, and their derivatives 193-G2-012 and 193-C2-002, the preferred combination of the 5' and 3'-terminal stretches is "X1X2GCGUG" (5'-terminal stretch) and "UACGCX3X4" (3'-terminal stretch), where X1 is "A" or absent, X2 is "G", X3 is "C", and X4 is "U" or absent.

[0238] However, when combining the 5' and 3' terminal stretches of all the tested CXCL12-binding nucleic acids, the general formula for the 5' terminal stretch of the CXCL12-binding nucleic acid is "X1X2SVNS", and the general formula for the 3' terminal stretch of the CXCL12-binding nucleic acid is "BVBSX3X4", where X1 is "A" or absent, X2 is "G", X3 is "C", and X4 is "U" or absent; or, X1 is absent, X2 is "G" or absent, X3 is "C" or absent, and X4 is absent.

[0239] To extend the plasma residence time of the Spiegelmer in vivo, as described in Chapter 2, Spiegelmer 193-G2-012 was covalently linked to a 40 kDa polyethylene glycol (PEG) moiety at the 5' end (PEGylated nucleic acid molecule: 193-G2-012-5'-PEG, also referred to as NOX-A12). The PEGylated Spiegelmer NOX-A12 was analyzed under cell culture in an in vitro chemotaxis assay to determine the inhibition of CXCL12-induced chemotaxis (IC of 0.2 nM 50 ). The PEGylated Spiegelmer NOX-A12 was analyzed by Biacore measurement to determine a binding constant (K D ) of 0.2 nM.

[0240] <Type C CXCL12-binding nucleic acid molecule> As depicted in Figure 5, all sequences of the Type C CXCL12-binding nucleic acid contain one central stretch of nucleotides flanked by 5' and 3' terminal stretches (also referred to as the first and second terminal stretches of nucleotides) that can hybridize to each other. However, within the molecule, such hybridization is not necessarily given.

[0241] Hereinafter, the terms "CXCL12-binding nucleic acid molecule of type C" and "type C CXCL12-binding nucleic acid" or "type C CXCL12-binding nucleic acid molecule" are used synonymously herein unless the contrary is indicated.

[0242] The sequences of the defined boxes or stretches can vary among CXCL12-binding nucleic acids of type C and affect their binding affinity to CXCL12. Based on the binding analysis of various CXCL12-binding nucleic acids summarized as type C CXCL12-binding nucleic acids, the central stretch of nucleotides and its nucleotide sequence described below are each, and more preferably as a whole, essential for binding to CXCL12.

[0243] The central stretch of nucleotides of all identified sequences of type C CXCL12-binding nucleic acids has the sequence

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0244] Type C CXCL12-binding nucleic acid 190-A3-001 contains a 17-nucleotide 5'-terminal stretch ("CGUGCGCUUGAGAUAGG", SEQ ID NO: 220) and a 12-nucleotide 3'-terminal stretch ("CUGAUUCUCACG", SEQ ID NO: 221), while 4 nucleotides at the 5'-end of the 5'-terminal stretch and 4 nucleotides at the 3'-end of the 3'-terminal stretch can hybridize to each other to form a terminal helix. Alternatively, the nucleotides "UGAGA" in the 5'-terminal stretch can hybridize to the nucleotides "UCUCA" in the 3'-terminal stretch to form a terminal helix. The reduction to 9 nucleotides ("UGAGAUAGG") of the 5'-terminal stretch and to 10 nucleotides ("CUGAUUCUCA", SEQ ID NO: 222) of the 3'-terminal stretch of molecule 190-A3-001 has no effect on the binding affinity for CXCL12 (190-A3-003; Figure 6). The reduction to 8 nucleotides ("GAGAUAGG") of the 5'-terminal stretch and to 9 nucleotides ("CUGAUUCUC") of the 3'-terminal stretch of molecule 190-A3-001 has no effect on the binding affinity for CXCL12 (190-A3-004; Figure 6). The equilibrium binding constant K D of 190-A3-004 was determined using a pull-down binding assay (K D = 4.6 nM) and by surface plasmon resonance measurement (K D = 4.7 nM). The 0.1 nM IC 50 (inhibitory concentration 50%) for 190-A3-004 was measured using a cell culture in vitro chemotaxis assay. However, shortening to 2 nucleotides in the 5'-terminal stretch leads to a very strong decrease in binding affinity (190-A3-007; Figure 6).

[0245] Type C CXCL12-binding nucleic acids 191-D5-001, 197-B2, and 197-H1 (central stretch of nucleotides:

Chemical formula

[0246] A truncated derivative of type C CXCL12-binding nucleic acid 191-D5-001 was constructed and tested in a competitive pull-down binding assay against the original molecule 191-D5-001 (FIGS. 7A, 7B). First, as depicted in FIG. 7A, the lengths of the 5' and 3' terminal stretches were shortened from 10 nucleotides each (191-D5-001) to 7 nucleotides each (191-D5-004), such that 9 out of 10 (191-D5-001) or 6 out of 7 nucleotides of the 5' and 3' terminal stretches, respectively, can hybridize to each other. The reduction to 7 nucleotides in the 5' and 3' terminal stretches (where 6 out of 7 nucleotides can hybridize to each other) led to a reduction in the binding affinity to CXCL12, respectively (191-D5-004). The terminal stretch of type C CXCL12-binding nucleic acid 191-D5-004 was modified by substituting the unpaired nucleotide "A" within the 3' terminal stretch of 191-D5-004 with "C" (191-D5-005). This modification led to an improvement in binding. This derivative type C CXCL12-binding nucleic acid 191-D5-005 showed binding to CXCL12 comparable to that of 191-D5-001. Further shortening of the 5' and 3' terminal stretches to 5 nucleotides each led to molecules having a total length of 29 nucleotides, respectively (191-D5-007). Due to the similarity between 191-D5-001 and type C CXCL12-binding nucleic acids 197-B2, 191-D5-001, 197-H1, 191-A5, 197-H3, 197-B1, 197-E3, 197-D1, 197-H2, and 197-D2, and due to the data shown for 191-D5-007, it was speculated that the 5' and 3' terminal stretches could be shortened to 5 nucleotides in principle, and we succeeded in testing the nucleotide sequence "CGGGA" for the 5' terminal stretch and "UCCCG" for the 3' terminal stretch (type C CXCL12-binding nucleic acid 191-D5-007).Type C CXCL12-binding nucleic acid 191-D5-007, surprisingly, binds somewhat better to CXCL12 than 191-D5-001 (determined at the aptamer level using a competitive binding assay). The equilibrium binding constant K of 191-D5-007. D was determined using a pull-down binding assay (K D = 2.2 nM), and by surface plasmon resonance measurement (K D = 0.8 nM). The 0.1 nM IC 50 (inhibitory concentration 50%) for 191-D5-007 was measured using a cell culture in vitro chemotaxis assay. Further shortening of the both-terminal stretches to 4 nucleotides (191-D5-010, Figure 7A).

[0247] Additional derivatives of type C CXCL12-binding nucleic acid 191-D5-001, each having 5'- and 3'-terminal stretches of 4 nucleotides (191-D5-017 / -024 / -029), also showed a decrease in binding affinity to CXCL12 in a competitive pull-down binding assay against 191-D5-007 (Figure 7B). Alternative 5'- and 3'-terminal stretches each having a length of 5 nucleotides were also additionally tested (191-D5-017-29a, 191-D5-017-29b, 191-D5-019-29a, 191-D5-024-29a, 191-D5-024-29b). The general formula for these derivatives with respect to the 5'-terminal stretch is "X S SSSV" (type C formula -7-5'), and for the 3'-stretch is "BSSSX S ", (type C formula -7-3'), where X Sis absent or is "S". Two of the five tested variants showed the same binding affinity to CXCL12 as 191-D5-007 (191-D5-024-29a, 191-D5-024-29b; Figure 7B). The sequences of the 5'-end and 3'-end stretches of the 191-D5-001 derivative (191-D5-007, 191-D5-024-29a, 191-D5-024-29b) that showed the highest binding affinity to CXCL12 and each contained a 5'-end and 3'-end stretch of five nucleotides can be summarized by the general formula (5'-end stretch: "SGGSR", type C formula-8-5'; 3'-end stretch: "YSCCS", type C formula-8-3').

