VEGF-BINDING MOLECULE AND ITS PHARMACEUTICAL APPLICATIONS

Claims

1. A VEGF-binding molecule comprising: a first VEGF-binding domain and a second VEGF-binding domain; wherein the first VEGF-binding domain comprises VEGFR2 immunoglobulin-like domain 2 (R2D2) and VEGFR2 immunoglobulin-like domain 3 (R2D3); the second VEGF-binding domain contains a VEGF trap or contains an anti-VEGF antibody or antigen-binding fragment thereof.

2. The VEGF-binding molecule of claim 1, wherein: The VEGF trap contains VEGFR1 immunoglobulin-like domain 2 (R1D2) and / or VEGFR2 immunoglobulin-like domain 3 (R2D3).

3. The VEGF-binding molecule according to claim 1 or 2, wherein: the first VEGF binding domain comprises an amino acid mutation at any one or more positions selected from the group consisting of position 162, position 221, position 222, position 274, position 276, position 277, position 280, position 284, position 288, position 289, and position 313; wherein the position of the amino acid mutation is determined relative to the natural sequence position of the amino acid sequence set forth in SEQ ID NO: 1; preferably, the first VEGF binding domain comprises an amino acid mutation at any one or more sets of positions selected from the group consisting of the following: - position 162; - position 162 and position 221; - position 162 and position 222; - position 162 and position 274; - position 162 and position 276; - position 162 and position 277; - position 162 and position 289; - position 162 and position 313; - position 221 and position 222; - position 277 and position 289; - position 162, position 221, and position 222; - position 162, position 277, and position 289; and - position 162, position 221, position 222, and position 274; more preferably, the first VEGF binding domain comprises an amino acid mutation at any one set of positions selected from the group consisting of the following: - position 162; and - position 162, position 221, position 222 and position 274.

4. A VEGF binding molecule according to any one of claims 1-3, wherein: the first VEGF binding domain comprises any one or more sets of amino acid mutations selected from the group consisting of the following: - 162A, 162V, 162S, 162I, or 162L; - 221S or 221N; - 222K; - 274F or 274I; - 276Q; - 277R; - 280E; - 284N; - 288Y; - 289Y; and - 313R; preferably the first VEGF binding domain comprises any one or more sets of amino acid mutations selected from the group consisting of the following: - 162V or 162I; - 162V / 274F;162V / 274I;162I / 274F; or 162I / 2741; - 221S / 222K; - 277R / 289Y; - 162V / 221S / 222K; - 162V / 276Q; - 162V / 289Y; - 162V / 277 / 289Y; - 162V / 313R; and - 162V / 221S / 222K / 274F; more preferably, the first VEGF binding domain comprises any one or more sets of amino acid mutations selected from the group consisting of the following: - 162V; and - 162V / 221S / 222K / 274F.

5. A VEGF binding molecule according to any one of claims 1-3, wherein: the first VEGF binding domain comprises an amino acid mutation at any one or more positions selected from the group consisting of position 135, position 197, position 199, position 213, position 215, position 219, position 257, position 275, position 277, position 278, position 283, position 289, position 313, and position 314; wherein the position of the amino acid mutation is determined relative to the natural sequence position of the amino acid sequence set forth in SEQ ID NO: 1; preferably, the first VEGF binding domain comprises an amino acid mutation at position 283.

6. The VEGF-binding molecule of claim 5, wherein: the first VEGF binding domain comprises any one or more sets of amino acid mutations selected from the group consisting of the following: - 135Y; - 197Y; - 199Q or 199T; - 213N; - 215F or 215H; - 2191; - 257Q or 257T; - 275L or 275E; - 277R; - 278D, 278S or 278E; - 283T; - 289Y; - 313Y; and - 314S or 314Q; preferably, the first VEGF-binding domain comprises a mutation at amino acid 283T.

7. A VEGF binding molecule according to any one of claims 1-6, wherein: the first VEGF binding domain comprises an amino acid mutation at any one or more positions selected from the group consisting of position 162, position 221, position 222, position 274, position 276, position 277, position 280, position 284, position 288, position 289, and position 313; and contains an amino acid mutation at any one or more positions selected from the group consisting of position 135, position 197, position 199, position 213, position 215, position 219, position 257, position 275, position 277, position 278, position 283, position 289, position 313, and position 314; preferably, the first VEGF binding domain comprises an amino acid mutation at any one set of positions selected from the group consisting of the following: - position 162 and position 275; - position 162 and position 283; - position 162 and position 313; - position 162, position 274 and position 283; - position 162, position 221, position 222 and position 283; and - position 162, position 221, position 222, position 274 and position 283; more preferably, the first VEGF binding domain comprises an amino acid mutation at any one set of positions selected from the group consisting of the following: - position 162 and position 283; And - position 162, position 221, position 222, position 274 and position 283.

