Method of treating tumor in subject

Abstract

The present invention relates to a C-X-C motif chemokine 12 (CXCL12) antagonist for use in a method of treating a brain tumor in a subject wherein the method comprises administering to the subject a C-X-C motif chemokine 12 (CXCL12) antagonist, a radiation therapy, and an angiogenic compound wherein the tumor is a brain tumor.

Description

Technical Field

[0001] The present invention relates to methods of treating a tumor in a subject. Background Art

[0002] Brain cancer is a disease of the brain in which cancer cells arise in brain tissue. Cancer cells grow to form a tumor, which is a mass of cancerous tissue that also contains supportive, noncancerous cells (tumor stroma) that interfere with the brain's functions, such as muscle control, sensation, memory, and other normal body functions. Tumors that contain cancer cells that grow uncontrollably and have the ability to invade nearby tissues and spread to other parts of the body through the blood and lymphatic systems are called malignant tumors, while those that do not have a tendency to invade nearby tissues or spread to distant parts of the body are called benign tumors. Tumors that develop from brain tissue are called primary brain tumors, while tumors that spread to the brain from other parts of the body are called metastatic or secondary brain tumors. Statistics show that brain cancer does not occur often (accounting for 1.4% of all new cancer patients each year), so according to estimates by the National Cancer Institute (NCI) and the American Cancer Society, brain cancer may occur in about 23,770 new patients each year and kill about 16,050 people. Only about 5% of brain tumors may be due to inherited genetic conditions such as neurofibromatosis, tuberous sclerosis and some other conditions.

[0003] Based on how the tumor cells in the tumor appear under a microscope, the National Cancer Institute (NCI) lists the following grades of brain tumors:

[0004] Grade I: The tissue is benign. The cells look almost like normal brain cells, and they grow slowly.

[0005] Grade II: The tissue is malignant. The cells look less like normal cells than the cells in a grade I tumor.

[0006] Grade III: Malignant tissue has cells that look very different from normal cells. The abnormal cells are actively growing and have a distinctly abnormal appearance (anaplastic).

[0007] Grade IV: Malignant tissue has cells that look the least normal and tends to grow rapidly.

[0008] Most symptoms caused by brain cancer are not specific to brain cancer and include symptoms such as difficulty walking and / or dizziness / vertigo, episodes of muscle weakness (such as weakness in the arms and legs), headaches (persistent and / or severe). Other common symptoms that may occur include nausea, vomiting, blurred vision, changes in a person's alertness, drowsiness, decreased mental abilities and / or confusion, memory problems, speech changes (such as difficulty speaking, loss of voice, or inability to speak), personality changes, hallucinations, weakness on one side of the body, problems with coordination, fatigue, and a tingling sensation and / or decreased sense of touch.

[0009] Treatment of brain tumors typically involves surgery, radiation therapy, and chemotherapy. Other treatment options include hyperthermia, immunotherapy, or steroid therapy. Depending on the individual brain tumor, a combination of two or more of the above options may be implemented in the treatment of a subject with a brain tumor. Summary of the invention

[0010] The problem addressed by the present invention is to provide a method for treating a tumor in a subject, preferably said tumor is a brain tumor.

[0011] Another problem addressed by the present invention is to provide a method for treating a malignant brain tumor in a subject.

[0012] Another problem addressed by the present invention is to provide a method for treating a brain tumor in a subject, wherein the brain tumor is or is treatable by radiotherapy.

[0013] Another problem of the present invention is to provide a method of treating a brain tumor in a subject, wherein the brain tumor is an inoperable tumor, typically a tumor that is inoperable due to the size of the tumor or its location near important adjacent tissue.

[0014] These and other problems are solved by the subject-matter of the attached independent claims; preferred embodiments can be taken from the attached dependent claims.

[0015] Regardless, the problem addressed by the present invention is solved by the subject matter of the following embodiments, whereby Embodiment 1 is also referred to as the first aspect of the present invention.

[0016] Embodiment 1. A method for treating a tumor in a subject, wherein the method comprises administering to the subject

[0017] -CXC motif chemokine 12 (CXCL12) antagonist,

[0018] - Radiation therapy, and

[0019] - anti-angiogenic compounds,

[0020] Wherein the tumor is a brain tumor.

[0021] Embodiment 2. The method of embodiment 1, wherein the tumor is selected from the group comprising glioblastoma, glioma, acoustic neuroma, astrocytoma, brain metastasis, choroid plexus carcinoma, craniopharyngioma, embryonal tumor, medulloblastoma, meningioma, childhood brain tumor, pineoblastoma and pituitary tumor.

[0022] Embodiment 3. The method of any one of Embodiments 1 and 2, wherein the tumor is selected from the group comprising glioblastoma and glioma.

[0023] Embodiment 4. The method of any one of Embodiments 1 to 3, wherein the tumor is a grade IV astrocytoma or a glioblastoma.

[0024] Embodiment 5. The method of embodiment 4, wherein the tumor is a supratentorial glioblastoma.

[0025] Embodiment 6. The method of any one of embodiments 4 and 5, wherein the glioblastoma is a cell type with unmethylated O 6 -methylguanine DNA methyltransferase (MGMT) promoter status in glioblastoma.

[0026] Embodiment 7. The method of any one of Embodiments 1 to 6, preferably any one of Embodiments 4 to 6, wherein the tumor is unresponsive to temozolomide.

[0027] Embodiment 8. The method of any one of Embodiments 1 to 7, preferably any one of Embodiments 4 to 7, wherein the tumor expresses CXCL12 and VEGF.

[0028] Embodiment 9. The method of Embodiment 8, wherein the tumor expresses CXCL12 and VEGF in different compartments of the tumor.

[0029] Embodiment 10. The method of any one of Embodiments 8 and 9, wherein CXCL12 is expressed at the leading edge of the tumor, in the proliferative blood vessels of the tumor, and / or in the microvascular hyperplasia zone of the tumor.

[0030] Embodiment 11. The method of any one of Embodiments 8 to 10 (preferably Embodiment 10), wherein VEGF is expressed in the cellular tumor compartment of the tumor, expressed by regions having pseudopalisading cells of the tumor and / or expressed in peri-necrotic regions of the tumor, and wherein preferably the tumor is a glioblastoma.

[0031] Embodiment 12. The method of any one of Embodiments 1 to 11, wherein the tumor is selected from the group comprising astrocytoma, ependymoma and oligodendroglioma.

[0032] Embodiment 13. The method of any one of Embodiments 1 to 12, preferably any one of Embodiments 4 to 11, wherein the tumor is an incompletely resected tumor.

[0033] Embodiment 14. The method of embodiment 13, wherein the tumor is an incompletely resected tumor that is detectable by each postoperative T1-weighted, contrast-enhanced MRI scan.

[0034] Embodiment 15. The method of any one of Embodiments 1 to 14, wherein the CXCL12 antagonist inhibits CXCL12 signaling through one or both of the CXCL12 receptor CXC chemokine receptor type 4 (CXCR4) and the CXCL12 receptor CXC chemokine receptor type 7 (CXCR7).

[0035] Embodiment 16. The method of embodiment 15, wherein the CXCL12 antagonist inhibits the binding of CXCL12 to the CXCL12 receptor CXC chemokine receptor type 4 (CXCR4) and / or the CXCL12 receptor CXC chemokine receptor type 7 (CXCR7).

[0036] Embodiment 17. The method of Embodiment 16, wherein the CXCL12 antagonist binds to CXCL12.

[0037] Embodiment 18. The method described in Embodiment 17, wherein the CXCL12 antagonist is a nucleic acid molecule that binds to CXCL12, wherein 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.

[0038] Embodiment 19. The method of embodiment 18, wherein the type-B SDF-1 binding nucleic acid molecule comprises a central nucleotide fragment, whereby the central nucleotide fragment comprises the following nucleotide sequence:

[0039] 5' GUGUGAUCUAGAUGUADWGGCUGWUCCUAGUYAGG 3' (SEQ ID NO: 52).

[0040] Embodiment 20. The method of embodiment 19, wherein the central nucleotide fragment comprises the following nucleotide sequence:

[0041] 5' GUGUGAUCUAGAUGUADUGGCUGAUCCUAGUCAGG 3' (SEQ ID NO: 53).

[0042] Embodiment 21. The method of any one of Embodiments 19 to 20, wherein the type-B SDF-1 binding nucleic acid molecule comprises a first terminal nucleotide segment, a central nucleotide segment, and a second terminal nucleotide segment in the 5'->3' direction.

[0043] Embodiment 22. The method of any one of Embodiments 19 to 20, wherein the type-B SDF-1 binding nucleic acid molecule comprises a second terminal nucleotide segment, a central nucleotide segment and a first terminal nucleotide segment in the 5'->3' direction.

[0044] Embodiment 23. The method of any one of embodiments 21 to 22, wherein the first terminal nucleotide fragment comprises 5' X 1 X 2 SVNS 3' nucleotide sequence, and the second terminal nucleotide fragment contains 5' BVBSX 3 X 4 3' nucleotide sequence, whereby

[0045] X 1 Does not exist or is A, X 2 It's G, X 3 is C, and X 4 does not exist or is U;

[0046] or

[0047] X 1 Does not exist, X 2 Does not exist or is G, X 3 does not exist or is C, and X 4 Does not exist.

[0048] Embodiment 24. Any one of embodiments 21 to 23, preferably the method of embodiment 50, wherein the first terminal nucleotide fragment comprises a nucleotide sequence of 5' X1X2CRWG 3', and the second terminal nucleotide fragment comprises a nucleotide sequence of 5' KRYSX3X4 3',

[0049] From this X 1 Does not exist or is A, X 2 It's G, X 3 is C, and X 4 Does not exist or is U.

[0050] Embodiment 25. Any one of embodiments 21 to 24, preferably the method of embodiment 23 or 24, wherein the first terminal nucleotide fragment comprises 5' X 1 X 2 CGUG 3' nucleotide sequence, and the second terminal nucleotide fragment contains 5' UACGX3 X 4 The nucleotide sequence of 3'

[0051] From this X 1 Does not exist or is A, X 2 It's G, X 3 is C, and X 4 Does not exist or is U,

[0052] Preferably, the first terminal nucleotide fragment comprises a nucleotide sequence of 5'AGCGUG 3', and the second terminal nucleotide fragment comprises a nucleotide sequence of 5'UACGCU 3'.

[0053] Embodiment 26. Any one of embodiments 21 to 23, preferably the method of embodiment 23, wherein the first terminal nucleotide fragment comprises a nucleotide sequence of 5' X1X2SSBS 3', and the second terminal nucleotide fragment comprises a nucleotide sequence of 5' BVSSX3X4 3',

[0054] From this X 1 Does not exist, X 2 Does not exist or is G, X 3 does not exist or is C, and X 4 Preferably, the first terminal nucleotide fragment comprises a nucleotide sequence of 5' GCGUG 3', and the second terminal nucleotide fragment comprises a nucleotide sequence of 5' UACGC 3'.

[0055] Embodiment 27. The method of any one of Embodiments 18 to 26, wherein the type-B SDF-1 binding nucleic acid molecule comprises a nucleotide sequence according to any one of SEQ ID NO: 5 to SEQ ID NO: 20 and SEQ ID NO: 22 to SEQ ID NO: 28, or a nucleotide sequence having at least 80% identity thereto,

[0056] Preferably 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 having at least 80% identity thereto,

[0057] More preferably any one of SEQ ID NO: 22 and SEQ ID NO: 28, or a nucleotide sequence having at least 80% identity thereto.

[0058] Embodiment 28. The method of embodiment 18, wherein the C-type SDF-1 binding nucleic acid molecule comprises a central nucleotide segment, whereby the central nucleotide segment comprises GGUYAGGGCUHRXA The nucleotide sequence of AGUCGG (SEQ ID NO: 108),

[0059] From this X A Does not exist or is A.

[0060] Embodiment 29. The method of embodiment 28, wherein the central nucleotide fragment comprises the nucleotide sequence of 5'GGUYAGGGCUHRAAGUCGG 3' (SEQ ID NO: 109), the nucleotide sequence of 5'GGUYAGGGCUHRAGUCGG 3' (SEQ ID NO: 110) or the nucleotide sequence of 5'GGUUAGGGCUHGAAGUCGG 3' (SEQ ID NO: 111), preferably the nucleotide sequence of 5'GGUUAGGGCUHGAAGUCGG 3' (SEQ ID NO: 111).

[0061] Embodiment 30. The method of any one of Embodiments 28 to 29, wherein the C-type SDF-1 binding nucleic acid molecule comprises a first terminal nucleotide fragment, the central nucleotide fragment and a second terminal nucleotide fragment in the 5'->3' direction.

[0062] Embodiment 31. The method of any one of Embodiments 28 to 29, wherein the C-type SDF-1 binding nucleic acid molecule comprises the second terminal nucleotide fragment, the central nucleotide fragment and the first terminal nucleotide fragment in the 5'->3' direction.

[0063] Embodiment 32. The method of any one of embodiments 30 to 31, wherein the first terminal nucleotide fragment comprises a nucleotide sequence of 5'RKSBUSNVGR 3' (SEQ ID NO: 138), and the second nucleotide fragment comprises a nucleotide sequence of 5'YYNRCASSMY 3' (SEQ ID NO: 139),

[0064] Preferably, the first terminal nucleotide fragment comprises a nucleotide sequence of 5' RKSBUGSVGR 3' (SEQ ID NO: 140), and the second terminal nucleotide fragment comprises a nucleotide sequence of 5' YCNRCASSMY 3' (SEQ ID NO: 141).

[0065] Embodiment 33. The method of any one of embodiments 30 to 31, wherein the first terminal nucleotide segment comprises a nucleotide sequence of 5'XSSSSV 3', and the second terminal nucleotide segment comprises a nucleotide sequence of 5'BSSSSXS 3', whereby Xs is absent or is S,

[0066] Preferably, the first terminal nucleotide fragment comprises a nucleotide sequence of 5' SGGSR 3', and the second terminal nucleotide fragment comprises a nucleotide sequence of 5' YSCCS 3'.

[0067] Embodiment 34. The method of any one of Embodiments 30 to 31, wherein

[0068] a) the first terminal nucleotide fragment comprises a nucleotide sequence of 5' GCCGG 3', and the second terminal nucleotide fragment comprises a nucleotide sequence of 5' CCGGC 3'; or

[0069] b) the first terminal nucleotide fragment comprises a nucleotide sequence of 5' CGUGCGCUUGAGAUAGG 3' (SEQID NO: 220), and the second terminal nucleotide fragment comprises a nucleotide sequence of 5' CUGAUUCUCACG 3' (SEQID NO: 221); or

[0070] c) the first terminal nucleotide fragment comprises a nucleotide sequence of 5' UGAGAUAGG 3', and the second terminal nucleotide fragment comprises a nucleotide sequence of 5' CUGAUUCUCA 3' (SEQ ID NO: 222); or

[0071] d) the first terminal nucleotide fragment comprises a nucleotide sequence of 5' GAGAUAGG 3', and the second terminal nucleotide fragment comprises a nucleotide sequence of 5' CUGAUUCUC 3'.

[0072] Embodiment 35. The method of any one of Embodiments 28 to 34, wherein the C-type SDF-1 binding nucleic acid molecule comprises 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,

[0073] Preferably 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.

[0074] Embodiment 36. The method of embodiment 18, wherein the A-type SDF-1 binding nucleic acid molecule comprises a central nucleotide fragment, whereby the central nucleotide fragment comprises 5' AAAGYRACAHGUMAAXA UGAAAGGUARC 3' nucleotide sequence (SEQ ID NO: 74),

[0075] From this X A Does not exist or is A.

[0076] Embodiment 37. The method of embodiment 36, wherein the nucleotide core fragment comprises

[0077] 5'AAAGYRACAHGUMAAUGAAAGGUARC 3' (SEQ ID NO: 75), or

[0078] 5' AAAGYRACAHGUMAAAUGAAAGGUARC 3' (SEQ ID NO: 76), or

[0079] 5' AAAGYAACAHGUCAAUGAAAGGUARC 3' nucleotide sequence (SEQ ID NO: 77), preferably the nucleotide center fragment comprises the nucleotide sequence 5' AAAGYAACAHGUCAAUGAAAGGUARC 3' (SEQ ID NO: 77).

[0080] Embodiment 38. The method of any one of Embodiments 36 to 37, wherein the A-type SDF-1 binding nucleic acid molecule comprises a first terminal nucleotide fragment, the central nucleotide fragment and a second terminal nucleotide fragment in the 5'->3' direction.

[0081] Embodiment 39. The method of any one of Embodiments 36 to 37, wherein the A-type SDF-1 binding nucleic acid molecule comprises the second terminal nucleotide fragment, the central nucleotide fragment and the first terminal nucleotide fragment in the 5'->3' direction.

[0082] Embodiment 40. The method of any one of embodiments 38 to 39, wherein the first terminal nucleotide fragment comprises 5'X 1 X 2 NNBV 3' nucleotide sequence, and the second terminal nucleotide fragment comprises 5'BNBNX 3 X 4 The nucleotide sequence of 3'

[0083] From this X 1 Does not exist or is R, X 2 It is S, X 3 is S, and X 4 does not exist or is Y;

[0084] or

[0085] X 1 Does not exist, X 2 Does not exist or is S, X 3 Does not exist or is S, and X 4 Does not exist.

[0086] Embodiment 41. Any one of embodiments 38 to 40, preferably the method of embodiment 40, wherein the terminal first nucleotide fragment comprises a nucleotide sequence of 5'RSHRYR 3', and the second terminal nucleotide fragment comprises a nucleotide sequence of 5'YRYDSY 3',

[0087] Preferably, the first terminal nucleotide fragment comprises a nucleotide sequence of 5' GCUGUG 3', and the second terminal nucleotide fragment comprises a nucleotide sequence of 5' CGCAGC 3'.

[0088] Embodiment 42. Any one of embodiments 38 to 24, preferably the method of embodiment 40, wherein the first terminal nucleotide fragment comprises 5' X 2 BBBS 3' nucleotide sequence, and the second terminal nucleotide fragment contains 5'SBBVX 3 The nucleotide sequence of 3'

[0089] From this X 2 Does not exist or is S, and X 3 does not exist or is S;

[0090] Preferably, the first terminal nucleotide fragment comprises a nucleotide sequence of 5' CUGUG 3', and the second terminal nucleotide fragment comprises a nucleotide sequence of 5' CGCAG 3';

[0091] Alternatively, the first terminal nucleotide fragment comprises a nucleotide sequence of 5' GCGUG 3', and the second terminal nucleotide fragment comprises a nucleotide sequence of 5' CGCGC 3'.

[0092] Embodiment 43. The method of any one of Embodiments 36 to 42, wherein the A-type SDF-1 binding nucleic acid molecule comprises a nucleotide sequence according to 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 with at least 80% identity thereof,

[0093] 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 having at least 80% identity thereto,

[0094] More preferably any one of SEQ ID NO: 84 and SEQ ID NO: 146, or a nucleotide sequence having at least 80% identity thereto.

[0095] Embodiment 44. The method of embodiment 18, wherein the D-type SDF-1 binding nucleic acid molecule comprises a nucleotide sequence according to any one of SEQ ID NO: 142 to SEQ ID NO: 144, or a nucleotide sequence having at least 80% identity thereto.

[0096] Embodiment 45. The method of any one of embodiments 18 to 44, wherein the nucleic acid molecule comprises a modification, whereby the modification is preferably a high molecular weight portion and / or whereby the modification preferably allows modification of the characteristics of the nucleic acid molecule in terms of residence time in an animal or human, preferably a human.

[0097] Embodiment 46. The method of Embodiment 45, wherein the modification is selected from the group consisting of a HES moiety, a PEG moiety, a biodegradable modification, and a combination thereof.

[0098] Embodiment 47. The method of embodiment 46, wherein the modification is a PEG moiety consisting of a linear PEG or a branched PEG, whereby preferably the molecular weight of the linear or branched PEG is from about 20,000 Da to 120,000 Da, more preferably from about 30,000 Da to 80,000 Da and most preferably about 40,000 Da.

[0099] Embodiment 48. The method of embodiment 46, wherein the modification is a HES moiety, whereby preferably the molecular weight of the HES moiety is from about 10,000 Da to 200,000 Da, more preferably from about 30,000 Da to 170.000 Da and most preferably about 150,000 Da.

[0100] Embodiment 49. The method of any one of Embodiments 45 to 48, wherein the modification is attached to the nucleic acid molecule via a linker, wherein preferably the linker is a biostable or biodegradable linker.

[0101] Embodiment 50. The method of any one of Embodiments 45 to 49, wherein the modification is attached to the nucleic acid molecule at the 5'-terminal nucleotide of the nucleic acid molecule and / or the 3'-terminal nucleotide of the nucleic acid molecule, and / or a nucleotide of the nucleic acid molecule between the 5'-terminal nucleotide of the nucleic acid molecule and the 3'-terminal nucleotide of the nucleic acid molecule.

[0102] Embodiment 51. The method of any one of Embodiments 18 to 50, wherein the nucleotides of the nucleic acid molecule or the nucleotides forming the nucleic acid molecule are L-nucleotides.

[0103] Embodiment 52. The method of any one of Embodiments 18 to 51, wherein the nucleic acid molecule is an L-nucleic acid molecule.

[0104] Embodiment 53. The method described in Embodiment 18, wherein the nucleic acid molecule is an L-ribonucleic acid molecule, and the L-ribonucleic acid molecule comprises a nucleotide sequence of GCGUGGUGUGAUCUAGAUGUAUUGGCUGAUCCUAGUCAGGUACGC (SEQ ID NO: 22).

[0105] Embodiment 54. The method of embodiment 53, wherein the L-ribonucleic acid molecule comprises a 40 kDa polyethylene glycol moiety, wherein the 40 kDa polyethylene glycol moiety is attached to the 5' end of the nucleotide sequence.

[0106] Embodiment 55. The method of embodiment 54, wherein N-[ω-methoxypoly(oxy-1,2-ethanediyl)], N'-[ω-methoxypoly(oxy-1,2-ethanediyl)-acetyl-9-amino-8-oxo-7-aza-nonyloxyphosphate is dispersed between the 5' terminal nucleotide and the 40 kDa polyethylene glycol moiety.

[0107] Embodiment 56. The method of embodiment 55, wherein the L-ribonucleic acid molecule is present as a sodium salt, preferably the L-nucleic acid molecule is NOX-A12.

[0108] Embodiment 57. The method of any one of Embodiments 1 to 16, wherein the CXCL12 antagonist is an aptamer that binds CXCL12.

[0109] Embodiment 58. The method of any one of Embodiments 1 to 16, wherein the CXCL12 antagonist is an antibody that binds CXCL12.

[0110] Embodiment 59. The method of Embodiment 58, wherein the antibody that binds CXCL12 is mAB-30D8, 1131-H12, or 1143-H1.

[0111] Embodiment 60. The method of any one of Embodiments 1 to 16, wherein the CXCL12 antagonist is a protein that binds CXCL12.

[0112] Embodiment 61. The method of Embodiment 60, wherein the protein is a TRAP protein or anticalin.

[0113] Embodiment 62. The method of any one of Embodiments 1 to 16, wherein the CXCL12 antagonist targets CXCR4.

[0114] Embodiment 63. The method of Embodiment 62, wherein the CXCL12 antagonist binds to CXCR4.

[0115] Embodiment 64. The method of any one of Embodiments 62 to 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.

[0116] Embodiment 65. The method of Embodiment 64, wherein the CXCR4-binding CXCL12 antagonist is a small molecule.

[0117] Embodiment 66. The method described in Embodiment 65, wherein the CXCR4-binding CXCL12 antagonist is a small molecule selected from the group consisting of plerixafor, mavorixafor, 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.

[0118] Embodiment 67. The method of embodiment 66, wherein the small molecule is selected from the group consisting of plerixafor, mavorixafor, Q-122, HPH-112, BKT-300, X4P-002, X4P-003, X4-136, GP-01CR01, GP-01CR11, GP-01CR21 and pentixather.

[0119] Embodiment 68. The method of Embodiment 64, wherein the CXCR4-binding CXCL12 antagonist is a peptide.

[0120] Embodiment 69. The method of embodiment 68, wherein the CXCR4-binding CXCL12 antagonist is motixafortide.

[0121] Embodiment 70. The method of Embodiment 64, wherein the CXCR4-binding CXCL12 antagonist is a synthetic peptide.

[0122] Embodiment 71. The method of embodiment 70, wherein the CXCR4-binding CXCL12 antagonist is a synthetic peptide selected from the group consisting of balixafortide, LY-2510924, ALB-408 and POL-5551.

[0123] Embodiment 72. The method of Embodiment 71, wherein the CXCR4-binding CXCL12 antagonist is selected from the group consisting of balixafortide and LY-2510924.

[0124] Embodiment 73. The method of Embodiment 64, wherein the CXCR4-binding CXCL12 antagonist is a recombinant protein.

[0125] Embodiment 74. The method of Embodiment 73, wherein the recombinant protein is selected from the group consisting of PTX-9098 and NNL-121.

[0126] Embodiment 75. The method of Embodiment 64, wherein the CXCR4-binding CXCL12 antagonist is a fusion protein.

[0127] Embodiment 76. The method of Embodiment 75, wherein the fusion protein is selected from the group consisting of AD-214 and AM-3114.

[0128] Embodiment 77. The method of Embodiment 64, wherein the CXCR4-binding CXCL12 antagonist is an antibody or an antigen-binding fragment of the antibody.

[0129] Embodiment 78. The method of Embodiment 77, wherein the antibody is a monoclonal antibody.

[0130] Embodiment 79. The method of any one of Embodiments 77 and 78, wherein the CXCR4 binding to CXCL12 antagonist is an antibody.

[0131] Embodiment 80. The method of embodiment 79, wherein the antibody is a monoclonal antibody selected from the group consisting of uloplumab, hz-515H7, KY-1051, PF-06747143, ALX-0651, AT-009, LY-2624587 and STIA-220X.

[0132] Embodiment 81. The method of Embodiment 80, wherein the antibody is a monoclonal antibody selected from the group consisting of uloplumab, hz-515H7 and KY-1051.

[0133] Embodiment 82. The method of any one of Embodiments 1 to 16, wherein the CXCL12 antagonist binds to CXCR7.

[0134] Embodiment 83. The method of embodiment 82, wherein the CXCR7-binding CXCL12 antagonist is selected from the group consisting of small molecules, synthetic peptides and antibodies.

[0135] Embodiment 84. The method of Embodiment 83, wherein the CXCR7 binding to CXCL12 antagonist is a small molecule.

[0136] Embodiment 85. The method of embodiment 84, wherein the CXCR7 binding to CXCL12 antagonist is a small molecule selected from the group consisting of ACT-10041239, CCX-650, CCX-662 and CCX-771.

[0137] Embodiment 86. The method of Embodiment 85, wherein the CXCR7 binding to CXCL12 antagonist is ACT-10041239.

[0138] Embodiment 87. The method of Embodiment 83, wherein the CXCR7-binding CXCL12 antagonist is a synthetic peptide.

[0139] Embodiment 88. The method of Embodiment 87, wherein the synthetic peptide is selected from the group consisting of LIH-383 and POL-6926.

[0140] Embodiment 89. The method of Embodiment 88, wherein the synthetic peptide is LIH-383.

[0141] Embodiment 90. The method of Embodiment 83, wherein the CXCR7-binding CXCL12 antagonist is an antibody or an antigen-binding fragment of the antibody.

[0142] Embodiment 91. The method of Embodiment 90, wherein the antibody is a monoclonal antibody.

[0143] Embodiment 92. The method of any one of Embodiments 90 and 91, wherein the antibody is a monoclonal antibody selected from the group comprising JT-07, X-7Ab and STIA-230X.

[0144] Embodiment 93. The method of Embodiment 92, wherein the antibody is a monoclonal antibody selected from the group consisting of JT-07 and X-7Ab.

[0145] Embodiment 94. The method of 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, wherein the amount is effective to inhibit neovascularization of the tumor.

[0146] Embodiment 95. The method of embodiment 94, wherein neovascularization is achieved through CXCL12-mediated recruitment of endothelial cells or bone marrow-derived pro-angiogenic cells.

[0147] Embodiment 96. The method of any one of Embodiments 94 to 95, wherein CXCL12-mediated recruitment is dependent on CXCR4 and / or CXCR7.

[0148] Embodiment 97. The method of any one of Embodiments 1 to 96, preferably any one of Embodiments 17 to 57, wherein the radiation therapy utilizes ionizing electromagnetic radiation or particle beams.

[0149] Embodiment 98. The method described in Embodiment 97, wherein the electromagnetic radiation is selected from the group consisting of X-rays and gamma rays.

[0150] Embodiment 99. The method of Embodiment 98, wherein the X-rays have a wavelength from about 10 picometers to about 10 nanometers.

[0151] Embodiment 100. A method as described in any of Embodiments 98 to 99, wherein the energy of the X-rays is from about 145 eV to about 124 keV.

[0152] Embodiment 101. The method of Embodiment 98, wherein the gamma rays have a wavelength of about 10 picometers or less.

[0153] Embodiment 102. The method described in Embodiment 101, wherein the energy of the gamma rays is 100 eV or higher.

[0154] Embodiment 103. The method described in Embodiment 97, wherein the radiation therapy utilizes a particle beam.

[0155] Embodiment 104. The method described in 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.

[0156] Embodiment 105. The method described in any one of Embodiments 97 to 104, wherein the radiation therapy utilizes intensity modulated radiation therapy (IMRT), helical tomotherapy (also known as image guided radiation therapy (IGRT)) and / or stereotactic radiosurgery.

[0157] Embodiment 106. The method of any one of Embodiments 97 to 105, wherein the radiation therapy is performed by external radiation therapy, preferably external beam radiation therapy.

[0158] Embodiment 107. The method of any one of Embodiments 97 to 106, wherein the radiation therapy is administered by brachytherapy.

[0159] Embodiment 108. The method of Embodiment 107, wherein the brachytherapy is provided by means of a low dose rate (LDH) implant, a high dose rate (HDR) implant, a permanent implant, a formulation (preferably a liquid formulation) containing a therapeutically active radionuclide, or a combination thereof.

[0160] Embodiment 109. The method of any one of Embodiments 97 to 108, wherein the radiation therapy is targeted to or directed to cells of the tumor.

[0161] Embodiment 110. The method of any one of Embodiments 97 to 109, wherein the radiation therapy targets or is directed to the microvasculature of the tumor.

[0162] Embodiment 111. The method of Embodiment 110, wherein the radiation therapy depletes the microvasculature of the tumor.

[0163] Embodiment 112. The method of any one of Embodiments 97 to 111, wherein the radiation therapy produces intratumoral hypoxia.

[0164] Embodiment 113. The method of any one of Embodiments 97 to 112, preferably Embodiment 112, wherein the tumor expresses CXCL12 during radiation therapy.

[0165] Embodiment 114. The method of any one of Embodiments 97 to 113, preferably any one of Embodiments 112 to 113, wherein the tumor is revascularized during radiotherapy.

[0166] Embodiment 115. The method of any one of Embodiments 97 to 114, preferably any one of Embodiments 112 to 114, wherein radiation therapy results in immunosuppression.

[0167] Embodiment 116. The method of any one of Embodiments 97 to 115, wherein the total absorbed radiation dose of the radiotherapy is from about 10 Gy to about 100 Gy, preferably from about 40 Gy to about 60 Gy, more preferably about 40 Gy or about 60 Gy.

[0168] Embodiment 117. The method described in Embodiment 116, wherein the radiation therapy is conventional fractionated radiation therapy.

[0169] Embodiment 118. The method of Embodiment 116, wherein the radiation therapy is administered as hypofractionated radiation therapy.

[0170] Embodiment 119. The method of any one of Embodiments 116 to 118, wherein the radiation absorbed by the individual for fractionated radiotherapy is from about 0.5 Gy to about 5 Gy, preferably from about 2 Gy to about 2.7 Gy, more preferably about 2 Gy or about 2.7 Gy.

[0171] Embodiment 120. The method of any one of Embodiments 97 to 119, wherein the radiation therapy comprises intraoperative radiation therapy (IORT).

[0172] Embodiment 121. The method of any one of Embodiments 1 to 120, wherein the anti-angiogenic compound inhibits angiogenesis.

[0173] Embodiment 122. The method of embodiment 121, wherein angiogenesis is the formation of new blood vessels from pre-existing blood vessels.

[0174] Embodiment 123. The method of any one of Embodiments 121 and 122, wherein the anti-angiogenic compound inhibits angiogenesis toward and / or in the tumor.

[0175] Embodiment 124. The method of any one of Embodiments 121 to 123, wherein the anti-angiogenic compound inhibits growth of the tumor.

[0176] Embodiment 125. The method of any one of Embodiments 121 to 124, wherein the anti-angiogenic compound inhibits the interaction between vascular endothelial growth factor (VEGF) and a VEGF receptor.

[0177] Embodiment 126. The method described in Embodiment 125, wherein the VEGF is human VEGF including any isoforms thereof and any proteolytic cleavage forms thereof.