[0248] A truncated derivative of type C CXCL12-binding nucleic acid 197-B2 was analyzed in a competitive pull-down binding assay against the original molecules 197-B2 and 191-D5-007 (Figure 8). Using the competitive pull-down binding assay against 191-D5-007, 197-B2 was shown to have the same binding affinity for CXCL12 as 191-D5-007. The 5' and 3' terminal stretches were shortened from 10 nucleotides each (197-B2) to 5 nucleotides each (197-B2-005) without loss of binding affinity, such that the nucleotides of the 5' terminal stretch and the 3' terminal stretch can hybridize completely to each other. When the 5' terminal ("GCGGG") and 3' terminal ("CCUGC") stretches of 197-B2-005 were replaced by "GCCGG" (5' terminal stretch) and "CCGGC" (3' terminal stretch) of 197-B2-006, the binding affinity for CXCL12 was completely sustained. Since 197-B2 and 191-D5-001 (and their derivatives) share the same core nucleotide sequence and several derivatives of 191-D5 with 5' and 3' terminal stretches each having a length of 4 nucleotides were tested, further shortening of the 5' and 3' terminal stretches was omitted. Two additional derivatives were designed that each contain 6 nucleotides at the 5' and 3' termini (5' and 3' terminal stretches). The binding affinity for CXCL12 of both molecules (197-B2-006-31a and 197-B2-006-31b) is the same as that shown for 191-D5-007 and 197-B2-006 (Figure 8). The sequences of the 5' and 3' terminal stretches of the 197-B2 derivative that show the highest binding affinity for CXCL12 and contain 5'-terminal and 3'-terminal stretches of 5 nucleotides each can be summarized by the general formula (5'-terminal stretch: "GCSGG", type C formula-9-5'; 3'-terminal stretch: "CCKGC", type C formula-9-3').

[0249] Combining the preferred 5' and 3' stretches of the shortened derivatives of Type C CXCL12-binding nucleic acids 191-D5-001 (5'-terminal stretch: "SGGSR", Type C formula -8-5'; 3'-terminal stretch: "YSCCS", Type C formula -8-3') and 197-B2 (5'-terminal stretch: "GCSGG", Type C formula -9-5'; 3'-terminal stretch: "CCKGC", Type C formula -9-3'), the common preferred general formulas for the 5'-terminal and 3'-terminal stretches are "SSSSR" (5'-terminal stretch, Type C formula -10-5') and "YSBSS" (3'-terminal stretch: Type C formula -10-3').

[0250] To extend the plasma residence time of the Spiegelmers in vivo, as described in Chapter 2, Spiegelmers 197-B2-006 and 191-D5-007 were covalently linked to a 40 kDa polyethylene glycol (PEG) moiety at their 5'-termini. The PEGylated Spiegelmers 197-B2-006 and 191-D5-007 were analyzed in cell culture for in vitro chemotaxis. The PEG moiety has no effect on the ability of the Spiegelmer to inhibit CXCL12-induced chemotaxis.

[0251] <CXCL12-binding nucleic acid molecule of Type D> Additionally, three additional CXCL12-binding nucleic acids that do not share the CXCL12-binding motifs of "Type A", "Type B", and "Type C" were identified and are referred to herein as "Type D". They were analyzed as aptamers using a pull-down binding assay (Figure 9).

[0252] Although the sequences shown in Figures 1-9 are nucleic acid molecules according to the present invention, it should be understood that such shortened and elongated nucleic acid molecules are also included if their shortened and elongated forms, not just their original forms, are still capable of binding to the target.

[0253] [Example 2: Determination of Binding Constant (Pull-down Binding Assay)] <Direct Pull-Down Binding Assay> The affinity of the aptamer for biotinylated human D-CXCL12 was measured in a pull-down binding assay format at 37°C. The aptamer was 5'-phosphoryl labeled with T4 polynucleotide kinase (Invitrogen, Karlsruhe, Germany) using [γ- 32 P] labeled ATP (Hartmann Analytic, Braunschweig, Germany). The specific radioactivity of the labeled aptamer was 200,000 - 800,000 cpm / pmol. The aptamer was incubated with various amounts of biotinylated human D-CXCL12 for 4 - 12 hours after denaturation and regeneration at concentrations of 10, 20, 30, or 40 pM at 37°C in selection buffer (20 mM Tris-HCl pH 7.4; 137 mM NaCl; 5 mM KCl; 1 mM MgCl2; 1 mM CaCl2; 0.1% [w / vol] Tween-20) to reach equilibrium at low concentrations. The selection buffer was supplemented with 10 μg / ml human serum albumin (Sigma-Aldrich, Steinheim, Germany), and 10 μg / ml yeast RNA (Ambion, Austin, USA) to prevent adsorption of binding partners to the surface of plastic products or immobilized matrices used. The concentration range of biotinylated human D-CXCL12 was set from 8 pM to 100 nM; the total reaction volume was 1 ml. Peptides and peptide-aptamer complexes were immobilized on 1.5 μl of Streptavidin Ultralink Plus particles (Pierce Biotechnology, Rockford, USA) pre-equilibrated with selection buffer and resuspended to a total volume of 6 μl. The particles were kept in suspension at each temperature for 30 minutes in a thermomixer. After removing the supernatant and washing appropriately, the immobilized radioactivity was quantified with a scintillation counter. The percentage of binding was plotted against the concentration of biotinylated human D-CXCL12, and the dissociation constant was obtained by using a software algorithm (GRAFIT; Erithacus Software; Surrey U.K.) assuming 1:1 stoichiometry.

[0254] <Competitive pull-down binding assay> To compare various D-CXCL12 binding aptamers, a competitive ranking assay was performed. For this purpose, the most closely related available aptamer was radiolabeled (see above), which served as a reference. After denaturation and regeneration, it was incubated with biotinylated human D-CXCL12 in 1 ml of selection buffer at 37 °C under conditions that resulted in approximately 5 - 10% binding to the peptide after immobilization and washing on NeutrAvidin agarose or Streptavidin Ultralink Plus (both from Pierce) without competition. An excess of denatured and regenerated unlabeled D-RNA aptamer variants was added to various concentrations (e.g., 2, 10, and 50 nM) together with the labeled reference aptamer to parallelize the binding reaction. The aptamers being tested competed with the reference aptamer for target binding and thus decreased the binding signal according to their binding characteristics. The aptamer found to be most active in this assay could then serve as a new reference for further comparative analysis of additional aptamer variants.

[0255] [Example 3: Binding analysis by surface plasmon resonance measurement] The binding of the Spiegelmer to human CXCL12α was analyzed using a Biacore 2000 instrument (Biacore AB, Uppsala, Sweden). When the coupling of human CXCL12α was to be achieved via an amine group, human CXCL12α was dialyzed against water for 1 - 2 hours (Millipore VSWP mixed cellulose ester; pore size, 0.025 μM) to remove interfering amines. Prior to protein coupling, the CM4 sensor chip (Biacore AB, Uppsala, Sweden) was activated by injection of 35 μl of a 1:1 dilution of 0.4 M NHS and 0.1 M EDC at a flow rate of 5 μl / min. Then, human MCP-1 or human CXCL12α was injected at a flow rate of 2 μl / min at a concentration of 0.1 - 1.5 μg / ml until the response of the instrument was in the range of 1000 - 2000 RU (relative units). Unreacted NHS ester was inactivated by injection of 35 μl of ethanolamine hydrochloride solution (pH 8.5) at a flow rate of 5 μl / min. The sensor chip was primed twice with binding buffer and equilibrated at 10 μl / min for 1 - 2 hours until the baseline appeared stable. For all proteins, kinetic parameters and dissociation constants were evaluated by a series of Spiegelmer injections at concentrations of 1000, 500, 250, 125, 62.5, 31.25, and 0 nM in the selected buffer (Tris-HCl, 20 mM; NaCl, 137 mM; KCl, 5 mM; CaCl2, 1 mM; MgCl2, 1 mM; Tween20, 0.1% [w / v]; pH 7.4). In all experiments, the analysis was performed at 37 °C using the Kinject command that defines an association time of 180 and a dissociation time of 360 seconds at a flow rate of 10 μl / min. Data analysis and calculation of the dissociation constant (K D ) were performed using the BIAevaluation 3.0 software (BIACORE AB, Uppsala, Sweden) with the Langmuir 1:1 stoichiometric fitting algorithm.

[0256] [Example 4: Analysis of the inhibition of CXCL12-induced chemotaxis by CXCL12-binding Spiegelmers] The human T cell leukemia cell line Jurkat, the human leukemic monocyte lymphoma cell line U937, the human pre-B cell leukemia cell line BV-173, and the human pre-B ALL cell line Nalm-6 express CXCR4. Although Jurkat cells do not express CXCR7, the leukemia cell lines BV-173 and U-937 were positive for CXCR7 expression upon testing. All cells used were obtained from DSMZ (Braunschweig). All cell lines were cultured at 37 °C and 5% CO2 in RPMI 1640 medium with Glutamax (Invitrogen, Karlsruhe, Germany) containing 10% fetal calf serum, 100 units / ml penicillin, and 100 μg / ml streptomycin (Invitrogen, Karlsruhe, Germany). One day before the experiment, the cells were seeded into new T175 flasks at a density of 0.3×10 6 / ml (Jurkat, U937, BV-173) or 0.75×10 6 / ml (Nalm-6).