8. The VEGF binding molecule of any one of claim 7, wherein: the first VEGF binding domain comprises: (i) any one or more sets of amino acid mutations selected from the group consisting of the following: - 162A, 162V, 162S, 162I or 162L; - 221S; - 222K; - 274F or 274I; - 276Q; - 277R; - 280E; - 284N; - 288Y; - 289Y; and - 313R; and (i) any one or more sets of amino acid mutations selected from the group consisting of the following: - 135Y; - 197Y; - 199Q or 199T; - 213N; - 215F or 215H; - 219I; - 257Q or 257T; - 275L or 275E; - 277R; - 278D, 278S or 278E; - 283T; - 289Y; - 313Y; and - 314S or 314Q; Preferably, the first VEGF binding domain comprises any one or more sets of amino acid mutations selected from the group consisting of the following: - 162V / 275L; - 162V / 283T; - 162V / 313Y; - 162V / 274F / 283T; - 162V / 221S / 222K / 283T; and - 162V / 221S / 222K / 274F / 283T; more preferably, the first VEGF binding domain comprises any one or more sets of amino acid mutations selected from the group consisting of the following: - 162V / 283T; and - 162V / 221S / 222K / 274F / 283T.

9. VEGF-binding molecule containing: at least one VEGF-binding domain comprising VEGFR2 immunoglobulin-like domain 2 (R2D2) and VEGFR2 immunoglobulin-like domain 3 (R2D3), wherein said VEGF binding domain comprises an amino acid mutation at one or more positions selected from the group consisting of position 162, position 221, position 222, position 274, position 276, position 277, position 280, position 284, position 288, position 289, and position 313; wherein the position of the amino acid mutation is determined relative to the natural sequence position of the amino acid sequence set forth in SEQ ID NO:

1.

10. The VEGF binding molecule of claim 9, wherein the VEGF binding domain comprises an amino acid mutation at any one or more sets of positions selected from the group consisting of the following: - position 162; - position 162 and position 221; - position 162 and position 222; - position 162 and position 274; - position 162 and position 276; - position 162 and position 277; - position 162 and position 289; - position 162 and position 313; - position 221 and position 222; - position 277 and position 289; - position 162, position 221 and position 222; - position 162, position 277 and position 289; And - position 162, position 221, position 222 and position 274; preferably, the VEGF binding domain comprises an amino acid mutation at any one set of positions selected from the group consisting of the following: - position 162; and - position 162, position 221, position 222 and position 274.

11. The VEGF binding molecule of claim 9 or 10, wherein the VEGF binding domain comprises any one or more sets of amino acid mutations selected from the group consisting of the following: - 162A, 162V, 162S, 162I or 162L; -221S or 221N; - 222K; - 274F or 274I; - 276Q; - 277R; - 280E; - 284N; - 288Y; - 289Y; and - 313R; Preferably, the VEGF binding domain comprises any one or more sets of amino acid mutations selected from the group consisting of the following: - 162V or 162I; - 162V / 274F; 162V / 274I; 162I / 274F; or 162I / 274I; -221S / 222K; - 277R / 289Y; - 162V / 221S / 222K; - 162V / 276Q; - 162V / 289Y; - 162V / 277 / 289Y; - 162V / 313R; and - 162V / 221S / 222K / 274F; more preferably, the VEGF binding domain comprises any one or more sets of amino acid mutations selected from the group consisting of the following: - 162V; and - 162V / 221S / 222K / 274F.

12. The VEGF binding molecule of claim 9 or 10, wherein the VEGF binding domain comprises an amino acid mutation at any one or more positions selected from the group consisting of position 135, position 197, position 199, position 213, position 215, position 219, position 257, position 275, position 277, position 278, position 283, position 289, position 313, and position 314; wherein the position of the amino acid mutation is determined relative to the natural sequence position of the amino acid sequence set forth in SEQ ID NO: 1; preferably, the VEGF binding domain comprises an amino acid mutation at position 283.

