VEGF-binding molecules and their pharmaceutical uses

Abstract

The present disclosure provides VEGF binding molecules and pharmaceutical uses thereof. In particular, the present disclosure relates to VEGF binding molecules, nucleic acids encoding said VEGF binding molecules, vectors comprising said nucleic acids, and pharmaceutical uses.

Description

[Technical Field]

[0001] This disclosure claims priority to Chinese patent application Ser. No. CN202210157037.X, filed on February 21, 2022, entitled "VEGF-binding molecule and pharmaceutical use thereof," and Chinese patent application Ser. No. CN202210156042.9, filed on February 21, 2022, entitled "Nucleic acid encoding a VEGF-binding molecule and pharmaceutical use thereof," the entire contents of which are incorporated herein by reference.

[0002] The present disclosure relates to the field of biomedicine, and in particular to VEGF binding molecules, nucleic acids encoding VEGF binding molecules, and pharmaceutical uses thereof. [Background technology]

[0003] The vascular endothelial growth factor (VEGF) family contains five members: VEGF-A, VEGF-B, VEGF-C, VEGF-D, and PGF (placental growth factor). VEGF receptors include three: flt-1 (fms-like tyrosine kinase, VEGFR1), Flk-1 / KDR (fetal liver kinase 1-murine homologue / kinase insert domain containing receptor-human homologue, VEGFR2), and flt-4 (VEGFR3). VEGFR1 and VEGFR2 are primarily distributed on the surface of vascular endothelial cells, while VEGFR3 is primarily distributed in lymphatic endothelium. Each VEGF family consists of an extracellular domain containing seven immunoglobulin-like structures, a membrane domain, and a tyrosine kinase domain. The tyrosine kinase activity is activated by receptor-ligand binding, and receptor phosphorylation triggers numerous intracellular enzymes and other reactions.

[0004] VEGF plays a key role in the normal embryonic angiogenesis process and in abnormal angiogenesis processes, such as in many diseases including cancer and hemangiomas, as well as in the pathological development of neovasculature. Solid tumors utilize blood vessels to obtain oxygen and nutrients and remove waste products. These tumors also produce matrix factors to induce the formation of new blood vessels to support their continued growth. Therefore, antiangiogenic strategies, for example, by blocking or inhibiting the VEGF signaling pathway, represent a viable cancer treatment option. One strategy is to use VEGF-binding proteins to sequester VEGF in circulating serum and reduce the number of circulating VEGF ligands using the VEGF-binding proteins as decoy receptors. VEGF-binding proteins include humanized monoclonal antibodies (e.g., Avastin®, bevacizumab, Lucentis®, ranibizumab), soluble VEGF receptors (e.g., Eylea®, aflibercept), or chimeric VEGF trap molecules.

[0005] Age-related macular degeneration (AMD) is a serious eye disease that has become increasingly prevalent as the human population ages. It is a common eye disease in people over 50 years of age and a leading cause of vision loss. Clinically, AMD is classified into two types: atrophic (dry) AMD and exudative (wet) AMD (wAMD), depending on the presence or absence of abnormal new blood vessels. AMD has already become the third leading cause of blinding eye disease and is one of the most widely explored and researched disease areas worldwide. Wet AMD is primarily characterized by the proliferation of abnormal new blood vessels under the retina in the macula, called choroidal neovascularization (CNV), which leads to localized edema or hemorrhage in the macular region, macular protuberance, and focal pigment epithelial detachment. Ultimately, this leads to scarring, damage to retinal photoreceptor cells, and consequently vision loss. Therefore, ocular fundus diseases can be effectively treated by inhibiting vascular endothelial growth factor (VEGF) and blocking the growth of lesional new blood vessels. The accepted first-line treatment for wet AMD is intravitreal injection of protein VEGF inhibitors. Currently, the main protein VEGF inhibitors used to treat wet AMD are Roche's bevacizumab, Roche / Novartis' Fab antibody fragment Lucentis, Regeneron's VEGFR-Fc (VEGF Trap) fusion protein aflibercept, and Sichuan Kanghong Pharmaceutical's VEGFR-Fc fusion protein Conbercept.

[0006] It is important to develop VEGF-binding molecules that have novel structures and target more members of the VEGF family (e.g., that can bind to both VEGF-A and VEGF-C). The present disclosure provides molecules that can simultaneously bind to VEGF-A and VEGF-C and have good efficacy, high stability, and good drug potential. Summary of the Invention

[0007] The present disclosure provides VEGF-binding molecules and pharmaceutical uses, wherein the VEGF-binding molecules have both VEGF-A and VEGF-C binding capabilities.

[0008] VEGF binding molecule The present disclosure provides VEGF-binding molecules, which comprise immunoglobulin-like domain 2 (R2D2) of VEGFR2 and / or immunoglobulin-like domain 3 (R2D3) of VEGFR2, or alternatively, the VEGF-binding molecules consist of R2D2 and / or R2D3.

[0009] In some embodiments, a VEGF-binding molecule is provided, which comprises a first VEGF-binding domain and a second VEGF-binding domain, wherein the first VEGF-binding domain comprises or consists of immunoglobulin-like domain 2 (R2D2) of VEGFR2 and immunoglobulin-like domain 3 (R2D3) of VEGFR2, and the second VEGF-binding domain comprises VEGF Trap, or comprises an anti-VEGF antibody or an antigen-binding fragment thereof, or consists of VEGF Trap or an anti-VEGF antibody, or an antigen-binding fragment thereof.

[0010] In some embodiments, the VEGF Trap in the second VEGF-binding domain is immunoglobulin-like domain 2 (R1D2) of VEGFR1 and / or immunoglobulin-like domain 3 (R2D3) of VEGFR2.

[0011] In some embodiments, the anti-VEGF antibody in the second VEGF binding domain is bevacizumab or ranibizumab.

[0012] In some embodiments, a VEGF binding molecule is provided, which comprises: a first VEGF-binding domain and a second VEGF-binding domain, wherein the first VEGF-binding domain comprises or consists of R2D2-R2D3; The second VEGF-binding domain comprises R1D2-R2D3, or consists of R1D2-R2D3, or comprises the heavy chain variable region (VH) and light chain variable region (VL) of an anti-VEGF antibody, illustratively the anti-VEGF antibody is bevacizumab or ranibizumab.

[0013] As a non-limiting explanation, R2D2-R2D3 refers to the VEGF-binding domain formed by R2D2 linking to R2D3, wherein R2D2 and R2D3 may be directly or indirectly linked (e.g., linked via a linker).

[0014] In some embodiments, a VEGF binding molecule is provided that comprises one or more selected from R1D2, R2D2, and R2D3, such as one R1D2, one R2D2, and two R2D3, or one R1D2, one R2D2, and two R2D3, such as one R2D2 and one R2D3, or one R2D2 and one R2D3.

[0015] In some embodiments, the VEGF binding molecule comprises an amino acid mutation that improves its stability.

[0016] In some embodiments, the VEGF binding molecule comprises an amino acid mutation that improves affinity for its ligand.

[0017] In some embodiments, the VEGF binding molecule comprises amino acid mutations that improve its affinity for its ligands VEGF-A and / or VEGF-C.

[0018] In some embodiments, the VEGF binding molecule comprises both amino acid mutations that improve its stability and amino acid mutations that improve its affinity for its ligands VEGF-A and / or VEGF-C.

[0019] In the first aspect, in some embodiments, R2D2 and / or R2D3 of the VEGF binding molecule comprise an amino acid mutation at any one or more positions selected from 162, 221, 222, 274, 276, 277, 280, 284, 288, 289, and 313.

[0020] The amino acid mutation sites are natural sites relative to the amino acid sequence shown in SEQ ID NO:1.

[0021] In some embodiments, R2D2 and / or R2D3 are: -162nd place, -162nd and 221st place, -162nd and 222nd place, -162nd and 274th place, -162nd and 276th place, -162nd and 277th place, -162nd and 289th place, -162nd and 313th place, -221st and 222nd place, -277th and 289th place, -162nd, 221st and 222nd places, -162nd, 277th and 289th place, -162nd, 221st, 222nd and 274th place, The amino acid mutation is at a position selected from the positions shown in any one or more of the groups:

[0022] In some embodiments, R2D2 and / or R2D3 are: -162nd place, -162nd, 221st, 222nd and 274th place, The amino acid mutation is at a position selected from any one of the following groups:

[0023] In some embodiments, R2D2 and / or R2D3 are: -162A, 162V, 162S, 162I or 162L, -221S or 221N, -222K, -274F or 274I, -276Q, -277R, -280E, -284H, -288Y, -289Y, -313R, The amino acid mutations include any one or more of the following groups:

[0024] In some embodiments, R2D2 and / or R2D3 are: -C162A, C162V, C162S, C162I or C162L, -Y221S or Y221N, -R222K, -N274F or N274I, -D276Q, -L277R, -Q280E, -E284H, -F288Y, -L289Y, -L313R, The amino acid mutations include any one or more of the following groups:

[0025] In some embodiments, R2D2 and / or R2D3 are: -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, -162V / 221S / 222K / 274F, The amino acid mutations include any one or more of the following groups:

[0026] In some embodiments, R2D2 and / or R2D3 are: -C162V or C162I, -C162V / N274F, C162V / N274I, C162I / N274F, or C162I / N274I, -Y221S / R222K, -L277R / L289Y, -C162V / Y221S / R222K, -C162V / D276Q, -C162V / L289Y, -C162V / L277R / L289Y, -C162V / L313R, -C162V / Y221S / R222K / N274F, The amino acid mutations include any one or more of the following groups:

[0027] In some embodiments, R2D2 and / or R2D3 are: -162V, -162V / 221S / 222K / 274F, The amino acid mutations include any one or more of the following groups:

[0028] In some embodiments, R2D2 and / or R2D3 are: -C162V, -C162V / Y221S / R222K / N274F, The amino acid mutations include any one or more of the following groups:

[0029] In some embodiments, a VEGF binding molecule comprising the amino acid mutation has improved stability compared to a wild-type molecule having the same molecular form.

[0030] In a second aspect, in some embodiments, R2D2 and / or R2D3 of the VEGF binding molecule comprise an amino acid mutation at one or more positions selected from 135, 197, 199, 213, 215, 219, 257, 275, 277, 278, 283, 289, 313, and 314.

[0031] The amino acid mutation sites are natural sites relative to the amino acid sequence shown in SEQ ID NO:1.

[0032] In some embodiments, the R2D2 and / or R2D3 comprises an amino acid mutation at position 283.

[0033] In some embodiments, R2D2 and / or R2D3 are: -135Y, -197Y, -199Q or 199T, -213N, -215F or 215H, -219I, -257Q or 257T, -275L or 275E, -277R, -278D, 278S or 278E, -283T, -289Y, -313Y, -314S or 314Q, The amino acid mutations include any one or more of the following groups:

[0034] In some embodiments, R2D2 and / or R2D3 are: -V135Y, -M197Y, -F199Q or F199T, -M213N, -I215F or I215H, -V219I, -D257Q or D257T, -R275L or R275E, -L277R, -K278D, K278S or K278E, -S283T, -L289Y, -L313Y, -M314S or M314Q, The amino acid mutations include any one or more of the following groups:

[0035] In some embodiments, R2D2 and / or R2D3 include 283T.

[0036] In some embodiments, the R2D2 and / or R2D3 comprises the amino acid mutation S283T.

[0037] In some embodiments, a VEGF binding molecule comprising the amino acid mutation has improved ligand binding activity compared to a wild-type molecule having the same molecular form.

[0038] In a third aspect, in some embodiments, R2D2 and / or R2D3 of the VEGF-binding molecule comprise an amino acid mutation at any one or more positions selected from 162, 221, 222, 274, 276, 277, 280, 284, 288, 289, and 313; and The amino acid mutation is present at one or more positions selected from 135, 197, 199, 213, 215, 219, 257, 275, 277, 278, 283, 289, 313, and 314.