[0257] For the experiment, the cells were centrifuged (300 g for 5 minutes), resuspended, counted, and washed once with 15 ml HBH (Hanks' balanced salt solution containing 1 mg / ml bovine serum albumin and 20 mM HEPES; Invitrogen, Karlsruhe, Germany). The cells were then resuspended at 1.33×10 6 / ml (Jurkat, U937, BV-173) or 2.67×10 6Resuspended in / ml (Nalm-6). Then, the cells were allowed to migrate through the porous membrane of the filter plate for 3 hours towards a solution containing CXCL12 and various amounts of Spiegelmer. The stimulation solution (CXCL12 + various concentrations of Spiegelmer) was prepared as a 10× solution in a 0.2 ml low-profile 96-tube plate. 212 μl of HBH was pipetted into the lower compartment of the transport plate, and 23.5 μl of the stimulation solution was added. All conditions were made in triplicate. After 20 - 30 minutes, the filter plate was inserted into the plate containing the stimulation solution, and 75 μl of the cell suspension having 1.33×10 6 / ml or 2.67×10 6 / ml was added to the wells of the filter plate (1×10 5 or 2×10 5 cells / well). Then, the cells were allowed to migrate at 37°C for 3 hours. For calibration, 0, 10, and 30 μl of the cell suspension were added to the wells of a separate 96-well plate in 235, 225, and 205 μl of HBH, respectively. After 3 hours of incubation, the insert plate was removed, and 30 μl of the resazurin working solution (440 μM in PBS) was added to the lower wells and the wells of the calibration plate. Then, the plate was incubated at 37°C for 2.5 hours. After incubation, 100 μl of each well was transferred to a black 96-well plate.

[0258] For evaluation, the fluorescence values were corrected against the background fluorescence (no cells in the well). Then, the difference between the experimental conditions with and without CXCL12 was calculated. The value for the sample without Spiegelmer (CXCL12 only) was set to 100%, and the value for the sample with Spiegelmer was calculated as a percentage of this. For the dose-response curve, the percentage values were plotted against the Spiegelmer concentration, and the IC 50 value (the concentration of Spiegelmer at which 50% of the activity without Spiegelmer is present) was determined graphically from the resulting curve.

[0259] <Results> Human CXCL12 has been found to stimulate the migration of Jurkat cells in a dose-dependent manner with a half-maximal stimulation of approximately 0.3 nM.

[0260] Human CXCL12 has been found to stimulate the migration of cells of the human leukemic monocyte lymphoma cell line U937 in a dose-dependent manner with a half-maximal stimulation of approximately 3 nM.

[0261] Human CXCL12 has been found to stimulate the migration of cells of the human pre-B cell leukemia cell line BV-173 in a dose-dependent manner with a half-maximal stimulation of approximately 3 nM.

[0262] Human CXCL12 has been found to stimulate the migration of cells of the human pre-B ALL cell line Nalm-6 in a dose-dependent manner with a half-maximal stimulation of approximately 0.3 nM.

[0263] When cells were migrated towards a solution containing increasing concentrations of CXCL12-binding Spiegelmer and human CXCL12, dose-dependent inhibition was observed. The IC50 of each of the tested Spiegelmers specified in Example 1 was determined in the human T cell leukemia cell line Jurkat cells. For example, for the CXCL12-binding Spiegelmer NOX-A12 (also referred to as 193-G2-012-5'-PEG), an IC50 of 0.2 nM was determined (Figure 10). When a non-specific control Spiegelmer was used instead of the CXCL12-binding Spiegelmer, no inhibitory effect was observed up to 1 μM.

[0264] Inhibition of CXCL12-induced chemotaxis by the CXCL12-binding Spiegelmer NOX-A12 was also observed in three other different leukemia cell types: the human leukemic monocyte lymphoma cell line U937 (Figure 11B), the human pre-B cell leukemia cell line BV-173 (Figure 12), and the human pre-B ALL cell line Nalm-6 (Figure 11A). Furthermore, the inventors have evidence that primary chronic lymphocytic leukemia cells migrate towards CXCL12 and that CXCL12-dependent chemotaxis is effectively blocked by NOX-A12.

[0265] The leukemia cell lines BV-173 and U-937 were also tested positive for CXCR7 expression. The efficacy of the SDF-binding Spiegelmer NOX-A12 that blocks the interaction between CXCL12 and CXCR7 was determined as shown in Example 5.

[0266] [Example 5: Inhibition of CXCR7 activation by CXCL12-binding Spiegelmer NOX-A12] In addition to CXCR4, CXCL12 also binds to the chemokine receptor CXCR7. The inhibitory potential of the CXCL12-binding Spiegelmer NOX-A12 against CXCR7 was tested in a complementation assay using CHO cells that stably express both CXCR7 and β-arrestin fused to a fragment of β-galactosidase (PathHunter™-β-arrestin assay, DiscoveRX, CA, USA). In the presence of CXCL12 binding, β-arrestin complexes with CXCR7, thus leading to the complementation and activation of β-galactosidase, which was measured using a chemiluminescent substrate.

[0267] [Method] PathHunter eXpress CHO-K1 human CXCR7 β-arrestin cells were seeded in OCC2 medium for 48 hours and stimulated with 10 nM CXCL12 and various concentrations of the CXCL12-binding Spiegelmer NOX-A12 for 90 minutes. After stimulation, signals were detected using the PathHUnter detection kit and the manufacturer's recommended protocol (DiscoveRX, CA, USA).

[0268] [Results] Stimulation of β-galactosidase with 10 nM human CXCL12, and thus CXCR7 activation, had an IC50 of 5.4 nM and was efficiently blocked by the CXCL12-binding Spiegelmer NOX-A12 (Figure 13).

[0269] [Example 6: Clinical investigation using triple therapy] The arms in the clinical phase 1 / 2 investigation were designed to assess the safety of NOX-A12 + bevacizumab when administered concomitantly with radiotherapy in glioblastoma patients, and to verify the benefit of these combinations on progression-free survival when administered in addition to irradiation, and to provide an estimate of the effect on overall survival.

[0270] The investigation was expected to advance understanding of the important role of CXCL12 in neovascularization by mobilizing endothelial cells and other bone marrow-derived angiogenesis-promoting cells through CXCR4- and CXCR7-dependent mechanisms.

[0271] The title of each investigation in which the NOX-A12 + bevacizumab arm is implemented is "Single-Arm Dose Escalation Phase 1 / 2 Investigation of Olaptesed Pegol (NOX-A12) in Combination with Irradiation in Patients with Inoperable or Subtotally Resected Primary Glioblastoma with Non-Methylated MGMT Promoter, Combined with a 3-Arm Expansion Cohort Including Bevacizumab or Pembrolizumab".

[0272] Investigation details are as follows.

[0273] Patient population: Patients with newly diagnosed glioblastoma (WHO grade IV) with a non-methylated MGMT promoter status after incomplete tumor resection or no tumor resection

[0274] Type of investigation: Multicenter, prospective, non-blinded

[0275] Investigation objectives: Primary: To examine the safety of olaptesed pegol in combination with radiotherapy, or olaptesed pegol in combination with radiotherapy and bevacizumab or pembrolizumab, in patients with newly diagnosed glioblastoma with a non-methylated MGMT promoter status. Secondary: (i) To examine the efficacy of olaptesed pegol in combination with radiotherapy and bevacizumab in patients with glioblastoma with unmethylated MGMT promoter status after a tumor resection that is not suitable for resection (biopsy only) or is incomplete; (ii) To examine the pharmacokinetics of olaptesed pegol during continuous administration; (iii) To monitor symptoms (NANO) and QoL.

[0276] Investigation assessment items: Primary: Safety (adverse events) Secondary: Progression-free survival at 6 months (PFS-6); Median progression-free survival (mPFS); Median overall survival (mOS); Tumor angiogenesis by vascular MRI scan at baseline and at 2, 4, and 6 months; Topographical features of recurrence; Determination of the maximum tolerated dose (MTD); Definition of the recommended phase 2 dose (RP2D); Steady-state NOX-A12 plasma levels; NANO assessment; Quality of life

[0277] Inclusion criteria: 1. Written informed consent 2. Age ≥ 18 years 3. Patient consent for diagnostic and scientific tests on glioblastoma tissue obtained during the previous surgery or biopsy (e.g., MGMT promoter analysis, cytogenetic markers such as IDH-1 mutation, etc.) 4. Patient consent for subcutaneous implant 5. Newly diagnosed, histologically confirmed WHO grade IV glioblastoma on the tent 6. Status after biopsy or incomplete resection (residual tumor detectable by postoperative T1-weighted contrast-enhanced MRI scan) (Arm A) 7. Unmethylated MGMT promoter status 8. Maximum US Eastern Cooperative Oncology Group (ECOG) score of 2 9. Estimated minimum life expectancy of 3 months 10. Stable or decreasing doses of corticosteroids during the week before inclusion 11. The following test parameters should be within the specified ranges: a. Total bilirubin ≤ 1.5 × upper limit of normal (ULN) b. Creatinine ≤ 1.5 × ULN or glomerular filtration rate ≥ 60 mL / min / 1.73 m² 2 c. ALT (alanine transaminase) ≤ 3 × ULN d. AST (aspartate transaminase) ≤ 3 × ULN 12. Female patients who may be pregnant must have a negative serum pregnancy test within 21 days before enrollment and must agree to use a highly effective method of birth control (such as an implant, vaginal ring, sterilization surgery, or abstinence from sexual intercourse, which, when used consistently and correctly, has a failure rate of less than 1% per year) during drug dosing and for 6 months after the last dose (more frequent pregnancy tests may be performed if required by local regulations). 13. Male patients who are sexually active with an FCBP must use an effective barrier method of contraception during the study and for 6 months after the last dose.