[0178] Embodiment 127. The method described in Embodiment 126, wherein the isoform is selected from the group comprising VEGF121, VEGF165, VEGF189, VEGF206, VEGF145, VEGF183 and VEGF165b, preferably the isoform is selected from the group comprising VEGF121, VEGF165, VEGF189, VEGF206 and VEGF 145.

[0179] Embodiment 128. The method of Embodiment 126, wherein the proteolytically cleaved form of VEGF is VEGF110.

[0180] Embodiment 129. The method of any one of Embodiments 125 to 128, wherein the anti-angiogenic compound inhibits the interaction between a VEGF receptor and at least one of human vascular endothelial growth factor (VEGF), an isoform of VEGF, and a proteolytically cleaved form of VEGF or an isoform of VEGF.

[0181] Embodiment 130. The method of any one of Embodiments 125 to 129, wherein the VEGF receptor is selected from the group comprising VEGFR1 and VEGFR2.

[0182] Embodiment 131. The method of Embodiment 130, wherein the VEGF receptor is VEGFR1.

[0183] Embodiment 132. The method of any one of Embodiments 125 to 131, wherein the anti-angiogenic compound binds to VEGF.

[0184] Embodiment 133. The method described in Embodiment 132, wherein the anti-angiogenic compound that binds to VEGF is selected from the group consisting of anti-VEGF antibodies, fragments of anti-VEGF antibodies, VEGF binding proteins, and VEGF binding nucleic acid molecules.

[0185] Embodiment 134. The method of Embodiment 133, wherein the anti-angiogenic compound is an anti-VEGF antibody.

[0186] Embodiment 135. The method of Embodiment 134, wherein the anti-VEGF antibody is a human anti-VEGF antibody, a humanized anti-VEGF antibody, or a human anti-VEGF antibody.

[0187] Embodiment 136. The method of any one of Embodiments 134 and 135, wherein the anti-VEGF antibody comprises the following light chain variable region CDRs: CDR1: QDISNY (SEQ ID NO: 226), CDR2: FTS and CDR3: QQYSTVPWT (SEQ ID NO: 227).

[0188] Embodiment 137. The method of any one of Embodiments 134 to 136, wherein the anti-VEGF antibody comprises the following heavy chain variable region CDRs: CDR1: GYTFTNYG (SEQ ID NO: 228), CDR2: INTYTGEP (SEQ ID NO: 229) and CDR3: AKYPHYYGSSHWYFDV (SEQ ID NO: 230).

[0189] Embodiment 138. The method of any one of Embodiments 136 and 137, wherein the antibody comprises the following light chain variable region CDRs: CDR1: QDISNY (SEQ ID NO: 226), CDR2: FTS and CDR3: QQYSTVPWT (SEQ ID NO: 227), and the following heavy chain variable region CDRs: CDR1: GYTFTNYG (SEQ ID NO: 228), CDR2: INTYTGEP (SEQ ID NO: 229) and CDR3: AKYPHYYGSSHWYFDV (SEQ ID NO: 230).

[0190] Embodiment 139. The method of any one of Embodiments 134 to 138, wherein the antibody comprises a light chain comprising the following amino acid sequence:

[0191] DIQMTQSPSSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 231).

[0192] Embodiment 140. The method of any one of Embodiments 134 to 139, wherein the antibody comprises a heavy chain comprising the following amino acid sequence:

[0193] EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 232).

[0194] Embodiment 141. The method of any one of Embodiments 134 to 140, wherein the antibody comprises a light chain and a heavy chain, the light chain comprising the following amino acid sequence:

[0195] DIQMTQSPSSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 231)

[0196] The heavy chain comprises the following amino acid sequence:

[0197] EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 232).

[0198] Embodiment 142. The method of any one of Embodiments 134 to 141, wherein the anti-VEGF antibody is an IgG1k isotype antibody.

[0199] Embodiment 143. The method of any one of Embodiments 134 to 142, wherein the anti-VEGF antibody is Bevacizumab.

[0200] Embodiment 144. The method of Embodiment 143, wherein the anti-VEGF antibody is a Fab fragment of bevacizumab.

[0201] Embodiment 145. The method of Embodiment 144, wherein the Fab fragment of bevacizumab is ranibizumab.

[0202] Embodiment 146. The method of Embodiment 133, wherein the anti-angiogenic compound is a VEGF binding protein.

[0203] Embodiment 147. The method described in Embodiment 146, wherein the VEGF binding protein is a functional derivative of the VEGF receptor.

[0204] Embodiment 148. The method described in Embodiment 147, wherein the VEGF receptor is selected from the group consisting of VEGFR1 and VEGFR2.

[0205] Embodiment 149. The method of any one of Embodiments 147 and 148, wherein the functional derivative of the VEGF receptor comprises at least one VEGF binding domain of VEGFR1 and / or VEGFR2.

[0206] Embodiment 150. The method of any one of Embodiments 147 to 149, wherein the VEGF binding protein is ziv aflibercerpt.

[0207] Embodiment 151. The method described in Embodiment 146, wherein the VEGF binding protein is a VEGF binding anticalin.

[0208] Embodiment 152. The method described in Embodiment 133, wherein the anti-angiogenic compound that binds VEGF is a VEGF binding nucleic acid molecule.

[0209] Embodiment 153. The method of Embodiment 152, wherein the VEGF binding nucleic acid molecule is a VEGF binding D-nucleic acid molecule.

[0210] Embodiment 154. The method of Embodiment 153, wherein the VEGF-binding D-nucleic acid molecule is a VEGF-binding aptamer.

[0211] Embodiment 155. The method of embodiment 154, wherein the VEGF-binding D-nucleic acid molecule is pegaptanib (Macugen), preferably binding to the VEGF isoform VEGF165.

[0212] Embodiment 156. The method described in Embodiment 152, wherein the VEGF binding nucleic acid molecule is a VEGF binding L-nucleic acid molecule.

[0213] Embodiment 157. The method described in Embodiment 156, wherein the VEGF binding nucleic acid molecule is a VEGF binding spiegelmer.

[0214] Embodiment 158. The method of any one of Embodiments 125 to 131, wherein the anti-angiogenic compound binds to VEGFR.

[0215] Embodiment 159. The method described in Embodiment 158, wherein the VEGFR is VEGFR1, VEGFR2, or both VEGFR1 and VEGFR2, preferably the VEGFR is VEGFR2.

[0216] Embodiment 160. The method of any one of Embodiments 158 to 159, wherein the anti-angiogenic compound that binds to VEGF is selected from the group consisting of anti-VEGFR antibodies, fragments of anti-VEGFR antibodies, VEGFR binding proteins, and VEGFR binding nucleic acid molecules.

[0217] Embodiment 161. The method of Embodiment 160, wherein the anti-angiogenic compound is an anti-VEGFR antibody.

[0218] Embodiment 162. The method of Embodiment 161, wherein the anti-VEGFR antibody is a human anti-VEGFR antibody, a humanized anti-VEGFR antibody, or a human anti-VEGFR antibody.

[0219] Embodiment 163. The method of any one of Embodiments 161 to 162, wherein the anti-VEGFR antibody comprises the following light chain variable region CDRs: CDR1: RASQGIDNWLG (SEQ ID NO: 233), CDR2: DASNLDT (SEQID NO: 234), CDR3: QQAKAFPPT (SEQ ID NO: 235).

[0220] Embodiment 164. The method of any one of Embodiments 161 to 163, wherein the anti-VEGFR antibody comprises the following heavy chain variable region CDRs: CDR1: GFTFS SYSMN (SEQ ID NO: 236), CDR2: SISSSSSYIYYADSVKG (SEQ ID NO: 237) and CDR3: VTDAFDI (SEQ ID NO: 238).

[0221] Embodiment 165. The method of any one of Embodiments 161 to 164, wherein the VEGFR antibody comprises the following light chain variable region CDRs: CDR1: RASQGID NWLG (SEQ ID NO: 233), CDR2: DASNLD (SEQ ID NO: 234), CDR3: QQAKAFPPT (SEQ ID NO: 2345; and the following heavy chain variable region CDRs: CDR1: GFTFSSYSMN (SEQ ID NO: 236), CDR2: SISSSSSYIYYADSVKG (SEQ ID NO: 237) and CDR3: VTDAFDI (SEQ ID NO: 238).

[0222] Embodiment 166. The method of any one of Embodiments 161 to 165, wherein the anti-VEGFR antibody comprises a light chain comprising the following amino acid sequence: DIQMTQSPSS VSASIGDRVT ITCRASQGIDNWLGWYQQKP GKAPKLLIYD ASNLDTGVPS RFSGSGSGTY FTLTISSLQA EDFAVYFCQQ AKAFPPTFGGGTKVDIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKDSTYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC (SEQ ID NO: 239).

[0223] Embodiment 167. The method of any one of Embodiments 161 to 166, wherein the anti-VEGFR antibody comprises a heavy chain comprising the following amino acid sequence: EVQLVQSGGG LVKPGGSLRL SCAASGFTFSSYSMNWVRQA PGKGLEWVSS ISSSSSYIYY ADSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCARVTDAFDIWGQGT MVTVSSASTK GPSVLPLAPS SKSTSGGTAA LGCLVKDYFP EPVTVSWNSG ALTSGVHTFPAVLQSSGLYS LSSVVTVPSS SLGTQTYICN VNHKPSNTKV DKRVEPKSCD KTHTCPPCPA PELLGGPSVFLFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYNSTYR VVSVLTVLHQDWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTL PPSREEMTKN QVSLTCLVKG FYPSDIAVEWESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK (SEQID NO: 240).

[0224] Embodiment 168. The method of any one of Embodiments 161 to 167, wherein the anti-VEGFR antibody comprises a light chain and a heavy chain, the light chain comprising the following amino acid sequence:

[0225] DIQMTQSPSS VSASIGDRVT ITCRASQGID NWLGWYQQKP GKAPKLLIYD ASNLDTGVPSRFSGSGSGTY FTLTISSLQA EDFAVYFCQQ AKAFPPTFGG GTKVDIKRTV AAPSVFIFPP SDEQLKSGTASVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQGLSSPVTKSFN RGEC (SEQ ID NO: 239);

[0226] The heavy chain comprises the following amino acid sequence:

[0227] EVQLVQSGGG LVKPGGSLRL SCAASGFTFS SYSMNWVRQA PGKGLEWVSS ISSSSSYIYYADSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCARVT DAFDIWGQGT MVTVSSASTK GPSVLPLAPSSKSTSGGTAA LGCLVKDYFP EPVTVSWNSG ALTSGVHTFP AVLQSSGLYS LSSVVTVPSS SLGTQTYICNVNHKPSNTKV DKRVEPKSCD KTHTCPPCPA PELLGGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDPEVKFNWYVDG VEVHNAKTKP REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKGQPREPQVYTL PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLTVDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK (SEQ ID NO: 240).

[0228] Embodiment 169. The method of any one of Embodiments 161 to 168, wherein the anti-VEGFR antibody is an IgG isotype antibody.

[0229] Embodiment 170. The method of any one of Embodiments 161 to 169, wherein the anti-VEGFR antibody is Ramucirumab (Cyramza).

[0230] Embodiment 171. The method of Embodiment 160, wherein the anti-angiogenic compound is a VEGFR binding protein.

[0231] Embodiment 172. The method described in Embodiment 171, wherein the VEGFR binding protein is a VEGFR binding anticalin.

[0232] Embodiment 173. The method described in Embodiment 160, wherein the anti-angiogenic compound that binds to VEGFR is a VEGFR binding nucleic acid molecule.

[0233] Embodiment 174. The method of Embodiment 173, wherein the VEGFR binding nucleic acid molecule is a VEGFR binding D-nucleic acid molecule.

[0234] Embodiment 175. The method of Embodiment 174, wherein the VEGFR binding D-nucleic acid molecule is a VEGFR binding aptamer.

[0235] Embodiment 176. The method of Embodiment 152, wherein the VEGFR binding nucleic acid molecule is a VEGFR binding L-nucleic acid molecule.

[0236] Embodiment 177. The method described in Embodiment 176, wherein the VEGF binding nucleic acid molecule is a VEGFR binding spiegelmer.

[0237] Embodiment 178. The method of any one of Embodiments 121 to 124, wherein the anti-angiogenic compound inhibits VEGFR signaling.

[0238] Embodiment 179. The method of Embodiment 178, wherein the VEGFR is VEGFR1, VEGFR2, VEGFR3, and any combination thereof.

[0239] Embodiment 180. The method of any one of Embodiments 178 to 179, wherein the anti-angiogenic compound inhibits intracellular signaling of VEGFR.

[0240] Embodiment 181. The method of any one of Embodiments 178 to 180, wherein the anti-angiogenic compound inhibits the signaling cascade triggered when VEGF binds to VEGFR.

[0241] Embodiment 182. The method of any one of Embodiments 180 and 181, wherein the anti-angiogenic compound is a tyrosine kinase inhibitor.

[0242] Embodiment 183. The method of Embodiment 182, wherein the tyrosine kinase inhibitor is a multi-tyrosine kinase inhibitor.

[0243] Embodiment 184. The method of any one of Embodiments 182 to 183, wherein the tyrosine kinase inhibitor is selected from the group comprising: pazopanib (Votrient), sunitinib (Sutent), sorafenib (Nexavar), axitinib (Inlyta), ponatinib (Iclusig), cabozantinib (Cometriq / Cabometyx), regorafenib (Stivarga), vandetanib (Caprelsa), and lenvatinib (Lenvima).

[0244] Embodiment 185. The method of any one of Embodiments 1 to 184, wherein the method further comprises administering chemotherapy to the subject.

[0245] Embodiment 186. The method of Embodiment 185, wherein the chemotherapy is chemotherapy for treating cancer.

[0246] Embodiment 187. The method of any one of Embodiments 185 to 186, wherein the tumor is a glioblastoma and the chemotherapy is chemotherapy for treating glioblastoma.

[0247] Embodiment 188. The method of any one of Embodiments 185 to 187, wherein the chemotherapy is selected from the group comprising temozolomide, lomustine, irinotecan, didehydrodulcitol, a combination of irinotecan and bevacizumab, and a combination of procarbazine, lomustine and vincristine (PCV).

[0248] Embodiment 189. The method of Embodiment 188, wherein the method comprises administering temozolomide to the subject.

[0249] Embodiment 190. The method of any one of Embodiments 1 to 189, wherein the CXCL12 antagonist is administered to the subject intravenously or subcutaneously, preferably if the CXCL12 antagonist is a spiegelmer or an aptamer, more preferably a spiegelmer.

[0250] Embodiment 191. The method of any one of Embodiments 1 to 190, preferably Embodiment 190, wherein the CXCL12 antagonist is administered to the subject continuously over a period of time.

[0251] Embodiment 192. The method described in Embodiment 191, wherein the period of time is 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks or more weeks.

[0252] Embodiment 193. The method of 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 comprising a 40 kDa PEG moiety.

[0253] Embodiment 194. The method of Embodiment 193, wherein the CXCL12 antagonist is NOX-A12.

[0254] Embodiment 195. The method of any one of Embodiments 193 to 194, wherein the weekly dose of the CXCL12 antagonist administered to the subject is between about 100 mg and about 1,000 mg, preferably between about 200 mg and about 600 mg, more preferably between about 400 mg and about 600 mg.

[0255] Embodiment 196. The method of 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 if the anti-angiogenic compound is bevacizumab, it is more preferably administered intravenously.

[0256] Embodiment 197. The method of any one of Embodiments 1 to 196, preferably any one of Embodiments 190 to 196, wherein the anti-angiogenic compound is administered once a week, every 2 weeks, or every 3 weeks over a period of time.

[0257] Embodiment 198. The method of Embodiment 197, wherein the period of time is 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or 6 weeks.

[0258] Embodiment 199. The method of any one of Embodiments 197 to 198, wherein

[0259] (a) the anti-angiogenic compound is administered once a week, and the period of time is 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or 6 weeks,

[0260] (b) the anti-angiogenic compound is administered once every 2 weeks, and the period of time is 2 weeks, 4 weeks or 6 weeks, or

[0261] (c) the anti-angiogenic compound is administered once every 3 weeks, and the period of time is 3 weeks or 6 weeks.

[0262] Embodiment 200. The method of any one of embodiments 1 to 199, preferably any one of embodiments 190 to 199, wherein the anti-angiogenic compound is an anti-VEGF antibody, preferably an IgG antibody, and more preferably bevacizumab.

[0263] Embodiment 201. The method of any one of embodiments 1 to 200, preferably any one of embodiments 190 to 200, wherein

[0264] (a) if the anti-angiogenic compound is administered once a week, the dose is 5 mg / kg body weight of the subject,

[0265] (b) if the anti-angiogenic compound is administered once every 2 weeks, the dose is 10 mg / kg body weight of the subject, or

[0266] (a) If the anti-angiogenic compound is administered once every 3 weeks, the dose is 15 mg / kg body weight of the subject.

[0267] Embodiment 202. The method of any one of Embodiments 1 to 201, preferably any one of Embodiments 190 to 201, wherein the chemotherapy is administered to the subject.

[0268] Embodiment 203. The method of any one of Embodiments 1 to 202, preferably any one of Embodiments 190 to 202, wherein the chemotherapy is administered to the subject daily.

[0269] Embodiment 204. The method of any one of Embodiments 1 to 203, preferably any one of Embodiments 190 to 203, wherein the chemotherapy is administered to the subject over a period of time, preferably the period of time is 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or 6 weeks, more preferably the chemotherapy is administered to the subject every day over a period of time.

[0270] Embodiment 205. The method of any one of embodiments 1 to 204, preferably any one of embodiments 190 to 203, more preferably any one of embodiments 202 to 204, wherein the chemotherapy comprises administering to the subject temozolomide.

[0271] Embodiment 206. The method of any one of Embodiments 1 to 205, preferably any one of Embodiments 190 to 205, wherein the period of time during which the chemotherapy is administered to the subject is the period of time during which the radiation therapy is administered to the subject.

[0272] Embodiment 207. The method of any one of embodiments 190 to 206, preferably any one of embodiments 202 to 206, wherein the daily dose of temozolomide administered to the subject is about 75 mg / m 2 Body surface area.

[0273] Embodiment 208. The method of any one of Embodiments 1 to 207, preferably any one of Embodiments 190 to 207, wherein the radiation therapy is administered to the subject several days a week over a period of time.

[0274] Embodiment 209. The method of Embodiment 208, wherein the radiation therapy is administered to the subject for 5 consecutive days over a period of 1 week.

[0275] Embodiment 210. The method of any one of Embodiments 208 to 209, wherein the period of time is 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or 6 weeks.

[0276] Embodiment 211. The method of any one of Embodiments 208 to 210, wherein the radiation therapy is fractionated radiation therapy.

[0277] Embodiment 212. The method of any one of Embodiments 208 to 211, wherein the absorbed radiation dose is about 2 Gy or about 2.7 Gy per time, preferably 5 consecutive times per day within 1 week.

[0278] Embodiment 213. The method of any one of Embodiments 191 to 212, wherein the period of time constitutes a first treatment phase.

[0279] Embodiment 214. The method of embodiment 213, wherein in the first stage of treatment

[0280] - the CXCL12 antagonist is NOX-A12, which is administered intravenously to the subject continuously over a period of 6 weeks, wherein the weekly dose administered to the subject is about 200 mg / kg of the subject's body weight, about 400 mg / kg of the subject's body weight, about 600 mg / kg of the subject's body weight, or about 800 mg / kg of the subject's body weight,

[0281] - the anti-angiogenic compound is bevacizumab, which is administered intravenously to the subject once every 2 weeks over a period of 6 weeks, wherein the once every 2 weeks dose administered to the subject is 10 mg / kg of the subject's body weight, and

[0282] - the radiation therapy is conventionally fractionated radiation therapy administered to the subject 5 consecutive days per week over a period of 6 weeks, wherein the daily absorbed radiation dose is about 2 Gy, or

[0283] The radiation therapy is hypofractionated radiation therapy administered to the subject for 5 consecutive days per week over a period of 3 weeks, wherein the daily absorbed radiation dose is about 2.7 Gy.

[0284] Embodiment 215. The method of embodiment 214, wherein chemotherapy is administered to the subject in the first phase of treatment, wherein the chemotherapy is temozolomide, which is administered orally or intravenously to the subject continuously over a period of 6 weeks, wherein the daily dose of temozolomide administered to the subject is 75 mg / m 2 The surface area of ​​the subject.

[0285] Embodiment 216. The method of any one of Embodiments 213 to 215, wherein the second treatment phase is subsequent to the first treatment phase.

[0286] Embodiment 217. The method of Embodiment 216, wherein the CXCL12 antagonist is administered intravenously to the subject during the second treatment phase.

[0287] Embodiment 218. The method of Embodiment 217, wherein the CXCL12 antagonist is continuously administered to the subject over a second period of time.

[0288] Embodiment 219. The method described in Embodiment 218, wherein the second time period is any time period from 1 week to 20 weeks or a time period longer than 20 weeks.

[0289] Embodiment 220. The method of any one of Embodiments 216 to 219, wherein the CXCL12 antagonist is a CXCL12 binding nucleic acid molecule comprising a 40 kDa PEG moiety.

[0290] Embodiment 221. The method of Embodiment 220, wherein the CXCL12 antagonist is NOX-A12.

[0291] Embodiment 222. The method of any one of Embodiments 220 to 221, wherein 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.

[0292] Embodiment 223. The method of any one of Embodiments 216 to 222, wherein the anti-angiogenic compound is administered intravenously to the subject.

[0293] Embodiment 224. The method of any one of Embodiments 216 to 223, wherein the anti-angiogenic compound is administered once a week, every 2 weeks, or every 3 weeks during the second time period.

[0294] Embodiment 225. The method described in Embodiment 224, wherein the second time period is any time period from 1 week to 20 weeks or a time period longer than 20 weeks.

[0295] Embodiment 226. The method of any one of Embodiments 224 to 225, wherein

[0296] (a) the anti-angiogenic compound is administered once a week, and the second time period is a multiple of 1 week.

[0297] (b) the anti-angiogenic compound is administered once every 2 weeks, and the second time period is a multiple of 2 weeks, or

[0298] (c) the anti-angiogenic compound is administered once every 3 weeks, and the second time period is a multiple of 3 weeks.

[0299] Embodiment 227. The method of any one of Embodiments 216 to 226, wherein the anti-angiogenic compound is an anti-VEGF antibody, preferably an IgG antibody, and more preferably bevacizumab.

[0300] Embodiment 228. The method of any one of Embodiments 216 to 227, wherein

[0301] (a) if the anti-angiogenic compound is administered once a week, the dose is about 5 mg / kg body weight of the subject,

[0302] (b) if the anti-angiogenic compound is administered once every 2 weeks, the dose is about 10 mg / kg body weight of the subject, or

[0303] (c) If the anti-angiogenic compound is administered once every 3 weeks, the dosage is about 15 mg / kg body weight of the subject.

[0304] Embodiment 229. The method of any one of Embodiments 216 to 228, wherein in the second treatment phase, chemotherapy is administered to the subject in 28-day cycles.

[0305] Embodiment 230. The method of any one of Embodiments 216 to 229, preferably Embodiment 229, wherein in the second phase, chemotherapy is administered in 1, 2, 3, 4, 5, 6 or more cycles, preferably 6 cycles.

[0306] Embodiment 231. The method of Embodiment 230, wherein the cycle consists of 28 days.

[0307] Embodiment 232. The method of Embodiment 231, wherein temozolomide is administered to the subject for the first 5 days of a cycle, followed by 23 days of rest.

[0308] Embodiment 233. The method of any one of Embodiments 229 to 232, wherein the chemotherapy comprises temozolomide.

[0309] Embodiment 234. The method of embodiment 233, wherein the daily dose of temozolomide is from about 150 to about 200 mg / m 2 The body surface area of ​​the subject.

[0310] Embodiment 235. The method of any one of Embodiments 216 to 234, wherein in the second treatment period

[0311] - the CXCL12 antagonist is NOX-A12, which is continuously administered intravenously to the subject over a period of about 1 to about 20 weeks, wherein the weekly dose administered to the subject is about 200 mg / kg of the subject's body weight, about 400 mg / kg of the subject's body weight, about 600 mg / kg of the subject's body weight, or about 800 mg / kg of the subject's body weight, and

[0312] - the anti-angiogenic compound is bevacizumab, which is administered intravenously to the subject once every 2 weeks over a period of about 1 to about 20 weeks, wherein the dose administered to the subject every 2 weeks is 10 mg / kg of the subject's body weight.

[0313] Embodiment 236. The method of any one of Embodiments 216 to 234, wherein in the second treatment period

[0314] - the CXCL12 antagonist is NOX-A12, which is continuously administered intravenously to the subject over a period of about 1 to about 20 weeks, wherein the weekly dose administered to the subject is about 200 mg / kg of the subject's body weight, about 400 mg / kg of the subject's body weight, about 600 mg / kg of the subject's body weight, or about 800 mg / kg of the subject's body weight,

[0315] - the anti-angiogenic compound is bevacizumab, which is administered intravenously to the subject once every 2 weeks over a period of 1 to about 20 weeks, wherein the dose administered to the subject every 2 weeks is about 10 mg / kg of the subject's body weight; and

[0316] - the chemotherapy is temozolomide, which is administered orally or intravenously to the subject for 6 cycles, wherein each cycle consists of 28 days, wherein temozolomide is administered to the subject on each of days 1, 2, 3, 4, and 5 of each cycle, and wherein the daily dose of temozolomide administered to the subject is about 150 to about 200 mg / m 2 The body surface area of ​​the subject.

[0317] Embodiment 237. The method of any one of Embodiments 226 to 236, preferably any one of Embodiments 235 and 236, wherein radiation therapy is not administered to the subject during the second treatment phase.

[0318] More specifically, the problem addressed by the present invention is solved in a first aspect by a method for treating a tumor in a subject, wherein the method comprises administering to the subject

[0319] -CXC motif chemokine 12 (CXCL12) antagonist,

[0320] - Radiation therapy, and

[0321] - Anti-angiogenic compounds

[0322] Wherein the tumor is a brain tumor.

[0323] More specifically, the problem addressed by the present invention is solved in a second aspect by a CXC motif chemokine 12 (CXCL12) antagonist for use in a method of treating a brain tumor in a subject, wherein the method comprises administering to the subject

[0324] - the CXC motif chemokine 12 (CXCL12) antagonist,

[0325] - Radiation therapy, and

[0326] - Anti-angiogenic compounds

[0327] Wherein the tumor is a brain tumor.

[0328] More specifically, the problem to which the present invention relates is solved in a third aspect by radiotherapy for use in a method of treating a brain tumor in a subject, wherein the method comprises administering to the subject

[0329] -CXC motif chemokine 12 (CXCL12) antagonist,

[0330] - said radiation therapy, and

[0331] - anti-angiogenic compounds,

[0332] Wherein the tumor is a brain tumor.

[0333] More specifically, the problem to which the present invention relates is solved in a fourth aspect by an anti-angiogenic compound for use in a method for treating a brain tumor in a subject, wherein the method comprises administering to the subject

[0334] -CXC motif chemokine 12 (CXCL12) antagonist,

[0335] - Radiation therapy, and

[0336] - said anti-angiogenic compound,

[0337] Wherein the tumor is a brain tumor.

[0338] More specifically, the problem to which the present invention relates is solved in a fifth aspect by a chemotherapy for use in a method for treating a brain tumor in a subject, wherein the method comprises administering to the subject

[0339] -CXC motif chemokine 12 (CXCL12) antagonist,

[0340] - Radiation therapy,

[0341] - anti-angiogenic compounds, and

[0342] - said chemotherapy,

[0343] Wherein the tumor is a brain tumor.

[0344] Those skilled in the art will appreciate that 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. Those skilled in the art will also appreciate that, unless expressly indicated in a different manner, embodiments of the present invention are embodiments of the first, second, third, fourth and fifth aspects of the present invention. This also applies when referring to embodiments of the present invention.

[0345] The method of the present invention for treating a brain tumor comprises administering to the subject

[0346] -CXC motif chemokine 12 (CXCL12) antagonist,

[0347] - Radiation therapy,

[0348] - anti-angiogenic compounds, and

[0349] -Chemotherapy

[0350] In principle, it can be applied to any brain tumor.

[0351] Brain tumors can be classified according to the cells that form such tumors or according to the microscopic appearance of the tumor cells. The most important brain tumors are gliomas, meningiomas, pituitary adenomas, vestibular schwannomas, and medulloblastomas. Gliomas include a variety of subtypes, such as astrocytomas, oligodendrogliomas, ependymomas, and choroid plexus papilloma. The classification of the microscopic appearance of the cells that form brain tumors is based on grades I to IV, as defined in the introduction section incorporated herein by reference.

[0352] In an embodiment of the invention, the brain tumor is a grade IV brain tumor. It is particularly preferred that the brain tumor is a glioblastoma which is a grade IV astrocytoma. Among glioblastomas, supratentorial glioblastomas and infratentorial glioblastomas can be further distinguished. Both forms can be treated by means of the method of the invention, with supratentorial glioblastomas occurring more frequently.

[0353] Brain tumors treatable by the methods of the invention may further or alternatively be characterized according to available treatment options or treatments already applied to subjects suffering from the tumor.

[0354] In embodiments of the invention, the tumors that can be treated with the methods of the invention are tumors that are sensitive to radiation therapy, tumors that can be treated with radiation, or tumors that have been treated with radiation prior to applying the methods of the invention to a subject with the brain tumor. Such radiation therapy is known to those skilled in the art. Those skilled in the art will appreciate that such radiation therapy can be the same type of radiation therapy administered to a subject according to the invention, the disclosures of each of which are incorporated herein by reference.

[0355] In an embodiment of the present invention, the tumor that can be treated by the method of the present invention is a residual tumor after surgery, usually an incompletely removed tumor. As mentioned above, surgery is still one of the most commonly used treatments for treating brain tumors, and it will be recognized that some tumors cannot be treated by surgery because the collateral tissue damage due to surgery may cause more serious overall damage or even death than the brain tumor itself. However, if a brain tumor is treated by surgery, some residual tumor tissue may escape surgical resection, which is usually due to its small size or its location in the subject's brain. The location in the subject's brain is, for example, a location that cannot be approached by surgery due to mechanical reasons (e.g., non-tumor brain tissue is not allowed to contact tumor tissue) or medical reasons (e.g., tumor tissue is too close to important structures and cannot be damaged for the patient's health). This residual tumor tissue can, in an embodiment of the present invention, be suitable for the method of the present invention.

[0356] A method for detecting incompletely resected tumors (usually postoperatively) is a magnetic resonance imaging (MRI) scan. Preferably, the MRI scan is a contrast-enhanced MRI scan, and 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 to its equilibrium value after excitation. T1-weighted magnetic resonance images are usually created 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 such images, but the proton density always contributes to the intensity of the image. The T1 dependence is mainly determined by the repetition time or any pre-pulses (e.g., in an inversion recovery pulse sequence). Accordingly, in an 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 T1-weighted, contrast-enhanced MRI scan.

[0357] Fat has a higher signal due to the greater longitudinal and transverse magnetization and will appear bright on T1-contrast MR images. In contrast, water has a lower longitudinal magnetization before the RF pulse and therefore a lower transverse magnetization after the RF pulse, producing a low signal that appears dark on T1-contrast images. Typically, a paramagnetic contrast agent (gadolinium compound) is administered, and both a pre-contrast T1-weighted image and a post-contrast T1-weighted image are obtained.

[0358] In embodiments of the present invention, the tumor to be treated may be characterized by certain biochemical characteristics. These biochemical characteristics include, but are not limited to, resistance to drugs and secretion of factors such as chemokines and / or angiogenic factors. Those skilled in the art will appreciate that the tumor may also display a combination of these biochemical characteristics.

[0359] In an embodiment of the present invention, the resistance to one or more drugs is resistance to temozolomide (TMZ). Temozolomide is an alkylating agent and is sold under trade names such as Temodar. It is used as a standard therapy for the treatment of brain tumors such as glioblastoma (first-line treatment) and anaplastic astrocytoma (second-line treatment). It is taken orally or intravenously. The most common side effects of temozolomide are nausea, vomiting, constipation, loss of appetite, alopecia (hair loss), headache, fatigue, convulsions (seizures), rash, neutropenia or lymphopenia (low white blood cell count) and thrombocytopenia (low platelet count).