[0039] In some embodiments, R2D2 and / or R2D3 of the VEGF binding molecule are: -162nd and 275th place, -162nd and 283rd place, -162nd and 313th place, -162nd, 274th and 283rd place, -162nd, 221st, 222nd and 283rd place, -162nd, 221st, 222nd, 274th and 283rd place, The amino acid mutation is at a position selected from any one of the following groups:

[0040] In some embodiments, R2D2 and / or R2D3 are: -162nd and 283rd place, -162nd, 221st, 222nd, 274th and 283rd place, The amino acid mutation is at a position selected from any one of the following groups:

[0041] In some embodiments, R2D2 and / or R2D3 are: (i) -162A, 162V, 162S, 162I or 162L, -221S, -222K, -274F or 274I, -276Q, -277R, -280E, -284H, -288Y, -289Y, -313R, and an amino acid mutation selected from any one or more of the following groups: (ii) -135Y, -197Y, -199Q or 199T, -213N, -215F or 215H, -219I, -257Q or 257T, -275L or 275E, -277R, -278D, 278S or 278E, -283T, -289Y, -313Y, -314S or 314Q, and an amino acid mutation selected from any one or more of the following groups:

[0042] In some embodiments, R2D2 and / or R2D3 are: (i) -C162A, C162V, C162S, C162I or C162L, -Y221S, -R222K, -N274F or N274I, -D276Q, -L277R, -Q280E, -E284H, -F288Y, -L289Y, -L313R, and an amino acid mutation selected from any one or more of the following groups: (ii) -V135Y, -M197Y, -F199Q or F199T, -M213N, -I215F or I215H, -V219I, -D257Q or D257T, -R275L or R275E, -L277R, -K278D, K278S or K278E, -S283T, -L289Y, -L313Y, -M314S or M314Q, and an amino acid mutation selected from any one or more of the following groups:

[0043] In some embodiments, R2D2 and / or R2D3 are: -162V / 275L, -162V / 283T, -162V / 313Y, -162V / 274F / 283T, -162V / 221S / 222K / 283T, -162V / 221S / 222K / 274F / 283T, The amino acid mutations include any one or more of the following groups:

[0044] In some embodiments, R2D2 and / or R2D3 are: -C162V / R275L, -C162V / S283T, -C162V / L313Y, -C162V / N274F / S283T, -C162V / Y221S / R222K / S283T, -C162V / Y221S / R222K / N274F / S283T, The amino acid mutations include any one or more of the following groups:

[0045] In some embodiments, R2D2 and / or R2D3 are: -162V / 283T, -162V / 221S / 222K / 274F / 283T, The amino acid mutations include any one or more of the following groups:

[0046] In some embodiments, R2D2 and / or R2D3 are: -C162V / S283T, -C162V / Y221S / R222K / N274F / S283T, The amino acid mutations include any one or more of the following groups:

[0047] In the context of the present disclosure, " / " indicates the simultaneous presence of said amino acid mutations in the same VEGF binding molecule.

[0048] In the context of the present disclosure, ordinal numbers such as "first", "second", etc. should not be understood to limit the number, level, or order of technical features, but are only used to distinguish between different technical features, steps, and elements.

[0049] In the above embodiments, the mutations in R2D2 and / or R2D3 of the VEGF binding molecule can contain or consist solely of the mutations.

[0050] In some embodiments, R2D2 and / or R2D3 of VEGFR2 are wild-type or functional variants having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the wild-type.

[0051] In some embodiments, immunoglobulin-like domain 2 and immunoglobulin-like domain 3 of wild-type VEGFR2 have the same amino acid sequences as R2D2 and R2D3 of the sequence set forth in SEQ ID NO:1.

[0052] In some embodiments, immunoglobulin-like domain 2 (R2D2) and immunoglobulin-like domain 3 (R2D3) of VEGFR2 contain one or more amino acid differences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid differences) compared to the wild-type, and the amino acid differences include amino acid substitutions, insertions, or deletions.

[0053] In some embodiments, the amino acid mutations are in the immunoglobulin-like domain 2 and the immunoglobulin-like domain 3 of wild-type VEGFR2, the amino acid sequence of which is set forth in SEQ ID NO:1.

[0054] In some embodiments, the immunoglobulin-like domain 2 and the immunoglobulin-like domain 3 of wild-type VEGFR2 have the amino acid sequence shown at positions 123 to 327 of SEQ ID NO:1.

[0055] In some embodiments, R2D2-R2D3 has the amino acid sequence shown at positions 123 to 327 of SEQ ID NO:1.

[0056] In some embodiments, the immunoglobulin-like domain 2 (R2D2) of wild-type VEGFR2 has the amino acid sequence shown at positions 123 to 225 (single underlined) of SEQ ID NO: 1, and the immunoglobulin-like domain 3 (R2D3) of wild-type VEGFR2 has the amino acid sequence shown at positions 226 to 327 (double underlined) of SEQ ID NO: 1.

[0057] In the context of the present disclosure, the amino acid position numbers associated with R2D2 and R2D3 all use the natural numbering of the amino acid sequence shown in SEQ ID NO:1, ie, counting from methionine (M) at position 1 of SEQ ID NO:1.

[0058] Wild-type VEGFR2 amino acid sequence: [ka]

[0059] In some embodiments, the first VEGF binding domain consists of R2D2-R2D3.

[0060] In some embodiments, the first VEGF-binding domain has the ability to bind to VEGF-C. In some embodiments, the first VEGF-binding domain has the ability to bind to VEGF-C and VEGF-A.

[0061] In some embodiments, the VEGF binding molecule has increased stability and / or ligand binding activity.

[0062] In some embodiments, the second VEGF binding domain is capable of binding to VEGF-A.

[0063] In some embodiments, the second VEGF-binding domain has VEGF-A binding ability and no or weak VEGF-C binding ability.

[0064] In some embodiments, the second VEGF binding domain comprises or consists of VEGF Trap.

[0065] In some embodiments, the VEGF Trap comprises immunoglobulin-like domain 2 (R1D2) of VEGFR1 and immunoglobulin-like domain 3 (R2D3) of VEGFR2.

[0066] In some embodiments, the immunoglobulin-like domain 2 (R1D2) of VEGFR1 is wild-type or a functional variant of R1D2 having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the wild-type.

[0067] In some embodiments, wild-type R1D2 has the same amino acid sequence as immunoglobulin-like domain 2 in wild-type VEGFR1 as set forth in SEQ ID NO:2.

[0068] In some embodiments, wild-type R1D2 has the amino acid sequence shown at positions 129 to 231 of SEQ ID NO:2.

[0069] In some embodiments, the R1D2 functional variant comprises one or more amino acid differences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid differences) compared to the wild-type, wherein the amino acid differences comprise amino acid substitutions, insertions, or deletions.

[0070] In the context of the present disclosure, all amino acid position numbers relative to R1D2 use the natural numbering of the amino acid sequence shown in SEQ ID NO:2, ie, counting from methionine (M) at position 1 of SEQ ID NO:2.

[0071] Wild-type VEGFR1 amino acid sequence: [ka]

[0072] In some embodiments, the VEGF Trap is aflibercept or conbercept. In some embodiments, the VEGF Trap is the VEGFR portion of aflibercept or the VEGFR portion of conbercept.

[0073] In some embodiments, an anti-VEGF-A antibody or antigen-binding fragment thereof. In some embodiments, the antigen-binding fragment is an scFv, Fv, Fab, or Fab' fragment.

[0074] In some embodiments, the anti-VEGF-A antibody is bevacizumab or ranibizumab. In some embodiments, the anti-VEGF-A antibody is the Fab of bevacizumab.

[0075] The present disclosure further provides a VEGF-binding molecule, which comprises immunoglobulin-like domain 2 (R2D2) of VEGFR2 and immunoglobulin-like domain 3 (R2D3) of VEGFR2, wherein the immunoglobulin-like domain 2 (R2D2) of VEGFR2 and / or the immunoglobulin-like domain 3 (R2D3) of VEGFR2 comprises an amino acid mutation at any one or more positions selected from 162, 221, 222, 274, 276, 277, 280, 284, 288, 289, and 313. The sites of the amino acid mutations are natural sequences relative to the amino acid sequence set forth in SEQ ID NO:1.

[0076] In some embodiments, R2D2 and / or R2D3 are: -162nd place, -162nd and 221st place, -162nd and 222nd place, -162nd and 274th place, -162nd and 276th place, -162nd and 277th place, -162nd and 289th place, -162nd and 313th place, -221st and 222nd place, -277th and 289th place, -162nd, 221st and 222nd places, -162nd, 277th and 289th place, -162nd, 221st, 222nd and 274th place, The amino acid mutation is present at a position selected from any one or more of the following groups:

[0077] In some embodiments, R2D2 and / or R2D3 are: -162nd place, -162nd, 221st, 222nd and 274th place, The amino acid mutation is at a position selected from any one of the following groups:

[0078] In some embodiments, R2D2 and / or R2D3 are: -162A, 162V, 162S, 162I or 162L, -221S or 221N, -222K, -274F or 274I, -276Q, -277R, -280E, -284H, -288Y, -289Y, -313R, The amino acid mutations include any one or more of the following groups:

[0079] In some embodiments, R2D2 and / or R2D3 are: -C162A, C162V, C162S, C162I or C162L, -Y221S, -R222K, -N274F or N274I, -D276Q, -L277R, -Q280E, -E284H, -F288Y, -L289Y, -L313R, The amino acid mutations include any one or more of the following groups:

[0080] In some embodiments, R2D2 and / or R2D3 are: -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, -162V / 221S / 222K / 274F, The amino acid mutations include any one or more of the following groups:

[0081] In some embodiments, R2D2 and / or R2D3 are: -C162V or C162I, -C162V / N274F, C162V / N274I, C162I / N274F, or C162I / N274I, -Y221S / R222K, -L277R / L289Y, -C162V / Y221S / R222K, -C162V / D276Q, -C162V / L289Y, -C162V / L277R / L289Y, -C162V / L313R, -C162V / Y221S / R222K / N274F, The amino acid mutations include any one or more of the following groups:

[0082] In some embodiments, R2D2 and / or R2D3 are: -162V, -162V / 221S / 222K / 274F, The amino acid mutations include any one or more of the following groups:

[0083] In some embodiments, R2D2 and / or R2D3 are: -C162V, -C162V / Y221S / R222K / N274F, The amino acid mutations include any one or more of the following groups:

[0084] In some embodiments, the R2D2 and / or R2D3 contain amino acid mutations at one or more positions selected from 135, 197, 199, 213, 215, 219, 257, 275, 277, 278, 283, 289, 313, and 314. The amino acid mutation sites are naturally occurring sites relative to the amino acid sequence set forth in SEQ ID NO:1.

[0085] In some embodiments, the R2D2 and / or R2D3 comprises an amino acid mutation at position 283.

[0086] In some embodiments, R2D2 and / or R2D3 are: -135Y, -197Y, -199Q or 199T, -213N, -215F or 215H, -219I, -257Q or 257T, -275L or 275E, -277R, -278D, 278S or 278E, -283T, -289Y, -313Y, -314S or 314Q, The amino acid mutations include any one or more of the following groups:

[0087] In some embodiments, R2D2 and / or R2D3 are: -V135Y, -M197Y, -F199Q or F199T, -M213N, -I215F or I215H, -V219I, -D257Q or D257T, -R275L or R275E, -L277R, -K278D, K278S or K278E, -S283T, -L289Y, -L313Y, -M314S or M314Q, The amino acid mutations include any one or more of the following groups:

[0088] In some embodiments, the R2D2 and / or R2D3 comprises the amino acid mutation 283T.

[0089] In some embodiments, the R2D2 and / or R2D3 comprises the amino acid mutation S283T.

[0090] In some embodiments, the R2D2 and / or R2D3 comprises an amino acid mutation at any one or more positions selected from 162, 221, 222, 274, 276, 277, 280, 284, 288, 289, and 313; and The amino acid mutation is present at one or more positions selected from 135, 197, 199, 213, 215, 219, 257, 275, 277, 278, 283, 289, 313, and 314.

[0091] In some embodiments, R2D2 and / or R2D3 are: -162nd and 275th place, -162nd and 283rd place, -162nd and 313th place, -162nd, 274th and 283rd place, -162nd, 221st, 222nd and 283rd place, -162nd, 221st, 222nd, 274th and 283rd place, The amino acid mutation is present at a position selected from any one or more of the following groups:

[0092] In some embodiments, R2D2 and / or R2D3 are: -162nd and 283rd place, -162nd, 221st, 222nd, 274th and 283rd place, The amino acid mutation is present at a position selected from any one or more of the following groups:

[0093] In some embodiments, R2D2 and / or R2D3 are: (i) -162A, 162V, 162S, 162I or 162L, -221S, -222K, -274F or 274I, -276Q, -277R, -280E, -284H, -288Y, -289Y, -313R, and an amino acid mutation selected from any one or more of the following groups: (ii) -135Y, -197Y, -199Q or 199T, -213N, -215F or 215H, -219I, -257Q or 257T, -275L or 275E, -277R, -278D, 278S or 278E, -283T, -289Y, -313Y, -314S or 314Q, and an amino acid mutation selected from any one or more of the following groups:

[0094] In some embodiments, R2D2 and / or R2D3 are: (i) -C162A, C162V, C162S, C162I or C162L, -Y221S, -R222K, -N274F or N274I, -D276Q, -L277R, -Q280E, -E284H, -F288Y, -L289Y, -L313R, and an amino acid mutation selected from any one or more of the following groups: (ii) -V135Y, -M197Y, -F199Q or F199T, -M213N, -I215F or I215H, -V219I, -D257Q or D257T, -R275L or R275E, -L277R, -K278D, K278S or K278E, -S283T, -L289Y, -L313Y, -M314S or M314Q, and an amino acid mutation selected from any one or more of the following groups:

[0095] In some embodiments, R2D2 and / or R2D3 are: -162V / 275L, -162V / 283T, -162V / 313Y, -162V / 274F / 283T, -162V / 221S / 222K / 283T, -162V / 221S / 222K / 274F / 283T, The amino acid mutations include any one or more of the following groups:

[0096] In some embodiments, R2D2 and / or R2D3 are: -C162V / R275L, -C162V / S283T, -C162V / L313Y, -C162V / N274F / S283T, -C162V / Y221S / R222K / S283T, -C162V / Y221S / R222K / N274F / S283T, The amino acid mutations include any one or more of the following groups:

[0097] In some embodiments, R2D2 and / or R2D3 are: -162V / 283T, -162V / 221S / 222K / 274F / 283T, It contains an amino acid mutation selected from any one of the following:

[0098] In some embodiments, R2D2 and / or R2D3 are: -C162V / S283T, -C162V / Y221S / R222K / N274F / S283T, It contains an amino acid mutation selected from any one of the following:

[0099] In some embodiments, the VEGF binding molecule consists of R2D2-R2D3.