[0278] Exclusion criteria: 1. Unable to understand and cooperate throughout the study, or unable or unwilling to comply with the study requirements 2. Participation in any clinical research study with administration of an investigational drug or therapy within 30 days from the screening visit or the observation period of a competing study 3. Contraindication or known hypersensitivity to MRI contrast agents, bevacizumab, olaptesed pegol, or polyethylene glycol 4. Planned hypofractionated radiotherapy 5. Cytostatic therapy (chemotherapy) within the past 5 years 6. History of other cancers (excluding appropriately treated basal cell or squamous cell skin cancer, cervical intraepithelial neoplasia, or other cancers for which the patient has been disease - free for ≥ 5 years) 7. Currently active secondary malignancies 8. a. Myocardial infarction within the past 12 months b. Uncontrolled angina c. Congestive heart failure (New York Heart Association functional classification of ≧ 2) d. Diagnosed or suspected congenital long QT syndrome e. QTc prolongation (> 470 ms) on electrocardiogram before participation f. Uncontrolled hypertension (blood pressure ≧ 160 / 95 mmHg) g. Heart rate < 50 beats / min on baseline electrocardiogram h. History of any clinically significant type of ventricular arrhythmia (ventricular tachycardia, ventricular fibrillation, or torsades de pointes, etc.) i. Cerebrovascular attack Clinically significant or uncontrolled cardiovascular diseases, including 9. Prior radiotherapy to the head 10. Any other previous or concurrent experimental glioblastoma treatment 11. Implantation of Gliadel® wafers, seeds, or ferromagnetic particles 12. Patients with a history of arterial or venous thrombosis (or any other disease) requiring permanent intake of anticoagulants 13. Pregnancy or lactation 14. Uncontrolled comorbidities, including but not limited to ongoing or active infections, chronic liver diseases (e.g., cirrhosis, hepatitis), diabetes, or any of the following: fasting blood glucose (FBG defined as fasting for at least 8 hours) ≧ 200 mg / dL (7.0 mmol / L), or HbA1c ≧ 8%, chronic kidney disease, pancreatitis, chronic lung disease, autoimmune diseases, or psychiatric / social situations that would limit compliance with study requirements. The patient must not have any clinically relevant diseases (other than glioblastoma) that would interfere with the conduct of the investigation or study evaluation in the opinion of the treating investigator. 15. Prolongation of coagulation factors ≧ 2.5 × ULN 16. Treatment has not been initiated within 6 weeks after the first biopsy or surgery for glioblastoma 17. Previous registration in this investigation

[0279] Treatment in the arm combining NOX-A12 with radiotherapy and bevacizumab:

[0280] JPEG2025522558000033.jpg25170

[0281] The treatment regimen is also shown in Figure 14.

[0282] <Results> All 6 planned patients targeted for treatment with NOX-A12, radiotherapy, and bevacizumab are included, and all are being treated with NOX-A12 and bevacizumab. MRI assessments of tumor response have already been performed up to week 27 for 1 patient, up to week 18 for 2 patients, and up to week 9 for 5 patients. A decrease in T1-enhanced signal (SPDP, sum of the products of the diameters of tumor lesions) of more than 50% has been observed for all patients for whom data are available, and thus all patients have shown a partial response. See Table 2 below.

[0283] JPEG2025522558000034.jpg111170

[0284] The treatment regimen is shown in Figure 14.

[0285] [Example 7: Clinical study using triple therapy further including chemotherapy] A similar clinical study in glioblastoma patients is conducted as described in Example 6, but in this study, in addition, the patients receive the chemotherapy drug temozolomide, which is standard care for glioblastoma. The title of the study is "Single-arm phase 1 / 2 study of olaptesed pegol (NOX-A12) in combination with irradiation, bevacizumab, and temozolomide in primary glioblastoma patients with methylated and unmethylated MGMT promoters".

[0286] The investigation assesses the safety of NOX-A12 + bevacizumab and temozolomide when administered concurrently with radiotherapy in glioblastoma patients, verifies the benefit of these combinations on progression-free survival when administered in addition to irradiation, and provides an estimate of the effect on overall survival.

[0287] The differences between this investigation and the investigation described in Example 6 are as follows.

[0288] Patient population: Patients newly diagnosed with glioblastoma (WHO grade IV) with methylated or unmethylated MGMT promoter status and any degree of tumor resection

[0289] Treatment: In this investigation, NOX-A12 is combined with radiotherapy, bevacizumab, and temozolomide.

[0290] JPEG2025522558000035.jpg40170 There is no reason to expect different results for this investigation compared to the investigation in Example 6, following the non-overlapping modes of action of NOX-A12, bevacizumab, or radiotherapy with temozolomide. However, since patients with MGMT methylated promoter status, who are sensitive to temozolomide, are also included in this investigation, some additional benefit may be seen for this group of patients.

[0291] The treatment regimen is shown in Figure 15.

[0292] [Example 8: Clinical Investigation Using Triple Therapy of NOX-A12, Radiotherapy, and Bevacizumab] The arm in the clinical phase 1 / 2 investigation was designed to assess the safety of NOX-A12 + bevacizumab when administered concurrently with radiotherapy in glioblastoma patients, verify the benefit of these combinations on progression-free survival when administered in addition to irradiation, and provide an estimate of the effect on overall survival.

[0293] The investigation was expected to advance the understanding of the important role of CXCL12 in neovascularization by mobilizing endothelial cells and other bone marrow-derived pro-angiogenic cells through CXCR4- and CXCR7-dependent mechanisms. Notably, irradiation further increased CXCL12 expression. Additionally, the data were expected to assist in the further clinical development of NOX-A12.

[0294] The title of each investigation in which the NOX-A12 + bevacizumab arm is conducted is "Single-Arm Dose-Escalating Phase 1 / 2 Investigation of Olaptesed Pegol (NOX-A12) in Combination with Irradiation in Patients with Inoperable or Sub-Totally Resected Primary Glioblastoma with Methylation-Deficient MGMT Promoter, in a 3-Arm Expansion Cohort Including Patients with Complete Resection and in Combination with Bevacizumab or Pembrolizumab".

[0295] Investigation details are as follows.

[0296] Patient population: Patients newly diagnosed with glioblastoma (WHO grade IV) with a methylation-deficient MGMT promoter status, after incomplete tumor resection or no tumor resection.

[0297] Type of investigation: Multicenter, prospective, non-blinded

[0298] Investigation objectives: Primary: To examine the safety of olaptesed pegol in combination with radiotherapy and bevacizumab in patients newly diagnosed with glioblastoma with a methylation-deficient MGMT promoter status. Secondary: (i) To examine the efficacy of olaptesed pegol in combination with radiotherapy and bevacizumab in patients with glioblastoma with a methylation-deficient MGMT promoter status; (ii) To examine the pharmacokinetics of olaptesed pegol during continuous dosing; (iii) To monitor symptoms (NANO) and QoL.

[0299] Investigation evaluation items: Primary: Safety (adverse events) Secondary: Progression-free survival at 6 months (PFS-6); Median progression-free survival (mPFS); Median overall survival (mOS); Tumor angiogenesis by vascular MRI scans at baseline and at 2, 4, and 6 months; Topographical features of recurrence; Determination of the maximum tolerated dose (MTD); Definition of the recommended Phase 2 dose (RP2D); Steady-state NOX-A12 plasma levels; NANO assessment; Quality of life

[0300] Inclusion criteria: 1. Written informed consent 2. Age ≥ 18 years 3. Patient consent for diagnostic and scientific tests on glioblastoma tissue obtained during previous surgery or biopsy (e.g., MGMT promoter analysis, cytogenetic markers such as IDH-1 mutation, etc.) 4. Patient consent for subcutaneous implant 5. Newly diagnosed, histologically confirmed WHO Grade IV glioblastoma on the tentorium 6. Post-biopsy or post-incomplete resection status (residual tumor detectable by postoperative T1-weighted contrast-enhanced MRI scan) 7. Non-methylated MGMT promoter status 8. Eastern Cooperative Oncology Group (ECOG) score of 2 at most 9. Estimated minimum life expectancy of 3 months 10. Stable or decreasing doses of corticosteroids during the week prior to inclusion 11. The following laboratory parameters should be within the specified ranges: a. Total bilirubin ≤ 1.5 × upper limit of normal (ULN) b. Creatinine ≤ 1.5 × ULN or glomerular filtration rate ≥ 60 mL / min / 1.73 m 2 c. Alanine transaminase (ALT) ≤ 3 × ULN d. Aspartate transaminase (AST) ≤ 3 × ULN 12. Female patients who may be pregnant must have a negative serum pregnancy test within 21 days prior to enrollment and must agree to use a highly effective method of birth control (such as an implant for contraception, a vaginal ring, sterilization surgery, or sexual abstinence, which, when used consistently and correctly, has a failure rate of less than 1% per year) during drug dosing and for 6 months after the last dose (more frequent pregnancy tests may be performed if required by local regulations). 13. Male patients who are sexually active with an FCBP must use an effective barrier method of contraception during the study and for 6 months after the last dose.