[0360] Resistance to temozolomide therapy is the main cause of treatment failure in gliomas and glioblastomas, and therefore, it is crucial to provide treatments for gliomas and glioblastomas that are resistant to temozolomide therapy. The demethylase O6-methylguanine-DNA methyltransferase (MGMT) is associated with intrinsic TMZ resistance (TMZ-R) and relapse by directly removing the alkyl group at the O6 position of guanine. However, epigenetic silencing of MGMT is common in gliomas, and gliomas with low MGMT levels (deficient and low expression) are still sufficient to confer TMZ-R, suggesting the existence of MGMT-independent mechanisms of acquired TMZ-R. At low MGMT levels, TMZ-R gliomas exhibit genetic mutations. For example, recurrent gliomas exhibit transcriptional silencing of the MGMT gene, followed by dysfunction of the mismatch repair (MMR) system and hyperfunction of the DNA repair system. When MMR is deficient, the DNA damage repair (DDR) system is activated, resulting in TMZ-R. Therefore, the functional availability of the DDR system may regulate the response of recurrent gliomas to TMZ. Therefore, it is hypothesized that the DDR signaling pathway is enhanced in TMZ-R glioma cells, which contributes to their phenotypic drug resistance. Studies have shown that Rho-associated kinase 2 (ROCK2) is overactivated in the TMZ-R model, and inhibition of ROCK2 can reverse TMZ-R and increase TMZ sensitivity, indicating that there is an association between ROCK2 and TMZ-R, but the mechanism remains unclear (Zhang X et al., Cell Death & Disease 13, Article number 138(2022)).

[0361] Accordingly, in an embodiment of the present invention, the tumor is a tumor that is unresponsive or resistant to temozolomide. In addition, in an embodiment of the present invention, the tumor is a tumor with unmethylated O 6 -methylguanine DNA methyltransferase (MGMT) promoter status in glioblastoma.

[0362] In an embodiment of the invention, the tumor is a tumor that secretes factors, wherein the factors are chemokines and / or angiogenic factors. It is particularly preferred if 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).

[0363] CXC motif chemokine 12 (CXCL12), also known as stromal cell-derived factor 1 (SDF-1), is a key chemokine responsible for the attraction or repulsion of many types of leukocytes through signaling through the receptor CXC motif chemokine receptor 4 (CXCR4) and potentially through its alternative receptor CXCR7.

[0364] CXCL12 is an angiogenic CXC motif chemokine that does not contain the typical ELR motif of IL-8-like chemokines, but binds and activates the G protein-coupled receptor CXCR4 (Salcedo, Wasserman et al. 1999; Salcedo and Oppenheim 2003). As a result of alternative splicing, there are other forms of CXCL12, such as CXCL12α (68 amino acids, SEQ ID NO: 1) and CXCL12β (SEQ ID NO: 2), which carries 5 more amino acids at the C-terminus compared to CXCL12α (Shirozu, Nakano et al. 1995).

[0365] The amino acid sequence conservation between CXCL12 from different species is remarkable: human CXCL12α (SEQ.ID.1) and mouse CXCL12α (SEQ ID NO: 3) are almost identical, with only a single conservative change from V to I at position 18 (Shirozu, Nakano et al. 1995).

[0366] Since the CXCL12 receptor CXCR4 is widely expressed on leukocytes, mature dendritic cells, endothelial cells, brain cells and megakaryocytes, the activity of CXCL12 is pleiotropic. This chemokine exhibits the broadest range of biological functions than any other chemokine discovered to date. The most notable functional effects of CXCL12 are:

[0367] -Homing and attachment of epithelial cells to neovascular sites in the choroidal portion of the retina;

[0368] -CXCL12 is required for the maintenance of stem and progenitor cells in the adult bone marrow, such as hematopoietic progenitor cells (usually CD34+ cells);

[0369] -SDF1 supports the proliferation of pre-B cells and increases the growth of bone marrow B cell progenitors. It induces specific migration of pre-B cells and progenitors, but does not act as a chemoattractant for mature B cells.

[0370] -CXCL12 is one of the most potent T cell chemoattractants; and

[0371] -CXCL12 and its receptor CXCR4 are essential for embryonic development.

[0372] Altered expression levels of CXCL12 or its receptor CXCR4, or altered responses to these molecules, are thought to be associated with a number of human diseases, such as 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 (Warts, Hypogammaglobulinemia, Infections, Myelokathexis (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 neovascularization (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. 2004).

[0373] Tumors, both solid and hematologic, and malignant, are more than just masses of cancer cells: infiltration of tumors with immune cells is a hallmark of cancer. Many human cancers have a complex network of chemokines that influence the extent and phenotype of this infiltration, as well as tumor growth, survival, migration, and angiogenesis. Most solid tumors contain many non-malignant stromal cells. In fact, sometimes stromal cells outnumber cancer cells. The main stromal cells found in cancer are macrophages, lymphocytes, endothelial cells, and fibroblasts.

[0374] Cells from different cancer types have different chemokine receptor expression profiles, but the CXCL12 receptor CXCR4 is most frequently found in tumor cells in mice and humans: 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 being constitutively expressed in the bone marrow and secondary lymphoid tissues, CXCL12 has been found in primary tumor sites of lymphomas (Corcione, Ottonello et al. 2000) and brain tumors of neuronal and astrocytic lineages. In addition, it is present at high levels in ovarian cancer (Scotton, Wilson et al. 2002) and pancreatic cancer (Koshiba, Hosotani et al. 2000), metastatic sites of breast cancer (Muller, Homey et al. 2001), metastatic sites of thyroid cancer (Hwang, Chung et al. 2003), neuroblastomas, and hematological malignancies (Geminder, Sagi-Assif et al. 2001).

[0375] 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 on prostate cancer cell lines have shown that altered CXCR7 / RDC1 expression is associated with enhanced adhesion and invasion activity in addition to a survival advantage. In vitro and in vivo studies have shown that the two receptors for CXCL12, CXCR4 and CXCR7, promote tumor growth, metastatic potential, and resistance to apoptosis (chemotherapy-induced) in many tumors, such as breast cancer, glioblastoma, ovarian cancer, neuroblastoma, lung cancer, colorectal cancer, and prostate cancer (Burns et al., 2006; Li et al., 2008; Scotton et al., 2002; Yang et al., 2008; Zagzag et al., 2008).

[0376] The expression of CXCR4 and CXCR7 thus appears to be a general feature of several tumors.

[0377] Vascular endothelial growth factor (VEGF) is a basic, heparin-binding homodimeric glycoprotein of 45,000 daltons. These properties correspond to those of VEGF165 (the major VEGF isoform). The human VEGF gene is located on chromosome 6p21.3 and consists of 8 exons separated by 7 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, followed by signal sequence cleavage (Ferrara & Henzel 1989, Biochem Biophys Res Comm 161:851; Houck et al. 1991, Mol Endocrinol 5:1806; Leung et al., 1989, Science 246:1306). Less common 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 (including all VEGF isoforms) are provided by a proteolytic cleavage mechanism, resulting in the VEGF110 form (Houck et al. 1992, J Biol Chem 267:26031).

[0378] VEGF is a major regulator of angiogenesis in normal and pathological processes, including angiogenesis 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 key regulatory function in developmental angiogenesis (Ferrara et al. 1996, Nature 380:439; Carmeliet et al., 1996, Nature 380:14756). VEGF's proven in vitro activity is the ability to promote the growth of vascular endothelial cells (EC) of arterial, venous and lymphatic origin (reviewed in Ferrara and Davis-Smyth 1997, Endocr Rev 18:4), as well as the growth of certain non-endothelial cells (reviewed in Matsumoto and Claesson-Welsh 2001, Sci STKE 2001 (112): p. RE21). VEGF has been shown to be a survival factor for endothelial cells 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 stimulates the production of surfactant proteins by cultured type 2 pneumocytes (Compernolle et al. 2002, Nat Med 8:702). VEGF induces vasodilation in vitro (Ku et al. 1993, Am J Physiol 265:H586), and due to its ability to induce vascular leakage, VEGF is believed to play a role in inflammation (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 involved in matrix degradation in endothelial cells. VEGF also has an effect on bone marrow-derived cells, promoting monocyte activation and chemotaxis (Clauss et al. 1990, J Exp Med 172:1535). VEGF enhances colony formation of mature subsets of granulocyte macrophages and erythroid progenitor cells that have been stimulated with colony stimulating factor (Bates et al., 2002, Cancer Res 62:4123).VEGF has also been shown to have immune effects by inhibiting the maturation of antigen-presenting dendritic cells (Gabrilovitch et al. 1996, NatMed 2:1096).

[0379] VEGF binds to two related receptor tyrosine kinases (RTKs), designated Flt-1 (VEGFR-1) (de Vries et al. 1992, Science 255:989; Shibuya et al. 1990, Oncogen 5:519), 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 RTK family, but is not a receptor for VEGF, instead binding to VEGFC and VEGFD8. In addition to these RTKs, VEGF also interacts with a family of coreceptors (neuropilins). Binding of VEGF to VEGFR-1 and VEGFR-2 induces homodimerization of the two receptor subunits, which in turn triggers autophosphorylation of their tyrosine kinase domains located in the cytoplasm. Autophosphorylation of the tyrosine kinase domain subsequently participates in a series of specific signaling events that ultimately regulate the multiple 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. VEGFR-2-mediated survival signaling 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 Cγ and mitogen-activated protein kinase MAPK p44 / 42 (Gille et al. 2001, J Biol Chem 276:3222).

[0380] In embodiments of the invention, CXCL12 is expressed at the leading edge of a tumor (the outermost tumor compartment where the tumor spreads into surrounding tissue), compartments with hyperplastic blood vessels (blood vessels with thickened walls due to endothelial cell proliferation), and / or compartments of the tumor with microvascular proliferation (see, e.g., Comba A et al. Front. Oncol., 05 August 2021; https: / / doi.org / 10.3389 / fonc.2021.703764).

[0381] In embodiments of the invention, VEGF is expressed in tumor cell compartments where tumor cell proliferation is most prominent, compartments where pseudopalisading cells (characterized by a rosette-like organization of tumor cells at the edge of areas of tumor necrosis) are present, and / or peri-necrotic zones of dying malignant cells of a tumor.

[0382] In particularly preferred embodiments of the invention, CXCL12 is expressed in the leading edge of the tumor, in the hyperplastic blood vessels of the tumor and / or in the microvascular proliferation zone of the tumor, and VEGF is expressed in the tumor cell compartment of the tumor in the pseudopalisading cells of the tumor and / or in the peri-necrotic zone of the tumor.

[0383] According to the method of the present invention, a CXCL12 antagonist is administered to a subject having the tumor. The CXCL12 antagonists used according to the present invention may use different modes of action. These modes of action include that the CXCL12 antagonist inhibits the signaling of CXCL12 through one or both of the CXCL12 receptor CXC chemokine receptor type 4 (CXCR4) and the CXCL12 receptor CXC chemokine receptor type 7 (CXCR7). In a preferred embodiment, the CXCL12 antagonist directly binds to CXCL12, such as through physical interaction, so that, preferably, one or more effects caused by CXCL12 will be reduced or eliminated. Preferably, this reduced or eliminated effect is effective for the treatment of the disease or contributes to 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 a CXCL12 receptor (e.g., CXCR4 and / or CXCR7). In alternative preferred embodiments, the CXCL12 antagonist directly binds to a CXCL12 receptor (e.g., CXCR4 and / or CXCR7), such as by physical interaction, so that CXCL12 no longer binds to such a CXCL12 receptor or binds at a reduced rate, reduced affinity, or shortened on-time. Preferably, due to such interference with the binding of CXCL12 to the CXCL12 receptor, one or more effects caused by CXCL12 will be reduced or eliminated. Preferably, such reduced or eliminated effects are effective for or contribute to the treatment of a disease.

[0384] Those skilled in the art will appreciate that such inhibition of CXCL12 signaling through one or both of the CXCL12 receptors CXCR4 and CXCR7 can be achieved by (a) providing a molecule that binds CXCL12 to inhibit the binding of CXCL12 to CXCR4 and / or CXCR7, providing a molecule that binds CXCR4 to inhibit the binding of CXCR4 to CXCL12, and (c) providing a molecule that binds CXCR7 to inhibit the binding of CXCL12 to CXCR7. Such a molecule that binds CXCL12 is preferably a molecule selected from the group comprising an aptamer (preferably a CXCL12 binding aptamer), a spiegelmer (preferably a CXCL12 binding spiegelmer), an antibody (preferably a CXCL12 binding antibody), a protein that binds CXCL12, a CXCL12 binding anticalin, a CXCL12 binding peptide, a fusion protein comprising a CXCL12 binding portion, and a CXCL12 binding small molecule.

[0385] Such a molecule that binds to CXCR4 to inhibit the signaling of CXCL12 is preferably a molecule selected from the group comprising an aptamer (preferably a CXCR4-binding aptamer), a spiegelmer (preferably a CXCR4-binding spiegelmer), an antibody (preferably a CXCR4-binding antibody), a protein that binds to CXCR4, a CXCR4-binding anticalin, a CXCR4-binding peptide, a fusion protein comprising a CXCR4-binding portion, and a CXCR4-binding small molecule.

[0386] Such a molecule that binds to CXCR7 is preferably a molecule selected from the group comprising an aptamer (preferably a CXCR7 binding aptamer), a spiegelmer (preferably a CXCR7 binding spiegelmer), an antibody (preferably a CXCR7 binding antibody), a protein that binds to CXCR7, a CXCR7 binding anticalin, a CXCR7 binding peptide, a fusion protein comprising a CXCR7 binding portion, and a CXCR7 binding small molecule.

[0387] Those skilled in the art will also appreciate that such inhibition of CXCL12 signaling through one or both of the CXCL12 receptors CXCR4 and CXCR7 may be achieved by compounds that bind to CXCR4 and / or CXCR7, whereby such binding of the compound interferes with signaling, preferably the signal transduction properties of CXCR4 and / or CXCR7, rather than interfering with the binding of CXCL12 to CXCR4 and / or CXCR7.

[0388] The molecule that binds to CXCR4 and interferes with the signal transduction properties of CXCR4 (preferably signal transduction triggered by the binding of CXCL12 to CXCR4) because of its binding to CXCR4 is preferably a molecule selected from the group comprising an aptamer (preferably a CXCR4-binding aptamer), a spiegelmer (preferably a CXCR4-binding spiegelmer), a protein that binds to CXCR4, an antibody or an antigen-binding portion of the antibody (preferably a CXCR4-binding antibody or a CXCR4-binding fragment of the CXCR4-binding antibody), anticalin (preferably CXCR4-binding anticalin), a CXCR4-binding peptide, a fusion protein comprising a CXCR4-binding portion, and a CXCR4-binding small molecule.

[0389] The production and identification of such aptamers, spiegelmers, antibodies, binding proteins, anticalins, peptides, fusion proteins and small molecules, respectively, are known to those skilled in the art and are also disclosed herein.

[0390] In a preferred embodiment of the invention, CXCL12 is human CXCL12, CXCR4 is human CXCR4, and CXCR7 is human CXCR7.

[0391] With respect to this part of the method of the invention, i.e., administering a CXCL12 antagonist to a subject with a tumor, it is acknowledged that animal studies have shown that radiation-induced blood supply blockage can be effectively restored by vasculogenesis after recruiting bone marrow-derived cells that lead to rapid recurrence of the tumor (Kioi et al. 2010, J Clin Invest 120:694). CXCL12 is speculated to play an important role in neovascularization by recruiting endothelial cells and other bone marrow-derived pro-angiogenic cells via a CXCR4- and CXCR7-dependent mechanism. 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).

[0392] In an embodiment of the present invention, the CXCL12 antagonist inhibits the signaling of CXCL12 through one or both of the CXCL12 receptor CXC chemokine receptor type 4 (CXCR4) and the CXCL12 receptor CXC chemokine receptor type 7 (CXCR7). In a preferred embodiment thereof, the CXCL12 antagonist inhibits the binding of CXCL12 to the CXCL12 receptor CXC chemokine receptor type 4 (CXCR4) and / or the CXCL12 receptor CXC chemokine receptor type 7 (CXCR7). The interaction between CXCL12 and its receptor CXCR4 is also referred to as the CXCL12-CXCR4 axis. This 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. This CXCL12-CXCR7 axis can be targeted by a CXCL12 antagonist. In an even more preferred embodiment, this binding of CXCL12 to the receptor is affected by a CXCL12 antagonist that binds to CXCL12.

[0393] CXCL12 antagonists that bind to CXCL12 include nucleic acid molecules that bind to CXCL12, etc. Such CXCL12 binding nucleic acid molecules can be CXCL12 binding aptamers or CXCL12 binding spiegelmers.

[0394] Aptamers are nucleic acid molecules composed of D-nucleotides as structural units, which specifically bind to target molecules by a mechanism different from Watson-Crick base pairing. Spiegelmers are nucleic acid molecules composed of L-nucleotides as structural units, which specifically bind to target molecules by a mechanism different from Watson-Crick base pairing. Aptamers and spiegelmers can both be composed of ribonucleotides, deoxyribonucleotides, and a combination of ribonucleotides and deoxyribonucleotides. Aptamers and spiegelmers are known to those skilled in the art, and in addition, they are described in "The Aptamer Handbook" (Klussmann, 2006), and spiegelmers containing both ribonucleotides and deoxyribonucleotides are disclosed in international patent application WO 2012 / 095303.

[0395] In an embodiment of the present invention, the CXCL12 antagonist is an L-nucleic acid molecule that binds to CXCL12, wherein the L-nucleic acid molecule is preferably selected from the group comprising a B-type CXCL12 binding nucleic acid molecule, a C-type CXCL12 binding nucleic acid molecule, an A-type CXCL12 binding nucleic acid molecule and a D-type CXCL12 binding nucleic acid molecule, as defined in more detail herein.

[0396] CXCL12 binding L-nucleic acid molecules can be characterized according to stretches of nucleotides also referred to herein as boxes (see Example 1).

[0397] Different types of CXCL12 binding nucleic acid molecules contain three different nucleotide fragments: a first terminal nucleotide fragment, a central nucleotide fragment, and a second terminal nucleotide fragment. In general, the CXCL12 binding nucleic acid molecules of the present invention contain terminal nucleotide segments at their 5' and 3' ends: a first terminal nucleotide fragment and a second terminal nucleotide fragment (also referred to as a 5' terminal fragment of nucleotides and a 3' terminal fragment of nucleotides). The first terminal nucleotide fragment and the second terminal fragment can (in principle due to the complementarity of their bases) hybridize with each other, thereby forming a double-stranded structure by hybridization. However, under physiological and / or non-physiological conditions, such hybridization is not necessarily achieved in the molecule. The three nucleotide fragments (the first terminal nucleotide fragment, the central nucleotide fragment, and the second terminal nucleotide fragment) of the CXCL12 binding nucleic acid molecule are arranged in a 5'-> 3'-direction: the first terminal nucleotide fragment-the central nucleotide fragment-the second terminal nucleotide fragment. However, alternatively, the second terminal nucleotide fragment, the central nucleotide fragment, and the first terminal nucleotide fragment are arranged with each other in a 5'-> 3'-direction.

[0398] Sequence differences in the defined boxes or segments between different CXCL12 binding nucleic acid molecules influence the binding affinity to CXCL12. Based on binding analysis of different XCL12 binding nucleic acid molecules used according to the invention, the central segment and the nucleotides forming it are individually, and more preferably as a whole, essential for binding to human CXCL12.

[0399] The terms "stretch" and "stretch of nucleotides" are used herein in a synonymous manner unless otherwise indicated.

[0400] In a preferred embodiment of the invention, the nucleic acid used according to the invention is a single nucleic acid molecule. In another embodiment, the single nucleic acid molecule is present as a multiple of said single nucleic acid molecule or as a multiple of said single nucleic acid molecule species.

[0401] It will be appreciated by the skilled person that the nucleic acid molecules used according to the present invention preferably consist of nucleotides covalently linked to each other, preferably via phosphodiester linkages or bonds.

[0402] Within the scope of the present invention, the nucleic acid used according to the present invention comprises two or more fragments or one or more parts thereof, which can hybridize with each other in principle. In the case of such hybridization, a double-stranded structure is formed. It is recognized by those skilled in the art that the hybridization may or may not occur, particularly under in vitro and / or in vivo conditions. In addition, in the case of such hybridization, the hybridization does not necessarily occur over the entire length of the two fragments, at least according to the rule of base pairing, the formation of such hybridization and thus the double-stranded structure may (in principle) occur. As preferably used herein, a double-stranded structure is a part of a nucleic acid molecule or a structure formed by two or more independent chains of a nucleic acid molecule or two spatially separated single-stranded fragments, whereby there is at least one, preferably two or more base pairs, and the base pairs are preferably paired according to the Watson-Crick base pair rule. It is also recognized by those skilled in the art that other base pairs (such as Hoogsten base pairing) may be present in or form such a double-stranded structure. It is also recognized that the feature of the hybridization of the two fragments preferably indicates that it is believed that such hybridization occurs due to the base complementarity of the two fragments.

[0403] In a preferred embodiment of the present invention, the term "arrangement" as used herein refers to the order or sequence of the structural or functional features or elements described herein in connection with the nucleic acids disclosed herein.

[0404] It is recognized by those skilled in the art that the nucleic acids used according to the present invention are capable of binding to CXCL12. Without wishing to be bound by any theory, the CXCL12 binding is caused by a combination of three-dimensional structural features or elements of the nucleic acid molecule, the binding being caused by the orientation and folding pattern of the primary sequence of the nucleotides forming these features or elements, whereby preferably these features or elements are the first terminal nucleotide fragment, the central nucleotide fragment and the second terminal nucleotide fragment of the CXCL12 binding nucleic acid molecule. Obviously, individual features or elements may be formed by various different individual sequences, the degree of variation of which may vary depending on the three-dimensional structure that such features or elements must form. The overall binding properties of the claimed nucleic acid are the result of the interaction of various elements and features, respectively, ultimately resulting in the interaction of the claimed nucleic acid with its target (i.e., CXCL12). Similarly, without wishing to be bound by any theory, the central nucleotide fragment (which is a feature of the CXCL12 binding nucleic acid) appears to be important for mediating the binding of the claimed nucleic acid molecule to CXCL12. Therefore, the nucleic acids according to the present invention are suitable for interacting with CXCL12. In addition, it is recognized by those skilled in the art that the nucleic acids used according to the present invention are antagonists to CXCL12. As such, the nucleic acids according to the invention are suitable for the treatment and prevention of brain cancer in the method according to the invention.

[0405] The nucleic acid molecules used according to the present invention should also include nucleic acids that are substantially homologous to the specific sequences disclosed herein. The term substantially homologous should be understood to mean, for example, that the homology is at least 75%, preferably 85%, more preferably 90% and most preferably greater than 95%, 96%, 97%, 98% or 99%.

[0406] The actual percentage of homologous nucleotides present in the nucleic acids used according to the invention will depend on the total number of nucleotides present in the nucleic acid.The percentage of modification may be based on the total number of nucleotides present in the nucleic acid.

[0407] Homology between two nucleic acid molecules can be as known to those skilled in the art.More specifically, based on the program parameters specified, a sequence comparison algorithm can be used to calculate the sequence homology percentage of a test sequence relative to a reference sequence.Described test sequence is preferably to be alleged to be homologous to different nucleic acid molecules or to be detected whether it is a homologous sequence or nucleic acid molecules, and if homologous (to what extent), thus this different nucleic acid molecules are also referred to as reference sequences. In an embodiment, the reference sequence is a nucleic acid molecule as 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. Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman (1981), the homology alignment algorithm of Needleman and Wunsch (1970), the similarity search method of Pearson and Lipman (1988), by computer implementations 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.

[0408] An 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 analyses is publicly available through the National Center for Biotechnology Information (hereinafter "NCBI"). Default parameters used in determining sequence identity using software provided by NCBI, such as BLASTN (for nucleotide sequences) and BLASTP (for amino acid sequences), are described in McGinnis et al. (McGinnis et al., 2004).

[0409] The nucleic acid molecules used according to the present invention should also include nucleic acid molecules that have a certain degree of identity with respect to the nucleic acid molecules disclosed herein and are defined by their nucleotide sequences. More preferably, this invention also includes those nucleic acid molecules that have at least 75%, preferably 85%, more preferably 90% and most preferably more than 95%, 96%, 97%, 98% or 99% identity with respect to the nucleic acid molecules disclosed herein and are defined by their nucleotide sequences or a portion thereof.

[0410] The terms nucleic acid and nucleic acid molecule are used interchangeably herein. In addition, the nucleic acid molecules used in accordance with the present invention shall also include those nucleic acid molecules comprising the nucleic acid sequences disclosed herein or portions thereof, for example, metabolites or derivatives of the nucleic acid molecules used in accordance with the present invention, preferably to the extent that the nucleic acid molecules or the portions are involved in binding to CXCL12 or are capable of binding to CXCL12. Such nucleic acid molecules may be derived from the nucleic acid molecules disclosed herein, for example by truncation. Truncation may be associated with either or both ends of the nucleic acid molecules disclosed herein. In addition, truncation may be associated with the internal sequence of the nucleotides, i.e., it may be associated with one or more nucleotides between the 5' and 3' terminal nucleotides, respectively. In addition, truncation shall include deletions as few as a single nucleotide from the nucleic acid sequences disclosed herein. Truncation may also be associated with more than one fragment of the nucleic acid molecules used in accordance with the present invention, whereby the fragments may be as short as one nucleotide long. The binding of the nucleic acid molecules used in accordance with the present invention may be determined by a person skilled in the art using routine experiments or by using or adopting methods as described herein (preferably as described herein in the Examples section).

[0411] In addition, within the scope of the present invention, one or more parts of the nucleic acid molecules used according to the present invention are present as D-nucleic acids, or at least one or more parts of the nucleic acid molecules are L-nucleic acids. The term "part" of a nucleic acid molecule should mean as little as one nucleotide. Such nucleic acid molecules are generally referred to as D-nucleic acids and L-nucleic acids, respectively, in this article. Therefore, in a particularly preferred embodiment, the nucleic acid molecules used according to the present invention consist of L-nucleotides and contain at least one D-nucleotide. Such D-nucleotides are preferably attached to parts of fragments different from the nucleic acid molecules used according to the definition of the present invention, preferably connected to those parts thereof, wherein interactions with other parts of the nucleic acid molecules are involved. Preferably, such D-nucleotides are attached to the ends of any fragments and any nucleic acid molecules used according to the present invention, respectively. In another preferred embodiment, such D-nucleotides can serve as spacers or linkers, preferably attaching modifications (such as PEG and HES) to the nucleic acids according to the present invention.

[0412] Also within the scope of the present invention, the nucleic acid molecule used according to the present invention is part of a longer nucleic acid molecule, whereby the longer nucleic acid molecule comprises several parts, whereby at least one such part is a nucleic acid molecule used according to the present invention or a part thereof. One or more other parts of these longer nucleic acid molecules may be one or more D-nucleic acids (one or more) or L-nucleic acids (one or more). Any combination may be used in conjunction with the present invention. One or more of these other parts of the longer nucleic acid molecule may exhibit a function different from binding (preferably binding to CXCL12). One possible function is to allow interaction with other molecules, whereby these other molecules are preferably different from CXCL12, for example for fixation, crosslinking, detection or amplification. In another embodiment of the present invention, the nucleic acid molecule used according to the present invention comprises several nucleic acid molecules used according to the present invention, as individual or combined parts. Such a nucleic acid molecule comprising several nucleic acid molecules used according to the present invention is also encompassed in the term longer nucleic acid.

[0413] As used herein, an L-nucleic acid molecule is a nucleic acid consisting of L-nucleotides, preferably consisting entirely of L-nucleotides.

[0414] As used herein, a D-nucleic acid molecule is a nucleic acid molecule consisting of D-nucleotides, preferably consisting entirely of D-nucleotides.

[0415] Unless clearly indicated to the contrary, the terms nucleic acid and nucleic acid molecule are used herein interchangeably.

[0416] Furthermore, unless indicated otherwise, any nucleotide sequence herein is listed in a 5'->3' orientation.

[0417] As preferably used herein, any position of determining or referring to a nucleotide is relative to the 5' end of a sequence, a fragment or a sub-fragment. Therefore, the second nucleotide is the second nucleotide counted from the 5' end of a sequence, a fragment and a sub-fragment, respectively. In addition, accordingly, the penultimate nucleotide is the second nucleotide counted from the 3' end of a sequence, a fragment and a sub-fragment, respectively.

[0418] It is also within the scope of the present invention that the nucleic acid molecules used according to the present invention, whether they are present as D-nucleic acids, L-nucleic acids or D,L-nucleic acids, or whether they are DNA or RNA, can exist as single-stranded or double-stranded nucleic acids. Usually, the nucleic acid molecules used according to the present invention are single-stranded nucleic acids, which exhibit a defined secondary structure due to the primary sequence and can therefore also form a tertiary structure. However, the nucleic acid molecules used according to the present invention can also be double-stranded, which means that two chains that are complementary or partially complementary to each other can hybridize with each other or at least be capable of hybridizing with each other according to their base complementarity.

[0419] The nucleic acid molecules used according to the present invention may be modified. Such modifications may be associated with individual nucleotides of the nucleic acid and are well known in the art. Venkatesan et al. and Kusser Examples of such modifications are described in, for example, . Such modifications may be H atoms, F atoms or O-CH located at the 2' position of the individual nucleotides constituting the nucleic acid. 3 Group or NH 2 Furthermore, the nucleic acid molecules used according to the invention may comprise at least one LNA nucleotide. In an embodiment, the nucleic acid according to the invention consists of LNA nucleotides.

[0420] In embodiments of the present invention, according to the nucleic acid molecules used according to the present invention can be multi-partite nucleic acid molecules.Multi-partite sub-nucleic acid molecules used herein are nucleic acid molecules composed of at least two independent nucleic acid chains.These at least two nucleic acid chains form functional units, and described functional units are the part of target molecules thus.By cutting nucleic acid molecules to produce two chains or by synthesizing a nucleic acid corresponding to the first part of the whole nucleic acid molecule and another nucleic acid molecule corresponding to the second part of the whole nucleic acid molecule, at least two nucleic acid chains can derive from any one nucleic acid molecule used according to the present invention.Should be recognized that cutting and synthesis can be used to produce multi-partite nucleic acid molecules, wherein there are two or more chains as shown above.In other words, at least two independent nucleic acid chains are usually different from two complementary and mutually hybridized chains, although there may be a certain degree of complementarity and therefore this complementarity between the described at least two independent nucleic acid chains can cause the hybridization of the described independent chain.

[0421] Finally, it is also within the scope of the present invention to achieve a completely closed (i.e. circular) structure of the nucleic acid molecule used according to the present invention, i.e. in an embodiment the nucleic acid molecule is closed, preferably by a covalent bond, whereby such a covalent bond is more preferably formed between the 5' end and the 3' end of the nucleic acid sequence as disclosed herein or any derivative thereof.

[0422] An option for determining the binding constants of the nucleic acid molecules used according to the invention is to use the method as described in Examples 3 and 4, which confirms the above findings that the nucleic acid molecules used according to the invention exhibit favorable K values ​​according to the invention. D To express the binding strength between a single nucleic acid molecule and a target (in this application CXCL12), an appropriate measure is the so-called K D The method for determining the value is also known to those skilled in the art.

[0423] Preferably, the nucleic acid molecules used according to the invention exhibit a K D The value is less than 1μM. The K of about 1µM D The K value is considered to be characteristic of non-specific binding of a nucleic acid to a target. As recognized by those skilled in the art, the K values ​​of a group of compounds (e.g., nucleic acid molecules used in accordance with the present invention) are D The value is within a certain range. The above K of about 1µM D is the K D The preferred upper limit of the value. D The lower limit of K can be as low as about 10 picomolar or can be higher. Within the scope of the present invention, the K of a single nucleic acid molecule binding to CXCL12 is D The value is preferably within this range. A preferred range can be defined by selecting any first number within this range and any second number within this range. The preferred upper limit K D The values ​​are 250 nM and 100 nM, with the preferred lower limit K D The values ​​are 50 nM, 10 nM, 1 nM, 100 pM and 10 pM. A more preferred upper limit K D The value is 2.5 nM, and the more preferred lower limit K D The value is 100 pM.

[0424] In addition to the binding properties 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 the respective target molecule, in this case CXCL12. Inhibition of the function of CXCL12 (e.g. stimulation of the respective receptor as described above) can be achieved by binding the nucleic acid molecules used according to the present invention to CXCL12 and forming a complex of the nucleic acid molecule and CXCL12. This complex of the nucleic acid molecule and CXCL12 is unable to stimulate receptors that are normally stimulated by CXCL12, such as CXCR4 and CXCR7. Thus, the inhibition of the receptor function by the nucleic acid molecule is independent of the respective receptor that can be stimulated by CXCL12, but is caused by preventing the stimulation of the receptor by CXCL12 by the nucleic acid molecule.

[0425] A possibility to determine the inhibition constant of the nucleic acid molecules used according to the invention is to use the methods as described in Examples 5 and 6 (for CXCR4 and CXCR7, respectively), which confirm the above findings that the nucleic acid molecules used according to the invention exhibit favorable inhibition constants, which allow the use of said nucleic acid molecules in the claimed method of treatment and in any treatment regimen comprising such a method of treatment. In order to express the strength of the inhibitory effect of a single nucleic acid molecule on the interaction of a target (in the present application CXCL12) with the corresponding receptor, a suitable measurement is the so-called half maximal inhibitory concentration (abbreviated as IC 50 ), and its determination method is also known to those skilled in the art.