[0100] In some embodiments, the VEGF-binding molecule comprises the amino acid sequence set forth in any one of SEQ ID NOs: 5 to 6, and SEQ ID NOs: 62 to 65, or an amino acid sequence having at least 80% sequence identity thereto.

[0101] In some embodiments, the VEGF-binding molecule consists of the amino acid sequence set forth in any one of SEQ ID NOs: 5 to 6, and SEQ ID NOs: 62 to 65, or consists of an amino acid sequence having at least 80% sequence identity thereto.

[0102] In some embodiments, the VEGF binding molecule has the ability to bind VEGF-C.

[0103] In some embodiments, the VEGF binding molecule has increased stability and / or ligand binding activity.

[0104] In some embodiments, compared to the wild-type, the VEGF binding molecule comprising the amino acid mutation has increased stability and / or ligand binding activity.

[0105] In some embodiments, any of the above VEGF binding molecules further optionally comprises a first VEGF binding domain, a second VEGF binding domain, and a multimeric component, including, but not limited to, an immunoglobulin Fc or a variant thereof.

[0106] In some embodiments, the Fc comprises a hinge region (e.g., upper, middle, and / or lower hinge regions), a CH2, a CH3 domain, or any combination thereof. In some embodiments, the Fc comprises at least a CH2 and a CH3 domain. In some embodiments, the Fc comprises a hinge region, a CH2, and a CH3 domain.

[0107] In some embodiments, the Fc is derived from an immunoglobulin of any species. In some embodiments, the Fc is derived from a human immunoglobulin. In some embodiments, the Fc is derived from an immunoglobulin of another mammalian species, including, but not limited to, rodent (e.g., mouse, rat, rabbit, guinea pig) or non-human primate (e.g., chimpanzee, macaque) species.

[0108] In some embodiments, the Fc is derived from any subtype of immunoglobulin, including, but not limited to, human IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibodies, hi some embodiments, the Fc is derived from human IgG1.

[0109] In some embodiments, the multimeric component is an Fc variant whose amino acid sequence has no more than 20 amino acid differences, e.g., no more than 15, no more than 10, no more than 5 amino acid differences, compared to a naturally occurring immunoglobulin Fc. In some embodiments, the amino acid differences comprise amino acid substitutions, insertions, or deletions. In some embodiments, the Fc variant has reduced effector function (e.g., FcγR binding).

[0110] In some embodiments, the multimer components cause the VEGF binding molecule to form a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, or decamer.

[0111] In some embodiments, the VEGF binding molecule is a dimer (including a homodimer or a heterodimer).

[0112] In some embodiments, the Fc comprises an IgG1 hinge region, CH2 and CH3 domains.

[0113] In some embodiments, the VEGF-binding domain is fused to a multimeric component (e.g., human immunoglobulin Fc), and the resulting fusion protein has excellent VEGF-binding activity, good stability, a long half-life, and the ability to bind to VEGF molecules in a multivalent manner.

[0114] In some embodiments, the VEGF binding molecule comprises, from N-terminus to C-terminus: R2D2-R2D3-Fc, R1D2-R2D3-R2D2-R2D3-Fc, R1D2-R2D3-linker-R2D2-R2D3-Fc, R2D2-R2D3-R1D2-R2D3-Fc, R2D2-R2D3-linker-R1D2-R2D3-Fc, It includes one selected from:

[0115] In some embodiments, the VEGF binding molecule comprises a heavy chain and a light chain, wherein the heavy chain comprises, from N- to C-terminus, in order: R2D2-R2D3-Fc-linker-VH-CH1, and the light chain comprises, from N- to C-terminus, in order: VL-CL.

[0116] In the formula, "-" indicates that the two domains before and after it are directly or indirectly linked, but the relative positions from the N-terminus to the C-terminus remain unchanged.

[0117] In some embodiments, any two adjacent domains of a VEGF binding molecule of the present disclosure may be directly linked or linked via a linker.

[0118] In some embodiments, the linker is a peptide linker.

[0119] In some embodiments, the linker has the formula (GS): a (GGS) b (GGGS) c (GGGGS) d (GGGG) e wherein a, b, c, d, and e are each independently an integer of 0 or greater, or the linker is selected from (EAAAK) (SEQ ID NO: 90), (EAAAR) (SEQ ID NO: 91), (EGGGK) (SEQ ID NO: 92), (EGGGR) (SEQ ID NO: 93), (DAAAR) (SEQ ID NO: 94), (DAAAK) (SEQ ID NO: 95), (DGGGR) (SEQ ID NO: 96), or (DGGGK) (SEQ ID NO: 97); or the linker is selected from (G x S) y wherein x is selected from integers of 1 to 5, and y is selected from integers of 1 to 6. GGGS corresponds to SEQ ID NO:98, and GGGGG corresponds to SEQ ID NO:99.

[0120] In some embodiments, the linker is GGGGS (SEQ ID NO: 100). In some embodiments, the VEGF binding molecule comprises, from N-terminus to C-terminus: R2D2-R2D3-Fc, R1D2-R2D3-R2D2-R2D3-Fc, R1D2-R2D3-linker-R2D2-R2D3-Fc, R2D2-R2D3-R1D2-R2D3-Fc, R2D2-R2D3-linker-R1D2-R2D3-Fc, Selected from.

[0121] In some embodiments, the VEGF binding molecule comprises a heavy chain and a light chain, wherein the heavy chain is, from N- to C-terminus, in order: R2D2-R2D3-Fc-linker-VH-CH1, and the light chain is, from N- to C-terminus, in order: VL-CL.

[0122] In some embodiments, the VEGF-binding molecule comprises an amino acid sequence set forth in any one of SEQ ID NOs: 3 to 4, 7 to 49, 52 to 61, and 66, or an amino acid sequence having at least 80% sequence identity thereto.

[0123] In some embodiments, the VEGF-binding molecule consists of the amino acid sequence set forth in any one of SEQ ID NOs: 3 to 4, 7 to 49, 52 to 61, and 66, or consists of an amino acid sequence having at least 80% sequence identity thereto.

[0124] In some embodiments, 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 80% sequence identity thereto, and the heavy chain of the VEGF-binding molecule comprises the light chain set forth in SEQ ID NO: 51, or an amino acid sequence having at least 80% sequence identity thereto.

[0125] In some embodiments, 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 80% 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 80% sequence identity thereto.

[0126] In some embodiments, the VEGF binding molecule comprises the amino acid sequence set forth in SEQ ID NO:44, or an amino acid sequence having at least 80% sequence identity thereto, or consists of the amino acid sequence set forth in SEQ ID NO:44.

[0127] In some embodiments, the VEGF binding molecule comprises the amino acid sequence set forth in SEQ ID NO:61, or an amino acid sequence having at least 80% sequence identity thereto, or consists of the amino acid sequence set forth in SEQ ID NO:61.

[0128] In some embodiments, the VEGF-binding molecule of the present disclosure includes a full-length or partial protein of any one of the above VEGF-binding molecules, or a mutein, functional derivative, functional fragment, biologically active peptide, fusion protein, isotype, or salt thereof obtained by further modification based on any one of the above VEGF-binding molecules. For example, a fusion protein containing a VEGF-binding molecule, a monomer, dimer, trimer, or multimer of the above VEGF-binding molecule, various modified forms of the above VEGF-binding molecule (e.g., PEGylation, PEGylation, glycosylation, albumin conjugation or fusion, albumin-antibody conjugation or fusion, hydroxyethylation, removal of O-glycosylation, etc.), and a homolog of the above VEGF-binding molecule in each species. Modification of the above VEGF-binding molecule does not adversely affect the therapeutically relevant protein activity.

[0129] In some embodiments, the VEGF binding molecule has increased stability and / or ligand binding activity.

[0130] In some embodiments, compared to the wild-type, the VEGF binding molecule comprising the amino acid mutation has increased stability and / or ligand binding activity.

[0131] In some embodiments, a VEGF binding molecule of the present disclosure has an HPLC purity of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% when stored at 40°C and 75% humidity for 2 weeks.

[0132] In some embodiments, a VEGF binding molecule of the disclosure has an HPLC purity of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% when stored at 40°C and 75% humidity for 4 weeks.

[0133] In some embodiments, the VEGF binding molecules of the present disclosure have an HPLC purity of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% when stored at room temperature and 75% humidity for two weeks.

[0134] In some embodiments, the VEGF binding molecules of the present disclosure have an HPLC purity of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% when stored at room temperature and 75% humidity for 4 weeks.

[0135] In some embodiments, HPLC purity detection can be performed using the method of Example 4.

[0136] In some embodiments, the binding activity of a VEGF binding molecule of the present disclosure to VEGF-A is an IC50 ≦10 nM, ≦5 nM, or ≦2 nM, e.g., an IC50 of 0.01 nM to 10 nM, 0.05 nM to 5 nM, 0.05 nM to 2 nM, 0.05 nM to 1.5 nM, 0.05 nM to 1.2 nM, or 0.05 nM to 1 nM; and / or The binding activity of the VEGF-binding molecule of the present disclosure to VEGF-C is IC50≦10 nM, ≦5 nM, or ≦2 nM, and for example, the binding activity IC50 of the VEGF-binding molecule of the present disclosure to VEGF-C is 0.01 nM to 10 nM, 0.05 nM to 5 nM, 0.05 nM to 2 nM, 0.05 nM to 1.5 nM, 0.05 nM to 1.2 nM, or 0.05 nM to 1 nM.

[0137] In some embodiments, the binding kinetics of a VEGF binding molecule of the present disclosure to VEGF-A is D ≦1×10 -9 For example, K D ≦1×10 -10 , K. D ≦9×10 -11 , K. D ≦8×10 -11 , K. D ≦7×10 -11 , K. D ≦6×10 -11 , K. D ≦5×10 -11 , K. D ≦4×10 -11 and The binding kinetics of the VEGF binding molecules of the present disclosure to VEGF-B are D ≦1×10 -9 For example, K D ≦4×10 -10 , K. D ≦3×10 -10 , K. D ≦2×10 -10 , K. D ≦1×10 -10 , K. D ≦9×10 -11 , K. D ≦8×10 -11 , K. D ≦7×10 -11 , K. D ≦6×10 -11 , K. D ≦5×10 -11 , K. D ≦4×10 -11 , K. D ≦3×10 -11 , K. D ≦2×10 -11and / or The binding kinetics of the VEGF binding molecules of the present disclosure to VEGF-C are D ≦1×10 -9 For example, K D ≦1×10 -10 , K. D ≦9×10 -11 , K. D ≦8×10 -11 , K. D ≦7×10 -11 , K. D ≦6×10 -11 , K. D ≦5×10 -11 , K. D ≦4×10 -11 , K. D ≦3×10 -11 , K. D ≦2×10 -11 is.

[0138] In some embodiments, the binding activity of a VEGF binding molecule of the present disclosure for a ligand can be measured using the methods described in Example 3 or Example 5.

[0139] Nucleic acids and vectors The present disclosure further provides nucleic acid molecules encoding the VEGF-binding molecules of the present disclosure. In some embodiments, the nucleic acids of the present disclosure may be RNA, DNA, or cDNA. According to some embodiments of the present disclosure, the nucleic acids of the present disclosure are isolated nucleic acids.

[0140] The present disclosure further provides a DNA molecule encoding any of the VEGF binding molecules of the present disclosure described above.

[0141] The nucleic acids of the present disclosure may be in the form of, present in, and / or part of a vector, such as a plasmid, cosmid, YAC, or viral vector. The vector may in particular be an expression vector, i.e., a vector that allows for the expression of a VEGF-binding molecule in vitro and / or in vivo (i.e., in a suitable host cell, host organism, and / or expression system). Such expression vectors typically contain at least one nucleic acid of the present disclosure, operably linked to one or more appropriate expression control elements (e.g., promoters, enhancers, terminators, etc.). The selection of such elements and their sequences for expression in a particular host is within the skill of the art. Regulatory elements and other elements useful or essential for the expression of a VEGF-binding molecule of the present disclosure are, for example, promoters, enhancers, terminators, integration factors, selectable markers, leader sequences, and reporter genes.

[0142] Nucleic acids according to the present disclosure may be prepared or obtained by known methods (e.g., automated DNA synthesis and / or recombinant DNA techniques) based on information about the amino acid sequence of a polypeptide according to the present disclosure, and / or may be isolated from a suitable natural source.

[0143] In some embodiments, the nucleic acid molecule comprises a recombinant or polynucleotide encoding any one of the VEGF binding molecules of the present disclosure.