13. The VEGF binding molecule of claim 12, wherein the VEGF binding domain comprises any one or more sets of amino acid mutations selected from the group consisting of the following: - 135Y; - 197Y; - 199Q or 199T; - 213N; - 215F or 215H; - 219I; - 257Q or 257T; - 275L or 275E; - 277R; - 278D, 278S or 278E; - 283T; - 289Y; - 313Y; and - 314S or 314Q; preferably, the VEGF binding domain comprises a mutation at amino acid 283T.

14. The VEGF binding molecule of any one of claims 9 to 13, wherein the VEGF binding domain comprises an amino acid mutation at any one or more positions selected from the group consisting of position 162, position 221, position 222, position 274, position 276, position 277, position 280, position 284, position 288, position 289, and position 313; and contains an amino acid mutation at any one or more positions selected from the group consisting of position 135, position 197, position 199, position 213, position 215, position 219, position 257, position 275, position 277, position 278, position 283, position 289, position 313, and position 314; preferably, the VEGF binding domain comprises an amino acid mutation at any one or more sets of positions selected from the group consisting of the following: - position 162 and position 275; - position 162 and position 283; - position 162 and position 313; - position 162, position 274 and position 283; - position 162, position 221, position 222 and position 283; and - position 162, position 221, position 222, position 274 and position 283; more preferably, the VEGF binding domain comprises an amino acid mutation in any one or more sets selected from the group consisting of the following: - position 162 and position 283; And - position 162, position 221, position 222, position 274 and position 283.

15. The VEGF binding molecule of claim 14, wherein the VEGF binding domain comprises: (i) any one or more sets of amino acid mutations selected from the group consisting of the following: - 162A, 162V, 162S, 162I or 162L; - 221S; - 222K; - 274F or 274I; - 276Q; - 277R; - 280E; - 284N; - 288Y; - 289Y; and - 313R; and (ii) any one or more sets of amino acid mutations selected from the group consisting of the following: - 135Y; - 197Y; - 199Q or 199T; - 213N; - 215F or 215H; - 219I; - 257Q or 257T; - 275L or 275E; - 277R; - 278D, 278S or 278E; - 283T; - 289Y; - 313Y; and - 314S or 314Q; preferably the VEGF binding domain comprises any one or more sets of amino acid mutations selected from the group consisting of the following: - 162V / 275L; - 162V / 283T; - 162V / 313Y; - 162V / 274F / 283T; - 162V / 221S / 222K / 283T; and - 162V / 221S / 222K / 274F / 283T; more preferably, the VEGF binding domain comprises any one or more sets of amino acid mutations selected from the group consisting of the following: - 162V / 283T; and - 162V / 221S / 222K / 274F / 283T.

16. The VEGF binding molecule of any one of claims 1-15, further comprising an Fc, wherein preferably the Fc is the Fc of human IgG1, human IgG2, human IgG3 or human IgG4.

17. The VEGF-binding molecule of any one of claims 1 to 8, wherein the VEGF-trap is aflibercept or conbercept; the anti-VEGF antibody or antigen-binding fragment thereof is an anti-VEGF-A antibody and antigen-binding fragment thereof, bevacizumab or antigen-binding fragment thereof, or ranibizumab or antigen-binding fragment thereof; preferably the antigen-binding fragment is an scFv, Fv, Fab or Fab' fragment.

18. A VEGF binding molecule according to any one of claims 1-17, wherein: said VEGF binding molecule comprises, from N-terminus to C-terminus, any one selected from the group consisting of the following: R2D2-R2D3-FC; R1D2-R2D3-R2D2-R2D3-FC; R1D2-R2D3-linker-R2D2-R2D3-Fc; R2D2-R2D3-R1D2-R2D3-Fc; and R2D2-R2D3-linker-R1D2-R2D3-Fc; or the VEGF binding molecule comprises a heavy chain and a light chain, wherein the heavy chain comprises from N-terminus to C-terminus: R2D2-R2D3-Fc-linker-VH-CH1, and the light chain comprises from N-terminus to C-terminus: VL-CL; preferably, VH and VL are VH and VL of bevacizumab or VH and VL of ranibizumab; preferably the linker is a peptide linker; more preferably, the linker is selected from the group consisting of: (GS)a(GGS)b(GGGS)c(GGGGS)d(GGGGG)e, where a, b, c, d and e are independently integers greater than or equal to 0; or the linker is selected from the group consisting of: (EAAAK)3, (EAAAR)3, (EGGGK)3, (EGGGR)3, (DAAAR)3, (DAAAK)3, (DGGGR)3, and (DGGGK)3; or The linker is (G x S) y , where x is selected from the group consisting of integers from 1 to 5, and y is selected from the group consisting of integers from 1 to 6.