[0426] Preferably, the nucleic acid molecules used according to the present invention exhibit an IC 50 The value is less than 1μM. The IC value is about 1µM 50 IC values ​​are considered to be characteristic of non-specific inhibition of target function by a nucleic acid molecule. As recognized by those skilled in the art, the IC values ​​of a group of compounds (e.g., nucleic acid molecules used in accordance with the present invention) are 50 The value is within a certain range. The IC above about 1µM 50 The IC 50 The preferred upper limit of the value. IC of the target binding nucleic acid molecule 50 The lower limit of may be as low as about 10 picomolar or may be higher. Within the scope of the present invention, the IC of a single nucleic acid molecule binding to CXCL12 is 50 The value is preferably within this range. A preferred range can be defined by selecting any first number within this range and any second number within this range. A preferred upper limit IC 50 The values ​​are 250 nM and 100 nM, with the preferred lower IC 50 The values ​​are 50 nM, 10 nM, 1 nM, 100 pM and 10 pM. A more preferred upper limit IC 50 The value is 2.5 nM, the more preferred lower limit IC 50The value is 100 pM.

[0427] The nucleic acid molecules used according to the present invention can have any length, as long as they still can bind the target molecule. It will be recognized in the art that there is a preferred length for the nucleic acid molecules used according to the present invention. Usually, the length is between 15 to 120 nucleotides. Those skilled in the art will recognize that for the nucleic acid molecules used according to the present invention, any integer between 15 to 120 is a possible length. The preferred scope of the nucleic acid molecules length used according to the present invention is a length 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.

[0428] In the scope of the present invention, the nucleic acid used according to the present invention comprises a part which is preferably a high molecular weight part and / or preferably allows modification of the properties of the nucleic acid in terms of residence time in the animal body (preferably the human body) and the like. Particularly preferred embodiments of such modifications are PEGylation and HESylation of the nucleic acid used according to the present invention. As used herein, PEG stands for poly(ethylene glycol) and HES stands for hydroxyethyl starch. PEGylation, as preferably used herein, is a modification of the nucleic acid used according to the present invention, whereby such modification consists of a PEG part attached to the nucleic acid used according to the present invention. HESylation, as preferably used herein, is a modification of the nucleic acid used according to the present invention, whereby such modification consists of a HES part attached to the nucleic acid used according to the present invention. These modifications and the process of modifying nucleic acids using such modifications are described in European patent application EP 1306382, the disclosure of which is incorporated herein by reference in its entirety.

[0429] In the case where PEG is such a high molecular weight portion, 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. In the case where HES is such a high molecular weight portion, 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 a molar substitution of 1 to 1.5, and presents a substitution pattern represented by a C2 / C6 ratio of about 0.1 to 15 (preferably about 3 to 10). The process of HES modification is described, for example, in German patent application DE 1 2004 006 249.8, the disclosure of which is incorporated herein by reference.

[0430] In principle, the nucleic acid molecule used according to the invention can be modified at any position thereof. Preferably, the modification is carried out at the 5' terminal nucleotide, the 3' terminal nucleotide and / or any nucleotide between the 5' nucleotide and the 3' nucleotide of the nucleic acid molecule.

[0431] The modification and preferably the PEG and / or HES moiety can be attached directly or indirectly (preferably through a joint) to the nucleic acid molecules used according to the present invention. Also within the scope of the present invention, the nucleic acid molecules used according to the present invention comprise one or more modifications, preferably one or more PEG and / or HES moieties. In an embodiment, a single joint molecule is attached to the nucleic acid molecules used according to the present invention by more than one PEG moiety or HES moiety. The joint itself used according to the present invention can be linear or branched. Such joints are known to those skilled in the art and are further described in patent applications WO2005 / 074993 and WO2003 / 035665.

[0432] In a preferred embodiment of the present invention, the linker is a biodegradable linker. Due to the release of the modification from the nucleic acid according to the present invention, the biodegradable linker allows the modification of the properties of the nucleic acid used according to the present invention in terms of the residence time in an animal body, preferably in a human body. The use of a biodegradable linker can allow for better control of the residence time of the nucleic acid molecules used according to the present invention. Preferred embodiments of such biodegradable linkers are biodegradable linkers as described in, but not limited to, international patent applications WO2006 / 052790, WO2008 / 034122, WO2004 / 092191 and WO2005 / 099768.

[0433] In the context of the present invention, the modification or modifying group is a biodegradable modification, whereby the biodegradable modification can be attached directly or indirectly (preferably via a linker) to the nucleic acid molecule used according to the present invention. Due to the release or degradation of the modification from the nucleic acid molecule used according to the present invention, the biodegradable modification allows the modification of the properties of the nucleic acid used according to the present invention with respect to aspects such as the residence time in an animal (preferably in the human body). The use of a biodegradable modification can allow a better control of the residence time of the nucleic acid molecule according to the present invention. Preferred embodiments of such biodegradable modifications are those described in, but not limited to, international patent applications WO2002 / 065963, WO2003 / 070823, WO2004 / 113394 and WO2000 / 41647 (preferably WO2000 / 41647, page 18, lines 4 to 24).

[0434] In addition to the modifications described above, other modifications may be used to modify the characteristics of the nucleic acids used according to the present invention, whereby such other modifications may be selected from the group of proteins, lipids (eg cholesterol) and sugar chains (eg amylase, dextran etc.).

[0435] Without wishing to be bound by any theory, it seems that by modifying the nucleic acid molecules used according to the present invention with a high molecular weight portion, the excretion kinetics are altered, the high molecular weight portion being for example a polymer, more specifically one or more polymers disclosed herein, which are preferably physiologically acceptable. More specifically, it seems that due to the increase in the molecular weight of the nucleic acid of this modification, and due to the fact that the nucleic acid molecules used according to the present invention are not metabolically restricted, particularly when in the L-form, the excretion from the animal body (preferably from the mammalian body, more preferably from the human body) is reduced. Since excretion usually occurs through the kidneys, the glomerular filtration rate of the nucleic acid molecules so modified is significantly reduced compared to nucleic acid molecules without such high molecular weight modifications, which results in an increase in the residence time in the animal body. In this connection, it is particularly noteworthy that, despite this high molecular weight modification, the specificity of the nucleic acid molecules used according to the present invention is not adversely affected. So far, the nucleic acid molecules used according to the present invention have surprising characteristics (usually not expected from pharmaceutically active compounds), and therefore do not necessarily require a pharmaceutical preparation for providing sustained release to provide sustained release of the nucleic acid molecules used according to the present invention. In contrast, the nucleic acid molecules used according to the invention in their modified form comprising high molecular weight parts can be used as sustained release preparations (just as they function (due to their modification)), just as if they were released from a sustained release preparation. So far, one or more modifications of the nucleic acid molecules used according to the invention as disclosed herein and thus the modified nucleic acid molecules used according to the invention and any composition comprising the same can provide their unique (preferably controlled) pharmacokinetics and biodistribution. This also includes the residence time in the circulation and the distribution to tissues. Such modifications are further described in patent application WO2003 / 035665.

[0436] However, within the scope of the invention, the nucleic acid molecules used according to the present invention do not comprise any modification, and particularly do not comprise high molecular weight modifications, such as PEGylation or HESization. When the nucleic acid molecules used according to the present invention show preferential distribution to any target organ or target tissue in the body, or when the nucleic acid molecules used according to the present invention need to be removed quickly from the body after administration, this embodiment is particularly preferred. The preferential distribution characteristics of the nucleic acid molecules used according to the present invention in any target organ or target tissue in the body will allow to set up effective local concentrations in the target tissue, while keeping the systemic concentration of the nucleic acid molecules lower. This will allow the use of low doses, which is not only useful from an economic point of view, but also reduces the unnecessary exposure of other tissues to nucleic acid agents, thereby reducing the potential risk of side effects. In the case of in vivo imaging or specific therapeutic dose requirements using the nucleic acid molecules used according to the present invention or the medicine containing it, among others, it may be necessary to remove quickly from the body after administration according to the nucleic acid molecules used according to the present invention.

[0437] In an embodiment of the present invention, the pharmaceutical composition comprising the nucleic acid molecules used according to the present invention comprises at least one of the nucleic acid molecules and a pharmaceutically acceptable excipient. This pharmaceutical composition may additionally comprise one or more other pharmaceutically active compounds. This pharmaceutically acceptable excipient may be, for example, water, buffer, PBS, glucose solution, preferably 5% glucose-salt balanced solution, starch, sugar, gelatin or any other acceptable carrier material. Such excipients are generally known to those skilled in the art. Those skilled in the art will recognize that any embodiment, use and aspect of the medicine of the present invention or aspects related thereto are also applicable to pharmaceutical compositions of the present invention, and vice versa.

[0438] The indications, diseases and conditions for which the nucleic acids, pharmaceutical compositions and medicaments used according to the invention or prepared according to the invention are used to treat and / or prevent are the result of the (direct or indirect) involvement of CXCL12 in the respective pathogenic mechanisms.

[0439] In a preferred embodiment of the present invention, the CXCL12 antagonist is NOX-A12. NOX-A12 (CAS-RN1322069-19-1) is a (L)-oligonucleotide of 45 (L)-ribonucleotides in length (molar weight 14.5 kDa), terminating in a hexylamino linker at the 5'-terminus, 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 a nucleic acid molecule of SEQ ID NO: 28. One skilled in the art will appreciate that a preferred nucleic acid molecule that binds to CXCL12 is an L-nucleic acid molecule that has at least 80% identity to an L-nucleic acid molecule of SEQ ID NO: 28 or SEQ ID NO: 22.

[0440] The chemical structure of NOX-A12 can also be described as L-guanylyl-(3'→5')-L-cytidyl-(3'→5')-L-guanylyl-(3'→5')-L-uridyl-(3'→5')-L-guanylyl-(3'→5')-L-uridyl-(3'→5')-L-guanylyl-(3'→5')-L-uridyl-(3'→5')-L-guanylyl-(3'→5')-L-uridyl-(3'→5')-L-guanylyl-(3'→5')-L-uridyl-(3'→5')-L-guanylyl-(3'→5')-L-adenylyl-(3'→5')-L-uridyl-(3'→5')-L-cytidyl-(3'→5')- →5')-L-uridylyl-(3'→5')-L-adenylyl-(3'→5')-L-guanylyl-(3'→5')-L-adenylyl-(3'→5')-L-uridylyl-(3'→5')-L-guanylyl-(3'→5')-L-uridylyl-(3'→5')-L-adenylyl-(3'→5')-L-uridylyl-(3'→5')-L-guanylyl-(3'→5')-L-cytidyl-(3'→5')-L-uridylyl-(3'→5')-L- 3'→5')-L-guanylyl-(3'→5')-L-adenylyl-(3'→5')-L-uridyl-(3'→5')-L-cytyl-(3'→5')-L-cytyl-(3'→5')-L-uridyl-(3'→5')-L-adenylyl-(3'→5')-L-guanylyl-(3'→5')-L-uridyl-(3'→5')-L-cytyl-(3'→5')-L-uridyl-(3'→5')-L-adenylyl-(3'→5')-L-guanylyl-(3'→5')-L-uridyl-(3'→5')-L-cytyl-(3'→5')-L-adenylyl-(3'→5')-L-guanylyl-(3'→5')-L-uridyl -(3'→5')-L-adenyl-(3'→5')-L-cytidylyl-(3'→5')-L-guanylyl-(3'→5')-L-cytidylyl-(3'→5'), with 5'-ester of N-[ω-methoxypoly(oxy-1,2-ethanediyl], N'-[ω-methoxypoly(oxy-1,2-ethanediyl]-acetyl-9-amino-8-oxo-7-aza-nonyloxyphosphate, sodium salt, whereby it further comprises a 40 kDa PEG moiety attached to the 5' end of the nucleotide sequence via the linker indicated above. In the case of NOX-A12, the 40 kDa PEG moiety increases the plasma half-life in humans from several minutes (for non-PEGylated L-nucleic acid molecules) to nearly two days.

[0441] In an embodiment of the invention, the CXCL12 antagonist is Olaptesed pegol. Preferably, the terms NOX-A12 and Olaptesed pegol are used synonymously.

[0442] In an embodiment of the present invention, the CXCL12 antagonist is a CXCL12 binding aptamer provided by Aptitude Medical Systems, which was originally used for the treatment of wet AMD.

[0443] In another embodiment of the present invention, the CXCL12 antagonist that binds to CXCL12 and inhibits the binding of CXCL12 to the CXCL12 receptor 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 receptor 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.

[0444] Monoclonal antibodies against CXCL12 are known in the art and include mAB-30D8 (provided by Genentech), 1131-H12 or 1143-H1 (both provided by iOnctura), etc. 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).

[0445] In another embodiment of the present invention, CXCL12 is bound by a protein, which may be a CXCL12-binding TRAP protein (provided by OncoTrap) or a CXCL12-binding anticalin. In an embodiment, the TRAP protein is provided by an mRNA encoding the TRAP protein delivered to the tumor. The expression product of the mRNA is secreted by the transfected cells and diffuses throughout the tumor environment, capturing CXCL12 with nanomolar binding affinity, so that CXCL12 is harvested and its function is eliminated in the tumor microenvironment. In an embodiment, and preferably as used herein, anticalin is a target binding polypeptide. This class of target binding polypeptides is described in German patent application DE 197 42 706, etc.

[0446] Those skilled in the art will appreciate that, in addition to the above-mentioned classes of compounds, in principle, small molecules can also be used as antagonists of CXCL12. The latter is due to the direct or indirect interaction of such small molecules, preferably direct physical interaction, and the binding of CXCL12 to its receptor CXCR4 and / or CXCR7 is inhibited. As preferably used herein, a small molecule is a molecule with a molecular weight of 900 Da or less. In another option, a small molecule is a molecule that meets the Lipinski five rules, that is, the compound violates no more than one of the following criteria: 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, and an octanol-water partition coefficient (log P) of no more than 5.

[0447] 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 a target and an antigen, respectively, which is bound by the CXCL12 antagonist of this embodiment. In the case where the CXCL12 antagonist binds to CXCL12 and inhibits the binding of CXCL12 to CXCR4 and / or CXCR7, the CXCL12 antagonist that binds to CXCR4 can be a compound selected from the group consisting of the following categories of compounds: aptamers, spiegelmers, antibodies, target binding proteins, target binding peptides, synthetic peptides, peptides, anticalins, small molecules, and fusion proteins.

[0448] Various small molecules are disclosed in the prior art that can be used as CXCL12 antagonists in the methods of the present invention because they bind to CXCR4. Such small molecules include, but are not limited to, plerixafor (Sanofi), mavorixafor (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).

[0449] Various peptides are disclosed in the prior art that can be used as CXCL12 antagonists in the methods of the present invention because they bind to CXCR4. Such peptides include, but are not limited to, motixafortide (BioLineRx).

[0450] Synthetic peptides are disclosed in the prior art that can be used as CXCL12 antagonists in the methods of the present invention because they bind to CXCR4. Such synthetic peptides include, but are not limited to, balixafortide (Spexis), LY-2510924 (Eli Lilly), ALB-408 (Pharis Biotec), and POL-5551 (Spexis).

[0451] Recombinant proteins are disclosed in the prior art that can be used as CXCL12 antagonists in the methods of the present invention because they bind to CXCR4. Such recombinant proteins include, but are not limited to, PTX-9098 (Pertinax Therapeutics) and NNL-121 (Nanoligent).

[0452] Fusion proteins are disclosed in the prior art that can be used as CXCL12 antagonists in the methods of the present invention because they bind to CXCR4. Such recombinant fusion proteins include, but are not limited to, AD-214 (Adalta) and AM-3114 (Adalta).

[0453] Antibodies and more specifically monoclonal antibodies are disclosed in the prior art that can be used as CXCL12 antagonists in the methods of the present invention because they bind to CXCR4. 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).

[0454] 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 a target and an antigen, respectively, which is bound by the CXCL12 antagonist of this embodiment. In the case where the CXCL12 antagonist binds to CXCL12 and inhibits the binding of CXCL12 to CXCR4 and / or CXCR7, the 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.

[0455] Various small molecules are disclosed in the prior art that can be used as CXCL12 antagonists in the methods of the present invention because they bind to CXCR7. Such small molecules include, but are not limited to, ACT-10041239 (Idorsia Pharmaceutical), CCX-650 (ChemoCentryx), CCX-662 (ChemoCentryx), and CCX-771 (ChemoCentryx).

[0456] Various synthetic peptides are disclosed in the prior art that can be used as CXCL12 antagonists in the methods of the present invention because they bind to CXCR7. Such small synthetic peptides include, but are not limited to, LIH-383 (Luxembourg Institute of Health) and POL-6926 (Spexis).

[0457] Various antibodies or antibody fragments are disclosed in the prior art, which can be used as CXCL12 antagonists in the methods of the present invention because they bind to CXCR7. 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).

[0458] Those skilled in the art will recognize that the amount and dosage of the CXCL12 antagonist administered to the subject will vary according to such factors, such as the patient's age, weight, height, sex, general medical condition and past medical history. However, such a dosage of the CXCL12 antagonist administered to the subject should preferably be effective in inhibiting neovascularization of the tumor. Preferably, such neovascularization is achieved by CXCL12-mediated recruitment of endothelial cells or bone marrow-derived pro-angiogenic cells. More preferably, CXCL12-mediated recruitment is dependent on CXCR4 and / or CXCR7.

[0459] Those skilled in the art will appreciate that the exact dosage administered to a subject in the practice of the methods of the present invention can be determined by routine practice.

[0460] The mode of administration of the CXCL12 antagonist (route of administration and / or formulation) is 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 is usually administered by intravenous administration. According to the present invention, the CXCL12 antagonist will be continuously administered to the subject. Even if the CXCL12 antagonist is not continuously administered to the subject, it is preferred to achieve a therapeutic activity level of the CXCL12 antagonist in the patient. Preferably, this level is a therapeutically active level in terms of anti-angiogenic and / or immunostimulatory effects in the tumor and / or tumor environment (e.g., tumor vasculature) and / or the vascular compartment of the subject.

[0461] In an embodiment of the invention, wherein the CXCL12 antagonist is a CXCL12 binding spiegelmer or a CXCL12 binding aptamer, the weekly dose of such CXCL12 antagonist administered to the subject is from about 100 to 1,000 mg, preferably from about 200 to about 600 mg, more preferably from about 400 to about 600 mg. This dose is particularly suitable if the CXCL12 antagonist is NOX-A12, which is preferably administered continuously by intravenous injection.

[0462] According to the present invention, radiation therapy is administered to a subject having said tumor.

[0463] Radiation therapy is the most commonly prescribed treatment for brain tumors, such as glioblastoma. It can be used as a primary treatment if the surgeon decides that removing the tumor is too risky, or to destroy any remaining cancer cells after surgery that the surgeon cannot see or reach. During radiation therapy for glioblastoma, ionizing magnetic radiation or particle beams are directed at the tumor to destroy the cancer cells and their supporting environment. As the cancer cells and their supporting environment are destroyed and cleared by the body's immune system, the tumor shrinks; this helps to relieve pressure on the brain.

[0464] Radiation therapy not only directly targets tumor cells but also depletes tumor microvasculature. The resulting intratumoral hypoxia initiates a cascade of events that ultimately leads to revascularization, immunosuppression, and, ultimately, tumor regrowth. A key component of this cascade is the overexpression of CXC-driven chemokine ligand 12 (CXCL12), formerly known as stromal cell-derived factor 1 (SDF-1).

[0465] In an embodiment of the invention, the radiation therapy administered to a subject with a tumor is standard of care radiation therapy. Generally speaking, for brain cancer, there are two forms of standard of care radiation therapy regimens, each of which may be consistent with a preferred embodiment of the method of the invention. One form is conventional fractionated radiation therapy (nfRT); the other form is hypofractionated radiation therapy (hfRT).

[0466] In a first embodiment of conventional fractionated radiotherapy (nfRT), a total of 60 Gy is administered to the subject in 30 fractions (2 Gy) over 6 weeks, wherein radiotherapy is administered to the subject for 5 consecutive days per week, and then no radiotherapy is administered to the subject during 2 days. Preferably, conventional fractionated radiotherapy is administered to the subject by means of external beam radiotherapy (EBRT). More preferably, this conventional 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 guidelines, the rough tumor volume (GTV) is defined as the resection cavity on the postoperative magnetic resonance imaging (MRI) and the planned computer tomography CT scan. In order to create a clinical target volume (CTV), the GTV should be expanded by up to 20 mm in all directions (preferably also covering abnormal fluid attenuation inversion recovery (FLAIR) signals). This may be reduced at anatomical barriers (e.g., ventricles, falx, tentorial bones: 5 mm; bones: 0 mm) or critical organs at risk (OARs, such as brainstem, chiasm, optic nerve: 0 mm). The planning target volume (PTV) is then defined as the CTV plus a 3-5 mm margin in all directions.

[0467] In a second embodiment of conventional fractionated radiotherapy (nfRT), a total of 60 Gy is also administered to the subject, however, 46 Gy is administered to the first planning target volume 1 (PTV1) in 2 Gy fractions in the first 23 fractions, followed by 14 Gy administered to the second planning target volume 2 (PTV2) in 2 Gy fractions in the next 7 fractions (which is also called a "boost" or "conedown"). More specifically and in accordance with the guidelines provided by the Radiation Therapy Oncology Group (RTOG), the gross tumor volume 1 (GTV1) resembles edema (if visible) in the T2-FLAIR sequence and must also cover the complete resection cavity and any contrast-enhancing lesions in the T1 post-contrast sequence. The clinical target volume 1 (CTV1) is then defined as the GTV1 plus a 2 cm margin. The CTV1 may be reduced around the natural margins of the tumor (e.g., skull, falx, etc.). Planning target volume 1 (PTV1) is similar to CTV1 plus an additional margin of 2-5 mm. All (T1-) contrast-enhancing lesions / abnormalities (must include cavitary margins) will be defined as the bonus volume (GTV2). The bonus clinical target volume (CTV2) will be the GTV2 plus a 2 cm margin. Around the natural margins of the tumor (e.g., skull, falx, etc.), the CTV2 may be reduced. The bonus planning target volume (PTV2) is similar to the CTV2 plus an additional margin of 2-5 mm. In conjunction with the above, the skilled person will appreciate that virtually any glioblastoma has edema. Edema is one of the main causes of increased intracranial pressure that is a typical symptom of glioblastoma. Because tumor cells are more likely to infiltrate this lesion, the edematous area is also irradiated. In addition, it is recognized that T2 FLAIR is particularly suitable for detecting edema because it can distinguish between free fluid and tissue-bound fluid. T2 represents longitudinal relaxation. The FLAIR (Fluid Attenuated Inversion Recovery) sequence is a special MRI sequence that is very similar to the T2 sequence, but it is normalized to the signal of the liquid in the brain, which is set to 0. Thus, the liquid will appear black.

[0468] In hypofractionated radiotherapy (hfRT), a subject with a tumor will receive a total dose of approximately 40 Gy, typically 40.05 Gy, administered in 15 fractions over 3 weeks. Typically, this means that 2.67 Gy is administered to the subject on five consecutive days, either weekly or in any of the three weeks, followed by two days without radiation therapy. More specifically, following guidelines provided by the Canadian Cancer Society Research Institute, the gross tumor volume (GTV) is defined as the volume of the contrast-enhanced area on the postoperative planning magnetic resonance imaging (MRI) scan and includes the surgical bed. The clinical target volume (CTV) is a 1.5 cm margin outside the gross tumor volume outline that takes into account the anatomical boundaries, and a 0.5 cm planning target volume (PTV) margin is applied.

[0469] Radiation therapy can utilize ionizing electromagnetic radiation or ionizing particle beams. Such ionizing electromagnetic radiation can be X-rays or gamma rays. Typically, X-rays have a wavelength from about 10 picometers to about 10 nanometers, and the energy of X-rays is from about 145 eV to about 124 keV. As for gamma rays, their wavelength is typically 10 picometers or less, and the energy of gamma rays is typically 100 keV or more.

[0470] Such ionizing particle beams may be proton beams, photon beams or electron beams, whereby, preferably, such particle beams are used for external beam radiation therapy (EBRT). In an embodiment of the present invention, EBRT, including any aspect thereof, is used in radiation therapy administered to a subject having a tumor according to the present invention.

[0471] In external beam radiation therapy (EBRT), the tumor is exposed to an external source of ionizing radiation, megavoltage X-rays are used to treat deep tumors such as GBM. Such X-ray or photon beams are produced in most radiation therapy machines. The photon beams can reach tumors deep in the body. As they pass through the body, the photon beams scatter small amounts of radiation along their path. These beams do not stop once they reach the tumor, but instead enter normal tissue in their path. Megavoltage X-rays have energies of 1 to 25 MeV. Such megavoltage X-rays are produced by linear accelerators (linacs). Commercially available medical linacs produce X-rays with energies ranging from 4 MeV to about 25 MeV, while in clinical practice, it is uncommon for nominal energies to exceed 15 MeV, so the energy range of 4-15 MeV is the preferred range. The X-rays themselves are produced by the rapid deceleration of electrons in a target material (usually a tungsten alloy), which produces the X-ray spectrum by bremsstrahlung. The shape and intensity of the beam produced by a linac can be modified or collimated in a variety of ways.

[0472] Additionally, like photon beams, proton beams can also reach tumors deep in the body and are sometimes used for recurrent brain tumors, particularly recurrent glioblastoma. However, proton beams do not scatter radiation on their way through the body, and they stop once they reach the tumor. Proton beams are therefore thought to reduce the amount of normal tissue exposed to radiation. In general, the high cost and size of the machines limit their use.

[0473] Those skilled in the art will appreciate that radiation therapy can also use "heavier" particles (e.g., protons or He ions) to produce a radiotherapeutic effect. The use of such heavier particles constitutes another embodiment of the method of the present invention.

[0474] EBRT can be administered to a subject in a variety of ways. Some of these ways are outlined below and constitute further embodiments of the methods of the invention, namely three-dimensional conformal radiation therapy, intensity modulated radiation therapy (IMRT), image guided radiation therapy (IGRT) and stereotactic radiosurgery.

[0475] 3D 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 radiation field is designed to conform to the shape of the tumor.

[0476] Intensity-modulated radiation therapy (IMRT) is a more advanced form of 3D conformal radiation therapy. It uses sophisticated software and 3D images from CT scans to focus radiation directly on the tumor. These beam-like thin beams vary in intensity (thus delivering higher doses 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 occurrence of adverse reactions

[0477] Image-guided radiation therapy (IGRT), or tomosynthesis, is even more advanced than IMRT and uses imaging scans not only for treatment planning before radiation therapy but also to guide the placement of radiation beams during radiation therapy fractions.

[0478] Stereotactic radiosurgery uses focused, high-energy beams to treat small, well-defined tumors in the brain and central nervous system. This precise treatment places the radiation source closer to the tumor than is possible with conventional radiation therapy. Therefore, it may be an option if surgery is too risky due to age or other health problems, or if surgery cannot safely reach the tumor. While traditional radiation therapy for glioblastoma involves multiple treatments over the course of several months, stereotactic radiosurgery is delivered in fewer sessions.

[0479] In another embodiment of the invention, the radiation therapy is brachytherapy. Brachytherapy is a type of localized internal irradiation therapy in which seeds, tapes or capsules containing a radioactive source, or a liquid formulation containing a radioactive source, are placed in or near the tumor, preferably in the resection cavity after surgery. It is commonly used to treat head and neck cancer, breast cancer, cervical cancer, prostate cancer and eye cancer, but is also suitable for the treatment of brain tumors. Basically, there are three types of brachytherapy: (a) low dose rate (LDR) implants, where the radiation source stays for 1 to 7 days, (b) high dose rate (HDR) implants, where the radiation source stays for only 10 to 20 minutes at a time; and (c) permanent implants, where the radiation source stays indefinitely, but the radiation decreases every day. In one embodiment, the radiation source is a therapeutically active radionuclide. In a preferred embodiment, the therapeutically active radionuclide is selected from the group consisting of 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. 211 At group, more preferably, the therapeutically active radionuclide is selected from the group comprising 186 Re, 47 Sc, 67 Cu, 90 Y. 177 Lu, 188 Re, 212 Pb, 213 Bi, 225 Ac, 227 Th, 131 I. 211 The group of At, and most preferably the therapeutically active radionuclide is selected from the group comprising 186 Re, 90 Y. 177 Lu, 225 Ac, 227 Th, 131 I and 211 At's group.

[0480] In an embodiment of the present invention, a modified brachytherapy is used. This modified brachytherapy does not use implanted solid radiation sources, but rather injects 186 Nanoliposomes of Re, 186 Re is a beta-emitting therapeutic radionuclide with a 90-hour half-life, a 1.8 mm radiation path range, and a high beta / gamma energy ratio, making it suitable for cancer brachytherapy. 186 Re nanoliposomes to enhance long half-life 186 Re energy delivery characteristics, thereby achieving long-term tumor retention and achieving very high absorbed radiation doses to the tumor. In preclinical, this nanoliposome infused by convection-enhanced delivery achieved very high targeted radiation doses and a wide therapeutic index. This improved brachytherapy is described, for example, in Floyd 2021, Int. J. Radiat. Oncol. Biol. Phys. Vol. 111, No. 3, Supplement, November 1, 2021, p. e589.

[0481] In another embodiment of the invention, the radiation therapy is intraoperative radiation therapy (IORT). Intraoperative radiation therapy (IORT) delivers radiation to a tumor / tumor bed when an area of ​​the tumor / tumor bed is exposed to radiation during surgery. IORT is able to deliver high doses of radiation, precisely to the tumor bed, with minimal exposure to surrounding healthy tissue. Those skilled in the art will appreciate that IORT is often combined with external beam radiation therapy and allows for the avoidance of tumor cell repopulation at a site where the tumor has been removed or otherwise treated.

[0482] IORT allows (i) precise localization of the tumor bed and targeted delivery of high-dose radiation to the tumor bed; (ii) minimal exposure of dose-limiting normal tissues that are distant from the tumor bed and shielded from radiation; (iii) the possibility of dose escalation beyond that achievable with EBRT; and (iv) the opportunity for re-irradiation, particularly in recurrent cancers where further irradiation may not be possible with EBRT. Thus, IORT is able to deliver a higher total effective dose to the tumor bed than with EBRT, facilitating dose escalation without significantly increasing normal tissue complications and improving the therapeutic ratio. Methods of IORT are (i) electron IORT / IOERT (electron beam), (ii) kV IORT (X-ray), and (iii) HDR IORT (high-dose-rate brachytherapy).

[0483] Within the scope of the present invention, the method for treating a tumor comprises anti-angiogenic therapy. Preferably, such anti-angiogenic therapy comprises administering an anti-angiogenic compound to a subject having a tumor.

[0484] It will be appreciated by those skilled in the art that this part of the method of the invention (i.e., administering an anti-angiogenic therapy to a subject with a tumor) should avoid or at least reduce angiogenesis. As preferably used herein, angiogenesis is the formation of new blood vessels within and / or toward a tumor. More preferably, angiogenesis begins with pre-existing blood vessels.

[0485] The formation of new blood vessels is an indispensable prerequisite for tumor growth, and malignant tissues also rely on nutrients and oxygen. Very small tumors can be supplied by diffusion, but once a tumor exceeds 1-2 mm in diameter, further growth requires its own vascular system. To generate such a system, tumors secrete various growth factors that stimulate blood vessel formation (angiogenic switch). The most important growth factor for angiogenesis in tumors is VEGF (vascular endothelial growth factor). Blocking VEGF signaling through the VEGF receptors VEGFR1 (Flt-1), VEGFR2 (KDR / Flk-1) signaling pathway inhibits endothelial cell proliferation and subsequent new blood vessel formation in tumors. 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.

[0486] In view of this, it is recognized by those skilled in the art 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, results in a therapeutically effective anti-angiogenic effect. Those skilled in the art will understand that 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, providing a molecule that binds to VEGFR1 to inhibit the binding of VEGFR1 to VEGF, and (c) providing a molecule that binds to VEGFR2 to inhibit the binding of VEGF to VEGFR2.

[0487] Such a molecule that binds to VEGF is preferably a molecule selected from the group comprising an aptamer (preferably a VEGF-binding aptamer), a spiegelmer (preferably a VEGF-binding antibody), a protein that binds to VEGF, a VEGF-binding anticalin, a VEGF-binding peptide, a fusion protein comprising a VEGF-binding portion, and a VEGF-binding small molecule.

[0488] Such a molecule binding to VEGFR1 is preferably a molecule selected from the group comprising an aptamer (preferably a VEGFR1 binding aptamer), a spiegelmer (preferably a VEGFR1 binding antibody), a VEGFR1 binding protein, a VEGFR1 binding anticalin, a VEGFR1 binding peptide, a fusion protein comprising a VEGFR1 binding portion and a VEGFR1 binding small molecule.