[0144] In some embodiments, the genetic modification comprises a nucleic acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, or 100% identity to the nucleic acid sequence of any one of SEQ ID NOs: 78-86, or a codon-optimized variant of the nucleic acid sequence of SEQ ID NOs: 78-86.

[0145] In some embodiments, the transgenic nucleic acid sequence is operably linked to a constitutive promoter. In other embodiments, the transgenic nucleic acid sequence is operably linked to an inducible promoter. In some cases, the transgenic nucleic acid sequence is operably linked to a tissue-specific or cell-type-specific regulatory element.

[0146] In the context of this disclosure, genetic modification refers to a target gene and a nucleic acid encoding any one of the VEGF binding molecules of the present disclosure.

[0147] In some embodiments, the isolated nucleic acid further comprises a promoter operably linked to the genetic modification.

[0148] In some embodiments, the promoter is selected from the group consisting of a cytomegalovirus (CMV) promoter, a Rous sarcoma virus (RSV) promoter, an MMT promoter, an EF-1alpha promoter, a UB6 promoter, a chicken beta-actin promoter, a CAG promoter, an RPE65 promoter, and an opsin promoter such as a CMV promoter.

[0149] In some embodiments, the isolated nucleic acid further comprises an enhancer, for example, the enhancer is a CMV enhancer.

[0150] In some embodiments, the isolated nucleic acid further comprises an intron.

[0151] In some embodiments, the isolated nucleic acid further comprises a Kozak sequence. In some embodiments, the Kozak sequence is located between an intron and the genetic sequence encoding the VEGF binding molecule.

[0152] In some embodiments, the isolated nucleic acid further comprises a 5'-UTR and a 3'-UTR.

[0153] In some embodiments, the isolated nucleic acid further comprises a WPRE sequence.

[0154] In some embodiments, the isolated nucleic acid further comprises a polyA sequence, for example, an sv40 polyA sequence.

[0155] The present disclosure further provides a vector comprising any one of the isolated nucleic acids. In some embodiments, the vector is a plasmid or a viral vector. In some embodiments, the virus is selected from the group consisting of adeno-associated virus (AAV), auxiliary-dependent adenovirus, retrovirus, herpes simplex virus, lentivirus, adenovirus, adeno-associated viral vector, poxvirus, Sendai virus-liposome (HVJ) complex, Moloney murine leukemia virus, and HIV-based viruses.

[0156] In some embodiments, the vector is a recombinant adeno-associated virus vector.

[0157] The present disclosure further provides a recombinant adeno-associated viral vector, which comprises: (i) a genetic modification encoding a VEGF binding molecule as defined in any one of the preceding disclosures; (ii) an enhancer (e.g., a CMV enhancer); and (iii) a promoter (e.g., a CMV promoter); (iv) a 5'-UTR and a 3'-UTR; (v) a post-transcriptional regulatory element (e.g., WPRE); (vi) a polyadenylation signal (polyA, e.g., SV40 polyA), optionally containing (vii) a Kozak sequence, and / or (viii) an intron; Any one or any combination of (ii) to (viii) above is operably linked to (i) above.

[0158] In some embodiments, the recombinant adeno-associated viral vector comprises a 5' ITR and / or a 3' ITR derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAV-DJ, or AAV-DJ8, e.g., AAV2, AAV9.

[0159] In some specific embodiments, the promoter is selected from CMV, CAG, CBh, EFS, EF1 (e.g., EF-1α), PGK, SV40, Ubi, RSV, or any combination thereof.

[0160] In some specific embodiments, the enhancer is selected from a Ubi, CMV, RSV enhancer, or any combination thereof.

[0161] In some specific embodiments, the intron is selected from MVM, SV40, β Globin, EF1 (eg, EF-1α), a hybrid intron, or any combination thereof.

[0162] In some specific embodiments, the polyA is selected from PA75 polyA, SV40 polyA, hGH polyA, BGH polyA, rbGlob polyA, or any combination thereof.

[0163] In some specific embodiments, the post-transcriptional regulatory element is selected from a WPRE, an HPRE, or a combination thereof.

[0164] In some embodiments, the recombinant adeno-associated virus vector comprises any one of the above isolated nucleic acids and capsid proteins.

[0165] In some embodiments, the capsid protein is selected from a serotype of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or a variant thereof.

[0166] In some embodiments, the capsid protein comprises or has the amino acid sequence set forth in SEQ ID NO:77, or an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:77.

[0167] The VEGF-binding molecules with novel structures provided by the present disclosure can be administered as novel VEGF inhibitors in the form of protein drugs or nucleic acid drugs. Functionally, they can simultaneously bind to VEGF-A and VEGF-C, and have higher VEGF inhibitory efficacy. In terms of efficacy, the VEGF-binding molecules have higher stability, and in particular, the nucleic acid drug form can effectively reduce the frequency of administration, making them drugs with good prospects for clinical development.

[0168] AAV capsids and AAV particles Any one of the above polynucleotides of the present disclosure can be packaged in the AAV capsid described below to form AAV particles.

[0169] Does the above AAV contain or derive from the AAV serotype, PHP.B, PHP.A, AAV1, AAV2, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3, AAV4, AAV4-4, AAV5, AAV6, AAV6.1, AAV AV6.2、AAV6.1.2、AAV7、AAV7.2、AAV8、AAV9、AAV9.11、AAV9.13、AAV9.16、AAV9.24、AAV9.45、AAV9.47、AAV9.61、AAV9.68、AAV9.84、AAV9.9、AAV10、AA V11、AAV12、AAV16.3、AAV24.1、AAV27.3、AAV42.12、AAV42-1b、AAV42-2、AAV42-3a、AAV42-3b、AAV42-4、AAV42-5a、AAV42-5b、AAV42-6b、AAV42-8、AAV 42-10、AAV42-11、AAV42-12、AAV42-13、AAV42-15、AAV42-aa、AAV43-1、AAV43-12、AAV43-20、AAV43-21、AAV43-23、AAV43-25、AAV43-5、AAV44.1、AAV44. 4.2、AAV44.5、AAV223.1、AAV223.2、AAV223.4、AAV223.5、AAV223.6、AAV223.7、AAV1-7 / rh.48、AAV1-8 / rh.49、AAV2-15 / rh.62、AAV2-3 / rh.61 4 / rh.50、AAV2-5 / rh.51、AAV3.1 / hu.6、AAV3.1 / hu.9、AAV3-9 / rh.52、AAV3-11 / rh.53、AAV4-8 / r11.64、AAV4-9 / rh.54、AAV4-19 / rh.55、AAV5-3 / rh.55 7、AAV5-22 / rh.58、AAV7.3 / hu.7、AAV16.8 / hu.10、AAV16.12 / hu.11、AAV29.3 / bb.1、AAV29.5 / bb.2、AAV106.1 / hu.37、AAV114.3 / hu.40、AAV127.2 / hu.41、AAV127.5 / hu.42、AAV128.3 / hu.44、AAV130.4 / hu.48、AAV145.1 / hu.53、AAV145.5 / hu.54、AAV145.6 / hu.55、AAV161.10 / hu.60、AAV161.6 / hu.61、AAV33.12 / hu.17、AAV33.4 / hu.15、AAV33.8 / hu.16、AAV52 / hu.19、AAV52.1 / hu.20、AAV58.2 / hu.25、AAVA3.3、AAVA3.4、AAVA3.5、AAVA3.7、AAVC1、AAV C2、AAVC5、AAV-DJ、AAV-DJ8、AAVF3、AAVF5、AAVH2、AAVrh.72、AAVhu.8、AAVrh.68、AAVrh.70、AAVpi.1、AAVpi.3、AAVpi.2、AAVrh.60、AAVrh.44、AAVrh. .65、AAVrh.55、AAVrh.47、AAVrh.69、AAVrh.45、AAVrh.59、AAVhu.12、AAVH6、AAVLK03、AAVH-1 / hu.1、AAVH-5 / hu.3、AAVLG-10 / rh.40、AAVLG-4 / rh.38、AAVLG-9 / hu.39、AAVN721-8 / rh.43、AAVCh.5、AAVCh.5R1、AAVcy.2、AAVcy.3、AAVcy.4、AAVcy.5、AAVCy.5R1、AAVCy.5R2、AAVCy.5R3、AAVCy.5R4、AAVc y.6、AAVhu.1、AAVhu.2、AAVhu.3、AAVhu.4、AAVhu.5、AAVhu.6、AAVhu.7、AAVhu.9、AAVhu.10、AAVhu.11、AAVhu.13、AAVhu.15、AAVhu.16、AAVhu.17、AA Vhu.18、AAVhu.20、AAVhu.21、AAVhu.22、AAVhu.23.2、AAVhu.24、AAVhu.25、AAVhu.27、AAVhu.28、AAVhu.29、AAVhu.29R、AAVhu.31、AAVhu.32、AAVhu. 34、AAVhu.35、AAVhu.37、AAVhu.39、AAVhu.40、AAVhu.41、AAVhu.42、AAVhu.43、AAVhu.44、AAVhu.44R1、AAVhu.44R2、AAVhu.44R3、AAVhu.45、AAVhu.4 6、AAVhu.47、AAVhu.48、AAVhu.48R1、AAVhu.48R2、AAVhu.48R3、AAVhu.49、AAVhu.51、AAVhu.52、AAVhu.54、AAVhu.55、AAVhu.56、AAVhu.57、AAVhu.58、AAVhu.60、AAVhu.61、AAVhu.63、AAVhu.64、AAVhu.66、AAVhu.67、AAVhu.68、AAVhu.14 / 9、AAVhu.t19、AAVrh.2、AAVrh.2R、AAVrh.8、AAVrh.8R、AAVrh.10、AAVrh.12、AAVhu. Vrh.13、AAVrh.13R、AAVrh.14、AAVrh.17、AAVrh.18、AAVrh.19、AAVrh.20、AAVrh.21、AAVrh.22、AAVrh.23、AAVrh.24、AAVrh.25、AAVrh.31、AAVrh.32、AAVrh.33、AAVrh. h.34、AAVrh.35、AAVrh.36、AAVrh.37、AAVrh.37R2、AAVrh.38、AAVrh.39、AAVrh.40、AAVrh.46、AAVrh.48、AAVrh.48.1、AAVrh.48.1.2、AAVrh.48.2、AAVrh.49、AAVrh. 51、AAVrh.52、AAVrh.53、AAVrh.54、AAVrh.56、AAVrh.57、AAVrh.58、AAVrh.61、AAVrh.64、AAVrh.64R1、AAVrh.64R2、AAVrh.67、AAVrh.73、AAVrh.74、AAVrh.88 A586R mutation、AAVrh8R R533A mutant、AAAV、BAAV、ヤギAAV、ウシAAV、ヒツジAAV、AAVhE1.1、AAVhEr1.5、AAVhER1.14、AAVhEr1.8、AAVhEr1.16、AAVhEr1.18、AAVhEr 1.35、AAVhEr1.7、AAVhEr1.36、AAVhEr2.29、AAVhEr2.4、AAVhEr2.16、AAVhEr2.30、AAVhEr2.31、AAVhEr2.36、AAVhEr1.23、AAVhEr2. r3.1、AAV2.5T、AAV-PAEC、AAV-LK01、AAV-LK02、AAV-LK03、AAV-LK04、AAV-LK05、AAV-LK06、AAV-LK07、AAV-LK08、AAV-LK09、AAV -LK10、AAV-LK11、AAV-LK12、AAV-LK13、AAV-LK14、AAV-LK15、AAV-LK16、AAV-LK17、AAV-LK18、AAV-LK19、AAV-PAEC2、AAV-PAEC4、AAV-PAEC6, AAV-PAEC7, AAV-PAEC8, AAV-PAEC11, AAV-PAEC12, AAV-2-pre-miRNA-101, AAV-8h, AAV-8b, AAV-h, AAV-b, AAV SM 10-2, AAVShuffle 100-1, AAV Shuffle 100-3, AAV Shuffle 100-7, AAV Shuffle 10-2, AAVShuffle 10-6, AAV Shuffle 10-8, AAV Shuffle 100-2, AAV SM 10-1, AAV SM 10-8, AAVSM 100-3, AAV SM 100-10, BNP61 AAV, BNP62 AAV, BNP63 AAV, AAVrh.50, AAVrh.43, AAVrh.62, AAVrh.48, AAVhu.19, AAVhu.11, AAVhu.53, AAV4-8 / rh.64, AAVLG-9 / hu.39, AAV54.5 / hu.23, AAV54.2 / hu.22, AAV54.7 / hu.24, AAV54.1 / hu.21, AAV54.4R / hu.27, AAV46.2 / hu.28, AAV46.6 / hu.29, AAV128.1 / hu.43, true type AAV (ttAAV), UPENNAAV 10, Japanese AAV 10 serotype, AAV CBr-7.1, AAV CBr-7.10, AAV CBr-7.2, AAV CBr-7.3, AAVCBr-7.4, AAV CBr-7.5, AAV CBr-7.7, AAV CBr-7.8, AAV CBr-B7.3, AAV CBr-B7.4, AAVCBr-E1, AAV CBr-E2, AAV CBr-E3, AAV CBr-E4, AAV CBr-E5, AAV CBr-e5, AAV CBr-E6, AAVCBr-E7, AAV CBr-E8, AAV CHt-1, AAV CHt-2, AAV CHt-3, AAV CHt-6.1, AAV CHt-6.10, AAVCHt-6.5, AAV CHt-6.6, AAV CHt-6.7, AAV CHt-6.8, AAV CHt-P1, AAV CHt-P2, AAV CHt-P5, AAV CHt-P6, AAV CHt-P8, AAV CHt-P9, AAV CKd-1, AAV CKd-10, AAV CKd-2, AAV CKd-3, AAVCKd-4AAV CKd-6、AAV CKd-7、AAV CKd-8、AAV CKd-B1、AAV CKd-B2、AAV CKd-B3、AAV CKd-B4、AAV CKd-B5、AAV CKd-B6、AAV CKd-B7、AAV CKd-B8、AAV CKd-H1、AAV CKd-H2、AAV CKd-H3、AAV CKd-H4、AAV CKd-H5、AAV CKd-H6、AAV CKd-N3、AAV CKd-N4、AAV CKd-N9、AAV CLg-F1、AAV CLg-F2、AAV CLg-F3、AAV CLg-F4、AAV CLg-F5、AAV CLg-F6、AAV CLg-F7、AAV CLg-F8、AAV CLv-1、AAV CLv1-1、AAV CLv1-10、AAV CLv1-2、AAV CLv-12、AAV CLv1-3、AAV CLv-13、AAV CLv1-4、AAV Clv1-7、AAV Clv1-8、AAV Clv1-9、AAV CLv-2、AAV CLv-3、AAV CLv-4、AAV CLv-6、AAV CLv-8、AAV CLv-D1、AAV CLv-D2、AAV CLv-D3、AAV CLv-D4、AAV CLv-D5、AAV CLv-D6、AAV CLv-D7、AAV CLv-D8、AAV CLv-E1、AAV CLv-K1、AAV CLv-K3、AAV CLv-K6、AAV CLv-L4、AAV CLv-L5、AAV CLv-L6、AAV CLv-M1、AAV CLv-M11、AAV CLv-M2、AAV CLv-M5、AAV CLv-M6、AAV CLv-M7、AAV CLv-M8、AAV CLv-M9、AAV CLv-R1、AAV CLv-R2、AAV CLv-R3、AAV CLv-R4、AAV CLv-R5、AAV CLv-R6、AAV CLv-R7、AAV CLv-R8、AAV CLv-R9、AAV CSp-1、AAV CSp-10、AAV CSp-11、AAV CSp-2、AAV CSp-3、AAV CSp-4、AAV CSp-6、AAV CSp-7、AAVCSp-8、AAV CSp-8.10、AAV CSp-8.2、AAV CSp-8.4、AAV CSp-8.5、AAV CSp-8.6、AAV CSp-8.7、AAV CSp-8.8, AAV CSp-8.9, AAV CSp-9, AAV.hu.48R3, AAV.VR-355, AAV3B, AAV4, AAV5, AAVF1 / HSC1, AAVF11 / HSC11, AAVF12 / HSC12, AAVF13 / HSC13, AAVF14 / HSC14, AAVF15 / , HSC15, AAVF16 / HSC16, AAVF17 / HSC17, AAVF2 / HSC2, AAVF3 / HSC3, AAVF4 / HSC4, AAVF5 / HSC5, AAVF6 / HSC6, AAVF7 / HSC7, AAVF8 / HSC8, AAVF9 / HSC9, PHP.B (AAV-PHP.B), PHP.A (AAV.PHP.A), G2B-26, G2B-13, TH1.1-32, TH1.1-35, AAVPHP.B2, AAVPHP.B3, AAVPHP.N / PHP.B-DGT, AAVPHP.B-EST, AAVPHP.B-GGT, AAVPHP.B-ATP, AAVPHP.B-ATT-T, AAVPHP.B-DGT-T, AAVPHP.B-GGT-T, AAVPHP.B-SGS, AAVPHP.B-AQP, AAVPHP.B-QQP, AAVPHP.B-SNP(3), AAVPHP.B-SNP, AAVPHP.B-QGT, AAVPHP.B-NQT, AAVPHP.B-EGS, AAVPHP.B-SGN, AAVPHP.B-EGT, AAVPHP.B-DST, AAVPHP.B-DST, AAVPHP.B-STP, AAVPHP.B-PQP, AAVPHP.B-SQP, AAVPHP.B-QLP, AAVPHP.B-TMP, AAVPHP.B-TTP, AAVPHP.S / G2A12, AAVG2A15 / G2A3, AAVG2B4, AAVG2B5, AAVDJ8 and variants thereof, including but not limited to these.