19. A VEGF binding molecule according to any one of claims 9-18, wherein: said VEGF binding molecule comprises an amino acid sequence set forth in any one of SEQ ID NOs: 5-6 and SEQ ID NOs: 62-65, or an amino acid sequence having at least 90% sequence identity thereto; or said VEGF binding molecule consists of an amino acid sequence set forth in any one of SEQ ID NOs: 5-6 and SEQ ID NOs: 62-65, or an amino acid sequence having at least 90% sequence identity thereto.

20. A VEGF binding molecule according to any one of claims 1-18, wherein: 1) said VEGF binding molecule comprises an amino acid sequence set forth in any of SEQ ID NOs: 3-4, SEQ ID NOs: 7-49, SEQ ID NOs: 52-61, and SEQ ID NO: 66, or an amino acid sequence having at least 90% sequence identity thereto; 2) the heavy chain of the VEGF binding molecule comprises the amino acid sequence set forth in SEQ ID NO: 50 or an amino acid sequence having at least 90% sequence identity thereto, and the light chain of the VEGF binding molecule comprises the amino acid sequence set forth in SEQ ID NO: 51 or an amino acid sequence having at least 90% sequence identity thereto; or 3) the heavy chain of the VEGF binding molecule comprises the amino acid sequence set forth in SEQ ID NO: 67, or an amino acid sequence having at least 90% sequence identity thereto, and the light chain of the VEGF binding molecule comprises the amino acid sequence set forth in SEQ ID NO: 68, or an amino acid sequence having at least 90% sequence identity thereto.

21. An isolated nucleic acid comprising a polynucleotide encoding a VEGF-binding molecule according to any one of claims 1-20.

22. The isolated nucleic acid of claim 21, wherein the polynucleotide comprises a nucleic acid sequence having at least 70% identity to the nucleic acid sequence set forth in any one of SEQ ID NOs: 78-86.

23. The isolated nucleic acid of any one of claims 21-22, further comprising a promoter and / or enhancer that is / are operably linked to the polynucleotide, wherein preferably the promoter is a CMV promoter; preferably the enhancer is a CMV enhancer.

24. The isolated nucleic acid of any one of claims 21-23, further comprising any one or any combination of the following: a Kozak sequence, a 5'-UTR and a 3'-UTR, a WPRE sequence, a polyA sequence, and an intron, wherein preferably the Kozak sequence is located between the intron and the polynucleotide encoding the VEGF binding molecule; preferably the polyA sequence is the polyA sv40 sequence.

25. The isolated nucleic acid according to any one of claims 21-24, which is DNA or RNA, preferably cDNA or mRNA.

26. A vector, wherein said vector comprises an isolated nucleic acid according to any one of claims 21-25; preferably, the vector is selected from the group consisting of any one of the following or a combination thereof: a plasmid, a lentiviral vector, an adenovirus, an adeno-associated virus (AAV) vector, a recombinant adeno-associated virus (rAAV) vector, and a retroviral vector; more preferably, the vector is a recombinant adeno-associated virus vector.

27. A vector based on a recombinant adeno-associated virus containing: (i) a polynucleotide encoding a VEGF binding molecule as defined in any one of claims 21 to 25; (ii) an enhancer, preferably a CMV enhancer; (iii) a promoter, preferably a CMV promoter; (iv) 5'-UTR and 3'-UTR; (v) Kozak sequence; (vi) the WPRE sequence; and (vii) the sv40 polyA sequence; where any one or any combination of (ii)-(vii) are functionally related to (i).

28. A particle of a recombinant adeno-associated virus comprising: an isolated nucleic acid according to any one of paragraphs 21-25 or a vector based on a recombinant adeno-associated virus according to claim 27 and a capsid protein; where preferably, the capsid protein is selected from the group consisting of any one of the following serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9 (hu14), AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAV-DJ and AAV-DJ8 or variants thereof; more preferably, the capsid protein comprises the amino acid sequence set forth in SEQ ID NO: 77, or an amino acid sequence having at least 70% sequence identity with SEQ ID NO:

77.