[0489] Such a molecule that binds to VEGFR2 is preferably a molecule selected from the group comprising an aptamer (preferably a VEGFR2 binding aptamer), a spiegelmer (preferably a VEGFR2 binding antibody), a VEGFR2 binding protein, a VEGFR2 binding anticalin, a VEGFR2 binding peptide, a fusion protein comprising a VEGFR2 binding portion, and a VEGFR2 binding small molecule.

[0490] Those skilled in the art will also understand that such inhibition of the interaction of VEGF through one or both of the VEGF receptors VEGFR1 and VEGFR2 can be achieved by compounds that bind to VEGFR1 and / or VEGFR2, whereby such binding of the compound interferes with the signaling of VEGFR1 and / or VEGFR2 rather than interfering with the binding of VEGF to VEGFR1 and / or VEGFR2.

[0491] The molecule that binds to VEGFR1 and interferes with the signal transduction properties of VEGFR1 (preferably signal transduction triggered by the binding of VEGF to VEGFR1) because of its binding to VEGFR1 is preferably a molecule selected from the group comprising an aptamer (preferably a VEGFR1 binding aptamer), a spiegelmer (preferably a VEGFR1 binding antibody), a VEGFR1 binding protein, a VEGFR1 binding anticalin, a VEGFR1 binding peptide, a fusion protein comprising a VEGFR1 binding portion, and a VEGFR1 binding small molecule.

[0492] The generation and identification of such aptamers, spiegelmers, antibodies, binding proteins, anticalins, peptides, fusion proteins and small molecules, respectively, are known to those skilled in the art and are also disclosed herein.

[0493] In an embodiment of the invention, VEGF is human VEGF, VEGFR1 is human VEGFR1, and VEGFR2 is human VEGF2.

[0494] VEGF exists in full-length form, but may also exist as isoforms produced by alternative splicing or proteolytic cleavage by the proteases plasmin, MMP3 and uPA. Such isoforms and proteolytic cleavage forms of VEGF are disclosed, for example, in Vempati et al. (Vempati P et al., Cytokine Growth Factor Rev. 2014 Feb; 25(1). 1-19) and Guyot M and Pagès G (Guyot M and Pagès G, Methods Mol. Biol 2015; 1332: 2-23). ​​In particular, isoforms are disclosed in Guyot M and Pagès G, and in Fig. 20 The amino acid sequences of different forms of VEGF are depicted in. The isoforms of VEGF are preferably selected from the group comprising VEGF121, VEGF165, VEGF189, VEGF206, VEGF145, VEGF183 and VEGF165b.

[0495] The proteolytic cleavage form of VEGF is VEGF110. Those skilled in the art will understand that whenever VEGF and compounds that bind to or interfere with the function of VEGF are mentioned herein, such VEGF (in embodiments) is any of the above-mentioned full-length VEGF, an isoform of VEGF, or a proteolytic cleavage form thereof. Those skilled in the art recognize that within the scope of the present invention, molecules that bind to VEGF may bind to the forms of VEGF used in the generation and identification of such binding molecules, respectively, and may further bind to one or more of the above-mentioned forms of VEGF (including any isoform or proteolytic cleavage form).

[0496] In a preferred embodiment of the present invention, the anti-angiogenic compound is an anti-VEGF antibody. The anti-VEGF antibody may be a human anti-VEGF antibody, a humanized anti-VEGF antibody or a chimeric anti-VEGF antibody. The term anti-VEGF antibody as preferably used herein also encompasses any fragment of such an anti-VEGF antibody, whereby such fragment still binds or is still capable of binding to VEGF.

[0497] 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 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). AKYPHYYGSSHWYFDV (SEQ ID NO: 230).

[0498] In another preferred embodiment of the present invention, the anti-angiogenesis antibody comprises the following amino acid sequence:

[0499] DIQMTQSPSSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQID NO: 239) light chain,

[0500] Contains the following amino acid sequence:

[0501] The heavy chain of EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 240), or

[0502] comprising the following amino acid sequence:

[0503] the light chain of DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 239) and

[0504] comprising the following amino acid sequence:

[0505] EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK A combination of heavy chains of (SEQ ID NO: 240).

[0506] 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 number: RO487-6646; CAS registration number: 216974-75-3; Bevacizumab is also known as rhuMAb VEGF and anti-VEGF.

[0507] Bevacizumab is an inhibitor of angiogenesis. As such, it prevents the formation of new vascular structures. Bevacizumab selectively binds to all isoforms of human VEGF (a key driver of angiogenesis) with high affinity, thereby inhibiting the binding of VEGF to its surface receptors (Flt-1, VEGFR1 and KDR, VEGFR2) on endothelial cells. It neutralizes the biological activity of VEGF by sterically blocking the binding of VEGF to its receptors, regressing tumor angiogenesis, normalizing the remaining tumor vascular structures, and inhibiting the formation of new tumor vascular structures, thereby inhibiting tumor growth. Receptor activation generally induces its tyrosine phosphorylation, and the subsequent series of signal transduction events causes signals of mitogenic and pro-survival activities of vascular endothelial cells. In most normal tissues (except glomeruli), since the expression of VEGF receptors is extremely low or undetectable, but significantly upregulated in the vascular structures of many tumors, the neutralization of VEGF by bevacizumab provides a theoretical basis for the relatively specific inhibition of tumor angiogenesis, thereby inhibiting tumor growth and metastasis.

[0508] Bevacizumab is a recombinant humanized monoclonal IgG1κ isotype antibody containing human framework regions (93%) and mouse complementarity-determining regions (7%). The antibody consists of two identical light chains (214 amino acid residues) and two heavy chains (453 residues) with a total molecular weight of 149 kDa. The heavy chain exhibits heterogeneity at the C-terminus (lysine variants) and also contains an N-linked glycosylation site at asparagine 303. The oligosaccharide has a complex biantennary structure with a core fucose and two branches terminating predominantly with 0 (G0), 1 (G1), or 2 (G2) galactose residues. The G0 glycoform predominates with a relative abundance of approximately 80%. Each light chain is covalently coupled to a heavy chain at cysteine ​​226 via a disulfide bond at cysteine ​​214. The two heavy chains are covalently coupled to each other via two interchain disulfide bonds, which is consistent with the structure of human IgG1. Bevacizumab was prepared by humanization of the mouse parent antibody A4.6.1, which was generated at Genentech using hybridomas from mice immunized with a 165-residue form of recombinant human vascular endothelial growth factor (rhuVEGF165) conjugated to keyhole limpet hemocyanin.

[0509] Humanization of the A4.6.1 antibody involved the insertion of the six CDRs of A4.6.1, replacing a selected human antibody Fab framework (pEMX1) with a common human kappa subgroup I light chain (domains VL-CL) and a truncated 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 contained eight substitutions of the human framework in addition to the CDRs. The VH and VL domains of Fab-12 were combined with 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 liposome transfection, and the cells were selected in the presence of increasing concentrations of methotrexate (MTX). Isolates that secreted active bevacizumab were selected. The isolated subclone 107N was used to produce Phase I and Phase II clinical material and served as a starting point for the development of the higher producing G7 cell line, which was used to produce Phase III clinical material and will be used to produce drug substance ready for marketing.

[0510] 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 with few changes (see IMGT Repertoire (IG and TR available at https: / / www.imgt.org / IMGTrepertoire / GenesClinical / humanized / bevacizumab / bevacizumab_ProteinDisplay.html). Ranibizumab is the Fab fragment of bevacizumab. In ranibizumab, in the CDRs, 4 out of 6 amino acids are modified. Ranibizumab is not glycosylated. More specifically, ranibizumab has the following amino acid changes compared to bevacizumab: one amino acid change in VH (CDR1-IMGT: T29→D, N36→H, CDR3-IMGT: H109→Y, S112→T) and in V-KAPPA (M4→L). Bevacizumab also has additional changes in the hinge (T10>L).

[0511] In another embodiment of the 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 known in the scientific literature as aflibercept or also known as VEGF TRAP, is a recombinant fusion protein consisting of a VEGF binding moiety derived from the extracellular domain of human VEGF receptors 1 and 2 fused to the Fc portion of human IgG1. Aflibercept is produced by recombinant DNA technology in the Chinese Hamster Ovary (CHO) K-1 mammalian expression system. Aflibercept is a dimeric glycoprotein with a protein molecular weight of 97 kilodaltons (kDa) and contains glycosylation, which accounts for 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 VEGF-A with higher affinity than its native receptor and related ligands PlGF and VEGF-B. By acting as a ligand trap, aflibercept prevents endogenous ligands from binding to their cognate receptors, thereby blocking receptor-mediated signaling. Aflibercept blocks activation of VEGF receptors and proliferation of endothelial cells, thereby inhibiting the growth of new blood vessels that supply tumors with oxygen and nutrients. Aflibercept binds to human VEGF-A (KD of 0.5 pM for VEGF-A165 and 0.36 pM for VEGF-A121), human PlGF (KD of 39 pM for PlGF-2), and human VEGF-B (KD of 1.92 pM) to form stable, inert complexes with no detectable biological activity (see European Medicines Agency, portal related to zaltrap: https: / / www.ema.europa.eu / en / medicines / human / EPAR / zaltrap).

[0512] In an embodiment of the present invention, the anti-angiogenic compound is a compound that binds to VEGFR. VEGFR may 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.

[0513] In an embodiment of the 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). The combination of SISSSSSYIYYADSVKG (SEQ ID NO: 237) and CDR3: VTDAFDI (SEQ ID NO: 238).

[0514] In an embodiment of the present invention, the anti-VEGFR antibody comprises the following light chain: DIQMTQSPSSVSASIGDRVTITCRASQGIDNWLGWYQQKPGKAPKLLIYDASNLDTGVPS RFSGSGSGTYFTLTISSLQAEDFAVYFCQQAKAFPPTFGGGTKVDIKRTVAAPSVFIFPP SDEQLKSGTA SVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHK VYACEVTHQGLSSPVTKSFN RGEC (SEQ ID NO: 239), or the following: EVQLVQSGGG LVKPGGSLRL SCAASGFTFS SYSMNWVRQA PGKGLEWVSS ISSSSSYIYYADSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCARVT DAFDIWGQGT MVTVSSASTK GPSVLPLAPSSKSTSGGTAA LGCLVKDYFP EPVTVSWNSG ALTSGVHTFP AVLQSSGLYS LSSVVTVPSS SLGTQTYICNVNHKPSNTKV DKRVEPKSCD KTHTCPPCPA PELLGGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDPEVKFNWYVDG VEVHNAKTKP REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKGQPREPQVYTL PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLTVDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK (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 a combination of 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 (SEQID NO: 238). ,

[0515] In an embodiment of the invention, the anti-VEGFR antibody comprises the following light chain DIQMTQSPSS VSASIGDRVTITCRASQGID NWLGWYQQKP GKAPKLLIYD ASNLDTGVPS RFSGSGSGTY FTLTISSLQA EDFAVYFCQQAKAFPPTFGG GTKVDIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC (SEQ ID NO: 239) and the following EVQLVQSGGG LVKPGGSLRL SCAASGFTFS SYSMNWVRQA PGKGLEWVSS ISSSSSYIYYADSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCARVT DAFDIWGQGT MVTVSSASTK GPSVLPLAPSSKSTSGGTAA LGCLVKDYFP EPVTVSWNSG ALTSGVHTFP AVLQSSGLYS LSSVVTVPSS SLGTQTYICNVNHKPSNTKV DKRVEPKSCD KTHTCPPCPA PELLGGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDPEVKFNWYVDG VEVHNAKTKP REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKGQPREPQVYTL PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLTVDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK (SEQ ID NO: 240).

[0516] In another embodiment of the present invention, the anti-angiogenic compound that binds to VEGF is an aptamer, i.e., a D-nucleic acid, more specifically pegaptanib (Macugen). Pegaptanib (Macugen) is an anti-VEGF aptamer developed as an anti-angiogenic drug for the treatment of neovascular (wet) age-related macular degeneration. Pegaptanib is a pegylated aptamer that specifically binds to the 165 isoform of VEGF.

[0517] More preferably, the anti-VEGFR antibody is Ramucirumab (Cyramza), which is a specific VEGFR2 inhibitor. The amino acid sequence of the heavy chain of Ramucirumab is

[0518] EVQLVQSGGG LVKPGGSLRL SCAASGFTFS SYSMNWVRQA PGKGLEWVSS ISSSSSYIYY

[0519] ADSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCARVT DAFDIWGQGT MVTVSSASTK

[0520] GPSVLPLAPS SKSTSGGTAA LGCLVKDYFP EPVTVSWNSG ALTSGVHTFP AVLQSSGLYS

[0521] LSSVVTVPSS SLGTQTYICN VNHKPSNTKV DKRVEPKSCD KTHTCPPCPA PELLGGPSVF

[0522] LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYNSTYR

[0523] VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTL PPSREEMTKN

[0524] QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN

[0525] VFSCSVMHEA LHNHYTQKSL SLSPGK (SEQ ID NO: 241);

[0526] And the light chain of ramucirumab is

[0527] DIQMTQSPSS VSASIGDRVT ITCRASQGID NWLGWYQQKP GKAPKLLIYD ASNLDTGVPS

[0528] RFSGSGSGTY FTLTISSLQA EDFAVYFCQQ AKAFPPTFGG GTKVDIKRTV AAPSVFIFPP

[0529] SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT

[0530] LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC (SEQ ID NO: 242).

[0531] These sequences can be obtained from the information provided at the following link: https: / / www.genome.jp / entry / D09371.

[0532] In another embodiment of the invention, the anti-angiogenic compound is a compound that inhibits signaling of VEGFR, preferably VEGFR1 and VEGFR2. However, it is also within the scope of the invention that signaling of one or more of the following receptors is inhibited: platelet-derived growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR) and angiopoietin-1 receptor (Tie-2).

[0533] In a preferred embodiment of the invention, the anti-angiogenic compound inhibits the signaling cascade triggered by the binding of VEGF to VEGFR. Assays for determining whether a compound inhibits the signaling cascade are known in the art. More specifically, cell culture assays have been used for this purpose and for the characterization of inhibitors of VEGFR. The assay detects the activation of intracellular signaling pathways (Ras / MAPK pathway in the case of VEGFR), regulating cell proliferation and gene expression; FAK / paxillin pathway, involved in the rearrangement of the cytoskeleton; PI3K / AKT pathway, regulating cell survival; PLCγ pathway, controlling vascular permeability.

[0534] 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.

[0535] In a more preferred embodiment of the present invention, the anti-angiogenic compound which is a tyrosine kinase inhibitor is selected from the group comprising tyrosine kinase inhibitors selected from the group comprising pazopanib (Votrient, inhibits VEGFR1-3, PDGFRα and β, FGFR1 and 3, KIT, ITK, LCK, c-FMS), sunitinib (Sutent, inhibits VEGFR1-3, PDGFRα and β, KIT, FLT-3, CSF-1R, RET), sorafenib (Nexavar inhibits intracellular c-RAF, BRAF and mutant BRAF) and cell surface kinases (KIT, FLT-3, RET, RET / PCT, VEGFR1-3, PDGFR-β), axitinib (Inlyta, inhibits VEGFR1-3), ponatinib (Iclusig, inhibits VEGFR, PDGFR, FGFR, EPH receptor, SCR kinase family, KIT, RET, TIE2 and FLT3), cabozantinib (Cometriq / Cabometyx, inhibits RET, MET, VEGFR1-3, KIT, TKRB, FLT-3, AXL, ROS1, TYRO3, MER, TIE-2), regorafenib (Stivarga, 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 The group that inhibits EGFR and VEGFR families, RET, BRK, TIE-2, EPHR, and members of the Src kinase family) and lenvatinib (Lenvima inhibits VEGFR1-3, FGFR1-4, PDGFRα, KIT, and RET).

[0536] Those skilled in the art will recognize that the amount and dosage of the anti-angiogenic compound administered to a subject will vary according to factors such as the patient's age, weight, height, sex, general medical condition and past medical history. However, such dosage of the anti-angiogenic compound administered to a subject should preferably be effective in inhibiting neovascularization of the tumor. Preferably, such neovascularization is affected by the interaction of VEGF with VEGFR1 and / or VEGFR2.

[0537] Those skilled in the art will appreciate that the exact dosage of anti-angiogenic compound administered to a subject in the practice of the methods of the present invention can be determined by routine practice.

[0538] The mode of administration of the anti-angiogenic compound (route of administration and / or formulation) is determined by the chemical nature of the anti-angiogenic compound. For example, if the anti-angiogenic compound is a spiegelmer, aptamer or antibody, it is typically administered by intravenous administration. According to the present invention, the anti-angiogenic compound will be administered to the subject discontinuously.

[0539] In an embodiment of the invention, where the anti-angiogenic compound is an antibody (e.g., an anti-VEGF antibody and in particular bevacizumab), this antibody is administered by intravenous infusion once a week, once every two weeks, or once every three weeks, preferably once every two weeks or once every three weeks. Those skilled in the art will appreciate that the dosing regimen of the anti-angiogenic compound and the anti-VEGF antibody (e.g., bevacizumab) is synchronized with the combination therapy of the anti-angiogenic compound.

[0540] In an embodiment of the invention, the method of the invention comprises chemotherapy. In a preferred embodiment, this chemotherapy is a chemotherapy specifically for the treatment of gliomas and glioblastomas, more preferably a chemotherapy used as standard of care in the treatment of gliomas and glioblastomas.

[0541] In an embodiment of the invention, the chemotherapy comprises temozolomide (TMZ). TMZ is preferably administered daily during radiation therapy (at 75 mg / m 2 Administered for 6 weeks), followed by 6 cycles after radiation therapy during the maintenance phase. Each cycle of the maintenance phase lasts 28 days, with temozolomide taken for the first 5 days of each cycle at a dose of 150-200 mg / m² per day, followed by a 23-day rest period. In a preferred embodiment, the subject's tumor is sensitive to temozolomide.

[0542] In another embodiment of the present invention, the chemotherapy comprises lomustine (CCNU). A common regimen for CCNU-based chemotherapy is 110 mg / m 2 on day 1 of a 42-day cycle according to local standards. 2 / day dose, and then continue treatment for up to 6 cycles. In another embodiment, CCNU is used in the treatment of recurrent glioblastoma.

[0543] In another embodiment of the invention, the chemotherapy comprises procarbazine / lomustine / vincristine (PCV) or bevacizumab / irinotecan (BI) (Carvalho 2015, Oncol Res Treat 38:348). In another embodiment, this chemotherapy is used for the treatment of recurrent glioblastoma.

[0544] In another embodiment of the invention, the chemotherapy includes dehydrogenated chloranol (VAL-083), which is an alkylating agent (DNA synthesis inhibitor). Subjects with brain tumors, particularly glioblastomas, may receive dehydrogenated chloranol in the maintenance period after radiation therapy and receive temozolomide (75 mg / m² TMZ taken orally daily during radiation therapy. Maintenance cycle: 30 mg / m² IV VAL-083 on days 1, 2, and 3 of a 21-day cycle).

[0545] In an embodiment of the invention, the subject is a human subject.

[0546] In an embodiment of the present invention, the antibody may be a human antibody, a humanized antibody or a chimeric antibody. Preferably, the antibody is a monoclonal antibody (MAb).

[0547] In an embodiment of the present invention, the antibody may be a human antibody, a humanized antibody or a chimeric antibody, but is not limited thereto.

[0548] In embodiments of the present invention and preferably as used herein, the term antibody includes any antigen 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 portion of the antibody. Exemplary antibody fragments include, but are not limited to, F(ab') 2 , Fab', Fab, Fv, sFv, and methods for preparing the same are known to those skilled in the art. As discussed below, antibody fragments may also include single domain antibodies and IgG4 half molecules. Regardless of the structure, antibody fragments may bind to the same antigen recognized by full-length antibodies. The term "antibody fragment" also includes isolated fragments consisting of antibody variable regions, such as "Fv" fragments consisting of heavy and light chain variable regions, and recombinant single-chain polypeptide molecules ("scFv proteins") in which the light and heavy chain variable regions are connected by a peptide linker.

[0549] In embodiments of the present invention, and preferably as used herein, human antibodies are, for example, antibodies that can be obtained from people or transgenic mice that have been "engineered" to produce specific human antibodies in response to antigenic challenges. In this technology, elements of human heavy and light chain loci are introduced into mouse strains of embryonic stem cell lines from targeted breaks containing endogenous mouse heavy and light chain loci. Transgenic mice can synthesize human antibodies against specific antigens, and mice can be used to produce hybridomas that secrete human antibodies.

[0550] In the embodiments of the present invention and preferably as used herein, humanized antibodies are recombinant proteins in which CDRs are derived from antibodies from one species; for example, from rodent antibodies, heavy variable chains and light variable chains of rodent antibodies are converted into human heavy variable domains and light variable domains (e.g., framework sequences). The constant domains of antibody molecules are derived from the constant domains of human antibodies. In certain embodiments, a limited number of framework amino acid residues from a parent (rodent) antibody can be substituted into a human antibody framework sequence.

[0551] In one embodiment and preferably as used herein, a chimeric antibody is a recombinant protein containing a variable domain comprising a complementarity determining region (CDR) of an antibody derived from one species (preferably a rodent antibody), while the constant domain of the antibody molecule is derived from the constant domain of a human antibody. For veterinary applications, the constant domain of the chimeric antibody may be derived from another species, such as a cat or dog.

[0552] Those skilled in the art will appreciate that antibodies (including therapeutically active antibodies) can be produced for any target and antigen respectively by conventional methods. This method is well known in the art. See, for example, Köhler and Milstein, Nature 256: 495 (1975) and Coligan et al. (eds.), CURRENT PROTOCOLS INIMMUNOLOGY, VOL. 1, 2.5.1-2.6.7 pages (John Wiley & Sons 1991). In short, monoclonal antibodies can be obtained by the following steps: injecting a composition containing an antigen into mice, removing the spleen to obtain B lymphocytes, fusing B lymphocytes with myeloma cells to produce hybridomas, cloning hybridomas, selecting positive clones for antibodies against antigens, cultivating clones for antibodies against antigens, and separating antibodies from hybridoma cultures.

[0553] MAbs can be isolated and purified from hybridoma cultures by various established techniques. Such separation techniques include affinity chromatography (performed with protein A or protein G agarose), size exclusion chromatography, and ion exchange chromatography. For example, see Coligan, pages 2.7.1-2.7.12 and 2.9.1-2.9.3. In addition, see Baines et al., "Purification of Immunoglobulin G (IgG)," METHODS IN MOLECULAR BIOLOGY, Vol. 10, pp. 79-104 (Humana Press, Inc. 1992).

[0554] After the initial production of antibodies to the immunogen, the antibodies can be sequenced and subsequently produced by recombinant techniques. Humanization and chimerization of murine antibodies and antibody fragments are well known to those skilled in the art and are described below.

[0555] Chimeric antibodies are recombinant proteins in which the variable regions of a human antibody have been replaced by variable regions (e.g., of a mouse antibody) (including the complementarity determining regions (CDRs) of the mouse antibody). When administered to a subject, chimeric antibodies exhibit reduced immunogenicity and increased stability. 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 skilled in the art. For example, Leung et al., Hybridoma 13:469 (1994), described chimeric antibodies by cloning the Vκ and Vκ encoding murine LL2 (anti-CD22 monoclonal antibody) into a chimeric antibody. H The DNA sequences of the domains are similar to those of the corresponding human κ and IgG 1 The constant region domains were combined to generate the LL2 chimera.

[0556] The 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 antibodies or murine monoclonal antibodies can be humanized by transferring mouse CDRs from the heavy and light chain variable chains of mouse immunoglobulins to the corresponding variable domains of human antibodies. The mouse framework regions (FRs) in chimeric monoclonal antibodies are also replaced by human FR sequences. Because simply transferring mouse CDR to human FR often leads to the reduction or even loss of antibody affinity, it may be necessary to carry out additional modification to restore the original affinity of mouse antibody. This can be achieved by replacing one or more human residues in the FR region with their mouse counterparts to obtain an antibody with good binding affinity to its epi-position. For example, see Tempest et al., Biotechnology 9:266 (1991) and Verhoeyen et al., Science 239:1534 (1988). Preferred residues for replacement include FR residues within 1,2 or 3 angstroms of the CDR residue side chain, FR residues near the CDR sequence or FR residues predicted to interact with the CDR residues.

[0557] Methods for producing fully human antibodies using combinatorial methods or transgenic animals transformed with human immunoglobulin loci 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, Curr. Opin. Pharmacol. 3:544-50). Fully human antibodies can also be constructed by genetic or chromosomal transfection methods and phage display technology, all of which are known in the art. For example, see McCafferty et al., Nature 348:552-553 (1990). In cases where the antibody will be used in vivo, such as in tumor treatment after detection of Trop-2 positive cancer, it is expected that such fully human antibodies will show fewer side effects than chimeric or humanized antibodies and will function essentially as endogenous human antibodies in vivo.

[0558] In an alternative, phage display technology can be used to generate human antibodies (e.g., Dantas-Barbosa et al., 2005, Genet. Mol. Res. 4:126-40). Human antibodies may be generated in normal people or in people who exhibit a specific disease state such as cancer (Dantas-Barbosa et al., 2005). The advantage of constructing human antibodies from sick individuals is that the circulating antibody repertoire may be biased towards antibodies against disease-associated antigens.

[0559] In a non-limiting example of this method, Dantas-Barbosa et al. (2005) constructed a phage display library of human Fab antibody fragments from osteosarcoma patients. Typically, total RNA is obtained from circulating blood lymphocytes (Id.). Recombinant Fabs are cloned from μ, γ and κ chain antibody libraries and inserted into phage display libraries (Id.). RNA is converted into cDNA, and a Fab cDNA library is prepared using specific primers for heavy and light chain immunoglobulin sequences (Marks et al., J. Mol. Biol. 222:581-97). Library construction is performed according to Andris-Widhopf et al. (2000, in: Phage Display Laboratory Manual, Barbas et al. (eds.), 1st edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pages 9.1 to 9.22). The final Fab fragments are digested with restriction endonucleases and inserted into the phage genome to prepare phage display libraries. As is known in the art, such libraries can be screened by standard phage display methods. Phage display can be performed in a variety of formats, for review, see, e.g., Johnson and Chiswell, Current Opinion in Structural Biology 3:5564-571 (1993).

[0560] Human antibodies can also be produced by in vitro activated B cells. See U.S. Pat. Nos. 5,567,610 and 5,229,275. Those skilled in the art will recognize that these techniques are exemplary and any known method for making and screening human antibodies or antibody fragments can be utilized.

[0561] In another alternative, using standard immunization protocols, transgenic animals that have been genetically engineered to produce human antibodies can be used to produce antibodies against essentially any immunogenic target. Green et al., Nature Genet.7:13 (1994), Lonberg et al., Nature 368:856 (1994) and Taylor et al., Int. Immun. 6:579 (1994) disclose methods for obtaining human antibodies from transgenic mice. A non-limiting example of such a system is the XenoMouse® from Abgenix (Fremont, CA) (e.g., Green et al., 1999, J. Immunol. Methods 231:11-23). ​​In the XenoMouse® and similar animals, mouse antibody genes have been inactivated and replaced by functional human antibody genes, while the rest of the mouse immune system remains intact.

[0562] XenoMouse® is transformed with a germline-configured YAC (yeast artificial chromosome) containing a portion of the human IgH and Igк loci, including most variable region sequences, as well as accessory genes and regulatory sequences. The human variable region library can be used to generate antibody-producing B cells, which can be processed into hybridomas by known techniques. XenoMouse® immunized with the target antigen will produce human antibodies through a normal immune response, which can be harvested and / or produced by the standard techniques described above. There are a variety of strains of XenoMouse® available, each of which is capable of producing different types of antibodies. Transgenic human antibodies have been shown to have therapeutic potential while retaining the pharmacokinetic properties of normal human antibodies (Green et al., 1999). The skilled artisan will recognize that the claimed compositions and methods are not limited to the use of the XenoMouse® system, but can utilize any transgenic animal that has been genetically engineered to produce human antibodies.

[0563] Antibody fragments are 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 produced by known techniques. F(ab') 2 fragments can be produced, for example, by digestion of antibody molecules with pepsin. These and other methods are described, for example, in Goldenberg, U.S. Patent Nos. 4,036,945 and 4,33,1647 and references contained therein. In addition, see Nisonoff et al., Arch Biochem. Biophys. 89: 230 (1960); Porter, Biochem. J. 73: 119 (1959), Edelman et al., METHODS INENZYMOLOGY VOL. 1, 422 pages (Academic Press 1967), and Coligan at 2.8.1-2.8.10 and 2.10.-2.10.4 pages. Alternatively, Fab' expression libraries can be constructed (Huse et al., 1989, Science, 246:1274-1281) to allow rapid and easy identification of monoclonal Fab' fragments with the desired specificity.

[0564] Single-chain Fv molecules (scFv) contain VL domains and VH domains. VL and VH domains associate to form a target binding site. The two domains are further covalently linked by a peptide linker (L). If the VL domain is the N-terminal portion of the scFv molecule, the scFv molecule is represented as VL-L-VH, and if the VH domain is the N-terminal portion of the scFv molecule, the scFv molecule is represented as VH-L-VL. Methods for preparing 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, "SingleChain Fvs." FASEB Vol 9:73-80 (1995) and RE Bird and BW Walker, SingleChain Antibody Variable Regions, TIBTECH, Vol 9: 132-137 (1991).

[0565] 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 (VHH) can be obtained, for example, from 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). VHH may have strong antigen binding capacity and can interact with new epitopes that are inaccessible to conventional VH-VL pairs. (Muyldermans et al., 2001). Alpaca serum IgG contains about 50% camelid heavy chain only IgG antibodies (HCAbs) (Maass et al., 2007). Alpacas can be immunized with known antigens (e.g., TNF-α) and VHHs that bind and neutralize the target antigen can be isolated (Maass et al., 2007). PCR primers that amplify nearly all alpaca VHH coding sequences have been identified and can be used to construct alpaca VHH phage display libraries that can be used for antibody fragment isolation by standard biopanning techniques well known in the art (Maass et al., 2007).

[0566] Antibody fragments can also be prepared by proteolysis of full-length antibodies or by expressing DNA encoding the fragment in E. coli or another host. Antibody fragments can be obtained by digesting full-length antibodies with pepsin or papain by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide an approximately 100 kD fragment expressed as F(ab')2. The fragment can be further cleaved using a thiol reducing agent and an optional blocking group of the sulfhydryl group produced by disulfide bond cleavage to produce an approximately 50 Kd Fab' monovalent fragment. Alternatively, enzymatic cleavage using papain directly produces two monovalent Fab fragments and an Fc fragment.

[0567] Other methods of cleaving antibodies, such as separation of the heavy chains to form monovalent light-heavy chain fragments, further cleavage of the fragments, or other enzymatic, chemical or genetic techniques may also be used, so long as the fragments bind the antigen recognized by the intact antibody.

[0568] The terms CXCL12 and SDF-1 are synonymous and are used interchangeably herein.

[0569] In an embodiment of the present invention and preferably as used herein, an antagonist of CXCL12.

[0570] Those skilled in the art will understand 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 embodiments thereof.

[0571] According to the various SEQ ID NOs: of the present invention, the chemical properties of the nucleic acid molecules and the target molecule CXCL12 used herein, their actual sequences and internal reference numbers are summarized in Table 1 below. It should be noted that the nucleic acids are characterized at the aptamer level, i.e., D-nucleic acid level (D-RNA) with biotinylated human D-CXCL12 (SEQ ID NO: 4) or at the Spiegelmer level, i.e., L-nucleic acid (L-RNA) with the natural configuration of CXCL12, i.e., L-CXCL12 (human CXCL12α, SEQ ID NO: 1). Different nucleic acids share an internal reference name, but there is one SEQ ID Nos: for D-RNA (aptamer) molecules and one SEQ ID Nos for L-RNA (Spiegelmer) molecules, respectively. Those skilled in the art will understand that the present application also encompasses, in particular, other sequences and amino acid sequences.

[0572] Table 1

[0573]

[0574]

[0575]

[0576]

[0577]

[0578]

[0579]

[0580]

[0581]

[0582]

[0583]

[0584]

[0585]

[0586]

[0587] BRIEF DESCRIPTION OF THE DRAWINGS

[0588] The present invention will be further illustrated by way of and with reference to the following figures and examples, from which further advantages, features and embodiments may be derived.