[0301] Exclusion criteria: 1. Unable to understand and cooperate throughout the study, or unable or unwilling to comply with the study requirements 2. Participation in any clinical research study involving the administration of investigational drugs or therapies within 30 days of screening visit or the observation period of a competing study 3. Contraindication or known hypersensitivity to MRI contrast agents, bevacizumab, olaptesed pegol, or polyethylene glycol 4. Planned hypofractionated radiation therapy 5. Cytostatic therapy (chemotherapy) within the past 5 years 6. History of other cancers (excluding appropriately treated basal cell or squamous cell skin cancer, cervical intraepithelial neoplasia, or other cancers for which the patient has been disease-free for ≥ 5 years) 7. Currently active secondary malignancies 8. a. Myocardial infarction within the past 12 months b. Uncontrolled angina pectoris c. Congestive heart failure (New York Heart Association functional classification ≥ 2) d. Diagnosed or suspected congenital long QT syndrome e. QTc prolongation (> 470 ms) on pre-participation electrocardiogram f. Uncontrolled hypertension (blood pressure ≥ 160 / 95 mmHg) g. Heart rate < 50 beats per minute on baseline electrocardiogram h. History of any clinically significant type of ventricular arrhythmia (ventricular tachycardia, ventricular fibrillation, torsades de pointes, etc.) i. Cerebrovascular accident Clinically significant or uncontrolled cardiovascular disease, including 9. Prior radiotherapy to the head 10. Any other previous or concurrent experimental glioblastoma treatment 11. Implantation of Gliadel® wafers, seeds, or ferromagnetic particles 12. Patients with a history of arterial or venous thrombosis (or any other disease) requiring permanent anticoagulant intake 13. Pregnancy or lactation 14. Uncontrolled comorbidities, including but not limited to ongoing or active infections, chronic liver disease (e.g., cirrhosis, hepatitis), diabetes, or any of the following: fasting blood glucose (FBG defined as at least 8 hours of fasting) ≥ 200 mg / dL (7.0 mmol / L), or HbA1c ≥ 8%, chronic kidney disease, pancreatitis, chronic lung disease, autoimmune disease, or psychiatric / social situations that would limit compliance with study requirements. The patient must be free of any clinically relevant disease (other than glioblastoma) that, in the opinion of the treating investigator, would interfere with the conduct of the study or study evaluation. 15. Prolongation of coagulation factors ≥ 2.5 × ULN 16. Treatment has not been initiated within 6 weeks of the first biopsy or surgery for glioblastoma 17. Prior enrollment in this study

[0302] Treatment in the arm combining NOX-A12 with radiotherapy and bevacizumab:

[0303] JPEG2025522558000036.jpg25170

[0304] The treatment regimen is also shown in Figure 14.

[0305] <Results> All six planned patients targeted for treatment with NOX-A12, radiotherapy, and bevacizumab were included, and four are still being treated with NOX-A12 and bevacizumab beyond the originally planned 26 weeks, according to the decisions of the treating physicians and patients. One patient died.

[0306] Safety: The treatment was well tolerated and safe, with no dose-limiting toxicities or treatment-related deaths.

[0307] Efficacy: MRI assessments of tumor response have already been performed up to week 43 for one patient, up to week 35 for one patient, up to week 27 for three patients, and up to week 18 for one patient. In only one patient (Patient 6), although control of the target lesion was maintained, there was progression (PD) after an initial stable disease (SD) at week 9 due to the formation of cerebrospinal fluid metastases (see Table 3).

[0308] Five out of six patients achieved a partial response (PR) by modified Response Assessment in Neuro-Oncology (mRANO) assessment at week 9, and all PRs were durable with respect to the assessments available so far: Patient 1 showed a partial response by week 43; Patient 2 showed a partial response by week 35; Patients 3 - 5 showed partial responses by week 27; the median current follow-up period was 7.6 months. The Neuro-Oncology Longitudinal Neurological Assessment (NANO) assessment, a quantifiable assessment of nine relevant neurological domains based on direct observation and examination by the principal investigator of the clinical trial, revealed stable neurological function in five out of six patients with respect to the assessments available so far: Patient 1 showed no changes up to week 43; Patient 2 showed no changes up to week 35; Patients 3 and 4 showed no changes up to week 27; Patient 5 showed improvement at week 9 and further improvement at week 27. These mRANO responses and NANO scores for treatment from week 9 to week 43 are depicted in Figure 20.

[0309] High - field MRI imaging, neurological function, and quality - of - life (QoL) assessments revealed the following: · An improvement in apparent diffusion coefficient (ADC) with a mean maximal change of +20.6% (-24.5% to +59.1%); · An improvement in relative cerebral blood volume (rCBV) with a mean maximal change of -77.9% (-50% to -100%); · An improvement in high - perfusion tumor fraction (FTB 高 ) with a mean maximal change of -87.2% (-61.3% to -100%); · A very good response in non - target lesions with a mean maximal change of -93.8% (-81.4% to -100%); · A stable or slightly improved clinician - reported NANO score with a mean maximal change of -5.6% (0% to -33.3%); · Improvements in patient - reported outcomes regarding the following 〇 Overall functional score (OFS) with a mean maximal change of 40.7% (-12.4% to 224.4%); 〇 Global health score (GHS) with a mean maximal change of 31.3% (-8.3% to 100%)

[0310] These results for high - field MRI imaging, neurological function, and quality - of - life (QoL) assessments are depicted graphically in Figure 21.

[0311] JPEG2025522558000037.jpg134170

[0312] [Example 9: Clinical trial in glioblastoma patients using triple therapy of NOX - A12, radiotherapy, and bevacizumab] The arms in the clinical phase 1 / 2 study were designed to assess the safety of NOX - A12 + bevacizumab when administered concurrently with radiotherapy in glioblastoma patients, and to verify the benefit of these combinations on progression - free survival when administered in addition to irradiation, and to provide an estimate of the effect on overall survival.

[0313] The investigation was expected to advance the understanding of the important role of CXCL12 in neovascularization by mobilizing endothelial cells and other bone marrow-derived pro-angiogenic cells through CXCR4- and CXCR7-dependent mechanisms. Notably, irradiation further increased CXCL12 expression. Additionally, the data were expected to assist in the further clinical development of NOX-A12. The title of each investigation in which the NOX-A12 + bevacizumab arm was implemented was "Single-Arm Dose Escalation Phase 1 / 2 Investigation of Olaptesed Pegol (NOX-A12) in Combination with Irradiation in Patients with Unresectable or Sub-Totally Resected Primary Glioblastoma with Unmethylated MGMT Promoter, in a 3-Arm Expansion Cohort Including Patients with Complete Resection and in Combination with Bevacizumab or Pembrolizumab".

[0314] Details of the investigation are as follows.

[0315] Patient population: Patients with newly diagnosed glioblastoma (WHO grade IV) with an unmethylated MGMT promoter status, after incomplete tumor resection or no tumor resection

[0316] Type of investigation: Multicenter, prospective, non-blinded

[0317] Investigation objectives: Primary: To examine the safety of olaptesed pegol in combination with radiotherapy and bevacizumab in patients with newly diagnosed glioblastoma with an unmethylated MGMT promoter status. Secondary: (i) To examine the efficacy of olaptesed pegol in combination with radiotherapy and bevacizumab in patients with glioblastoma with an unmethylated MGMT promoter status; (ii) To examine the pharmacokinetics of olaptesed pegol during continuous dosing; (iii) To monitor symptoms (NANO) and QoL.