29. A host cell comprising an isolated nucleic acid according to any one of claims 21-25, or a vector according to any one of claims 26, 27.

30. A pharmaceutical composition comprising a VEGF-binding molecule according to any one of claims 1-20, an isolated nucleic acid according to any one of claims 21-25, a vector according to any one of claims 26, 27 or a particle of a recombinant adeno-associated virus according to claim 28, wherein preferably the pharmaceutical composition additionally comprises at least one pharmaceutically acceptable carrier, diluent or excipient.

31. Use of a VEGF-binding molecule according to any one of claims 1-20, an isolated nucleic acid according to any one of claims 21-25 or a vector according to any one of claims 26, 27 or a recombinant adeno-associated virus particle according to claim 28 for the production of a medicinal product for the treatment of a disease associated with pathological angiogenesis, wherein: preferably, the disease associated with pathological angiogenesis is an angiogenic eye disease or cancer; more preferably, the angiogenic eye disease is selected from the group consisting of macular degeneration (e.g., age-related macular degeneration), macular edema (e.g., macular edema after retinal vein occlusion or diabetic macular edema), retinal vein occlusion (e.g., central retinal vein occlusion, branch retinal vein occlusion), choroidal neovascularization, iris neovascularization, neovascular glaucoma, postoperative fibrosis in glaucoma, proliferative vitreoretinopathy, optic disc neovascularization, corneal neovascularization, retinal neovascularization, vitreous neovascularization, pannus, pterygium, choroidal retinopathy, retinopathy of prematurity, vascular retinopathy, diabetic retinopathy, non-proliferative diabetic retinopathy and proliferative diabetic retinopathy. the cancer is selected from the group consisting of small cell lung cancer, renal cell cancer, uterine cancer, prostate cancer, bladder cancer, ovarian cancer, colon cancer, breast cancer, leukemia, lymphoma, myeloma, sarcoma (e.g. fibrosarcoma, myxosarcoma, liposarcoma, lymphatic endotheliosarcoma, angiosarcoma, endotheliosarcoma, chondrosarcoma, osteosarcoma, chordoma, lymphangiosarcoma, synovioma, mesothelioma, leiomyosarcoma, or rhabdomyosarcoma), neuroglioma, pancreatic cancer, squamous cell sarcoma, basal cell carcinoma, adenocarcinoma, sweat gland cancer, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary cancer, bronchopulmonary carcinoma, choriocarcinoma, renal cell carcinoma, liver cancer, bile duct cancer, seminoma, embryonal carcinoma, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, astrocytoma, ependymoma,pinealoma, hemangioblastoma, acoustic neuroma, medulloblastoma, craniopharyngioma, oligodendroglioma, meningioma, melanoma, neutrophilic tumor and retinoblastoma., 32. An rAAV production system for producing a recombinant adeno-associated virus particle according to claim 28, wherein said production system comprises: (a) a nucleic acid sequence encoding the amino acid sequence of the AAV capsid protein; (b) an isolated nucleic acid according to any one of claims 21 to 25 or a vector according to any one of claims 26, 27; and (c) a helper packaging element having sufficient AAV rep function and helper function to enable packaging of the isolated nucleic acid of any one of claims 21-25 or the vector of any one of claims 26, 27 into an AAV capsid; wherein the AAV capsid is selected from the group consisting of any of the following: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9 (hu14), AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAV-DJ and AAV-DJ8 or variants thereof, preferably AAV2.

33. The rAAV production system of claim 32, wherein: the helper packaging element is provided by a packaging cell; preferably, the packaging cell comprises a plasmid selected from a group consisting of any one or combination of the following: pHelper, pRC9, and pGOI.

34. A method for producing a particle of a recombinant adeno-associated virus according to claim 28, comprising: a step of packaging the isolated nucleic acid of any one of claims 21-25 or the vector of any one of claims 26-27 into an AAV capsid using a packaging cell, wherein: the AAV capsid is selected from the group consisting of any one of the following: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9 (hu14), AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAV-DJ and AAV-DJ8 or variants thereof, preferably AAV2; the packaging cell comprises a plasmid selected from the group consisting of any one of the following or a combination thereof: pHelper, pRC9, and pGOI.