[0589] Figure 1 An alignment of the sequences of "type A" CXCL12 binding nucleic acid molecules is shown;

[0590] Figure 2A +B shows a derivative of the CXCL12 binding nucleic acid molecule 192-A10-001 (“Type A” CXCL12 binding nucleic acid molecule);

[0591] Figure 3 An alignment of the sequences of "type B" CXCL12-binding nucleic acid molecules is shown;

[0592] Figure 4A +B shows derivatives of CXCL12 binding nucleic acid molecules 193-C2-001 and 193-G2-001 (type B CXCL12 binding nucleic acid molecules);

[0593] Figure 5 An alignment of the sequences of “type C” CXCL12-binding nucleic acid molecules is shown;

[0594] Figure 6 Derivatives of the CXCL12 binding nucleic acid molecule 190-A3-001 (“Type C” CXCL12 binding nucleic acid molecule) are shown;

[0595] Fig. 7A +B shows a derivative of the CXCL12 binding nucleic acid molecule 190-D5-001 (“Type C” CXCL12 binding nucleic acid molecule);

[0596] Figure 8 Derivatives of the CXCL12 binding nucleic acid molecule 197-B2 (“Type C” CXCL12 binding nucleic acid molecule) are shown;

[0597] Fig. 9 Additional CXCL12 binding nucleic acid molecules are shown, which are also referred to as "type D" CXCL12 binding nucleic acid molecules, in addition to other CXCL12 binding nucleic acid molecules;

[0598] Fig.10Shown is a graph illustrating the efficiency of CXCL12 binding to Spiegelmer 193-G2-012-5'-PEG (also known as NOX-A12), 197-B2-006-5'-PEG, 191-D5-007-5'-PEG, and 191-A10-008-5'-PEG in a chemotaxis assay of the human T-cell leukemia cell line Jurkat, thereby allowing cells to migrate toward 0.3 nM human CXCL12 preincubated at 37°C with various amounts of Spiegelmer 93-G2-012-5'-PEG, 197-B2-006-5'-PEG, 191-D5-007-5'-PEG, and 191-A10-008-5'-PEG as controls relative to Spiegelmer The concentrations of 193-G2-012-5′-PEG, 197-B2-006-5′-PEG, 191-D5-007-5′-PEG, and 191-A10-008-5′-PEG are expressed as percentages;

[0599] Fig.11 A shows a graph illustrating the efficiency of CXCL12 binding to Spiegelmer NOX-A12 in a chemotaxis assay of the human pre-B ALL cell line Nalm-6, thereby allowing the cells to migrate towards 0.3 nM human CXCL12 pre-incubated with different amounts of Spiegelmer NOX-A12 at 37°C, expressed as a percentage of the control relative to the concentration of Spiegelmer NOX-A12;

[0600] Fig.11 B shows a graph illustrating the efficiency of CXCL12 binding to Spiegelmer NOX-A12 in a chemotaxis assay of the human leukemic monocytic lymphoma cell line U937, thereby allowing the cells to migrate towards 3 nM human CXCL12 pre-incubated with different amounts of Spiegelmer NOX-A12 at 37°C, expressed as a percentage of the control relative to the concentration of Spiegelmer NOX-A12;

[0601] Fig.12 shows a graph illustrating the efficiency of CXCL12 binding to Spiegelmer NOX-A12 in a chemotaxis assay of the human pre-B cell leukemia cell line BV-173, thereby allowing the cells to migrate towards 3 nM human CXCL12 pre-incubated with varying amounts of Spiegelmer NOX-A12 at 37° C., expressed as a percentage of the control relative to the concentration of Spiegelmer NOX-A12;

[0602] Fig.13 shows a graph illustrating the efficiency of CXCL12 binding to Spiegelmer NOX-A12 in a complementation assay with CHO cells stably expressing CXCR7 and β-arrestin, both fused to a fragment of β-galactosidase, thereby activating the cellular CXCR7 towards 10 nM human CXCL12 pre-incubated with different amounts of Spiegelmer NOX-A12 at 37° C., expressed as a percentage of the control relative to the concentration of Spiegelmer NOX-A12;

[0603] Fig.14 is a diagram illustrating the treatment regimen in a clinical study conducted using triple therapy as performed in Example 6;

[0604] Fig.15 is a diagram illustrating a treatment regimen in a clinical study conducted using triple therapy as performed in Example 7 further including chemotherapy;

[0605] Fig.16 The amino acid sequence of human CXCR4 is shown (SEQ ID NO: 243);

[0606] Fig.17 The amino acid sequence of human CXCR7 is shown (SEQ ID NO: 244);

[0607] Fig.18 The amino acid sequence of human VEGFR1 is shown (SEQ ID NO: 245);

[0608] Fig.19 The amino acid sequence of human VEGFR2 is shown (SEQ ID NO: 246);

[0609] Fig. 20 Lane plots are shown illustrating the progression of disease in patients 1 to 6 according to the mRANO response and NANO score assessments (BL, baseline; W, week; PR, partial response; SD, stable disease; PD, progressive disease; mRANO, modified response assessment in neuro-oncology; NANO, neurologic function assessment in neuro-oncology);

[0610] Fig.21 Box plots are shown illustrating the maximum change from baseline in advanced MRI imaging parameters (ADC, apparent diffusion coefficient; rCBV, relative cerebral blood volume; FTB, relative cerebral blood volume). 高 , hyperperfused tumor segmentation; NTL, nontarget lesion), neurological function (NANO, Neurological Assessment in Neuro-Oncology), and quality of life (QoL) assessment (OFS, Overall Functional Score; GHS, Global Health Score);

[0611] Fig. 22 Lane plots are shown illustrating the progression of disease in patients 1 to 6 according to the mRANO response and NANO score assessments (BL, baseline; W, week; PR, partial response; SD, stable disease; PD, progressive disease; mRANO, modified response assessment in neuro-oncology; NANO, neurologic function assessment in neuro-oncology);

[0612] Fig.23 Box plots are shown illustrating the maximum optimal change from baseline in advanced MRI imaging parameters (ADC, apparent diffusion coefficient; rCBV, relative cerebral blood volume; FTB, relative cerebral blood volume). 高 , hyperperfused tumor segmentation; NTL, nontarget lesion), neurological function (NANO, Neurological Assessment in Neuro-Oncology), and quality of life (QoL) assessment (OFS, Overall Functional Score; GHS, Global Health Score);

[0613] Fig.24 Shown is a bar graph illustrating the best response (SPDP, sum of products of perpendicular diameters; white bars, more than 50% decrease in SPDP, i.e., partial response) of target lesions in patients with glioblastoma treated with the triple combination of NOX-A12, radiation therapy, and bevacizumab;

[0614] Fig.25 A bar graph is shown illustrating the best response (SPDP, sum of products of perpendicular diameters; white bars, more than 50% reduction in SPDP, i.e., partial response; shaded bars, less than 50% reduction in SPDP, i.e., stable disease; black bars, more than 25% increase in SPDP, i.e., progressive disease) of target lesions in glioblastoma patients treated with the double combination of NOX-A12 and radiation therapy;

[0615] Fig.26 A bar graph is shown illustrating the best response of target lesions for a historical reference cohort of glioblastoma patients treated with standard of care (i.e., radiotherapy and temozolomide for chemotherapy) (white bars, more than 50% SPDP reduction, i.e., partial response; shaded bars, less than 50% SPDP reduction, i.e., stable disease; gray bars, maximum 25% SPDP increase, i.e., stable disease; black bars, more than 25% SPDP increase, i.e., progressive disease);

[0616] Fig. 27 A swim-lane plot showing the follow-up of patients 1 to 6 based on survival is shown;

[0617] Fig.28 A swim lane graph is shown showing further follow-up of patients 1 to 6 based on survival; and

[0618] Fig.29Graphs are shown illustrating the percentage change from baseline in tumor response over time in glioblastoma patients 1 to 6 assessed by MRI; PD, progressive disease; SD, stable disease; PR, partial response; black lines / symbols, during treatment with NOX-A12 / bevacizumab; gray lines / symbols, during follow-up. DETAILED DESCRIPTION

[0619] Example 1 Nucleic Acids Binding to Human CXCL12

[0620] In the following, if there is no indication to the contrary, the terms "nucleic acid" and "nucleic acid molecule" are used in a synonymous manner herein. In addition, if there is no indication to the contrary, the terms "stretch" and "stretch of nucleotides" are used in a synonymous manner herein.

[0621] L-nucleic acid molecules that bind to human CXCL12 and the corresponding nucleotide sequences are described in Figures 1 to 9 Nucleic acids were characterized at the aptamer (i.e., D-nucleic acid) level using competitive or direct pull-down binding assays with biotinylated human D-CXCL12 (protocol, see Example 3). Spiegelmers were tested with the native configuration of CXCL12 (L-CXCL12) by surface plasmon resonance measurements using a Biacore 2000 instrument (protocol, see Example 5) and in vitro chemotaxis assays in cell culture (protocol, see Example 4).

[0622] Nucleic acid molecules that bind CXCL12 exhibit different sequence motifs, Figure 1 , 2A and 2B (Type A), Figure 3 , 4A and 4B (Type B), Figure 5 Three major types are defined in , 4, 7A, 7B and 8 (type C). Nucleic acid molecules exhibit different sequence motifs. To define nucleotide sequence motifs, IUPAC abbreviations are used for undefined nucleotides:

[0623] S strong G or C;

[0624] W weak A or U;

[0625] R purine G or A;

[0626] Y pyrimidine C or U;

[0627] K keto G or U;

[0628] M imino group A or C;

[0629] B is not AC or U or G;

[0630] D is not CA or G or U;

[0631] H is not GA or C or U;

[0632] V is not UA or C or G;

[0633] N All A or G or C or U

[0634] If not indicated to the contrary, the sequence of any nucleic acid sequence or fragment and frame, respectively, is indicated in the 5'→3' direction.

[0635] Type A CXCL12 binding nucleic acid molecules

[0636] like Figure 1 All sequences of the described A-type CXCL12 binding nucleic acid molecules comprise a central nucleotide stretch flanked by a first (5'-) terminal and a second (3'-) terminal stretch of nucleotides (also referred to as the first and second terminal nucleotide stretches), whereby the two stretches can hybridize to each other. However, such hybridization is not necessarily present in the molecule.

[0637] In the following, if there is no contrary indication, the terms "A-type CXCL12-binding nucleic acid molecule" and "A-type CXCL12-binding nucleic acid" or A-type CXCL12-binding nucleic acid molecule are used herein in a synonymous manner.

[0638] The sequence of the defined nucleotide frame or fragment may differ between type A CXCL12 binding nucleic acids, which affects the binding affinity to CXCL12. Based on the binding analysis of different CXCL12 binding nucleic acids (summarized as type A CXCL12 binding nucleic acids), the central nucleotide fragment and its nucleotide sequence described below are essential for binding to CXCL12 individually and more preferably in their entirety.

[0639] The central nucleotide fragment of all identified sequences of type A CXCL12 binding nucleic acids shares the sequence AAAGYRACAHGUMAAX A UGAAAGGUARC (Formula A-1, SEQ ID NO: 74), wherein X AAbsent or "A". If "A" is absent, the sequence of the central nucleotide sequence can be summarized as A-formula-2 (AAAGYRACAHGUMAA-UGAAAGGUARC, SEQ ID NO: 75). The A-type CXCL12 binding nucleic acid 191-A6 (central nucleotide sequence: AAAGUAACACGUAAAAUGAAAGGUAAC, SEQ ID NO: 54) carries an additional nucleotide "A" within the central nucleotide sequence and still binds to CXCL12, resulting in an alternative central nucleotide sequence (AAAGYRACAHGUMAAAUGAAAGGUARC, A-formula-3, SEQ ID NO: 76). Exemplarily, for all other nucleic acids of the A-type CXCL12 binding nucleic acid, the A-type CXCL12 binding nucleic acid 192-A10-001 is characterized by its binding affinity to human CXCL12. The equilibrium binding constant K D was used in a pull-down binding assay (K D = 1.5 nM) and surface plasmon resonance measurements (K D = 1.0 nM). IC of 192-A10-001 measured using a cell culture in vitro chemotaxis assay 50 (50% inhibitory concentration) was 0.12 nM. Therefore, the Figure 1 All A-type CXCL12 binding nucleic acids shown. In these competition experiments, the A-type CXCL12 binding nucleic acids 192-B11 and 192-C10 showed the same binding affinity as 192-A10-001. The binding affinities of the A-type CXCL12 binding nucleic acids 192-G10, 192-F10, 192-C9, 192-E10, 192-D11, 192-G11, 192-H11 and 191-A6 were determined to be weaker. The binding affinities of the A-type CXCL12 binding nucleic acids 192-D10, 192-E9 and 192-H9 were much weaker than 192-A10-001.

[0640] As described above, the A-type CXCL12 binding nucleic acids 192-B11 and 192-C10 exhibited the same binding affinity to CXCL12 as 192-A10-001 to CXCL12. However, they showed a small amount of difference in the nucleotide sequence of the central nucleotide fragment. Therefore, the consensus sequence of the three molecules that bind to CXCL12 with almost the same high affinity can be summarized as the nucleotide sequence AAAGYAACAHGUCAAUGAAAGGUARC (A-type-4, SEQ ID NO: 77), whereby the nucleotide sequence of the central nucleotide fragment of 192-A10-001 (nucleotide sequence: AAAGCAACAUGUCAAUGAAAGGUAGC, SEQ ID NO: 84) represents the nucleotide sequence with the best binding affinity of the A-type CXCL12 binding nucleic acid.

[0641] Five or six of the six nucleotides of the 5' terminal fragment (also referred to as the first terminal fragment) of the A-type CXCL12 binding nucleic acid can hybridize to corresponding five or six of the six nucleotides of the 3' terminal fragment (also referred to as the second terminal fragment) to form a terminal helix. Although these nucleotides are variable at several positions, different nucleotides allow hybridization of five or six of the six nucleotides of each of the 5' and 3' terminal fragments. Figure 1 As shown, the 5' and 3' terminal fragments of the A-type CXCL12 binding nucleic acids can be summarized as the general formula for the 5' terminal fragment ("RSHRYR", A-type formula-5-5') and the general formula for the 3' terminal fragment ("YRYDSY", A-type formula-5-3'). Truncated derivatives of the A-type CXCL12 binding nucleic acid 192-A10-001 ( Figure 2A and 2B These experiments showed that reducing the 6 terminal nucleotides of 192-A10-001 (5' end: GCUGUG; 3' end: CGCAGC) to 5 nucleotides of the derivative 192-A10-002 (5' end: CUGUG; 3' end: CGCAG) did not reduce binding affinity. However, truncation to 4 terminal nucleotides (5' end: UGUG; 3' end: CGCA; 192-A10-003) or less (192-A10-004 / -005 / -006 / -007) resulted in reduced binding affinity for CXCL12 ( Figure 2A ). The 5' and 3' terminal fragments were determined to be 5 and 4 nucleotides in length of the derivative of the A-type CXCL12 binding nucleic acid 192-A10-001 (eg Figure 2A and 2BAs shown in the figure, the 5'-terminal fragment can be represented by the general formula ("X 2 BBBS", A-6-5') and the general formula of the 3'-terminal fragment ("SBBVX 3 "; A-6-3') description, where X 2 Does not exist or is "S", and X 3 Not present or "S".

[0642] The nucleotide sequences of the 5' and 3' terminal fragments have an influence on the binding affinity of the A-type CXCL12 binding nucleic acids. This was demonstrated not only by the nucleic acids 192-F10 and 192-E10, but also by the derivatives of 192-A10-001 ( Figure 2B ). The central fragments of 192-F10 and 192-E10 are identical to 192-B11 and 192-C10, but contain minor differences at the 3' end of the 5' terminal fragment and at the 5' end of the 3' terminal fragment, which result in reduced binding affinity.

[0643] Substitution of the 5' and 3' terminal nucleotides "CUGUG" and "CGCAG" of the A-type CXCL12 binding nucleic acid 192-A10-002 with "GCGCG" and "CGCGC" (192-A10-015) resulted in decreased binding affinity, whereas substitution with "GCGUG" and "CGCGC" (192-A10-008) resulted in the same binding affinity as 192-A10-002 ( Figure 2B In addition, the binding affinity of nine derivatives of the A-type CXCL12 binding nucleic acid 192-A10-001 (192-A10-014 / -015 / -016 / -017 / -018 / -019 / -020 / -021 / -022 / -023) with four 5'- and 3'-terminal nucleotides, respectively, as aptamers was tested in comparison with 192-A10-001 or its derivative 192-A10-008 (both of which had the same binding affinity for CXCL12). The binding affinity of all molecules for CXCL12 was weaker, very weaker, or extremely weaker than that of 192-A10-001 (with six nucleotides forming the terminal helix) or 192-A10-008 with five terminal nucleotides, respectively. Figure 2B). Therefore, the sequence and number of nucleotides of the 5' and 3' terminal fragments are crucial for effective binding to CXCL12. As shown for the A-type CXCL12 binding nucleic acids 192-A10-002 and 192-A10-08, the preferred combinations of the 5' and 3' terminal fragments are "CUGUG" and "CGCAG" (the 5' and 3' terminal fragments of the A-type CXCL12 binding nucleic acid 192-A10-002) and "GCGUG" and "CGCGC" (the 5' and 3' terminal fragments of the A-type CXCL12 binding nucleic acid 192-A10-008).

[0644] However, combining the 5'-terminal and 3'-terminal fragments of all tested A-type CXCL12 binding nucleic acids, the general formula of the 5'-terminal fragment of the A-type CXCL12 binding nucleic acid is "X 1 X 2 NNBV" (Type A-7-5'), the general formula of the 3' terminal fragment of Type A CXCL12 binding nucleic acid is "BNBNX 3 X 4 ” (Type A-7-3'), and

[0645] X 1 Is "R" or does not exist, X 2 It is "R", X 3 is "R", and X 4 is "Y" or absent;

[0646] or

[0647] X 1 Does not exist, X 2 Is "S" or not present, X 3 is "S" or not present, and X 4 Does not exist.

[0648] To prolong the plasma residence time of Spiegelmer in vivo, Spiegelmer 192-A10-008 was covalently coupled to 40 kDa polyethylene glycol (PEG) at the 5' terminal moiety as described in Chapter 2. The PEG moiety had no effect on the potency of Spiegelmer in inhibiting CXCL12-induced chemotaxis.

[0649] Type B CXCL12 binding nucleic acid molecules

[0650] like Figure 3 All sequences of type B CXCL12 binding nucleic acids described herein comprise a central stretch of nucleotides flanked by 5' and 3' terminal stretches (also referred to as first and second terminal stretches of nucleotides) that can hybridize to each other. However, such hybridization is not necessarily present in the molecule.

[0651] In the following, if there is no indication to the contrary, 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 herein in a synonymous manner.

[0652] The sequence of the defined box or fragment may differ between CXCL12 binding nucleic acids, which affects the binding affinity to CXCL12. Based on the binding analysis of different CXCL12 binding nucleic acids, the central nucleotide fragment and its nucleotide sequence described below are essential individually and more preferably as a whole for binding to CXCL12.

[0653] The central nucleotide fragment of all identified 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 shared the sequence GUGUGAUCUAGAUGUADWGGCUGWUCCUAGUYAGG (Formula B-1, SEQ ID NO: 52). CXCL12 binding nucleic acids 193-G2-001, 193-C2-001 and 193-F2-001 differed at one position in the nucleotide center segment (consensus sequence of the central nucleotide segment: GUGUGAUCUAGAUGUADUGGCUGAUCCUAGUCAGG (Formula B-2, SEQ ID NO: 53) were analyzed in a competitive pull-down binding assay and compared to CXCL12 binding nucleic acid 192-A10-001 (K determined in the pull-down binding assay). D 1.5 nM, IC 50 0.12 nM). Each of the CXCL12 binding nucleic acids 193-G2-001, 193-C2-001, and 193-F2 showed superior binding to human CXCL12 compared to the CXCL12 binding nucleic acid 192-A10-001, with 193-G2-001 binding affinity being as good as that of 193-C2-001 and 193-F2-001 ( Figure 3). The data indicate that the nucleotide sequence differences in the central nucleotide fragments of CXCL12 binding nucleic acids 193-G2-001, 193-C2-001, and 193-F2-001 have no effect on the binding affinity to CXCL12. CXCL12 binding nucleic acids 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 show reduced binding to human CXCL12 compared to CXCL12 binding nucleic acid 193-G2-001. CXCL12 binding nucleic acid 193-G2-001 is characterized by its binding affinity to human CXCL12. Equilibrium binding constant K D Use pull-down binding assay to determine (K D = 0.3 nM). The IC of 193-G2-001 was measured using a cell culture in vitro chemotaxis assay. 50 (50% inhibitory concentration) is 0.08nM.

[0654] Four, five, or six of the six nucleotides of the 5' terminal segment of the CXCL12 binding nucleic acid can hybridize to corresponding four, five, or six of the six nucleotides of the 3' terminal segment of the CXCL12 binding nucleic acid to form a terminal helix. Although the nucleotides are variable at several positions, different nucleotides allow hybridization of four, five, or six of the six nucleotides of each of the 5' and 3' terminal segments. Figure 3 As shown, the 5' and 3' terminal segments of the CXCL12-binding nucleic acid can be summarized as the general formula of the 5' terminal segment ("X 1 X 2 GCRWG", and X 1 Is "A" or not present, X 2 is "G") and the 3' terminal fragment has the general formula ("KRYSCX 3 X 4 ", and X 3 It is "G", X 4 is "U" or absent). CXCL12 binding nucleic acids 193-G1-002, 193-D2-002, 193-A1-002, and 193-D3-002 have weak binding affinity to CXCL12, although they share the same central nucleotide fragment with 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 nucleotides in the 5' and 3' terminal fragments.

[0655] Truncated derivatives of the CXCL12 binding nucleic acids 193-G2-001 and 193-C2-001 were analyzed in a competitive pull-down binding assay and compared to 193-G2-001 and 193-G2-012, respectively ( Figure 4A and 4B These experiments showed that reducing the 6 terminal nucleotides (5' end: AGCGUG; 3' end: UACGCU) of the CXCL12 binding nucleic acids 193-G2-001 and 193-C2-001 to 5 nucleotides (5' end: GCGUG; 3' end: UACGC) resulted in molecules with similar binding affinities (193-C2-002 and 193-G2-012). The equilibrium dissociation constant K D Using pull-down binding assay (K D = 0.3 nM). Truncation to 4 (5' end: CGUG; 3' end: UACG; 193-C2-003) or fewer nucleotides (193-C2-004, 193-C2-005, 193-C2-006, 193-C2-007) resulted in decreased binding affinity to CXCL12, as measured using a competitive pull-down binding assay ( Figure 4A ). The nucleotide sequence of the 5 terminal nucleotides at the 5' and 3' ends had an effect on the binding affinity of the CXCL12 binding nucleic acid, respectively. Replacement of the 5'- and 3'-terminal nucleotides "GCGUG" and "UACGC" (193-C2-002, 193-G2-12) by "GCGCG" and "CGCGC" (193-G2-013) resulted in a decreased binding affinity. In addition, 4 different derivatives of the CXCL12 binding nucleic acid 193-G2-001, whose terminal helices had a length of 4 base-paired nucleotides (193-G2-014 / -015 / -016 / -017), were tested. They all showed a decreased binding affinity to CXCL12 ( Figure 4B ). Therefore, the sequence and length of the 5' and 3' terminal fragment nucleotides are critical for effective binding to CXCL12. The 5' and 3' terminal fragments, with lengths of CXCL12 binding nucleic acids 193-C2-003 and 193-G2-012 (e.g. Figure 4A and 4B ) of derivatives of 5 and 4 nucleotides, which can be represented by the general formula of the 5'-terminal fragment ("X 1 X 2 SSBS”) (where X 1 Does not exist, X 2 absent or "G") and the general formula of the 3'-terminal fragment ("BVSSX 3 X 4 ”) describes (where X3 does not exist or is "C", and X 4 As shown for CXCL12 binding nucleic acids 193-G2-001 and 193-C2-01 and their derivatives 193-G2-012 and 193-C2-002, a preferred combination of 5'- and 3'-terminal fragments is "X 1 X 2 GCGUG" (5'-terminal fragment) and "UACGCX 3 X 4 ” (3'-terminal fragment), while X 1 Is "A" or not present, X 2 It is "G", X 3 is "C", and X 4 Is "U" or not present.

[0656] However, combining the 5' and 3' terminal fragments of all tested CXCL12 binding nucleic acids, the general formula of the 5' terminal fragment of the CXCL12 binding nucleic acid is "X 1 X 2 SVNS”, the general formula of the 3' terminal fragment of CXCL12 binding nucleic acid is "BVBSX 3 X 4 ",and

[0657] X 1 Is "A" or not present, X 2 It is "G", X 3 is "C", and X 4 It is "U" or not present;

[0658] or X 1 Does not exist, X 2 Is "G" or does not exist, X 3 is "C" or does not exist, and X 4 Does not exist.

[0659] To prolong the plasma residence time of Spiegelmer in vivo, Spiegelmer 193-G2-012 was covalently coupled to 40 kDa polyethylene glycol (PEG) at the 5' end as described in Chapter 2 (PEGylated nucleic acid molecule: 193-G2-012-5'-PEG also known as NOX-A12). In an in vitro chemotaxis assay, PEGylated Spiegelmer NOX-A12 was analyzed in cell culture and the inhibitory effect of CXCL12-induced chemotaxis (IC 50 PEGylated Spiegelmer NOX-A12 was analyzed by Biacore measurement and the binding constant (K D ) is 0.2 nM.

[0660] C-type CXCL12 binding nucleic acid molecules

[0661] like Figure 5 All sequences of the described C-type CXCL12 binding nucleic acid molecules contain a central nucleotide stretch flanked by 5' and 3' terminal stretches (also referred to as first terminal stretch and second terminal stretch of nucleotides) that can hybridize to each other. However, such hybridization is not necessarily present in the molecule.

[0662] In the following, if there is no indication to the contrary, the terms "C-type CXCL12-binding nucleic acid molecule" and "C-type CXCL12-binding nucleic acid" or C-type CXCL12-binding nucleic acid molecule are used herein in a synonymous manner.

[0663] The sequence of the defined box or fragment may differ between CXCL12 binding nucleic acids of type C, which affects the binding affinity to CXCL12. Based on the binding analysis of different CXCL12 binding nucleic acids (summarized as type C CXCL12 binding nucleic acids), the central nucleotide fragment and its nucleotide sequence described below are essential individually and more preferably as a whole for binding to CXCL12.

[0664] The central nucleotide fragment of all identified sequences of C-type CXCL12 binding nucleic acids shares the sequence GGUYAGGGCUHRX A AGUCGG (C-form 1, SEQ ID NO: 108), wherein X A Absent or "A". The central nucleotide fragment of all identified sequences of the C-type CXCL12 binding nucleic acids, except the C-type CXCL12 binding nucleic acid 197-D1, shared the nucleotide sequence GGUYAGGGCUHRAAGUCGG (C-type-2, SEQ ID NO: 109). The C-type CXCL12 binding nucleic acid 197-D1 (central nucleotide fragment: GGUUAGGGCUAA-AGUCGG (SEQ ID NO: 56)) lacked one nucleotide "A" within the central nucleotide fragment and still bound CXCL12, resulting in an alternative central nucleotide fragment (GGUYAGGGCUHR-AGUCGG, C-type-3, SEQ ID NO: 110). Initially, the competitive pull-down binding assay was analyzed as Figure 5 All C-type CXCL12 binding nucleic acids shown, K determined by pull-down assay and surface plasmon resonance measurements compared to the A-type CXCL12 binding nucleic acid 192-A10-001 D =1.5 nM; IC 50= 0.12 nM. In these competition experiments, C-type CXCL12 binding nucleic acids 191-D5-001, 197-B2, 190-A3-001, 197-H1, 197-H3, and 197-E3 showed weaker binding affinities than 192-A10-001. For 191-A5, 197-B1, 197-D1, 197-H2, and 197-D2, weaker binding affinities were determined ( Figure 5 The molecules or their derivatives were further characterized by further competitive pull-down binding assays, plasmon resonance measurements and in vitro chemotaxis assays. The C-type CXCL12 binding nucleic acid 191-D5-001 was characterized by its binding affinity to human CXCL12, while the equilibrium binding constant K D is measured by surface plasmon resonance (K D = 0.8 nM). The IC of 191-D5-001 was measured using a cell culture in vitro chemotaxis assay 50 The binding affinity (K) of C-type CXCL12 binding nucleic acid 197-B2 to human CXCL12 was determined by surface plasmon resonance measurement. D =0.9 nM), and its IC 50 (50% inhibitory concentration) was 0.2 nM. These data indicate that the C-type CXCL12 binding nucleic acids 191-D5-001 and 197-B2 have similar binding affinities for CXCL12 ( Figure 5 and 8 ).

[0665] Type C CXCL12 binding nucleic acid 190-A3-001 comprises a 17 nucleotide 5' terminal segment ("CGUGCGCUUGAGAUAGG", SEQ ID NO: 220) and a 12 nucleotide 3' terminal segment ("CUGAUUCACG", SEQ ID NO: 221), whereby in one aspect, the 4 nucleotides at the 5' end of the 5' terminal segment and the 4 nucleotides at the 3' end of the 3' terminal segment can hybridize with each other to form a terminal helix. Alternatively, the nucleotides "UGAGA" in the 5' terminal segment can hybridize to the nucleotides "UCUCA" in the 3' terminal segment to form a terminal helix. Reduction of the 5' terminal segment to 9 nucleotides ("UGAGAUAGG") and the 3' terminal segment ("CUGAUUCUC") to 10 nucleotides ("CUGAUUCUCA", SEQ ID NO: 222) of molecule 190-A3-001 had no effect on the binding affinity of CXCL12 (190-A3-003; Figure 6). Reduction of the 5' terminal fragment to 8 nucleotides ("GAGAUAGG") and the 3' terminal fragment to 9 nucleotides ("CUGAUUCUC") of molecule 190-A3-001 had no effect on the binding affinity of CXCL12 (190-A3-004; Figure 6 ). The equilibrium binding constant K of 190-A3-004 D was used in a pull-down binding assay (K D =4.6 nM) and surface plasmon resonance measurements (K D =4.7 nM). The IC of 190-A3-004 was measured using a cell culture in vitro chemotaxis assay. 50 (50% inhibitory concentration) was 0.1 nM. However, truncation of the 5'-terminal fragment to 2 nucleotides resulted in a very strong decrease in binding affinity (190-A3-007; Figure 6 ).

[0666] C-type CXCL12 binding nucleic acids 191-D5-001, 197-B2 and 197-H1 (central nucleotide fragment: GGUUAGGGCUAGAAGUCGG, SEQ ID 57), 197-H3 / 191-A5 (central nucleotide fragment: GGUUAGGGCUCGAAGUCGG, SEQ ID NO: 58) and 197-E3 / 197-B1 (central nucleotide fragment: GGUUAGGGCUUGAAGUCGG, SEQ ID NO: 59) share an almost identical central nucleotide fragment (C-type-4; nucleotide sequence: GGUUAGGGCUHGAAGUCGG SEQ ID NO: 111). 191-D5-001, 197-B2 and 197-H1 do not share similar 5' and 3' terminal fragments (197-H3 and 197-E3 have the same 5' and 3' terminal fragments as 197-B2). However, the corresponding 10 nucleotides (197-B2, 197-E3, 197-H3) or 9 nucleotides (191-D5-001, 197-H1) among the 10 nucleotides of the 5' terminal fragment can hybridize to the corresponding 10 nucleotides (197-B2, 197-E3, 197-H3) or 9 nucleotides (191-D5-001, 197-H1) among the 10 nucleotides of the 3' terminal fragment. Thus, the 5' terminal fragments of the C-type CXCL12 binding nucleic acids 197-B2, 191-D5-001, 197-H1, 197-E3 and 197-H3 plus 191-A5, 197-B1, 197-H2, 197-D1 and 197-D2 as described above contain the common universal nucleotide sequence of "RKSBUSNVGR" (C-type-5-5', SEQ ID NO: 138). The 3' terminal fragments of the C-type CXCL12 binding nucleic acids 197-B2, 191-D5-001, 197-H1, 197-E3 and 197-H3 plus 191-A5, 197-B1, 197-H2, 197-D1 and 197-D2 as described above contain the common universal nucleotide sequence of "YYNRCASSMY" (C-type 5-3', SEQ ID NO: 139), thereby preferably the 5' and 3' terminal fragments of the C-type CXCL12 binding nucleic acids 197-B2, 191-D5-001, 197-H1, 197-E3 and 197-H3.These preferred 5' and 3' terminal fragments of C-type CXCL12 binding nucleic acids 197-B2, 191-D5-001, 197-H1, 197-E3 and 197-H3 can be summarized in the general formula "RKBUGSVGR" (C-type -6-5'; 5' terminal fragment, SEQ ID NO: 140) and "YCNRCASSMY" (C-type -6-3'; 3' terminal fragment, SEQ ID NO: 141).