[0318] Investigation evaluation items: Primary: Safety (adverse events) Secondary: Progression-free survival at 6 months (PFS-6); Median progression-free survival (mPFS); Median overall survival (mOS); Tumor angiogenesis by vascular MRI scans at baseline and at 2, 4, and 6 months; Topographical features of recurrence; Determination of the maximum tolerated dose (MTD); Definition of the recommended phase 2 dose (RP2D); Steady-state NOX-A12 plasma levels; NANO assessment; Quality of life

[0319] Inclusion criteria: 1. Written informed consent 2. Age ≥ 18 years 3. Patient consent for diagnostic and scientific tests on glioblastoma tissue obtained during previous surgery or biopsy (e.g., MGMT promoter analysis, cytogenetic markers such as IDH-1 mutation, etc.) 4. Patient consent for subcutaneous implant 5. Newly diagnosed, histologically confirmed WHO grade IV glioblastoma on the tentorium 6. Post-biopsy or post-incomplete resection status (residual tumor detectable by postoperative T1-weighted contrast-enhanced MRI scan) 7. Non-methylated MGMT promoter status 8. Maximum Eastern Cooperative Oncology Group (ECOG) score of 2 9. Estimated minimum life expectancy of 3 months 10. Stable or decreasing dose of corticosteroids during the week prior to inclusion 11. The following laboratory parameters should be within the specified ranges: a. Total bilirubin ≤ 1.5 × upper limit of normal (ULN) b. Creatinine ≤ 1.5 × ULN or glomerular filtration rate ≥ 60 mL / min / 1.73 m² 2 c. Alanine transaminase (ALT) ≤ 3 × ULN d. Aspartate transaminase (AST) ≤ 3 × ULN 12. Female patients with a potential for pregnancy must have a negative serum pregnancy test within 21 days prior to enrollment and must agree to use a highly effective method of birth control (such as an implant, vaginal ring, sterilization, or sexual abstinence, which, when used consistently and correctly, has a failure rate of less than 1% per year) during drug dosing and for 6 months after the last dose (more frequent pregnancy tests may be performed if required by local regulations). 13. Male patients who are sexually active with an FCBP must use an effective barrier method of contraception during the study and for 6 months after the last dose.

[0320] Exclusion Criteria: 1. Unable to understand and cooperate throughout the study, or unable or unwilling to comply with study requirements 2. Participation in any clinical research investigation involving the administration of investigational drugs or therapies within 30 days of screening visit or during the observation period of a competing investigation 3. Contraindication or known hypersensitivity to MRI contrast agents, bevacizumab, olaptesed pegol, or polyethylene glycol 4. Planned hypofractionated radiation therapy 5. Cytostatic therapy (chemotherapy) within the past 5 years 6. History of other cancers (excluding appropriately treated basal cell or squamous cell skin cancer, cervical intraepithelial neoplasia, or other cancers for which the patient has been disease-free for ≥ 5 years) 7. Currently active secondary malignancy 8. a. Myocardial infarction within the past 12 months b. Uncontrolled angina c. Congestive heart failure (New York Heart Association functional class ≥ 2) d. Diagnosed or suspected congenital long QT syndrome e. QTc prolongation (> 470 ms) on pre-participation electrocardiogram f. Uncontrolled hypertension (blood pressure ≥ 160 / 95 mmHg) g. Heart rate < 50 beats per minute on baseline electrocardiogram h. History of any clinically significant type of ventricular arrhythmia (ventricular tachycardia, ventricular fibrillation, torsades de pointes, etc.) i. Cerebrovascular accident Clinically significant or uncontrolled cardiovascular disease, including 9. Prior radiation therapy to the head 10. Any other previous or concurrent experimental glioblastoma treatment 11. Implantation of Gliadel® wafers, seeds, or ferromagnetic particles 12. Patients with a history of arterial or venous thrombosis (or any other disease) requiring persistent intake of anticoagulants 13. Pregnancy or lactation 14. Uncontrolled comorbidities, including but not limited to ongoing or active infections, chronic liver disease (e.g., cirrhosis, hepatitis), diabetes, or any of the following: fasting blood glucose (FBG defined as at least 8 hours of fasting) ≥ 200 mg / dL (7.0 mmol / L), or HbA1c ≥ 8%, chronic kidney disease, pancreatitis, chronic lung disease, autoimmune disease, or psychiatric / social situations that would limit compliance with study requirements. The patient must be free of any clinically relevant disease (other than glioblastoma) that, in the opinion of the treating investigator, would interfere with the conduct of the study or study evaluation. 15. Prolongation of coagulation factors ≥ 2.5 × ULN 16. Treatment has not been initiated within 6 weeks of the first biopsy or surgery for glioblastoma 17. Prior enrollment in this study

[0321] Treatment in the arm combining NOX-A12 with radiotherapy and bevacizumab:

[0322] JPEG2025522558000038.jpg26170

[0323] The treatment regimen is also shown in Figure 14.

[0324] <Results> All six planned patients who were to be treated with NOX-A12, radiotherapy, and bevacizumab were included, and four are still being treated with NOX-A12 and bevacizumab beyond the originally planned 26 weeks, according to the decision of the treating physician and patient. One patient died.

[0325] Safety: The treatment was well tolerated and safe, with no dose-limiting toxicity or treatment-related deaths.

[0326] Efficacy: MRI assessments of tumor response have already been performed up to week 43 for one patient, up to week 35 for one patient, up to week 27 for three patients, and up to week 18 for one patient. Target lesion control was maintained in only one patient (Patient 6), but progression (PD) occurred after initial stability (SD) at week 9 due to the formation of cerebrospinal fluid metastases (see Table 4).

[0327] Five of the six patients achieved partial response (PR) by modified Response Assessment in Neuro-Oncology (mRANO) assessment at week 9, and all PRs were durable with respect to the assessments available so far: Patient 1 showed partial response by week 43; Patients 2 and 3 showed partial response by week 35; Patients 4 and 5 showed partial response by week 27; the median current follow-up period was 7.6 months. The Longitudinal Neurological Assessment in Neuro-Oncology (NANO) assessment, a quantifiable assessment of nine relevant neurological areas based on direct observation and examination by the principal investigator of the clinical trial, revealed stable neurological function in four of the six patients with respect to the assessments available so far: Patient 1 showed no change by week 43; Patient 2 showed no change by week 35; Patient 3 showed no change by week 27, but an increase in score at week 35, probably due to intracranial hemorrhage in an old infarct; Patient 4 showed no change by week 27; Patient 5 showed a decrease in score at week 9 and a further decrease at week 27. These mRANO responses and NANO scores for the treatment from week 9 to week 43 are depicted in Figure 22.

[0328] Advanced MRI imaging, neurological function, and quality of life (QoL) assessments for the maximum change from baseline revealed the following: · Improvement in apparent diffusion coefficient (ADC) with an average maximum change of +20.6% (-24.5% to +59.1%); · Improvement in relative cerebral blood volume (rCBV) with an average maximum change of -77.9% (-50% to -100%); · Improvement in high perfusion tumor fraction (FTB 高 ) with an average maximum change of -87.2% (-61.3% to -100%); · Very good response in non-target lesions with an average maximum change of -93.8% (-81.4% to -100%); · Stable or slightly improved clinician-reported NANO score with an average maximum change of -5.6% (0% to -33.3%); · Improvement in patient-reported outcomes regarding the following 〇 Overall functional score (OFS) with an average maximum change of 40.7% (-12.4% to 224.4%); 〇 Global health score (GHS) with an average maximum change of 31.3% (-8.3% to 100%)

[0329] These results for advanced MRI imaging, neurological function, and quality of life (QoL) assessments are depicted graphically in Figure 23 as the maximum maximum change from baseline.

[0330] Patients treated with the triple combination of NOX-A12, radiotherapy, and bevacizumab demonstrated a deeper response than patients treated with the dual combination of NOX-A12 and radiotherapy alone, and a much deeper response than patients receiving standard care: Target lesion response was expressed as the maximum change from baseline for the sum of products of perpendicular diameters (SPDP) and was depicted in Figure 24 for patients treated with the triple combination of NOX-A12, radiotherapy, and bevacizumab; in Figure 25 for patients treated with the dual combination of NOX-A12 and radiotherapy; and in Figure 26 for the historical reference cohort of patients treated with standard care (radiotherapy and the chemotherapeutic agent temozolomide).

[0331] JPEG2025522558000039.jpg134170

[0332] [Example 10: Clinical Trial in Glioblastoma Patients Using Triple Therapy of NOX-A12, Radiotherapy, and Bevacizumab - Follow-up Based on Survival Period] The arms in the clinical phase 1 / 2 study were designed to assess the safety of NOX-A12 + bevacizumab when administered concurrently with radiotherapy in glioblastoma patients, and to verify the benefit of these combinations on progression-free survival when administered in addition to irradiation, and to provide an estimate of the effect on overall survival.

[0333] The study was expected to advance the understanding of the important role that CXCL12 plays in angiogenesis by mobilizing endothelial cells and other bone marrow-derived pro-angiogenic cells through CXCR4- and CXCR7-dependent mechanisms. Notably, irradiation further increases CXCL12 expression. Additionally, the data were expected to assist in the further clinical development of NOX-A12.

[0334] The title of each investigation in which the NOX-A12 + bevacizumab arm is implemented is "Single-Arm Dose Escalation Phase 1 / 2 Study of Olaptesed Pegol (NOX-A12) in Combination with Radiation in Patients with Inoperable or Subtotal Resected Primary Glioblastoma with Unmethylated MGMT Promoter, in Association with a 3-Arm Expansion Cohort Including Complete Resection Patients and in Combination with Bevacizumab or Pembrolizumab".