[0170] In some embodiments, the AAV capsid may be modified or mutated, for example, containing one or more mutations of Y252F, Y272F, Y444F, Y500F, Y700F, Y704F, Y730F, Y275F, Y281F, Y508F, Y576F, Y612G, Y673F, and Y720F, for example, containing one or more mutations of F129L, D418E, K531E, L584F, V598A, and H642N, for example, AAV2 At least one of the Tyr residues at positions 252, 272, 444, 500, 700, 704, and 730 is substituted with a Phe residue or the like; for example, the AAV2 capsid contains an N587A, E548A, or N708A mutation; for example, the AAV9 capsid contains a T446F mutation; for example, the AAV capsid contains a V708K mutation; for example, the above AAV capsid is produced by an AAV9 capsid library having mutations in amino acids at positions 390 to 627 (VP1 numbering).

[0171] The present disclosure relates to WO2018232055 (e.g., Table 1 thereof), WO2005033321, WO2015168666, WO2015121501, WO2015038958, WO2016065001, WO2016130589, WO2016049230, WO2016134375, WO2017100671, WO2017083722, WO2017015102, WO2017058892, WO2017066764, US9546112, US7198951, US9233131, US6156303, US96 24274, US9475845, US8734809, US20130224836, US20140359799, US20150315612, US20150376240, US20150159173, US20150376607, US20150238550, US20160369298, US20160361439, US20170145405, and N Pulicherla et al. (Molecular Therapy 19(6):1070-1078(2011)). In some specific embodiments, the AAV capsids used in the present disclosure are the AAV2 and AAV9 capsids in the above-mentioned prior art.

[0172] In some embodiments, the AAV capsid is engineered, e.g., a hybrid AAV capsid derived from two or more parent serotypes. For example, the AAV capsid may be AAV2G9, which contains sequences from AAV2 and AAV9, incorporating all of the AAV2G9 sequences in US20160017005.

[0173] In some embodiments, the AAV capsid is an AAV2 or AAV9 capsid.

[0174] In some embodiments, other than the encoding heterologous payload, the AAV particles may comprise all or part of the nucleotide sequence of any naturally occurring and / or recombinant AAV capsid or mutant vector genome.

[0175] In some embodiments, the AAV particles may be replication-deficient (e.g., lacking sequences encoding functional Rep and Cap proteins in the vector genome). In some specific embodiments, the replication-deficient AAV particles may lack most or all of the parent coding sequences and possess essentially only one or two AAV ITR sequences and a nucleic acid of interest for delivery to a cell, tissue, organ, or organism.

[0176] In some embodiments, the AAV particles may be recombinant AAV (rAAV) particles.

[0177] In some embodiments, the AAV particles may be selected from single-stranded AAV particles (e.g., ssAAV), self-complementary AAV particles (e.g., scAAV), which achieve rapid expression in cells by skipping second strand synthesis.

[0178] In some embodiments, the AAV particles are encapsulated with a nucleic acid encoding a VEGF-binding molecule comprising the amino acid sequence set forth in any one of SEQ ID NOs: 3-4, 7-49, 52-61, and 66, wherein the VEGF-binding molecule comprises a heavy chain set forth in SEQ ID NO: 50 and a light chain set forth in SEQ ID NO: 51, or the VEGF-binding molecule comprises a heavy chain set forth in SEQ ID NO: 67 and a light chain set forth in SEQ ID NO: 68.

[0179] In some embodiments, the AAV particles are packaged with a nucleic acid having at least 95% identity to a sequence set forth in any one of SEQ ID NOs: 78-86.

[0180] In some embodiments, the AAV particles are packaged with a nucleic acid comprising a sequence set forth in any one of SEQ ID NOs: 78-86.

[0181] In some embodiments, the capsid protein of the AAV particle comprises or has the amino acid sequence set forth in SEQ ID NO: 77, or an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 77.

[0182] Regulatory elements The vector genome (or viral genome) of any one of the above nucleic acids or AAV particles of the present disclosure comprises at least one regulatory element to enable replication, transcription, and translation of the target gene (e.g., a VEGF binding molecule) in the payload.

[0183] In some embodiments, the regulatory elements include, but are not limited to, sequences for transcription initiation and / or termination, promoter and / or enhancer sequences, efficient RNA processing signals (e.g., splicing and polyadenylation signals), sequences that stabilize cytoplasmic mRNA, sequences that enhance translation efficiency (e.g., Kozak consensus sequences), sequences that enhance protein stability, and / or sequences that enhance protein processing and / or secretion. Exemplary regulatory elements include, but are not limited to, promoters, enhancers, introns, endogenous miRNAs, post-transcriptional regulatory elements (PREs), polyadenylation (PolyA) signal sequences, and upstream enhancers (USEs).

[0184] In some embodiments, the regulatory element (e.g., promoter) drives expression of the payload in the target tissue (e.g., in the eye of a subject) for a period of time, such as 1, 2, 3, 4, 5, 6, 7 days, 1, 2, 3, 4 weeks, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60 years or more. Also, for example, 1 to 2 weeks, 1 to 3 weeks, 1 to 4 weeks, 1 to 2 months, 1 to 4 months, 1 to 6 months, 2 to 6 months, 3 to 6 months, 3 to 9 months, 4 to 8 months, 6 to 12 months, 1 to 2 years, 1 to 5 years, 2 to 5 years, 3 to 6 years, 3 to 8 years, 4 to 8 years, 5 to 10 years, 10 to 20 years, 10 to 30 years, 20 to 40 years, 20 to 50 years, or the entire lifetime of the subject.

[0185] inverted terminal repeat (ITR) The vector genome (or viral genome) of any one of the above polynucleotides or AAV particles of the present disclosure comprises at least one ITR, e.g., two ITRs flanking the payload at the 5' and 3' ends, respectively.

[0186] In some embodiments, the ITRs have an origin of replication function.

[0187] In some embodiments, the ITRs comprise complementary, symmetrically alignable sequence regions.

[0188] In some embodiments, the ITRs may consist of naturally occurring or recombinantly derived polynucleotide sequences.

[0189] In some embodiments, the ITRs may be derived from the same or a different serotype as the capsid. In some embodiments, the 5' ITR and 3' ITR may be derived from the same serotype or from different serotypes. For example, the 5' ITR and 3' ITR may both be derived from AAV2 or both from AAV9. Also, for example, the 5' ITR may comprise the sequence set forth in SEQ ID NO:76 and / or the 3' ITR may comprise the nucleotide sequence set forth in SEQ ID NO:87.

[0190] In some embodiments, the length of each ITR can be about 100 to about 150 nucleotides, e.g., 100 to 105 nucleotides, 106 to 110 nucleotides, 111 to 115 nucleotides, 116 to 120 nucleotides, 121 to 125 nucleotides, 126 to 130 nucleotides, 131 to 135 nucleotides, 136 to 140 nucleotides, 141 to 145 nucleotides, or 146 to 150 nucleotides. In one embodiment, the length of the ITR is 140 to 142 nucleotides, e.g., 141 nucleotides. Non-limiting examples of ITR lengths are 102, 140, 141, 142, and 145 nucleotides, and lengths with at least 95% identity thereto.

[0191] promoter The vector genome (or viral genome) of any one of the above polynucleotides or AAV particles of the present disclosure comprises at least one promoter, including, but not limited to, a species-specific, inducible, tissue-specific, or cell cycle-specific promoter.

[0192] In some embodiments, the promoter drives expression of a protein or polypeptide (e.g., a VEGF-binding molecule) encoded in the vector genome payload of the AAV particle.

[0193] In some embodiments, the promoter is target tissue specific or tropic, for example, a promoter capable of expressing the payload in ocular tissue.

[0194] In some embodiments, the promoter may be a viral promoter, a plant promoter, a mammalian promoter, or a human promoter.

[0195] In some embodiments, the promoter includes, but is not limited to, a CMV, CBA (including derivatives CAG, CBh, etc.), EF-1α, PGK, UBC, RSV, EFS, EF1, GUSB (hGBp), UCOE (promoter of HNRPA2B1-CBX3), NSE, Synapsin, MeCP2, MeP418, MeP426, VMD2, MRHO, TRE, Ac5, Polyhedrin, CaMKIIa, Gall, TEF1, GDS, ADH1, Ubi, GFAP, or PKG promoter, and may be selected from a neurofilament light chain (NFL) promoter, a neurofilament heavy chain (NFH) promoter, an SCN8A promoter, a frataxin (FXN) promoter (also referred to as an FRDA promoter), an H1 promoter, an RNA pol III promoter (e.g., U6 or H1), or a small nuclear RNA (ULB or ULA) promoter. In some embodiments, the promoter is a liver or skeletal muscle promoter, such as human alpha-1-antitrypsin (hAAT) and thyroxine-binding globulin (TBG), and skeletal muscle promoters such as desmin, MCK, or synthetic C5-12.