[0667] A truncated derivative of the C-type CXCL12 binding nucleic acid 191-D5-001 was constructed and tested in a competitive pull-down binding assay (compared to the original molecule 191-D5-001). Fig. 7A , Figure 7B ). First, if Fig. 7AAs shown, the length of the 5' and 3' terminal fragments was shortened from 10 nucleotides each (191-D5-001) to 7 nucleotides each (191-D5-004), whereby 9 of the 10 nucleotides (191-D5-001) or 6 of the 7 nucleotides (191-D5-004) of the 5' terminal fragment and the 3' terminal fragment, respectively, could hybridize with each other. The reduction of the 5' and 3' terminal fragments to 7 nucleotides, respectively (whereas 6 of the 7 nucleotides could hybridize with each other) resulted in a decrease in binding affinity to CXCL12 (191-D5-004). The terminal fragment of the C-type CXCL12 binding nucleic acid 191-D5-004 was modified, whereby the non-paired nucleotide "A" within the 3' terminal fragment of 191-D5-004 was replaced by "C" (191-D5-005). This modification resulted in an improvement in binding. This derivative (C-type CXCL12 binding nucleic acid 191-D5-005) showed similar binding to CXCL12 as 191-D5- 001. The 5'- and 3'-terminal fragments were further truncated to 5 nucleotides each, resulting in a molecule with a total length of 29 nucleotides (191-D5-007). Due to the similarity of 191-D5-001 to the C-type 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 for 191-D5-007, it could be assumed that in principle the 5' and 3' terminal fragments could be truncated to 5 nucleotides, whereby the nucleotide sequences "CGGGA" for the 5' terminal fragment and "UCCCG" for the 3' terminal fragment were successfully tested (C-type CXCL12 binding nucleic acid 191-D5-007). The C-type CXCL12 binding nucleic acid 191-D5-007 surprisingly bound CXCL12 somewhat better than 191-D5-001 (determined at the aptamer level using a competition binding assay). Equilibrium binding constant K of 191-D5-007 D was used in a pull-down binding assay (K D = 2.2 nM) and surface plasmon resonance measurements (K D = 0.8 nM). The IC of 191-D5-007 was measured using a cell culture in vitro chemotaxis assay 50 (50% inhibitory concentration) was 0.1 nM. The two terminal fragments were further shortened to 4 nucleotides (191-D5-010, Fig. 7A ).

[0668] Further derivatives of the C-type CXCL12 binding nucleic acid 191-D5-001 (191-D5-017 / -024 / -029) carrying 4 nucleotide 5' and 3' terminal fragments, respectively, also showed reduced binding affinity for CXCL12 compared to 191-D5-007 in a competitive pull-down binding assay ( Figure 7B ). Alternative 5' and 3' terminal fragments of 5 nucleotides in length were also tested (191-D5-017-29a, 191-D5-017-29b, 191-D5-019-29a, 191-D5-024-29a, 191-D5-024-29b). The 5' terminal fragments of these derivatives are generally of the formula "X S SSSV" (C-7-5'), the 3' fragment formula is "BSSSX S "(Type C-7-3'), whereby X S Absent or “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'-terminal and 3'-terminal fragments of the 191-D5-001-derivatives showed the best binding affinity to CXCL12 and contained 5 nucleotides of 5'-terminal and 3'-terminal fragments, respectively (191-D5-007, 191-D5-024-29a, 191-D5-024-29b), which can be summarized as a general formula (5'-terminal fragment: "SGGSR", C-type 8-5'; 3'-terminal fragment: "YSCCS", C-type 8-3').

[0669] Truncated derivatives of the C-type CXCL12 binding nucleic acid 197-B2 ( Figure 8). Using a competitive pull-down binding assay (compared to 191-D5-007), it was demonstrated that 197-B2 had the same binding affinity to CXCL12 as 191-D5-007. The 5' and 3' terminal fragments were each shortened from 10 nucleotides (197-B2) to 5 nucleotides each (197-B2-005) without loss of binding affinity, whereby the nucleotides of the 5' terminal fragment and the 3' terminal fragment could fully hybridize with each other. If the 5' terminal ("GCGGG") and 3' terminal ("CCUGC") fragments of 197-B2-005 were replaced by "GCCGG" (5' terminal fragment) and "CCGGC" (3' terminal fragment) of 197-B2-006, the binding affinity to CXCL12 was fully maintained. Since 197-B2 and 191-D5-001 (and its derivatives) share the same core nucleotide sequence and several derivatives of 191-D5 were tested with 5' and 3' terminal fragments of 4 nucleotides in length, further truncation of the 5' and 3' terminal fragments was omitted. Two additional derivatives were designed, containing 6 nucleotides at the 5' and 3' ends (5' and 3' terminal fragments), respectively. The binding affinity of both molecules (197-B2-006-31a and 197-B2-006-31b) to CXCL12 was the same as shown for 191-D5-007 and 197-B2-006 ( Figure 8 ). The sequences of the 5'-terminal and 3'-terminal fragments of the 197-B2 derivative showed the best binding affinity to CXCL12, and the 5'-terminal and 3'-terminal fragments, respectively, comprised 5 nucleotides, which can be summarized as a general formula (5'-terminal fragment: "GCSGG", C-type-9-5'; 3'-terminal fragment: "CCKGC", C-type-9-3').

[0670] Preferred 5' and 3' fragments of the truncated derivatives of the C-type CXCL12 binding nucleic acid 191-D5-001 (5' terminal fragment: "SGGSR", C-type-8-5'; 3' terminal fragment: "YSCCS", C-type-8-3') and preferred 5' and 3' fragments of the truncated derivatives of 197-B2 (5' terminal fragment: "GCSGG", C-type-9-5'; 3' terminal fragment: "CCKGC", C-type-9-3'), the common preferred general formulas of the 5' terminal and 3' terminal fragments are "SSSSR" (5' terminal fragment, C-type-10-5') and "YSBSS (3' terminal fragment: C-type-10-3').

[0671] To prolong the plasma residence time of Spiegelmer in vivo, Spiegelmer 197-B2-006 and 191-D5-007 were covalently coupled to a 40 kDa polyethylene glycol (PEG) moiety at their 5' end as described in Chapter 2. PEGylated Spiegelmer 197-B2-006 and 191-D5-007 were analyzed in cell culture for in vitro chemotaxis. The PEG moiety had no effect on the potency of Spiegelmer to inhibit CXCL12-induced chemotaxis.

[0672] D-type CXCL12 binding nucleic acid molecules

[0673] In addition, three additional CXCL12-binding nucleic acids that do not share the CXCL12-binding motifs of "Type A", "Type B" and "Type C" were identified, referred to herein as "Type D". They were analyzed as aptamers using a pull-down binding assay ( Fig. 9 ).

[0674] It should be understood that Figures 1 to 9 Any sequence shown in is a nucleic acid molecule according to the invention, including truncated forms thereof, but also those extended forms thereof with the proviso that such truncated and extended (respectively) nucleic acid molecules are, however, still capable of binding to the target.

[0675] Example 2 Determination of binding constant (pull-down binding assay)

[0676] Direct pull-down binding assay

[0677] The affinity of the aptamers to biotinylated human D-CXCL12 was measured in a pull-down binding assay at 37°C. The aptamers were stained with [γ- 32 P]-labeled ATP (Hartmann Analytic, Braunschweig, Germany) was 5'-phosphate labeled by T4 polynucleotide kinase (Invitrogen, Karlsruhe, Germany). The specific radioactivity of the labeled aptamers was 200,000-800,000 cpm / pmol. The aptamers were expressed in selection buffer (20 mM Tris-HCl pH 7.4; 137 mM NaCl; 5 mM KCl; 1 mM MgCl 2 ; 1 mM CaCl 2After denaturation and renaturation of the aptamers at 10, 20, 30, or 40 pM in 1% [w / vol] Tween-20 (0.1% [w / vol] Tween-20) at 37°C, the aptamers were incubated with different amounts of biotinylated human D-CXCL12 for 4 to 12 h 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 used plastics or immobilization matrices. 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 UltralinkPlus particles (Pierce Biotechnology, Rockford, USA) that had been pre-equilibrated with selection buffer and resuspended in a total volume of 6 µl. The particles were kept in suspension for 30 min at the corresponding temperature in a thermomixer. After separation of the supernatant and appropriate washing, the immobilized radioactivity was quantified in a scintillation counter. The percentage of binding was plotted against the concentration of biotinylated human D-CXCL12 and the dissociation constant was obtained using a software algorithm (GRAFIT; Erithacus Software; Surrey, UK) assuming a 1:1 stoichiometry.

[0678] Competitive pull-down binding assay

[0679] To compare different D-CXCL12 binding aptamers, a competitive ranking assay was performed. For this purpose, the most affinity aptamer available was radiolabeled (see above) and used as a reference. After dedenaturation and renaturation, it was incubated with biotinylated human D-CXCL12 in 1 ml selection buffer at 37 °C, incubation 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) and no competition. Excess amounts of delabeled and renatured unlabeled D-RNA aptamer variants were added to labeled reference aptamers at different concentrations (e.g. 2, 10 and 50 nM) for parallel binding reactions. The aptamer to be tested competes with the reference aptamer for target binding, thereby reducing the binding signal according to its binding properties. The most active aptamer found in this assay can serve as a new reference for comparative analysis of additional aptamer variants.

[0680] Example 3 Surface Plasmon Resonance Measurement Binding Analysis

[0681] The Biacore 2000 instrument (Biacore AB, Uppsala, Sweden) was used to analyze the binding of Spiegelmer to human CXCL12α. When coupling of human CXCL12α was achieved via amine groups, human CXCL12α was dialyzed against water for 1–2 h (Millipore VSWP mixed cellulose esters; pore size 0.025 µM) to remove interfering amines. Prior to protein coupling, a CM4 sensor chip (Biacore AB, Uppsala, Sweden) was activated by injecting 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. Human MCP-1 or human CXCL12α was then injected at a concentration of 0.1–1.5 µg / ml at a flow rate of 2 µl / min until the instrument response was in the range of 1000–2000 RU (relative units). Unreacted NHS esters were deactivated by injecting 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 h until the baseline was stable. For all proteins, the 1:1 ratio was 5.1:1 in the selection buffer (Tris-HCl, 20 mM; NaCl, 137 mM; KCl, 5 mM; CaCl 2 , 1 mM; MgCl 2 Kinetic parameters and dissociation constants were evaluated by injecting a series of Spiegelmers at concentrations of 1000, 500, 250, 125, 62.5, 31.25, and 0 nM in 4% paraformaldehyde (pH 7.4). In all experiments, analysis was performed at 37°C using the Kinject command, which defined an association time of 180 s and a dissociation time of 360 s at a flow rate of 10 µl / min. Data analysis and dissociation constants (K) were performed using the Langmuir 1:1 stoichiometric fit algorithm using BIAevaluation 3.0 software (BIACORE AB, Uppsala, Sweden). D ) calculation.

[0682] Example 4 Analysis of the inhibition of CXCL12-induced chemotaxis by CXCL12 combined with Spiegelmer

[0683] The human T-cell leukemia cell line Jurkat, the human leukemic monocytic 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. While Jurkat cells do not express CXCR7, the leukemia cell lines BV-173 and U-937 tested positive for CXCR7 expression. All cells used were obtained from DSMZ (Braunschweig). All cell lines were cultured in RPMI 1640 medium with Glutamax (Invitrogen, Karlsruhe, Germany) containing 10% fetal bovine serum, 100 units / ml penicillin, and 100 µg / ml streptomycin (Invitrogen, Karlsruhe, Germany) at 37°C and 5% CO2. The day before the experiment, cells were plated at 0.3 × 10 6 / ml (Jurkat, U937, BV-173) or 0.75×10 6 / ml (Nalm-6) were inoculated in new T175 flasks.

[0684] For the experiments, cells were centrifuged (5 min at 300 g), 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). 6 / ml (Jurkat, U937, BV-173) or 2.67×10 6 / ml (Nalm-6). The cells were then allowed to migrate through the porous membrane of the filter plate towards a solution containing CXCL12 and various amounts of Spiegelmer for 3 hours. The stimulation solution (CXCL12 + various concentrations of Spiegelmer) was prepared as a 10x solution in a 0.2 ml low-profile 96-tube plate. 212µl HBH was pipetted into the lower compartment of the transport plate and 23.5µl of the stimulation solution was added. All conditions were set up in triplicate. After 20 to 30 minutes, the filter plate was inserted into the plate containing the stimulation solution and 1.33×10 6 / ml or 2.67×10 6 75 μl of cell suspension (1×10 5 or 2×10 5cells / well). The cells were then allowed to migrate for 3 hours at 37°C. For calibration, 0, 10 and 30µl of the cell suspension were added to 235, 225 and 205µl of HBH, respectively, in the wells of a separate 96-well plate. After 3 hours of incubation, the insert was removed and 30µl of Resazurin working solution (440µM in PBS) was added to the lower wells and the wells of the calibration plate. The plate was then incubated at 37°C for 2.5 hours. After incubation, 100μl from each well was transferred to a black 96-well plate.

[0685] For evaluation, fluorescence values ​​were corrected for background fluorescence (no cells in the well). The difference between experimental conditions with and without CXCL12 was then calculated. The values ​​for samples without Spiegelmer (CXCL12 only) were set to 100% and the values ​​for samples with Spiegelmer were calculated as a percentage of CXCL12. For dose-response curves, the percentage values ​​were plotted against the Spiegelmer concentration and the IC was determined graphically from the resulting curves. 50 value (Spiegelmer concentration with 50% of the activity in the absence of Spiegelmer).

[0686] result

[0687] Human CXCL12 was found to stimulate the migration of Jurkat cells in a dose-dependent manner, with half-maximal stimulation at approximately 0.3 nM.

[0688] Human CXCL12 was found to stimulate the migration of cells of the human leukemic monocytic lymphoma cell line U937 in a dose-dependent manner, with half-maximal stimulation at approximately 3 nM.

[0689] Human CXCL12 was found to stimulate the migration of cells of the human pre-B cell leukemia cell line BV-173 in a dose-dependent manner, with half-maximal stimulation at approximately 3 nM.

[0690] Human CXCL12 was found to stimulate the migration of cells of the human pre-B ALL cell line Nalm-6 in a dose-dependent manner, with half-maximal stimulation at approximately 0.3 nM.

[0691] When cells were allowed to migrate towards solutions containing human CXCL12 plus increasing concentrations of CXCL12 binding Spiegelmers, a dose-dependent inhibition was observed. The respective IC50s of the tested Spiegelmers were determined as specified in Example 1 in Jurkat cells, a human T-cell leukemia cell line. For example, for the CXCL12 binding Spiegelmer NOX-A12 (also known as 193-G2-012-5'-PEG), the determined IC50 was 0.2 nM ( Fig.10When a nonspecific control Spiegelmer was used instead of the CXCL12-binding Spiegelmer, no inhibitory effect was observed up to 1 µM.

[0692] Inhibition of CXCL12-induced chemotaxis by CXCL12 combined with Spiegelmer NOX-A12 was also observed in three other different leukemic cell types: the human leukemic monocytic lymphoma cell line U937 ( Fig.11 B), human pre-B cell leukemia cell line BV-173 ( Fig.12 ) and the human pre-B ALL cell line Nalm-6 ( Fig.11 A). In addition, the inventors have evidence that primary chronic lymphocytic leukemia cells migrate towards CXCL12 and that CXCL12-dependent chemotaxis is effectively blocked by NOX-A12.

[0693] The leukemia cell lines BV-173 and U-937 also tested positive for CXCR7 expression.As described in Example 5, the efficacy of SDF in combination with Spiegelmer NOX-A12 to block the interaction of CXCL12 and CXCR7 was determined.

[0694] Example 5 Inhibition of CXCR7 activation by CXCL12 combined with Spiegelmer NOX-A12

[0695] In addition to CXCR4, CXCL12 also binds to the chemokine receptor CXCR7. The inhibitory potential of CXCL12 in combination with Spiegelmer NOX-A12 against CXCR7 was tested in a complementation assay in which CHO cells stably expressed CXCR7 and β-arrestin, both fused to a fragment of β-galactosidase (PathHunterTM-β-arrestin assay, DiscoveRX, CA, USA). Upon CXCL12 binding, β-arrestin complexes with CXCR7 and thus leads to complementation and activation of β-galactosidase measured with a chemiluminescent substrate.

[0696] method

[0697] PathHunter eXpress CHO-K1 human CXCR7β-arrestin cells were plated in OCC2 medium for 48 h and stimulated for 90 min with 10 nM CXCL12 and various concentrations of CXCL12 combined with Spiegelmer NOX-A12. After stimulation, signals were detected using the PathHunter assay kit and the manufacturer's recommended protocol (DiscoveRX, CA, USA).

[0698] result

[0699] Stimulation of β-galactosidase and hence CXCR7 activation by 10 nM human CXCL12 can be effectively blocked by CXCL12 combined with Spiegelmer NOX-A12 with an IC50 of 5.4 nM ( Fig.13 ).

[0700] Example 6 Clinical study using triple therapy

[0701] The arms in the Phase 1 / 2 study were designed to evaluate the safety of NOX-A12 plus bevacizumab when administered concurrently with radiation therapy in patients with glioblastoma, to explore the benefit of these combinations on progression-free survival when administered in addition to radiation therapy, and to provide an estimate of the effect on overall survival.

[0702] This study is expected to promote the understanding of the important role of CXCL12 in neovascularization by recruiting endothelial cells and other bone marrow-derived pro-angiogenic cells through a CXCR4- and CXCR7-dependent mechanism.

[0703] The corresponding study that conducted the NOX-A12 plus bevacizumab arm is titled “A single-arm, dose-escalation phase 1 / 2 study of olaptesed pegol (NOX-A12) plus radiation in patients with first-line glioblastoma who have an unmethylated MGMT promoter and are inoperable or partially resectable, with a three-arm expansion group that includes completely resected patients and combination with bevacizumab or pembrolizumab.”

[0704] Details of the study are as follows.

[0705] Patient Population: Patients with newly diagnosed glioblastoma (WHO grade IV) with unmethylated MGMT promoter status after incomplete tumor resection or no tumor resection

[0706] Study type: Multicenter, prospective, open-label

[0707] Research objectives:

[0708] Primary: To investigate the safety of olaptesed pegol plus radiation therapy or olaptesed pegol plus radiation therapy and bevacizumab or pembrolizumab in patients with newly diagnosed glioblastoma with unmethylated MGMT promoter status

[0709] Secondary objectives: (i) to investigate the efficacy of olapted pegol in combination with radiotherapy and bevacizumab in patients with glioblastoma with unmethylated MGMT promoter status who are not amenable to tumor resection (biopsy only) or after incomplete tumor resection; (ii) to investigate the pharmacokinetics of olapted pegol during continuous administration; (iii) to monitor symptoms (NANO) and QoL

[0710] Study endpoints:

[0711] Primary: Safety (adverse events)

[0712] Secondary: PFS at 6 months (PFS-6); median progression-free survival (mPFS); median overall survival (mOS); tumor vascularization on each vascular MRI scan at baseline and at 2, 4, and 6 months; topography of recurrence; determination of maximum tolerated dose (MTD); definition of recommended phase 2 dose (RP2D); NOX-A12 plasma levels at steady state; NANO assessment; quality of life

[0713] Inclusion criteria:

[0714] 1. Written Informed Consent

[0715] 2. Age ≥ 18 years old

[0716] 3. The patient consents to diagnostic and scientific examination of glioblastoma tissue obtained during previous surgery or biopsy (e.g., MGMT promoter analysis, cytogenetic markers such as IDH-1 mutation, etc.)

[0717] 4. Patient consent for subcutaneous port implantation

[0718] 5. Newly diagnosed, histologically confirmed, supratentorial WHO grade IV glioblastoma

[0719] 6. Status after biopsy or incomplete resection (based on detectable residual tumor on postoperative T1-weighted, contrast-enhanced MRI scan) (Arm A)

[0720] 7. Unmethylated MGMT promoter status

[0721] 8. Maximum Eastern Cooperative Oncology Group (ECOG) score of 2

[0722] 9. Estimated minimum life expectancy 3 months

[0723] 10. The dose of corticosteroids has been stable or reduced in the week before inclusion

[0724] 11. The following laboratory parameters should be within the specified range:

[0725] a. Total bilirubin ≤ 1.5 times the upper limit of normal (ULN)

[0726] b. Creatinine ≤ 1.5 times ULN or glomerular filtration rate ≥ 60 mL / min / 1.73m²

[0727] c.ALT (alanine aminotransferase) ≤ 3 times ULN

[0728] d. AST (aspartate aminotransferase) ≤ 3 times ULN

[0729] 12. Female patients of childbearing potential must have a negative serum pregnancy test within 21 days before enrollment and agree to use a highly effective birth control method (with a failure rate of less than 1% per year when used continuously and correctly, such as contraceptive implants, vaginal rings, sterilization, or abstinence) during and within 6 months after the last dose of medication (more frequent pregnancy testing may be performed if required by local regulations)

[0730] 13. If sexually active and have FCBP, male patients must use an effective barrier contraceptive method during the study and for 6 months after the last dose.

[0731] Exclusion criteria:

[0732] 1. Failure to understand and cooperate throughout the research process, or inability or unwillingness to comply with research requirements

[0733] 2. Participation in any clinical study administering an investigational drug or investigational therapy within 30 days of the screening visit or observation period of a competing study

[0734] 3. Contraindications or known allergic reactions to MRI contrast agents, bevacizumab, olaptesed pegol, or polyethylene glycol

[0735] 4. Planned Hypofractionated Radiotherapy

[0736] 5. Cytostatic therapy (chemotherapy) within the past 5 years

[0737] 6. History of other cancers (except adequately treated basal or squamous cell skin cancer, cervical cancer in situ, or other cancers from patients who have been disease-free for ≥5 years)

[0738] 7. Currently active secondary malignancies

[0739] 8. Clinically significant or uncontrolled cardiovascular disease, including

[0740] a. Myocardial infarction in the past 12 months

[0741] b. Uncontrolled angina

[0742] c. Congestive heart failure (New York Heart Association functional class ≥2)

[0743] d. Diagnosed or suspected congenital long QT syndrome

[0744] e. QTc prolongation (>470 ms) on electrocardiogram before entering the study

[0745] f. Uncontrolled hypertension (blood pressure ≥160 / 95 mmHg)

[0746] g. Baseline ECG heart rate <50 / min

[0747] h. History of any clinically significant type of ventricular arrhythmia (eg, ventricular tachycardia, ventricular fibrillation, or torsade de pointes)

[0748] i. Cerebrovascular accident

[0749] 9. Previous radiation therapy to the head

[0750] 10. Any other previous or concomitant experimental glioblastoma treatment

[0751] 11. Placement of Gliadel® wafers, seeds or ferromagnetic nanoparticles

[0752] 12. Patients with a history of arterial or venous thrombosis (or any other disease) requiring permanent anticoagulant therapy

[0753] 13. Pregnancy or breastfeeding

[0754] 14. Uncontrolled concurrent illness, including but not limited to ongoing or active infection, chronic liver disease (e.g., cirrhosis, hepatitis), diabetes, or subjects have 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 disease, or psychiatric illness / social conditions that would limit compliance with study requirements. Patients must not have any clinically relevant (in the opinion of the treating investigator) illness (other than glioma) that would interfere with the conduct of the study or study assessments.

[0755] 15. Prolongation of coagulation factors ≥ 2.5 times ULN

[0756] 16. Treatment not started within 6 weeks after the first biopsy or surgery for glioblastoma

[0757] 17. Have you been enrolled in this study before?

[0758] Treatments in the arms to combine NOX-A12 with radiation therapy and bevacizumab:

[0759]

[0760] Treatment options also see Fig.14 .

[0761] result

[0762] All 6 patients planned to receive NOX-A12, radiation therapy, and bevacizumab have been enrolled, and all patients are receiving NOX-A12 and bevacizumab. MRI assessment of tumor response has been performed for up to 27 weeks in 1 patient, up to 18 weeks in 2 patients, and up to 9 weeks in 5 patients. For all patients with available data, a reduction of more than 50% in the T1 enhanced signal (SPDP, the sum of the products of the diameters of the tumor lesions) was observed, and therefore all patients showed partial responses, see Table 2 below.

[0763] Table 2:

[0764]

[0765] CFB = change from baseline; CFN = change from nadir; pPR = preliminary partial response; cPR = confirmed partial response; SPDP = sum of products of diameters of tumor lesions

[0766] Treatment options such as Fig.14 shown.

[0767] Example 7 Clinical study using triple therapy also including chemotherapy

[0768] A similar clinical study as described in Example 6 was conducted on glioblastoma patients, but in this study, the patients also received the chemotherapeutic agent temozolomide, which is the standard of care for glioblastoma. The title of the study was "A single-arm phase 1 / 2 study of olaptated pegol (NOX-A12) combined with radiation therapy, bevacizumab, and temozolomide in first-line glioblastoma patients with methylated and unmethylated MGMT promoters."

[0769] The study evaluated the safety of NOX-A12 in combination with bevacizumab and temozolomide when administered concurrently with radiation therapy in patients with glioblastoma, explored the benefit of these combinations on progression-free survival when administered in addition to radiation therapy, and provided an estimate of the effect on overall survival.

[0770] The differences between this study and the study described in Example 6 are:

[0771] Patient population: Patients with newly diagnosed glioblastoma (WHO grade IV) with methylated or unmethylated MGMT promoter status and all extents of tumor resection.

[0772] Treatment: In this study, NOX-A12 was combined with radiation therapy, bevacizumab, and temozolomide.

[0773]

[0774] Based on the non-overlapping modes of action of NOX-A12, bevacizumab or radiation therapy with temozolomide, there is no reason to expect relatively different results for this study compared to the study in Example 6. However, since patients with temozolomide-sensitive promoter status with MGMT methylation were also included in this study, some additional benefit was seen for this group of patients.

[0775] Treatment options such as Fig.15 shown.

[0776] Example 8 Clinical study using triple therapy with NOX-A12, radiation therapy and bevacizumab

[0777] The arms in the Phase 1 / 2 study were designed to evaluate the safety of NOX-A12 combined with bevacizumab when administered concurrently with radiation therapy in patients with glioblastoma, to explore the benefit of these combinations on progression-free survival when administered in addition to radiation therapy, and to provide an estimate of the effect on overall survival.

[0778] This study is expected to advance the understanding of the important role that CXCL12 plays in neovascularization by recruiting endothelial cells and other bone marrow-derived proangiogenic cells through a CXCR4- and CXCR7-dependent mechanism. Notably, irradiation also increased CXCL12 expression. In addition, this data is expected to guide the further clinical development of NOX-A12.

[0779] The corresponding study that conducted the NOX-A12 plus bevacizumab arm is titled “A single-arm, dose-escalation phase 1 / 2 study of olaptesed pegol (NOX-A12) plus radiation in patients with first-line glioblastoma who have an unmethylated MGMT promoter and are inoperable or partially resectable, with a three-arm expansion group that includes completely resected patients and combination with bevacizumab or pembrolizumab.”

[0780] Details of the study are as follows.

[0781] Patient Population: Patients with newly diagnosed glioblastoma (WHO grade IV) with unmethylated MGMT promoter status after incomplete tumor resection or no tumor resection

[0782] Study type: Multicenter, prospective, open-label

[0783] Research objectives:

[0784] Primary: To investigate the safety of olaptesed pegol combined with radiation therapy and bevacizumab in patients with newly diagnosed glioblastoma with unmethylated MGMT promoter status

[0785] Secondary: (i) To investigate the efficacy of olapted pegol combined with radiotherapy and bevacizumab in patients with glioblastoma with unmethylated MGMT promoter status; (ii) To investigate the pharmacokinetics of olapted pegol during continuous administration; (iii) To monitor symptoms (NANO) and QoL

[0786] Study endpoints:

[0787] Primary: Safety (adverse events)

[0788] Secondary: PFS at 6 months (PFS-6); median progression-free survival (mPFS); median overall survival (mOS); tumor vascularization on each vascular MRI scan at baseline and at 2, 4, and 6 months; topography of recurrence; determination of maximum tolerated dose (MTD); definition of recommended phase 2 dose (RP2D); NOX-A12 plasma levels at steady state; NANO assessment; quality of life

[0789] Inclusion criteria:

[0790] 1. Written Informed Consent

[0791] 2. Age ≥ 18 years old

[0792] 3. The patient consents to diagnostic and scientific examination of glioblastoma tissue obtained during previous surgery or biopsy (e.g., MGMT promoter analysis, cytogenetic markers such as IDH-1 mutation, etc.)

[0793] 4. Patient consent for subcutaneous port implantation

[0794] 5. Newly diagnosed, histologically confirmed, supratentorial WHO grade IV glioblastoma

[0795] 6. Status after biopsy or incomplete resection (based on detectable residual tumor on postoperative T1-weighted, contrast-enhanced MRI scan)

[0796] 7. Unmethylated MGMT promoter status

[0797] 8. Maximum Eastern Cooperative Oncology Group (ECOG) score of 2

[0798] 9. Estimated minimum life expectancy 3 months

[0799] 10. The dose of corticosteroids has been stable or reduced in the week before inclusion

[0800] 11. The following laboratory parameters should be within the specified range:

[0801] a. Total bilirubin ≤ 1.5 times the upper limit of normal (ULN)

[0802] b. Creatinine ≤ 1.5 times ULN or glomerular filtration rate ≥ 60 mL / min / 1.73m²

[0803] c.ALT (alanine aminotransferase) ≤ 3 times ULN

[0804] d. AST (aspartate aminotransferase) ≤ 3 times ULN

[0805] 12. Female patients of childbearing potential must have a negative serum pregnancy test within 21 days before enrollment and agree to use a highly effective birth control method (with a failure rate of less than 1% per year when used continuously and correctly, such as contraceptive implants, vaginal rings, sterilization, or abstinence) during and within 6 months after the last dose of medication (more frequent pregnancy testing may be performed if required by local regulations)

[0806] 13. If sexually active and have FCBP, male patients must use an effective barrier contraceptive method during the study and for 6 months after the last dose.

[0807] Exclusion criteria:

[0808] 1. Failure to understand and cooperate throughout the research process, or inability or unwillingness to comply with research requirements

[0809] 2. Participation in any clinical study administering an investigational drug or investigational therapy within 30 days of the screening visit or observation period of a competing study

[0810] 3. Contraindications or known allergic reactions to MRI contrast agents, bevacizumab, olaptesed pegol, or polyethylene glycol

[0811] 4. Planned Hypofractionated Radiotherapy

[0812] 5. Cytostatic therapy (chemotherapy) within the past 5 years

[0813] 6. History of other cancers (except adequately treated basal or squamous cell skin cancer, cervical cancer in situ, or other cancers from patients who have been disease-free for ≥5 years)

[0814] 7. Currently active secondary malignancies

[0815] 8. Clinically significant or uncontrolled cardiovascular disease, including

[0816] a. Myocardial infarction in the past 12 months

[0817] b. Uncontrolled angina

[0818] c. Congestive heart failure (New York Heart Association functional class ≥2)

[0819] d. Diagnosed or suspected congenital long QT syndrome

[0820] e. QTc prolongation (>470 ms) on electrocardiogram before study entry

[0821] f. Uncontrolled hypertension (blood pressure ≥160 / 95 mmHg)

[0822] g. Baseline ECG heart rate <50 / min

[0823] h. History of any clinically significant type of ventricular arrhythmia (eg, ventricular tachycardia, ventricular fibrillation, or torsade de pointes)

[0824] i. Cerebrovascular accident

[0825] 9. Previous radiation therapy to the head

[0826] 10. Any other previous or concomitant experimental glioblastoma treatment

[0827] 11. Placement of Gliadel® wafers, seeds or ferromagnetic nanoparticles

[0828] 12. Patients with a history of arterial or venous thrombosis (or any other disease) requiring permanent anticoagulant therapy

[0829] 13. Pregnancy or breastfeeding

[0830] 14. Uncontrolled concurrent illness, including but not limited to ongoing or active infection, chronic liver disease (e.g., cirrhosis, hepatitis), diabetes, or subjects have 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 disease, or psychiatric illness / social conditions that would limit compliance with study requirements. Patients must not have any clinically relevant (in the opinion of the treating investigator) illness (other than glioma) that would interfere with the conduct of the study or study assessments.

[0831] 15. Prolongation of coagulation factors ≥ 2.5 times ULN

[0832] 16. Treatment not started within 6 weeks after the first biopsy or surgery for glioblastoma

[0833] 17. Have you been enrolled in this study before?

[0834] Treatments in the arms to combine NOX-A12 with radiation therapy and bevacizumab:

[0835]

[0836] Treatment options also see Fig.14 .

[0837] result

[0838] All 6 patients who were scheduled to receive NOX-A12, radiation therapy, and bevacizumab were enrolled, with 4 patients continuing to receive NOX-A12 and bevacizumab beyond the originally planned 26 weeks at the discretion of the treating physician and the patient. One patient died.

[0839] Safety: The treatment was well tolerated and safe, with no dose-limiting toxicities and no treatment-related deaths.

[0840] Efficacy: MRI-evaluable tumor response was achieved up to 43 weeks in 1 patient, up to 35 weeks in 1 patient, up to 27 weeks in 3 patients, and up to 18 weeks in 1 patient. Only 1 patient (patient 6) experienced progressive disease (PD) after initial stable disease (SD) at week 9 (due to the formation of CSF metastases), whereas target lesion control was maintained (see Table 3).