[0335] Investigation details are as follows.

[0336] Patient population: Patients with newly diagnosed glioblastoma (WHO grade IV) with an unmethylated MGMT promoter status after incomplete tumor resection or after no tumor resection

[0337] Type of investigation: Multicenter, prospective, non-blinded

[0338] Investigation objectives: Primary: To examine the safety of olaptesed pegol in combination with radiotherapy and bevacizumab in patients with newly diagnosed glioblastoma with an unmethylated MGMT promoter status. Secondary: (i) To examine the efficacy of olaptesed pegol in combination with radiotherapy and bevacizumab in patients with glioblastoma with an unmethylated MGMT promoter status; (ii) To examine the pharmacokinetics of olaptesed pegol during continuous administration; (iii) To monitor symptoms (NANO) and QoL.

[0339] Investigation evaluation items: Primary: Safety (adverse events) Secondary: PFS at 6 months (PFS-6); Median progression-free survival (mPFS); Median overall survival (mOS); Tumor angiogenesis by vascular MRI scan at baseline and at 2, 4, and 6 months; Local characteristics of recurrence; Determination of the maximum tolerated dose (MTD); Definition of the recommended phase 2 dose (RP2D); Steady-state NOX-A12 plasma levels; NANO assessment; Quality of life

[0340] Inclusion Criteria: 1. Written informed consent 2. Age ≥ 18 years 3. Patient consent for diagnostic and scientific tests on glioblastoma tissue obtained during previous surgery or biopsy (e.g., MGMT promoter analysis, cytogenetic markers such as IDH-1 mutation, etc.) 4. Patient consent for subcutaneous implant 5. Newly diagnosed, histologically confirmed glioblastoma, WHO grade IV on the tentorium 6. Post-biopsy or post-incomplete resection status (residual tumor detectable by postoperative T1-weighted contrast-enhanced MRI scan) 7. Non-methylated MGMT promoter status 8. Eastern Cooperative Oncology Group (ECOG) performance status ≤ 2 9. Estimated life expectancy of at least 3 months 10. Stable or decreasing dose of corticosteroids during the week prior to inclusion 11. The following laboratory parameters should be within the specified ranges: a. Total bilirubin ≤ 1.5 × upper limit of normal (ULN) b. Creatinine ≤ 1.5 × ULN or glomerular filtration rate ≥ 60 mL / min / 1.73 m² 2 c. Alanine aminotransferase (ALT) ≤ 3 × ULN d. Aspartate aminotransferase (AST) ≤ 3 × ULN 12. Female patients of childbearing potential must have a negative serum pregnancy test within 21 days prior to enrollment and agree to use a highly effective method of birth control (e.g., contraceptive implant, vaginal ring, sterilization, or sexual abstinence, which, when used consistently and correctly, has a failure rate of less than 1% per year) during drug dosing and for 6 months following the last dose (more frequent pregnancy tests may be required as regulated by the site). 13. Male patients who are sexually active with a female partner must use an effective barrier method of contraception during the study and for 6 months following the last dose.

[0341] Exclusion criteria: 1. Unable to understand and cooperate throughout the investigation, or unable to comply with or unwilling to comply with the investigation requirements 2. Participation in any clinical research investigation with administration of investigational drug or therapy within 30 days from screening visit or observation period of competing investigation 3. Taboo or known hypersensitivity to MRI contrast agent, bevacizumab, olaptesed pegol, or polyethylene glycol 4. Planned hypofractionated radiotherapy 5. Cytostatic therapy (chemotherapy) within the past 5 years 6. History of other cancers (excluding appropriately treated basal cell or squamous cell skin cancer, cervical intraepithelial neoplasia, or other cancers for which the patient has been disease-free for ≥ 5 years) 7. Currently active secondary malignancy 8. a. Myocardial infarction within the past 12 months b. Unstable angina c. Congestive heart failure (New York Heart Association functional classification ≥ 2) d. Diagnosed or suspected congenital long QT syndrome e. QTc prolongation (> 470 ms) on electrocardiogram prior to participation f. Uncontrolled hypertension (blood pressure ≥ 160 / 95 mmHg) g. Heart rate < 50 beats / min on baseline electrocardiogram h. History of any clinically significant type of ventricular arrhythmia (ventricular tachycardia, ventricular fibrillation, torsades de pointes, etc.) i. Cerebrovascular accident Clinically significant or uncontrolled cardiovascular diseases, including 9. Prior radiotherapy to the head 10. Any other previous or concurrent experimental glioblastoma treatment 11. Implantation of Gliadel® wafer, seed, or ferromagnetic particle 12. Patients with a history of arterial or venous thrombosis (or any other disease) requiring permanent intake of anticoagulants 13. Pregnancy or lactation 14. Uncontrolled comorbidities, including but not limited to ongoing or active infectious diseases, chronic liver diseases (e.g., cirrhosis, hepatitis), diabetes, or any of the following: fasting blood glucose (FBG, defined as fasting for at least 8 hours) ≥ 200 mg / dL (7.0 mmol / L), or HbA1c ≥ 8%, chronic kidney disease, pancreatitis, chronic lung disease, autoimmune diseases, or psychiatric / social situations that would limit compliance with study requirements. The patient must be free of any clinically relevant diseases (other than glioblastoma) that would interfere with the conduct of the study or study assessment, in the opinion of the treating investigator. 15. Prolongation of coagulation factors ≥ 2.5 × ULN 16. Treatment has not been initiated within 6 weeks of the first biopsy or surgery for glioblastoma 17. Previous enrollment in this study

[0342] Treatment in the arm combining NOX-A12 with radiotherapy and bevacizumab:

[0343] JPEG2025522558000040.jpg24170

[0344] The treatment regimen is also shown in Figure 14.

[0345] <Results> All 6 planned patients who were to be treated with NOX-A12, radiotherapy, and bevacizumab are included. 3 patients are still being treated with NOX-A12 and bevacizumab beyond the originally planned 26 weeks, according to the decision of the treating physician and the patient. 1 patient died.

[0346] Safety: The treatment was well tolerated and safe, with no dose-limiting toxicity or treatment-related deaths.

[0347] Efficacy: On January 19, 2023, the median follow-up period of 10 months was reached. At that time, 5 out of 6 patients (83.3%) were still alive (see Figure 27). The median progression-free survival (mPFS) and the median overall survival (mOS) have not yet been reached.

[0348] [Example 11: Clinical Trial in Glioblastoma Patients Using Triple Therapy of NOX-A12, Radiation Therapy, and Bevacizumab - Further Follow-up Based on Survival] The arms in the clinical phase 1 / 2 investigation were designed to assess the safety of NOX-A12 + bevacizumab when administered concurrently with radiation therapy in glioblastoma patients, and to verify the benefit of these combinations on progression-free survival when administered in addition to irradiation, and to provide an estimate of the effect on overall survival.

[0349] The investigation was expected to advance the understanding of the important role of CXCL12 in angiogenesis by mobilizing endothelial cells and other bone marrow-derived pro-angiogenic cells through CXCR4- and CXCR7-dependent mechanisms. Notably, irradiation further increases CXCL12 expression. Furthermore, the data were expected to assist in the further clinical development of NOX-A12. The title of each investigation in which the NOX-A12 + bevacizumab arm is implemented is "Single-Arm Dose-Escalating Phase 1 / 2 Investigation of Olaptesed Pegol (NOX-A12) in Combination with Irradiation in Patients with Inoperable or Subtotal Resected Primary Glioblastoma with Unmethylated MGMT Promoter, Combined with a 3-Arm Expansion Cohort Including Patients with Complete Resection and Combination with Bevacizumab or Pembrolizumab".

[0350] Investigation details are as follows.

[0351] Patient population: Patients newly diagnosed with glioblastoma (WHO grade IV) with an unmethylated MGMT promoter status after incomplete tumor resection or no tumor resection.

[0352] Type of investigation: multi-center, prospective, non-blinded

[0353] Purpose of investigation: Primary: To examine the safety of olaptesed pegol in combination with radiotherapy and bevacizumab in patients newly diagnosed with glioblastoma with non-methylated MGMT promoter status. Secondary: (i) To examine the efficacy of olaptesed pegol in combination with radiotherapy and bevacizumab in patients with glioblastoma with non-methylated MGMT promoter status; (ii) To examine the pharmacokinetics of olaptesed pegol during continuous dosing; (iii) To monitor symptoms (NANO) and QoL.