[0196] In some embodiments, the AAV vector genome of the present disclosure comprises two promoters, for example, the CMV and CBA promoters, whose CMV sequence is set forth in SEQ ID NO: 75, or, for example, the EF1α promoter and the CMV promoter, or any two of the above promoters paired together.

[0197] In some embodiments, the promoter is a tissue-specific expression element that can limit expression to certain cell types, including, but not limited to, a muscle-specific promoter, a B-cell promoter, a monocyte promoter, a leukocyte promoter, a macrophage promoter, a pancreatic acinar cell promoter, an endothelial cell promoter, a lung tissue promoter, an astrocyte promoter, or a nervous system promoter, and can be used to limit expression to neurons, astrocytes, or oligodendrocytes.

[0198] In some embodiments, the promoter is a truncation or mutant of the above promoters and is less than 1 kb in length, for example, 200-300, 200-400, 300-400, 200-500, 200-600 or more.

[0199] In some embodiments, the promoter may be a combination of two or more components of the same or different initiating or parent promoters, for example, CMV and CBA.

[0200] Enhancer The vector genome (or viral genome) of any one of the above polynucleotides or AAV particles of the present disclosure comprises at least one enhancer, including, but not limited to, a species-specific, inducible, tissue-specific, or cell cycle-specific enhancer.

[0201] In some embodiments, the enhancer is target tissue specific or tropic, for example, an enhancer that can regulate expression of the payload in neural tissue.

[0202] In some embodiments, the enhancer may be or be derived from a viral enhancer, a plant enhancer, a mammalian enhancer, or a human enhancer.

[0203] In some embodiments, the enhancer can be located upstream or downstream of the promoter and operably linked to the promoter, such that the presence of the enhancer increases expression of the target gene (e.g., a VEGF binding molecule) by at least 20%, at least 50%, at least 80%, at least 100%, or at least several-fold (including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10-fold or more) compared to the absence of the enhancer.

[0204] In some embodiments, the enhancer includes, but is not limited to, EF-lα, Ubc, human b-actin, CAG, TRE, Ac5, Polyhedrin, CaMKIIa, Gall, TEF1, GDS, ADH1, Ubi, and α-1-antitrypsin (hAAT), or a variant or fragment thereof. In some embodiments, the enhancer is selected from IRBP, RSV, or CMV enhancers, or a variant or fragment thereof.

[0205] In some embodiments, the enhancer comprises or is the enhancer set forth in SEQ ID NO: 74, or a variant or fragment thereof.

[0206] Introns Any one of the above polynucleotides or vector genomes (or viral genomes) of an AAV particle of the present disclosure may include an intron or a portion thereof.

[0207] In some embodiments, the introns include, but are not limited to, MVM (67-97 bp), intron 1 of F.IX cleavage (approximately 300 bp), β-globulin SD / immunoglobulin heavy chain splicing acceptor (approximately 250 bp), adenovirus splicing donor / immunoglobulin splicing acceptor (approximately 500 bp), SV40 late splicing donor / splice acceptor (19S / 16S) (180 bp), and hybrid adenovirus splicing donor / IgG splicing acceptor (approximately 230 bp), hemoglobin intron, hybrid intron, β-globin intron, or variants or fragments of the above introns.

[0208] In some embodiments, the length of the intron is 100 to 800 nucleotides, for example, about 100, about 200, about 300, about 400, about 500, or about 600 nucleotides, or, for example, 200 to 250, 200 to 300, 100 to 200, 300 to 500, 500 to 600, 400 to 600, or 100 to 200 nucleotides.

[0209] In some embodiments, the intron in the technical solution of the present disclosure is selected from a hybrid intron and a β-globin intron.

[0210] Untranslated Regions (UTRs) The vector genome (or viral genome) of any one of the above polynucleotides or AAV particles of the present disclosure may contain untranslated regions (UTRs) selected from 5' UTRs and / or 3' UTRs to adjust (e.g., increase, decrease, or reduce) polynucleotide stability and protein production. Typically, the 5' UTR begins at the transcription initiation site and ends at the start codon, while the 3' UTR extends from immediately after the stop codon to the transcription termination signal. The UTRs may be wild-type, mutants thereof, or artificial UTRs.

[0211] In some embodiments, the 5'UTR comprises a Kozak sequence. In other embodiments, the 5'UTR does not comprise a Kozak sequence.

[0212] In some embodiments, the 3'UTR is AU-rich. In some embodiments, the 3'UTR is selected from type I AREs, which contain multiple interspersed copies of the AUUUA motif in a U-rich region, such as, but not limited to, c-Myc and MyoD; type II AREs, which have two or more overlapping UUAUUUA(U / A)(U / A) nonamers, such as, but not limited to, GM-CSF and TNF-α; and type III AREs, such as, but not limited to, c-Jun and myogenic proteins.

[0213] In some embodiments, the 3'UTR may contain an oligo(dT) sequence to template the addition of a polyadenylation sequence (PolyA).

[0214] Polyadenylation sequence (PolyA) The vector genome (or viral genome) of any one of the above polynucleotides or AAV particles of the present disclosure may comprise a sequence encoding PolyA, which may be wild-type, a mutant thereof, or a modified PolyA such that protein translation occurs.

[0215] In some embodiments, the PolyA-encoding sequence is between the 3' end of the payload coding sequence and the 5' end of the 3' ITR.

[0216] In some embodiments, the length of the sequence encoding PolyA is 0 to 500 nucleotides, for example, about 75, about 100, about 110, about 120, about 150, about 160, about 200, or about 300 nucleotides, or for example, 50 to 100, 50 to 150, 50 to 160, 50 to 200, 60 to 100, 60 to 150, 60 to 160, 60 to 200, 70 to 100, 70 to 150, 70 to 160, 70 to 200, 80 to 100, 80 to 150, 80 to 160, 80 to 200, 90 to 100, 90 to 150, 90 to 160, or 90 to 200 nucleotides.

[0217] In some embodiments, the coding PolyA can encode a continuous or discontinuous PolyA. When encoding a discontinuous PolyA, the coding sequence can be interrupted or separated by other nucleotides. For example, the polyA has at least two 60 adenylate fragments, and the at least two 60 adenylate fragments are separated by a sequence containing 10 to 90 nucleotides.

[0218] In some embodiments, the sequence encoding PolyA or polyA in the technical solution of the present disclosure is selected from β-globin polyA, SV40 polyA, bGH polyA, PA75 polyA, MeCP2 polyA, RDH1 polyA, BGH polyA, SPA49 polyA, sNRP-TK65 polyA, sNRP polyA, TK65 polyA, or a variant or fragment of the above PolyA.

[0219] In some embodiments, the polyA is set forth in SEQ ID NO: 89 in the technical solution of the present disclosure, or a variant or fragment thereof.

[0220] Here, polyA or its coding sequence in WO2016005324, WO2016005004, WO2016091391, WO2019036513, and WO2020074642 are all incorporated, and all of them can be used in the technical solution of the present disclosure.

[0221] Filler Arrangement The vector genome (or viral genome) of any one of the above polynucleotides or AAV particles of the present disclosure may include filler sequences to optimize the length of the vector genome for packaging, for example, to optimize the length of the vector genome to about 2.3 kb, about 4.6 kb, about 4.7 kb, or about 5.1 kb.

[0222] In some embodiments, the vector genome is a single-stranded or double-stranded genome, and the packaged AAV particles are ssAAV or scAAV.

[0223] In some embodiments, a vector genome may contain one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) filler sequences.

[0224] In some embodiments, the filler sequence may be located between or within multiple regulatory elements, for example, between two introns, 3' of the 5'ITR sequence, 5' of the 5'ITR sequence, 5' of the 3'ITR sequence, 3' of the 3'ITR sequence, and before or after regions such as promoters, introns, enhancers, polyA, polyclonal site (MCS) regions, exons, etc.

[0225] Methods for producing or preparing recombinant adeno-associated virus (rAAV) particles The present disclosure provides methods for producing, preparing, and / or modifying rAAV particles, which incorporate all of the methods for producing, preparing, and / or modifying rAAV particles in WO200028004, WO200123001, WO2004112727, WO2005005610, WO2005072364, WO2013123503, WO2015191508, and US20130195801. The rAAV particles can have properties that enhance delivery efficiency, can be packaged efficiently, and can successfully infect target cells (e.g., mammalian or human cells) at high frequency and with minimal toxicity.

[0226] In some embodiments, methods are provided for producing rAAV particles, comprising packaging any nucleic acid or vector genome (or viral genome) or vector according to the present disclosure into an AAV capsid. 1) co-transfecting a baculovirus vector and a viral construct vector and / or an AAV payload construct vector into competent cells; 2) isolating the resulting viral construct expression vector and AAV payload construct expression vector and transfecting them into virus-replicating cells, respectively; 3) isolating and purifying the resulting payload and viral construct particles comprising the viral construct expression vector or AAV payload construct expression vector; 4) co-transfecting both the AAV payload and the viral construct expression vector or the viral construct particle comprising the AAV payload construct expression vector into a viral replicating cell; 5) collecting and purifying the AAV particles containing the viral genome.

[0227] In some embodiments, a method for producing rAAV particles is provided, comprising: 1) co-transfecting any nucleic acid or vector genome (or viral genome) or vector of the present disclosure, and a construct expressing Rep and Cap genes and an auxiliary construct (to achieve auxiliary functions) into mammalian cells (e.g., HEK293 cells); 2) collecting and purifying the rAAV particles containing the viral genome.

[0228] In some embodiments, the viral genome of the rAAV particle optionally encodes a selectable marker, which may include a cell surface marker, e.g., any protein expressed on the cell surface, including, but not limited to, receptor CD markers, lectins, integrins, or truncated forms thereof.

[0229] In some embodiments, the present disclosure provides an AAV production system for producing rAAV particles, wherein the production system comprises: 1) a nucleic acid sequence encoding an AAV capsid; 2) any nucleic acid or vector genome (or viral genome) or vector of the present disclosure; 3) auxiliary packaging elements having sufficient AAV rep and accessory functions to enable packaging of the nucleic acid or vector genome (or viral genome) or vector in (b) into an AAV capsid.

[0230] The nucleic acid or vector genome (or viral genome) or vector comprises a nucleic acid encoding any of the above-described VEGF binding molecules of the present disclosure.

[0231] The AAV capsid is selected from any AAV capsid of the present disclosure, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAV-DJ, AAV-DJ8, or a variant of any one of the above.

[0232] In some embodiments, the auxiliary packaging plasmid contains sufficient AAV rep and accessory functions of the elements provided by the packaging cell, which may contain any one or more of three plasmids: pHelper, pRC9, and pGOI.

[0233] Illustratively, "auxiliary functions" or "auxiliary viral functions" refer to functions encoded in an auxiliary viral genome that enable AAV replication and packaging (in combination with other requirements for replication and packaging as described herein). In the present disclosure, "auxiliary viral functions" can be provided in many ways, including by providing an auxiliary virus or, for example, by providing polynucleotide sequences encoding the essential functions to a producer cell during transport.

[0234] Illustratively, "AAV rep function" refers to the function provided by the Rep gene and / or the Cap gene.

[0235] In some embodiments, the Rep gene encodes a nonstructural protein that regulates functions such as AAV genome replication, and may be selected from Rep78, Rep68, Rep52, and Rep40. Rep78 and Rep68 are typically transcribed from the p5 promoter, while Rep52 and Rep40 are typically transcribed from the p19 promoter. The Cap gene encodes the structural proteins VP1, VP2, and / or VP3, which assemble to form the viral capsid. The Cap gene is typically transcribed from the p40 promoter.

[0236] In some embodiments, rAAV particles produced by the above-described AAV production system are provided.

[0237] Host cells and preparation methods The present disclosure further provides a host cell, e.g., an isolated (genetically modified) host cell, comprising any one of the above nucleic acids. The host cell may be an isolated cell, e.g., a cell cultured in vitro. The host cell may express one or more of the VEGF-binding molecules or complexes thereof of the present disclosure and / or contain a nucleic acid or vector of the present disclosure.

[0238] In some embodiments, host cells include prokaryotic microorganisms (e.g., E. coli) or various eukaryotic cells (e.g., Chinese hamster ovary cells (CHO), human embryonic kidney (HEK) cells, or lymphocytes (e.g., Y0, NS0, Sp20 cells), insect cells, etc.).