[0841] Five of the six patients achieved a partial response (PR) at week 9 based on modified response assessment in neuro-oncology (mRANO), and all PRs remain durable at assessments to date: patient 1 showed a partial response until week 43; patient 2 showed a partial response until week 35; and patients 3 to 5 showed partial responses until week 27; the current median follow-up is 7.6 months. Longitudinal Neurological Assessment in Neuro-Oncology (NANO) is a quantitative assessment of nine relevant neurologic domains based on direct observation and testing by the investigator - Five of the six patients showed stable neurologic function at assessments to date: patient 1 had no change until week 43; patient 2 had no change until week 35; patients 3 and 4 had no change until week 27, and patient 5 showed improvement at week 9 with further improvement at week 27. These mRANO responses and NANO scores from treatment weeks 9 to 43 were consistent with the results from the 2018–2019 study. Fig. 20 Described in.

[0842] Advanced MRI imaging, neurological function, and quality of life (QoL) assessments showed:

[0843] Improved apparent diffusion coefficient (ADC), with a mean best change of +20.6% (-24.5% to +59.1%);

[0844] Improved relative cerebral blood volume (rCBV), with a mean best change of -77.9% (-50% to -100%);

[0845] Improved segmentation of highly perfused tumors (FTB 高 ), the average best change was -87.2% (-61.3% to -100%);

[0846] Non-target lesion responses were excellent, with a mean best change of -93.8% (-81.4% to -100%);

[0847] Clinician-reported NANO scores were stable or slightly improved, with a mean best change of -5.6% (0% to -33.3%);

[0848] Improved patient-reported outcomes

[0849] o Overall functional score (OFS), with a mean best change of 40.7% (-12.4% to 224.4%);

[0850] o Global Health Score (GHS), with a mean best change of 31.3% (-8.3% to 100%)

[0851] The results of these advanced MRI imaging, neurological function, and quality of life (QoL) assessments are presented graphically in Fig.21Described in.

[0852] Table 3 Tumor size determined by MRI evaluation (SPDP)

[0853]

[0854] CFB = change from baseline; CFN = change from nadir; pPR = preliminary partial response; cPR = confirmed partial response; SD = stable disease; PD = progressive disease; SPDP = sum of products of diameters of tumor lesions; nya = not available

[0855] Example 9 Clinical study of triple therapy using NOX-A12, radiation therapy and bevacizumab in patients with glioblastoma

[0856] The arms in the Phase 1 / 2 study were designed to evaluate the safety of NOX-A12 combined with bevacizumab when administered concurrently with radiation therapy in patients with glioblastoma, to explore the benefit of these combinations on progression-free survival when administered in addition to radiation therapy, and to provide an estimate of the effect on overall survival.

[0857] This study is expected to advance the understanding of the important role that CXCL12 plays in neovascularization by recruiting endothelial cells and other bone marrow-derived proangiogenic cells through a CXCR4- and CXCR7-dependent mechanism. Notably, irradiation also increased CXCL12 expression. In addition, this data is expected to guide the further clinical development of NOX-A12.

[0858] The corresponding study that conducted the NOX-A12 plus bevacizumab arm is titled “A single-arm, dose-escalation phase 1 / 2 study of olaptesed pegol (NOX-A12) plus radiation in patients with first-line glioblastoma who have an unmethylated MGMT promoter and are inoperable or partially resectable, with a three-arm expansion group that includes completely resected patients and combination with bevacizumab or pembrolizumab.”

[0859] Details of the study are as follows.

[0860] Patient population: Patients with newly diagnosed glioblastoma (WHO grade IV) with unmethylated MGMT promoter status after incomplete or no tumor resection

[0861] Study type: Multicenter, prospective, open-label

[0862] Research objectives:

[0863] Primary: To investigate the safety of olaptesed pegol combined with radiation therapy and bevacizumab in patients with newly diagnosed glioblastoma with an unmethylated MGMT promoter status

[0864] Secondary: (i) To investigate the efficacy of olapted pegol combined with radiotherapy and bevacizumab in patients with glioblastoma with unmethylated MGMT promoter status; (ii) To investigate the pharmacokinetics of olapted pegol during continuous administration; (iii) To monitor symptoms (NANO) and QoL

[0865] Study endpoints:

[0866] Primary: Safety (adverse events)

[0867] Secondary: PFS at 6 months (PFS-6); median progression-free survival (mPFS); median overall survival (mOS); tumor vascularization on each vascular MRI scan at baseline and at 2, 4, and 6 months; topography of recurrence; determination of maximum tolerated dose (MTD); definition of recommended phase 2 dose (RP2D); NOX-A12 plasma levels at steady state; NANO assessment; quality of life

[0868] Inclusion criteria:

[0869] 1. Written Informed Consent

[0870] 2. Age ≥ 18 years old

[0871] 3. The patient consents to diagnostic and scientific examination of glioblastoma tissue obtained during previous surgery or biopsy (e.g., MGMT promoter analysis, cytogenetic markers such as IDH-1 mutation, etc.)

[0872] 4. Patient consent for subcutaneous port implantation

[0873] 5. Newly diagnosed, histologically confirmed, supratentorial WHO grade IV glioblastoma

[0874] 6. Status after biopsy or incomplete resection (based on detectable residual tumor on postoperative T1-weighted, contrast-enhanced MRI scan)

[0875] 7. Unmethylated MGMT promoter status

[0876] 8. Maximum Eastern Cooperative Oncology Group (ECOG) score of 2

[0877] 9. Estimated minimum life expectancy 3 months

[0878] 10. The dose of corticosteroids has been stable or reduced in the week before inclusion

[0879] 11. The following laboratory parameters should be within the specified range:

[0880] a. Total bilirubin ≤ 1.5 times the upper limit of normal (ULN)

[0881] b. Creatinine ≤ 1.5 times ULN or glomerular filtration rate ≥ 60 mL / min / 1.73m²

[0882] c.ALT (alanine aminotransferase) ≤ 3 times ULN

[0883] d. AST (aspartate aminotransferase) ≤ 3 times ULN

[0884] 12. Female patients of childbearing potential must have a negative serum pregnancy test within 21 days before enrollment and agree to use a highly effective birth control method (with a failure rate of less than 1% per year when used continuously and correctly, such as contraceptive implants, vaginal rings, sterilization, or abstinence) during and within 6 months after the last dose of medication (more frequent pregnancy testing may be performed if required by local regulations)

[0885] 13. If sexually active and have FCBP, male patients must use an effective barrier contraceptive method during the study and for 6 months after the last dose.

[0886] Exclusion criteria:

[0887] 1. Failure to understand and cooperate throughout the research process, or inability or unwillingness to comply with research requirements

[0888] 2. Participation in any clinical study administering an investigational drug or investigational therapy within 30 days of the screening visit or observation period of a competing study

[0889] 3. Contraindications or known allergic reactions to MRI contrast agents, bevacizumab, olaptesed pegol, or polyethylene glycol

[0890] 4. Planned Hypofractionated Radiotherapy

[0891] 5. Cytostatic therapy (chemotherapy) within the past 5 years

[0892] 6. History of other cancers (except adequately treated basal or squamous cell skin cancer, cervical cancer in situ, or other cancers from patients who have been disease-free for ≥5 years)

[0893] 7. Currently active secondary malignancies

[0894] 8. Clinically significant or uncontrolled cardiovascular disease, including

[0895] a. Myocardial infarction in the past 12 months

[0896] b. Uncontrolled angina

[0897] c. Congestive heart failure (New York Heart Association functional class ≥2)

[0898] d. Diagnosed or suspected congenital long QT syndrome

[0899] e. QTc prolongation (>470 ms) on electrocardiogram before study entry

[0900] f. Uncontrolled hypertension (blood pressure ≥160 / 95 mmHg)

[0901] g. Baseline ECG heart rate <50 / min

[0902] h. History of any clinically significant type of ventricular arrhythmia (eg, ventricular tachycardia, ventricular fibrillation, or torsade de pointes)

[0903] i. Cerebrovascular accident

[0904] 9. Previous radiation therapy to the head

[0905] 10. Any other previous or concomitant experimental glioblastoma treatment

[0906] 11. Placement of Gliadel® wafers, seeds or ferromagnetic nanoparticles

[0907] 12. Patients with a history of arterial or venous thrombosis (or any other disease) requiring permanent anticoagulant therapy

[0908] 13. Pregnancy or breastfeeding

[0909] 14. Uncontrolled concurrent illness, including but not limited to ongoing or active infection, chronic liver disease (e.g., cirrhosis, hepatitis), diabetes, or subjects have 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 disease, or psychiatric illness / social conditions that would limit compliance with study requirements. Patients must not have any clinically relevant (in the opinion of the treating investigator) illness (other than glioma) that would interfere with the conduct of the study or study assessments.

[0910] 15. Prolongation of coagulation factors ≥ 2.5 times ULN

[0911] 16. Treatment not started within 6 weeks after the first biopsy or surgery for glioblastoma

[0912] 17. Have you been enrolled in this study before?

[0913] Treatments in the arms to combine NOX-A12 with radiation therapy and bevacizumab:

[0914]

[0915] Treatment options also see Fig.14 .

[0916] result

[0917] All 6 patients who were scheduled to receive NOX-A12, radiation therapy, and bevacizumab were enrolled, and 4 of them continued to receive NOX-A12 and bevacizumab beyond the originally planned 26 weeks at the discretion of the treating physician and the patient. One patient died.

[0918] Safety: The treatment was well tolerated and safe, with no dose-limiting toxicities and no treatment-related deaths.

[0919] Efficacy: MRI-evaluable tumor response was achieved up to 43 weeks in 1 patient, up to 35 weeks in 1 patient, up to 27 weeks in 3 patients, and up to 18 weeks in 1 patient. Only 1 patient (patient 6) maintained target lesion control after progressive disease (PD) at week 9 due to the development of CSF metastases after initial stable disease (SD) (see Table 4).

[0920] Five of the six patients achieved a partial response (PR) at week 9 based on modified response assessment in neuro-oncology (mRANO), and all PRs remain durable at assessments to date: patient 1 showed a partial response until week 43; patients 2 and 3 showed a partial response until week 35; and patients 4 and 5 showed a partial response until week 27; the current median follow-up is 7.6 months. Longitudinal Neurological Assessment in Neuro-Oncology (NANO) is a quantitative assessment of nine relevant neurologic domains based on direct observation and testing by the investigator - Four of the six patients showed stable neurological function at assessments to date: patient 1 had no change until week 43; patient 2 had no change until week 35; patient 3 had no change until week 27, with an increase in score at week 35, which may be due to intracranial hemorrhage from old infarction; patient 4 had no change until week 27, and patient 5 showed a decrease in score at week 9, with a further decrease at week 27. These mRANO responses and NANO scores from treatment weeks 9 to 43 were consistent with the results of the current study. Fig. 22 Described in.

[0921] Advanced MRI imaging, neurological function, and quality of life (QoL) assessments compared with baseline showed:

[0922] Improved apparent diffusion coefficient (ADC), with a mean best change of +20.6% (-24.5% to +59.1%);

[0923] Improved relative cerebral blood volume (rCBV), with a mean best change of -77.9% (-50% to -100%);

[0924] Improved segmentation of highly perfused tumors (FTB 高 ), the average best change was -87.2% (-61.3% to -100%);

[0925] Non-target lesion responses were excellent, with a mean best change of -93.8% (-81.4% to -100%);

[0926] Clinician-reported NANO scores were stable or slightly improved, with a mean best change of -5.6% (0% to -33.3%);

[0927] Improved patient-reported outcomes

[0928] o Overall functional score (OFS), with a mean best change of 40.7% (-12.4% to 224.4%);

[0929] o Global Health Score (GHS), with a mean best change of 31.3% (-8.3% to 100%)

[0930] The results of these advanced MRI imaging, neurological function, and quality of life (QoL) assessments are presented graphically in Fig.23 is described as the maximum optimal change compared to baseline.

[0931] Patients treated with the triple combination of NOX-A12, radiation therapy, and bevacizumab showed deeper responses than those treated with the double combination of NOX-A12 and radiation therapy alone, and deeper responses than those treated with standard therapy: Target lesion response is expressed as the maximum change from baseline in the sum of the perpendicular diameter products (SPDP) in patients treated with the triple combination of NOX-A12, radiation therapy, and bevacizumab. Fig.24 Description: For patients treated with a double combination of NOX-A12 and radiation therapy Fig.25 description; and a historical reference cohort of patients treated with standard of care (radiotherapy and the chemotherapeutic agent temozolomide) in Fig.26 Described in.

[0932] Table 4 Tumor size determined by MRI evaluation (SPDP)

[0933]

[0934] CFB = change from baseline; CFN = change from nadir; pPR = preliminary partial response; cPR = confirmed partial response; SD = stable disease; PD = progressive disease; SPDP = sum of products of diameters of tumor lesions; nya = not available

[0935] Example 10 Clinical study of triple therapy with NOX-A12, radiotherapy and bevacizumab in patients with glioblastoma - follow-up based on survival

[0936] The arms in the Phase 1 / 2 study were designed to evaluate the safety of NOX-A12 combined with bevacizumab when administered concurrently with radiation therapy in patients with glioblastoma, to explore the benefit of these combinations on progression-free survival when administered in addition to radiation therapy, and to provide an estimate of the effect on overall survival.

[0937] This study is expected to advance the understanding of the important role that CXCL12 plays in neovascularization by recruiting endothelial cells and other bone marrow-derived proangiogenic cells through a CXCR4- and CXCR7-dependent mechanism. Notably, irradiation also increased CXCL12 expression. In addition, this data is expected to guide the further clinical development of NOX-A12.

[0938] The corresponding study that conducted the NOX-A12 plus bevacizumab arm is titled “A single-arm, dose-escalation phase 1 / 2 study of olaptesed pegol (NOX-A12) plus radiation in patients with first-line glioblastoma who have an unmethylated MGMT promoter and are inoperable or partially resectable, with a three-arm expansion group that includes completely resected patients and combination with bevacizumab or pembrolizumab.”

[0939] Details of the study are as follows.

[0940] Patient population: Patients with newly diagnosed glioblastoma (WHO grade IV) with unmethylated MGMT promoter status after incomplete or no tumor resection

[0941] Study type: Multicenter, prospective, open-label

[0942] Research objectives:

[0943] Primary: To investigate the safety of olaptesed pegol combined with radiation therapy and bevacizumab in patients with newly diagnosed glioblastoma with an unmethylated MGMT promoter status

[0944] Secondary: (i) To investigate the efficacy of olapted pegol combined with radiotherapy and bevacizumab in patients with glioblastoma with unmethylated MGMT promoter status; (ii) To investigate the pharmacokinetics of olapted pegol during continuous administration; (iii) To monitor symptoms (NANO) and QoL

[0945] Study endpoints:

[0946] Primary: Safety (adverse events)

[0947] Secondary: PFS at 6 months (PFS-6); median progression-free survival (mPFS); median overall survival (mOS); tumor vascularization on each vascular MRI scan at baseline and at 2, 4, and 6 months; topography of recurrence; determination of maximum tolerated dose (MTD); definition of recommended phase 2 dose (RP2D); NOX-A12 plasma levels at steady state; NANO assessment; quality of life

[0948] Inclusion criteria:

[0949] 1. Written Informed Consent

[0950] 2. Age ≥ 18 years old

[0951] 3. The patient consents to diagnostic and scientific examination of glioblastoma tissue obtained during previous surgery or biopsy (e.g., MGMT promoter analysis, cytogenetic markers such as IDH-1 mutation, etc.)

[0952] 4. Patient consent for subcutaneous port implantation

[0953] 5. Newly diagnosed, histologically confirmed, supratentorial WHO grade IV glioblastoma

[0954] 6. Status after biopsy or incomplete resection (based on detectable residual tumor on postoperative T1-weighted, contrast-enhanced MRI scan)

[0955] 7. Unmethylated MGMT promoter status

[0956] 8. Maximum Eastern Cooperative Oncology Group (ECOG) score of 2

[0957] 9. Estimated minimum life expectancy 3 months

[0958] 10. The dose of corticosteroids has been stable or reduced in the week before inclusion

[0959] 11. The following laboratory parameters should be within the specified range:

[0960] a. Total bilirubin ≤ 1.5 times the upper limit of normal (ULN)

[0961] b. Creatinine ≤ 1.5 times ULN or glomerular filtration rate ≥ 60 mL / min / 1.73m²

[0962] c.ALT (alanine aminotransferase) ≤ 3 times ULN

[0963] d. AST (aspartate aminotransferase) ≤ 3 times ULN

[0964] 12. Female patients of childbearing potential must have a negative serum pregnancy test within 21 days before enrollment and agree to use a highly effective birth control method (with a failure rate of less than 1% per year when used continuously and correctly, such as contraceptive implants, vaginal rings, sterilization, or abstinence) during and within 6 months after the last dose of medication (more frequent pregnancy testing may be performed if required by local regulations)

[0965] 13. If sexually active and have FCBP, male patients must use an effective barrier contraceptive method during the study and for 6 months after the last dose.

[0966] Exclusion criteria:

[0967] 1. Failure to understand and cooperate throughout the research process, or inability or unwillingness to comply with research requirements

[0968] 2. Participation in any clinical study administering an investigational drug or investigational therapy within 30 days of the screening visit or observation period of a competing study

[0969] 3. Contraindications or known allergic reactions to MRI contrast agents, bevacizumab, olaptesed pegol, or polyethylene glycol

[0970] 4. Planned Hypofractionated Radiotherapy

[0971] 5. Cytostatic therapy (chemotherapy) within the past 5 years

[0972] 6. History of other cancers (except adequately treated basal or squamous cell skin cancer, cervical cancer in situ, or other cancers from patients who have been disease-free for ≥5 years)

[0973] 7. Currently active secondary malignancies

[0974] 8. Clinically significant or uncontrolled cardiovascular disease, including

[0975] a. Myocardial infarction in the past 12 months

[0976] b. Uncontrolled angina

[0977] c. Congestive heart failure (New York Heart Association functional class ≥2)

[0978] d. Diagnosed or suspected congenital long QT syndrome

[0979] e. QTc prolongation (>470 ms) on electrocardiogram before study entry

[0980] f. Uncontrolled hypertension (blood pressure ≥160 / 95 mmHg)

[0981] g. Baseline ECG heart rate <50 / min

[0982] h. History of any clinically significant type of ventricular arrhythmia (eg, ventricular tachycardia, ventricular fibrillation, or torsade de pointes)

[0983] i. Cerebrovascular accident

[0984] 9. Previous radiation therapy to the head

[0985] 10. Any other previous or concomitant experimental glioblastoma treatment

[0986] 11. Placement of Gliadel® wafers, seeds or ferromagnetic nanoparticles

[0987] 12. Patients with a history of arterial or venous thrombosis (or any other disease) requiring permanent anticoagulant therapy

[0988] 13. Pregnancy or breastfeeding

[0989] 14. Uncontrolled concurrent illness, including but not limited to ongoing or active infection, chronic liver disease (e.g., cirrhosis, hepatitis), diabetes, or subjects have 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 disease, or psychiatric illness / social conditions that would limit compliance with study requirements. Patients must not have any clinically relevant (in the opinion of the treating investigator) illness (other than glioma) that would interfere with the conduct of the study or study assessments.

[0990] 15. Prolongation of coagulation factors ≥ 2.5 times ULN

[0991] 16. Treatment not started within 6 weeks after the first biopsy or surgery for glioblastoma

[0992] 17. Have you been enrolled in this study before?

[0993] Treatments in the arms to combine NOX-A12 with radiation therapy and bevacizumab:

[0994]

[0995] Treatment options are also shown in Fig.14 .

[0996] result

[0997] All 6 patients who were scheduled to receive NOX-A12, radiation therapy, and bevacizumab were enrolled, with 3 patients continuing to receive NOX-A12 and bevacizumab beyond the originally planned 26 weeks at the discretion of the treating physician and the patient. One patient died.

[0998] Safety: The treatment was well tolerated and safe, with no dose-limiting toxicities and no treatment-related deaths.

[0999] Efficacy: Median follow-up of 10 months was January 19, 2023. At that time, 5 of 6 patients (83.3%) were still alive (see Fig. 27 ). The median progression-free survival (mPFS) and median overall survival (mOS) have not yet been reached.

[1000] Example 11 Clinical study of triple therapy with NOX-A12, radiotherapy and bevacizumab in patients with glioblastoma - further follow-up based on survival

[1001] The arms in the Phase 1 / 2 study were designed to evaluate the safety of NOX-A12 combined with bevacizumab when administered concurrently with radiation therapy in patients with glioblastoma, to explore the benefit of these combinations on progression-free survival when administered in addition to radiation therapy, and to provide an estimate of the effect on overall survival.

[1002] This study is expected to advance the understanding of the important role that CXCL12 plays in neovascularization by recruiting endothelial cells and other bone marrow-derived proangiogenic cells through a CXCR4- and CXCR7-dependent mechanism. Notably, irradiation also increased CXCL12 expression. In addition, this data is expected to guide the further clinical development of NOX-A12.

[1003] The corresponding study that conducted the NOX-A12 plus bevacizumab arm is titled “A single-arm, dose-escalation phase 1 / 2 study of olaptesed pegol (NOX-A12) plus radiation in patients with first-line glioblastoma who have an unmethylated MGMT promoter and are inoperable or partially resectable, with a three-arm expansion group that includes completely resected patients and combination with bevacizumab or pembrolizumab.”

[1004] Details of the study are as follows.

[1005] Patient population: Patients with newly diagnosed glioblastoma (WHO grade IV) with unmethylated MGMT promoter status after incomplete or no tumor resection

[1006] Study type: Multicenter, prospective, open-label

[1007] Research objectives:

[1008] Primary: To investigate the safety of olaptesed pegol combined with radiation therapy and bevacizumab in patients with newly diagnosed glioblastoma with an unmethylated MGMT promoter status

[1009] Secondary: (i) To investigate the efficacy of olapted pegol combined with radiotherapy and bevacizumab in patients with glioblastoma with unmethylated MGMT promoter status; (ii) To investigate the pharmacokinetics of olapted pegol during continuous administration; (iii) To monitor symptoms (NANO) and QoL

[1010] Study endpoints:

[1011] Primary: Safety (adverse events)

[1012] Secondary: PFS at 6 months (PFS-6); median progression-free survival (mPFS); median overall survival (mOS); tumor vascularization on each vascular MRI scan at baseline and at 2, 4, and 6 months; topography of recurrence; determination of maximum tolerated dose (MTD); definition of recommended phase 2 dose (RP2D); NOX-A12 plasma levels at steady state; NANO assessment; quality of life

[1013] Inclusion criteria:

[1014] 1. Written Informed Consent

[1015] 2. Age ≥ 18 years old

[1016] 3. The patient consents to diagnostic and scientific examination of glioblastoma tissue obtained during previous surgery or biopsy (e.g., MGMT promoter analysis, cytogenetic markers such as IDH-1 mutation, etc.)

[1017] 4. Patient consent for subcutaneous port implantation

[1018] 5. Newly diagnosed, histologically confirmed, supratentorial WHO grade IV glioblastoma

[1019] 6. Status after biopsy or incomplete resection (based on detectable residual tumor on postoperative T1-weighted, contrast-enhanced MRI scan)

[1020] 7. Unmethylated MGMT promoter status

[1021] 8. Maximum Eastern Cooperative Oncology Group (ECOG) score of 2

[1022] 9. Estimated minimum life expectancy 3 months

[1023] 10. The dose of corticosteroids has been stable or reduced in the week before inclusion

[1024] 11. The following laboratory parameters should be within the specified range:

[1025] a. Total bilirubin ≤ 1.5 times the upper limit of normal (ULN)

[1026] b. Creatinine ≤ 1.5 times ULN or glomerular filtration rate ≥ 60 mL / min / 1.73m²

[1027] c.ALT (alanine aminotransferase) ≤ 3 times ULN

[1028] d. AST (aspartate aminotransferase) ≤ 3 times ULN

[1029] 12. Female patients of childbearing potential must have a negative serum pregnancy test within 21 days before enrollment and agree to use a highly effective birth control method (with a failure rate of less than 1% per year when used continuously and correctly, such as contraceptive implants, vaginal rings, sterilization, or abstinence) during and within 6 months after the last dose of medication (more frequent pregnancy testing may be performed if required by local regulations)

[1030] 13. If sexually active and have FCBP, male patients must use an effective barrier contraceptive method during the study and for 6 months after the last dose.

[1031] Exclusion criteria:

[1032] 1. Failure to understand and cooperate throughout the research process, or inability or unwillingness to comply with research requirements

[1033] 2. Participation in any clinical study administering an investigational drug or investigational therapy within 30 days of the screening visit or observation period of a competing study

[1034] 3. Contraindications or known allergic reactions to MRI contrast agents, bevacizumab, olaptesed pegol, or polyethylene glycol

[1035] 4. Planned Hypofractionated Radiotherapy

[1036] 5. Cytostatic therapy (chemotherapy) within the past 5 years

[1037] 6. History of other cancers (except adequately treated basal or squamous cell skin cancer, cervical cancer in situ, or other cancers from patients who have been disease-free for ≥5 years)

[1038] 7. Currently active secondary malignancies

[1039] 8. Clinically significant or uncontrolled cardiovascular disease, including

[1040] a. Myocardial infarction in the past 12 months

[1041] b. Uncontrolled angina

[1042] c. Congestive heart failure (New York Heart Association functional class ≥2)

[1043] d. Diagnosed or suspected congenital long QT syndrome

[1044] e. QTc prolongation (>470 ms) on electrocardiogram before study entry

[1045] f. Uncontrolled hypertension (blood pressure ≥160 / 95 mmHg)

[1046] g. Baseline ECG heart rate <50 / min

[1047] h. History of any clinically significant type of ventricular arrhythmia (eg, ventricular tachycardia, ventricular fibrillation, or torsade de pointes)

[1048] i. Cerebrovascular accident

[1049] 9. Previous radiation therapy to the head

[1050] 10. Any other previous or concomitant experimental glioblastoma treatment

[1051] 11. Placement of Gliadel® wafers, seeds or ferromagnetic nanoparticles

[1052] 12. Patients with a history of arterial or venous thrombosis (or any other disease) requiring permanent anticoagulant therapy

[1053] 13. Pregnancy or breastfeeding

[1054] 14. Uncontrolled concurrent illness, including but not limited to ongoing or active infection, chronic liver disease (e.g., cirrhosis, hepatitis), diabetes, or subjects have 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 disease, or psychiatric illness / social conditions that would limit compliance with study requirements. Patients must not have any clinically relevant (in the opinion of the treating investigator) illness (other than glioma) that would interfere with the conduct of the study or study assessments.

[1055] 15. Prolongation of coagulation factors ≥ 2.5 times ULN

[1056] 16. Treatment not started within 6 weeks after the first biopsy or surgery for glioblastoma

[1057] 17. Have you been enrolled in this study before?

[1058] Treatments in the arms to combine NOX-A12 with radiation therapy and bevacizumab:

[1059]

[1060] Treatment options are also shown in Fig.14 .

[1061] result

[1062] All 6 patients who were scheduled to receive NOX-A12, radiation therapy, and bevacizumab were enrolled, with 2 patients continuing to receive NOX-A12 and bevacizumab beyond the originally planned 26 weeks at the discretion of the treating physician and the patient. One patient died.

[1063] Safety: The treatment was well tolerated and safe, with no dose-limiting toxicities and no treatment-related deaths.

[1064] Efficacy: Median follow-up of 14 months was May 21, 2023. At that time, 5 of 6 patients (83.3%) were still alive (see Fig.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. Five of the six patients achieved an objective response (83.3%), including one patient who achieved a complete response (100% tumor reduction), and two patients who achieved >99% tumor reduction as best response (see Fig.29 ).

[1065] References

[1066] Complete bibliographic data for documents cited herein (unless indicated to the contrary) are set forth below, the disclosures of which are hereby incorporated by reference.

[1067] Altschul SF, Gish W. et al (1990) Basic local alignment search tool. JMol Biol. 215(3):403-10.

[1068] Altschul SF, Madden TL et al (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25(17):3389-402.

[1069] 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 Chem280(42): 35760-35766.

[1070] Balabanian, K., Lagane B. et al (2005) WHIM syndromes with differentgenetic anomalies are accounted for by impaired CXCR4 desensitization toCXCL12. Blood 105(6): 2449-57.

[1071] Balkwill F. (2004) Cancer and the chemokine network. Nat Rev Cancer 4(7): 540-50.

[1072] 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.

[1073] Buckley C.D., Amft N. et al. (2000) Persistent induction of the chemokine receptor CXCR4 by TGF-beta 1 on synovial T c...

Claims

1. A CXC motif chemokine 12 (CXCL12) antagonist for use in a method of treating a tumor in a subject, wherein the method comprises administering to the subject - CXC motif chemokine 12 (CXCL12) antagonist, - Radiation therapy, and - Anti-angiogenic compounds, Wherein the tumor is a brain tumor.

2. The CXC motif chemokine 12 (CXCL12) antagonist for use according to claim 1, wherein the tumor is grade IV astrocytoma or glioblastoma.

3. The CXC motif chemokine 12 (CXCL12) antagonist for use according to any one of claims 1 to 2, wherein the CXCL12 antagonist inhibits the binding of CXCL12 to the CXCL12 receptor CXC chemokine receptor type 4 (CXCR4) and / or the CXCL12 receptor CXC chemokine receptor type 7 (CXCR7), preferably the CXCL12 antagonist binds to CXCL12.

4. The CXC motif chemokine 12 (CXCL12) antagonist for use according to any one of claims 1 to 3, wherein the CXCL12 antagonist is a nucleic acid molecule that binds to CXCL12, wherein the nucleic acid molecule that binds to CXCL12 comprises a central nucleotide fragment, whereby the central nucleotide fragment comprises the following nucleotide sequence: 5' GUGUGAUCUAGAUGUADWGGCUGWUCCUAGUYAGG 3' (SEQ ID NO: 52).

5. The CXC motif chemokine 12 (CXCL12) antagonist for use according to claim 4, wherein the nucleic acid molecule that binds to CXCL12 comprises a nucleotide sequence of any one of SEQ ID NO: 22 to SEQ ID NO: 28 and SEQ ID NO: 5 to SEQ ID NO: 20, or a nucleotide sequence having at least 80% identity thereto, Preferably the nucleotide sequence of any one of SEQ ID NO: 22, SEQ ID NO: 28, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 16, or a nucleotide sequence having at least 80% identity thereto, More preferably, the nucleotide sequence of any one of SEQ ID NO: 22 and SEQ ID NO: 28, or a nucleotide sequence having at least 80% identity thereto.

6. The CXC motif chemokine 12 (CXCL12) antagonist for use according to claim 5, wherein the nucleic acid molecule that binds to CXCL12 is an L-ribonucleic acid molecule comprising a nucleotide sequence of GCGUGGUGUGAUCUAGAUGUAUUGGCUGAUCCUAGUCAGGUACGC (SEQ ID NO: 22), preferably the L-ribonucleic acid molecule comprises a 40 kDa polyethylene glycol moiety, wherein the 40 kDa polyethylene glycol moiety is attached to the 5' end of the nucleotide sequence.

7. CXC motif chemokine 12 (CXCL12) for use according to any one of claims 1 to 6, wherein the radiation therapy is administered by external beam radiation therapy or internal radiation therapy (brachytherapy).

8. The CXC motif chemokine 12 (CXCL12) for use according to claim 7, wherein the radiotherapy is external beam radiotherapy, wherein the radiotherapy is fractionated radiotherapy and wherein the total absorbed radiation dose of the radiotherapy is from about 10 Gy to about 100 Gy, preferably from about 40 Gy to about 60 Gy, more preferably about 40 Gy or about 60 Gy.

9. The CXC motif chemokine 12 (CXCL12) for use according to claim 7, wherein the radiotherapy is external beam radiotherapy, wherein the radiotherapy is fractionated radiotherapy and wherein the radiation dose absorbed by the individual is from about 0.5 Gy to about 5 Gy, preferably from about 2 Gy to about 2.7 Gy, more preferably about 2 Gy or about 2.7 Gy.

10. CXC motif chemokine 12 (CXCL12) for use according to any one of claims 1 to 9, wherein the anti-angiogenic compound is an anti-VEGF antibody.

11. The CXC motif chemokine 12 (CXCL12) for use according to claim 10, wherein the anti-VEGF antibody comprises the following CDRs of the light chain variable region: CDR1: QDISNY (SEQ ID NO: 226), CDR2: FTS and CDR3: QQYSTVPWT (SEQ ID NO: 227), and / or the following CDRs of the heavy chain variable region: CDR1: GYTFTNYG (SEQ ID NO: 228), CDR2: INTYTGEP (SEQ ID NO: 229) and CDR3: AKYPHYYGSSHWYFDV (SEQ ID NO: 230).

12. CXC motif chemokine 12 (CXCL12) for use according to any one of claims 10 to 11, wherein the anti-VEGF antibody is bevacizumab.

13. The CXC motif chemokine 12 (CXCL12) for use according to any one of claims 1 to 12, wherein the method further comprises administering chemotherapy to the subject.

14. CXC motif chemokine 12 (CXCL12) for use according to claim 13, wherein the chemotherapy is or comprises temozolomide.

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