[0354] Investigation evaluation items: Primary: Safety (adverse events) Secondary: Progression-free survival at 6 months (PFS-6); Median progression-free survival (mPFS); Median overall survival (mOS); Tumor angiogenesis by vascular MRI scan at baseline and at 2, 4, and 6 months; Local characteristics of recurrence; Determination of the maximum tolerated dose (MTD); Definition of the recommended phase 2 dose (RP2D); Steady-state NOX-A12 plasma levels; NANO assessment; Quality of life

[0355] Inclusion criteria: 1. Written informed consent 2. Age ≥ 18 years 3. Patient consent for diagnostic and scientific tests on glioblastoma tissue obtained during previous surgery or biopsy (e.g., MGMT promoter analysis, cytogenetic markers such as IDH-1 mutation, etc.) 4. Patient consent for subcutaneous implant 5. Newly diagnosed and histologically confirmed WHO grade IV glioblastoma on the tent 6. Post-biopsy or post-incomplete resection state (residual tumor detectable by postoperative T1-weighted contrast-enhanced MRI scan) 7. Non-methylated MGMT promoter status 8. Maximum US Eastern Cooperative Oncology Group (ECOG) score 2 9. Presumed minimum remaining life of 3 months 10. Stable or decreasing doses of corticosteroids during the week before incorporation 11. The following test parameters should be within the specified ranges: a. Total bilirubin ≤ 1.5 × upper limit of normal (ULN) b. Creatinine ≤ 1.5 × ULN or glomerular filtration rate ≥ 60 mL / min / 1.73 m² 2 c. ALT (alanine transaminase) ≤ 3 × ULN d. AST (aspartate transaminase) ≤ 3 × ULN 12. Female patients who may be pregnant must have a negative serum pregnancy test within 21 days before enrollment and must agree to use a highly effective method of birth control (such as an implant, vaginal ring, sterilization, or sexual abstinence, which, when used consistently and correctly, has a failure rate of less than 1% per year) during drug dosing and for 6 months after the last dose (more frequent pregnancy tests may be performed if required by local regulations). 13. Male patients who are sexually active with an FCBP must use an effective barrier method of contraception during the study and for 6 months after the last dose.

[0356] Exclusion criteria: 1. Inability to understand and cooperate throughout the study, or inability or unwillingness to comply with study requirements 2. Participation in any clinical research study with administration of investigational drugs or therapies within 30 days of screening visit or the observation period of a competing study 3. Contraindication or known hypersensitivity to MRI contrast agents, bevacizumab, olaptesed pegol, or polyethylene glycol 4. Planned hypofractionated radiotherapy 5. Cytostatic therapy (chemotherapy) within the past 5 years 6. History of other cancers (excluding appropriately treated basal cell or squamous cell skin cancer, cervical intraepithelial neoplasia, or other cancers for which the patient has been disease-free for ≥ 5 years) 7. Currently active secondary malignancies 8. a. Myocardial infarction within the past 12 months b. Uncontrolled angina pectoris c. Congestive heart failure (New York Heart Association functional classification of ≥ 2) d. Diagnosed or suspected congenital long QT syndrome e. QTc prolongation (> 470 ms) on pre - study electrocardiogram f. Uncontrolled hypertension (blood pressure ≥ 160 / 95 mmHg) g. Heart rate < 50 beats per minute on baseline electrocardiogram h. History of any clinically significant type of ventricular arrhythmia (ventricular tachycardia, ventricular fibrillation, torsades de pointes, etc.) i. Cerebrovascular accident Clinically significant or uncontrolled cardiovascular diseases, including 9. Prior radiotherapy to the head 10. Any other previous or concurrent experimental glioblastoma treatment 11. Implantation of Gliadel® wafers, seeds, or ferromagnetic particles 12. Patients with a history of arterial or venous thrombosis (or any other disease) requiring permanent anticoagulant intake 13. Pregnancy or lactation 14. Uncontrolled co - morbidities, including but not limited to ongoing or active infections, chronic liver diseases (e.g., cirrhosis, hepatitis), diabetes mellitus, or any of the following: fasting blood glucose (FBG, defined as fasting for at least 8 hours) ≥ 200 mg / dL (7.0 mmol / L), or HbA1c ≥ 8%, chronic kidney disease, pancreatitis, chronic lung disease, autoimmune diseases, or psychiatric / social situations that would limit compliance with study requirements. The patient must not have any clinically relevant diseases (other than glioblastoma) that would, in the opinion of the treating study physician, interfere with the conduct of the investigation or study evaluation. 15. Prolongation of coagulation factors ≥ 2.5 × ULN 16. Treatment has not been initiated within 6 weeks of the first biopsy or surgery for glioblastoma 17. Previous registration for this investigation

[0357] Treatment in the arm combining NOX-A12 with radiotherapy and bevacizumab:

[0358] JPEG2025522558000041.jpg24170

[0359] The treatment regimen is also shown in Figure 14.

[0360] <Results> All 6 planned patients who were to be treated with NOX-A12, radiotherapy, and bevacizumab were included. Two patients are still being treated with NOX-A12 and bevacizumab beyond the originally planned 26 weeks, according to the decision of the treating physician and the patient. One patient died.

[0361] Safety: The treatment was well-tolerated and safe, with no dose-limiting toxicity or treatment-related deaths.

[0362] Efficacy: On May 21, 2023, the median follow-up period of 14 months was reached. At that time, 5 out of 6 patients (83.3%) were still alive (see Figure 28). The median progression-free survival (mPFS) was 9.1 months, and the median overall survival (mOS) had not yet been reached. Tumor response was assessed by MRI. 5 out of 6 patients (83.3%) achieved an objective response, of which 1 patient reached a complete response (100% tumor reduction), and 2 patients reached >99% tumor reduction as the best response (see Figure 29).

[0363] [References] The complete bibliographic data of the documents cited in this specification are as follows, unless otherwise indicated, and the disclosures of the said references are incorporated herein by reference. Altschul S.F., Gish W., et al. (1990) Basic local alignment search tool. J Mol Biol. 215(3):403-10. Altschul S.F., Madden T.L., et al. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25(17):3389-402. Balabanian K., Lagane B., et al. (2005) The chemokine SDF-1 / CXCL12 binds to and signals through the orphan receptor RDC1 in T lymphocytes. J Biol Chem 280(42): 35760-35766 Balabanian, K., Lagane B., et al. (2005) WHIM syndromes with different genetic anomalies are accounted for by impaired CXCR4 desensitization to CXCL12. Blood 105(6): 2449-57. Balkwill F. (2004) Cancer and the chemokine network. Nat Rev Cancer 4(7): 540-50. Brooks H.L. Jr., Caballero S. Jr., et al. (2004) Vitreous levels of vascular endothelial growth factor and stromal-derived factor 1 in patients with diabetic retinopathy and cystoid macular edema before and after intraocular injection of triamcinolone. Arch Ophthalmol 122(12): 1801-7. Buckley C.D., Amft N., et al. (2000) Persistent induction of the chemokine receptor CXCR4 by TGF-beta 1 on synovial T cells contributes to their accumulation within the rheumatoid synovium. J Immunol 165(6): 3423-9. Burger J.A., Tsukada N., et al. (2000) Blood-derived nurse-like cells protect chronic lymphocytic leukemia B cells from spontaneous apoptosis through stromal cell-derived factor-1. Blood. 96(8):2655-63. Burns J.M., Summers B.C., et al. (2006) A novel chemokine receptor for SDF-1 and I-TAC involved in cell survival, cell adhesion, and tumor development. J Exp Med 203(9): 2201-2213 Butler J.M., Guthrie S.M., et al. (2005) SDF-1 is both necessary and sufficient to promote proliferative retinopathy. J Clin Invest 115(1): 86-93 Cabioglu, N., Sahin A., et al. (2005) Chemokine receptor CXCR4 expression in breast cancer as a potential predictive marker of isolated tumor cells in bone marrow. Clin Exp Metastasis 22(1): 39-46. Corcione A., Ottonello L., et al. (2000) Stromal cell-derived factor-1 as a chemoattractant for follicular center lymphoma B cells. J Natl Cancer Inst 92(8): 628-35. Fedyk E.R., Jones D., et al. (2001) Expression of stromal-derived factor-1 is decreased by IL-1 and TNF and in dermal wound healing. J Immunol. 166(9):5749-54. Geminder H., Sagi-Assif O., et al. (2001) A possible role for CXCR4 and its ligand, the CXC chemokine stromal cell-derived factor-1, in the development of bone marrow metastases in neuroblastoma. J Immunol 167(8): 4747-57. Grassi F., Cristino S., et al. (2004) CXCL12 chemokine up-regulates bone resorption and MMP-9 release by human osteoclasts: CXCL12 levels are increased in synovial and bone tissue of rheumatoid arthritis patients. J Cell Physiol 199(2): 244-51. Grunewald M., Avraham I., et al. (2006) VEGF-induced adult neovasculariza...

Claims

[Claim 1] C-X-C motif chemo for use in methods for treating tumors in subjects Cain-12 (CXCL12) antagonist, and the method is applied to the subject, - The aforementioned C-X-C motif chemokine 12 (CXCL12) antagonist, - Radiation therapy, and - Anti-angiogenic compounds The step includes administering a C-X-C motif for use, wherein the tumor is a brain tumor. Chemokine 12 (CXCL12) antagonist.

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