[0239] In some embodiments, host cells expressing glycosylated polypeptides from multicellular organisms (e.g., including invertebrates and vertebrates), such as plant and insect cells, can be used. Vertebrate cells can also be used as host cells, such as suspension-grown mammalian cell lines, monkey kidney CV1 line (COS-7), human embryonic kidney line (293 or 293T cells), baby hamster kidney cells (BHK), mouse Sertoli cells (TM4 cells), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo rat hepatocytes (BRL3A), human lung cells (W138), human hepatocytes (Hep G2), mouse mammary tumor cells (MMT060562), TRI cells (see, e.g., Mather et al., Annals of Pharmacology, NY, 2001). Sci 383, 44-68 (1982), MRC5 cells and FS4 cells, Chinese hamster ovary (CHO) cells, myeloma cell lines such as YO, NS0, P3X63 and Sp2 / 0, and cells contained in transgenic animals, transgenic plants, or cultured plant or animal tissues can be used.

[0240] As described below, host cells can be used to produce the subject rAAV virions. The host cells used to produce the subject rAAV virions are referred to as "packaging cells." In some embodiments, the host cells are stably genetically modified with any one of the above. In other embodiments, the host cells are transiently genetically modified with any one of the above.

[0241] Any one of the above is stably or transiently introduced into host cells using established techniques including, but not limited to, electroporation, calcium phosphate precipitation, liposome-mediated transfection, etc. To stabilize the transformation, any one of the above typically further includes an optional marker, e.g., any of several well-known optional markers such as neomycin resistance.

[0242] The subject host cells are generated by introducing any of the above into any of a number of cells, e.g., mammalian cells, including (for example) murine cells and primate cells (e.g., human cells). Suitable mammalian cells include, but are not limited to, primary cells and cell lines, where suitable cell lines include, but are not limited to, 293 cells, COS cells, HeLa cells, Vero cells, 3T3 mouse fibroblasts, C3H10T1 / 2 fibroblasts, CHO cells, etc. Non-limiting examples of suitable host cells include (for example) HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse L cells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, and the like. The subject host cells (see, eg, US Patent No. 7,271,002, US Patent Publication No. 12 / 297,958) can also be prepared into insect cells, such as Sf9 cells, that produce AAV using baculovirus infection.

[0243] In some embodiments, the subject genetically modified host cells further comprise a nucleic acid comprising a nucleotide sequence encoding one or more AAV rep proteins, in addition to a nucleic acid comprising a nucleotide sequence encoding a mutant AAV capsid protein as described above. In other embodiments, the host cells further comprise an rAAV vector. rAAV virions can be produced using the host cells. Methods for producing rAAV virions are described, for example, in U.S. Patent Publication No. 2005 / 0053922 and U.S. Patent Publication No. 2009 / 0202490.

[0244] In some embodiments, a unit dose of the pharmaceutical composition contains 0.01 to 99% by weight of the polynucleotide (containing the VEGF-binding molecule) or rAAV particles. In other specific embodiments, the copy number of the genetic modification containing the VEGF-binding molecule in a unit dose of the pharmaceutical composition is 0.1 to 10 x 10 13 is.

[0245] Preparation method The present disclosure further provides a method for preparing a VEGF binding molecule of the present disclosure, comprising: - culturing a host cell of the present disclosure under conditions that allow expression of a VEGF binding molecule of the present disclosure; - recovering from the culture the VEGF binding molecule expressed by the host cell; - optionally further purifying and / or modifying the VEGF binding molecules of the disclosure.

[0246] The VEGF-binding molecules of the present disclosure may be produced intracellularly (e.g., in the cytoplasm, periplasm, or inclusion bodies) in cells as described above, then isolated from the host cells, and optionally further purified; alternatively, they may be produced extracellularly (e.g., in the culture medium in which the host cells are cultured), then isolated from the culture medium, and optionally further purified. For example, they may be purified on an A or G Sepharose FF column containing a conditioned buffer, nonspecifically bound components washed away, and the bound antibody eluted using a pH gradient, detected by SDS-PAGE, and collected. Optionally, they may be filtered and concentrated using conventional methods. Soluble compounds and polymers may be removed using conventional methods such as molecular sieving or ion exchange. The resulting product should be immediately frozen, e.g., at -70°C, or lyophilized.

[0247] Methods and reagents for recombinantly producing polypeptides are known in the art, such as particular suitable expression vectors, transformation or transfection methods, selectable markers, methods for inducing protein expression, culture conditions, etc. Similarly, protein isolation and purification techniques applicable to methods for preparing VEGF binding molecules of the present disclosure are well known to those of skill in the art.

[0248] composition The present disclosure further provides compositions, e.g., pharmaceutical compositions, comprising an effective amount of a VEGF binding molecule, isolated nucleic acid, or recombinant adeno-associated viral vector of the present disclosure, and one or more pharmaceutically acceptable vectors, diluents, buffers, or excipients.

[0249] In some specific embodiments, a unit dose of the pharmaceutical composition may contain 0.01 to 99% by weight of the isolated nucleic acid or recombinant adeno-associated viral vector, or the amount of the isolated nucleic acid or recombinant adeno-associated viral vector contained in a unit dose of the pharmaceutical composition is 0.1 to 2000 mg (e.g., 1 to 1000 mg).

[0250] In some specific embodiments, the pharmaceutical composition may be a lyophilized formulation or an injectable solution.

[0251] Treatment methods and medicinal uses The present disclosure further provides the use of any one of the above-described VEGF binding molecules, isolated nucleic acids, or recombinant adeno-associated viral vectors in the preparation of a medicament for the treatment and / or prevention of a disease associated with abnormal angiogenesis.

[0252] The present disclosure further provides a method of inhibiting VEGF receptor ligand activity in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of any one of the above-described VEGF binding molecules, isolated nucleic acids, recombinant adeno-associated viral vectors, or pharmaceutical compositions.

[0253] The present disclosure further provides a method for treating and / or preventing a disease associated with abnormal angiogenesis, comprising administering a therapeutically effective amount of any one of the above-mentioned VEGF-binding molecules, isolated nucleic acids, recombinant adeno-associated viral vectors, or pharmaceutical compositions to a subject in need thereof.

[0254] The present disclosure further provides a VEGF binding molecule, an isolated nucleic acid, or a recombinant adeno-associated viral vector for use as a medicament.

[0255] The present disclosure further provides a VEGF binding molecule, an isolated nucleic acid, or a recombinant adeno-associated viral vector, or a pharmaceutical composition comprising the same, for use in the treatment and / or prevention of a disease associated with abnormal angiogenesis.

[0256] In some embodiments, the subject is a mammal.

[0257] In some embodiments, the subject is a human.

[0258] In some embodiments, the disease associated with abnormal angiogenesis is a vascular eye disease or cancer; In some embodiments, the vascular eye disease is selected from the group consisting of macular degeneration (e.g., age-related macular degeneration (AMD), e.g., wet age-related macular degeneration, dry age-related macular degeneration), macular edema (e.g., macular edema after retinal vein occlusion, diabetic macular edema), retinal vein occlusion (e.g., central retinal vein occlusion, branch retinal vein occlusion), choroidal neovascularization, iris neovascularization, neovascular glaucoma, post-operative fibrosis of glaucoma, proliferative vitreoretinopathy, optic nerve head 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.

[0259] In some embodiments, the disease associated with abnormal angiogenesis is wet AMD or dry AMD.

[0260] In some embodiments, the cancer is selected from the group consisting of non-small cell lung cancer, renal cancer, uterine cancer, prostate cancer, bladder cancer, ovarian cancer, colon cancer, breast cancer, leukemia, lymphoma, myeloma, sarcoma (e.g., fibrosarcoma, myxosarcoma, liposarcoma, lymphangioendothelial sarcoma, angiosarcoma, endothelial sarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, lymphangiosarcoma, synovium, mesothelioma, leiomyosarcoma, rhabdomyosarcoma), glioma, pancreatic cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma ... leukemia, leukemia, leukemia, leukemia, leukemia, leukemia, leukemia, leukemia, leukemia, leukemia, leukemia, leukemia, leukemia, leukemia, leukemia, leukemia, leukemia, leukemia, leukemia, leukemia, leukemia, leukemia, leukemia, leukemia, leukemia, leukemia, leukemia, leuk Cancer, including, but not limited to, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchopulmonary carcinoma, choriocarcinoma, renal cell carcinoma, hepatocarcinoma, cholangiocarcinoma, seminoma, embryonal carcinoma, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, medulloblastoma, craniopharyngioma, oligodendroglioma, meningioma, melanoma, neutrophiloma, and retinoblastoma.

[0261] The present disclosure further provides a method of inhibiting abnormal angiogenesis, comprising administering to a subject in need thereof a therapeutically effective amount of any one of the above-described VEGF-binding molecules, isolated nucleic acids, or recombinant adeno-associated viral vectors, or pharmaceutical compositions.

[0262] The methods and uses described herein can be practiced in combination with other therapeutic agents or therapies (eg, radiation therapy).

[0263] Reagent kits and equipment The present disclosure provides reagent kits and devices comprising any of the VEGF binding molecules, nucleic acids, rAAV particles, and pharmaceutical compositions of the present disclosure.

[0264] In some embodiments, the reagent kit includes a container device, typically at least one vial, test tube, flask, bottle, syringe, or other container device, into which the nucleic acid, rAAV particles, or pharmaceutical composition can be placed. The reagent kit may also include second, third, or other separate containers into which separate components can be placed, typically for containing, for example, sterile, pharmaceutically acceptable buffers and / or other diluents. In some embodiments, the reagent kit includes a pharmaceutical package insert.

[0265] In some embodiments, the VEGF-binding molecules, nucleic acids, rAAV particles, and pharmaceutical compositions disclosed herein can be combined with, coated on, or embedded in a device. The device may include, but is not limited to, a stent, a pump, and / or other implantable therapeutic device. Furthermore, the nucleic acids, rAAV particles, and pharmaceutical compositions can be delivered to a subject using, for example, but not limited to, a compression device, to reduce the likelihood of deep vein thrombosis (DVT) in the subject.

[0266] In some embodiments, the nucleic acids, rAAV particles, and pharmaceutical compositions of the present disclosure may be delivered using devices including, but not limited to, stents, tubes (including catheters, pipes, and pipettes), and particle guns. The nucleic acids, rAAV particles, and pharmaceutical compositions of the present disclosure may be administered to a subject using a delivery system that integrates imaging and image-guided therapy, including, but not limited to, lasers, MRgFUS, endoscopes, and robotic surgical devices. The nucleic acids, rAAV particles, and pharmaceutical compositions of the present disclosure may be administered to a subject using an automated delivery system. The nucleic acids, rAAV particles, and pharmaceutical compositions of the present disclosure may be delivered using instruments localized to a target point on the subject, an enhanced delivery device, or an MIR-guided device. [Brief explanation of the drawings]

[0267] [Figure 1] FIG. 1 is a schematic diagram of the molecular form of a VEGF-binding molecule. [Figure 2] FIG. 1 is a schematic diagram of the structure of a vector containing a nucleic acid molecule that expresses a VEGF-binding molecule. [Figure 3] FIG. 1 is a schematic diagram of an AAV packaging plasmid. [Figure 4] 1 shows the results of detecting the stability of VEGF-binding molecules in rabbit double vitreous tissue. [Figure 5A] These are the results of fluorescein fundus angiography (FFA) detection 7 days after construction of the rat CNV model and 14 days after administration of the rat CNV model. [Figure 5B] This shows the results of detecting inhibition of choroidal neonatal fluorescein sodium leakage 14 days after administration in a rat CNV model. [Figure 6] Detected expression of AAV-1174 and ADVM-022 in the aqueous humor, vitreous, retina, and choroid of rabbits. [Figure 7] This shows the results of detecting inhibition of choroidal neonatal sodium fluorescein leakage by administration of AAV-1174 and ADVM-022 to a mouse CNV model. DETAILED DESCRIPTION OF THE INVENTION

[0268] definition In order that the present disclosure may be more readily understood, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this disclosure, all other technical and scientific terms used in this disclosure have the meanings commonly understood by those of ordinary skill in the art.

[0269] VEGFR-1, VEGFR1, Flt1, and Flt-1 may be used interchangeably and all refer to the VEGF receptor, and the amino acid sequence of wild-type VEGFR-1 is as shown in SEQ ID NO:2.

[0270] VEGFR-2, VEGFR2, Flk-1, and KDR may be used interchangeably and all refer to different VEGF receptors, and the wild-type VEGFR-2 amino acid sequence is as shown in SEQ ID NO:1.

[0271] "Wild type" refers to a VEGF-binding molecule that is the same as a VEGF-binding molecule of the present disclosure in other respects (e.g., domain composition, sequence length, linkage scheme, etc.), except that the VEGF-binding molecule has the molecular form of the wild-type amino acid at each amino acid position. For example, a VEGF-binding molecule having molecular form 1 (VEGFR1 D2+VEGFR2 D3+ VEGFR2 D2D3 + hinge region and CH2CH3 constant region of human IgG1), the corresponding wild type is a polypeptide having said molecular form 1 and in which each domain is a natural amino acid sequence.

[0272] "Amino acid mutation" includes amino acid substitution, deletion, insertion, modification, and any combination thereof, such that a final construct is achieved and the final construct has desired properties, such as enhanced stability. Deletions and insertions in the amino acid sequence include amino acid and / or carboxy-terminal deletions and insertions. For example, to alter the binding characteristics of a VEGF-binding molecule (or VEGFR fusion protein), non-conservative amino acid substitutions can be made, i.e., one amino acid can be replaced with another amino acid having different structural and / or chemical properties. Preferred amino acid substitutions include the substitution of a hydrophilic amino acid for a hydrophobic amino acid. Amino acid substitutions include substitutions with non-naturally occurring amino acids or naturally occurring amino acid derivatives of the 20 standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated by genetic or chemical methods known in the art, including site-directed mutagenesis, PCR, gene synthesis, chemical modification, and other methods. In the case of amino acid substitution, the substitution is preferably a conservative amino acid substitution, which means that an amino acid residue is substituted with another amino acid residue having a similar chemical structure, and has little or no effect on the function, activity, or other biological properties of the polypeptide. The conservative amino acid substitution is known in the art, and for example, a conservative amino acid substitution is preferably a substitution of one amino acid residue in the following groups (i) to (v) with another amino acid residue in the same group: (i) relatively small aliphatic non-polar or weakly polar residues: Ala, Ser, Thr, Pro, and Gly; (ii) polar negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (iii) polar positively charged residues: His, Arg and Lys, (iv) relatively large aliphatic non-polar residues: Met, Leu, Ile, Val and Cys, and (v) Aromatic residues: Phe, Tyr and Trp.

[0273] For example, conservative amino acid substitutions are Ala replaced with Gly or Ser, Arg replaced with Lys, Asn replaced with Gln or His, Asp replaced with Glu, Cys replaced with Ser, Gln replaced with Asn, Glu replaced with Asp, Gly replaced with Ala or Pro, His replaced with Asn or Gln, Ile replaced with Leu or Val, Leu replaced with Ile or Val, Lys replaced with Arg, Gln or Glu, Met replaced with Leu, Tyr or Ile, Phe replaced with Met, Leu or Tyr, Ser replaced with Thr, Thr replaced with Ser, Trp replaced with Tyr, Tyr replaced with Trp or Phe, and Val replaced with Ile or Leu.

[0274] "Homology," "identity," or "sequence identity" refers to the overall correlation between polymer (e.g., oligonucleotide, polynucleotide, or polypeptide) molecules. For example, two polynucleotide sequences can be aligned to calculate the percentage of identity between them. Because of gaps and penalties introduced in the calculation, the identity value may vary, but an optimal method can be selected (e.g., gaps can be introduced into one or both of the two sequences to be aligned to achieve optimal alignment, and different sequences can be ignored to achieve comparison, where one of the two sequences is the target sequence to be aligned and the other is the reference sequence). In some embodiments, the length of the aligned target sequence may be at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence, and then the nucleotides at corresponding nucleotide positions are compared. If a position in the aligned target sequence is occupied by the same nucleotide as the corresponding position in the reference sequence, the two are considered to be identical at that position. The percentage of identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced to achieve optimal alignment of the two sequences. The comparison of sequences and determination of the percentage identity between two sequences can be accomplished by a mathematical algorithm.For example, the percent identity between two polynucleotide sequences can be determined by methods such as those described in Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991 (all of which are incorporated herein in their entireties). For example, the percentage identity between two nucleotide sequences can be measured using the Meyers and Miller (CABIOS, 1989, 4:11-17) algorithm incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percentage identity between two nucleotide sequences can be measured using the GAP program in the GCG software package using the NWSgapdna.CMP matrix. In general, methods for measuring the percentage identity between sequences include, but are not limited to, those described by Carillo, H., and Lipman, D., SIAM J Applied Math., 48:1073.

[0275] "Avidity" or "affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., a receptor or a binding molecule of the present disclosure) and its binding partner (e.g., a ligand). Unless otherwise indicated, "avidity" herein refers to the intrinsic affinity that reflects a 1:1 interaction between members of a binding pair (e.g., a receptor and a ligand). The avidity (affinity) of a molecule X for its partner Y can usually be expressed by a dissociation constant (KD), which is a function of the association rate constant (KD, respectively). d and K. a The binding activity (affinity) is the ratio of dissociation to dissociation (where α is the binding activity). Thus, the same binding activity (affinity) can include different rate constants as long as the ratio of the rate constants remains the same. The binding activity (affinity) can be measured by conventional methods in the art, including the methods described in Example 3 or Example 5 herein.

[0276] "Stability" refers to the ability to achieve a stabilizing effect in which a VEGF-binding molecule essentially retains its physical and / or chemical stability and / or biological activity after storage for a certain period of time (e.g., 2 weeks, 4 weeks, 8 weeks, 12 weeks, or longer). Currently, several analytical techniques are available for measuring polypeptide stability, and stability can be measured after storage at a given temperature for a given period of time. A stable VEGF-binding molecule is one in which no significant changes are observed when stored at refrigerated temperatures (2-8°C) for at least 1 month, at least 3 months, at least 6 months, at least 1 year, at least 2 years, or longer. A stable VEGF-binding molecule further includes exhibiting desired characteristics after storage at 25°C or 40°C for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or longer. A typical acceptable criterion for stability is that, upon visual analysis, the aqueous solution or formulation of the VEGF-binding molecule is colorless or clear to slightly opalescent. The concentration, pH, and osmolality of the formulation vary by no more than ±10%. Typically, no more than about 10%, preferably no more than about 5%, of fragmentation is observed. Typically, no more than about 10%, preferably no more than about 5%, of aggregates are formed. A polypeptide is considered to have "retained its physical stability" if it shows no significant increase in aggregation, precipitation, and / or denaturation as measured by visual inspection of color and / or clarity or by UV light scattering, size exclusion chromatography (SEC), and dynamic light scattering (DLS). Conformational changes in the polypeptide can be assessed by fluorescence spectroscopy (which determines the tertiary structure of the polypeptide) and FTIR spectroscopy (which determines the secondary structure of the polypeptide). A polypeptide is considered to have "retained its chemical stability" if it shows no significant chemical changes. Chemical stability can be assessed by detecting and quantifying chemically altered polypeptides.Degradation processes that constantly change the chemical structure of a polypeptide include hydrolysis or cleavage (assessed by methods such as size exclusion chromatography and SDS-PAGE), oxidation (assessed by methods such as peptide mapping combined with mass spectrometry or MALDI / TOF / MS), deamidation (assessed by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, and measurement of isoaspartic acid content), and isomerization (assessed by measurement of isoaspartic acid content, peptide mapping, etc.). A polypeptide "retains its biological activity" if its biological activity at a given time falls within a predetermined range. The biological activity of a polypeptide can be determined, for example, by antigen binding assays.

[0277] The term "antibody" is used herein in the broadest sense and encompasses various antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antigen-binding fragments, as long as they exhibit the desired antigen-binding activity. Antibodies can include mouse antibodies, human antibodies, humanized antibodies, chimeric antibodies, and camelid antibodies. Exemplarily, an antibody may refer to an immunoglobulin, which has a tetrapeptide chain structure consisting of two heavy chains and two light chains linked by interchain disulfide bonds. Immunoglobulins differ in the amino acid composition and sequence of the heavy chain constant region, resulting in different antigenicities. Therefore, immunoglobulins can be divided into five types, or immunoglobulin isotypes, namely, IgM, IgD, IgG, IgA, and IgE, and the corresponding heavy chains are μ, δ, γ, α, and ε chains, respectively. Ig of the same type can be further divided into different subclasses depending on the amino acid composition of the hinge region and the number and position of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are divided into κ chains and λ chains depending on the constant region. Each of the five types of Ig may have either κ chains or λ chains.

[0278] "Antigen-binding fragment" refers to a Fab fragment, Fab' fragment, F(ab')2 fragment, single-chain Fv (i.e., sFv), nanobody (i.e., VHH), or VH / VL domain that has antigen-binding activity. An Fv fragment contains the heavy chain variable region and the light chain variable region of an antibody but lacks the constant region and contains the minimum antigen-binding fragment of all antigen-binding sites. Generally, an Fv antibody further contains a polypeptide linker between the VH and VL domains and is capable of forming the structure necessary for antigen binding. Two antibody variable regions may be linked by different linkers to form a single polypeptide chain called a single-chain antibody or single-chain Fv (sFv).

[0279] "Fc" refers to a polypeptide comprising an antibody constant region but not the first constant region immunoglobulin domain; i.e., Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, the last three constant region immunoglobulin domains of IgE and IgM, and the elastic hinge region at the N-terminus of these domains. For IgA and IgM, Fc may include the J chain. For IgG, Fc includes immunoglobulin domains Cγ2 and Cγ3 and the hinge region between Cγ1 and Cγ2. Although the boundaries of the Fc region are variable, the human IgG heavy chain Fc region is usually defined as the carboxyl-terminus including residues C226 or P230, numbered according to the EU index as described by Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, VA). The "EU index as described in Kabat" refers to Kabat et al. and is identical to the residue numbering of the human IgG1 EU antibody described above. Fc can refer to this region alone, or to this region in an antibody, antibody fragment, or Fc fusion protein. The Fc variant protein may be an antibody, an Fc fusion protein, or any protein or protein domain containing an Fc region. Proteins containing mutant Fc regions, i.e., non-naturally occurring Fc variants, are particularly preferred. Note that polymorphisms are observed at many Fc positions, including, but not limited to, Kabat 270, 272, 312, 315, 356, and 358, and therefore slight variations between the above sequence and those of the prior art are acceptable.

[0280] "Nucleic acid molecule" and "nucleic acid" refer to DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, preferably double-stranded DNA. A nucleic acid is "operatively linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operatively linked to a coding sequence if the promoter or enhancer affects the transcription of the coding sequence.

[0281] An "expression vector" or "expression construct" or "cassette" or "plasmid" or simply "vector" can include any type of genetic construct, including an AAV or rAAV vector, containing a nucleic acid or polynucleotide encoding a gene product, where part or all of the nucleic acid coding sequence can be transcribed and is suitable for gene therapy. The transcript can be translated into a protein. In some cases, it may be partially translated or not. In some embodiments, expression includes both gene transcription and translation of mRNA into a gene product. In some embodiments, expression includes only transcription of the nucleic acid encoding the gene of interest. An expression vector can further include control elements operably linked to the coding region to facilitate expression of the protein in a target cell. The combination of control elements and one or more genes operably linked thereto for expression is sometimes referred to as an "expression cassette," and a large number of these are known and available in the art or can be readily constructed from components available in the art.

[0282] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells, including "transformants" and "transformed cells," include the originally transformed cell and its progeny (without considering the number of passages). The progeny may not be completely identical in nucleic acid content to the parent cell, but may contain mutations. The specification includes mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell.

[0283] A "pharmaceutical composition" refers to a mixture containing one or more compounds described in the present disclosure or physiologically / pharmaceutically acceptable salts or prodrugs thereof, and other chemical components such as physiologically / pharmaceutically acceptable vectors and excipients. The pharmaceutical composition is intended to facilitate administration to a living body and contribute to the absorption of the active ingredients to further exert their biological activity.

[0284] "Administration," when used with animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refers to the contact of an exogenous agent, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid. "Administration" can refer, for example, to therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of cells includes contact of a reagent with the cell and contact of a reagent with a fluid, where the fluid contacts the cell. "Administration" also refers to ex vivo and in vitro treatment of, for example, cells with a reagent, diagnostic, binding composition, or through another cell. "Administration," when used with humans, veterinary, or research subjects, refers to therapeutic treatment, preventative or prophylactic measures, research, and diagnostic uses.

[0285] "Treatment" refers to administering an oral or topical therapeutic agent, for example, comprising a composition of any one of the disclosed VEGF binding molecules, to a subject diagnosed with, suspected of having, or susceptible to infection, one or more disease symptoms, for which the known therapeutic agent has a therapeutic effect. Typically, the therapeutic agent is administered to the subject or population being treated in an amount that effectively alleviates one or more disease symptoms, whether by inducing regression of such symptoms or by inhibiting such symptoms so that they do not progress to any clinically unmeasurable extent.

[0286] The amount of a therapeutic agent that effectively alleviates any specific disease symptom (also referred to as a "therapeutically effective amount") can vary depending on several factors, such as the subject's disease state, age and weight, and the ability of the drug to produce the desired therapeutic effect in the subject. Reduction of disease symptoms can be assessed by any clinical detection method commonly used by physicians or other professional healthcare providers to assess the severity or progression of the condition. While embodiments of the present disclosure (e.g., treatment methods or products) may be ineffective in alleviating the target disease symptom present in each patient, any statistical test known in the art, such as Student's t-test, chi-square test, Mann and Whitney U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test, can determine that the target disease symptom can be reduced in a statistically significant number of subjects.

[0287] An "effective amount" includes an amount sufficient to ameliorate or prevent the symptoms or pathology of a medical condition. An effective amount also refers to an amount sufficient to permit or facilitate diagnosis. The effective amount used for a particular subject or veterinary subject can vary depending on factors such as the condition being treated, the subject's overall health, the method, route and dose of administration, and the severity of side effects. An effective amount may be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.

[0288] The three-letter and one-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem, 243, p. 3558 (1968).

[0289] The terms "contain", "have", or "include" encompass "comprise", "mainly consisting of", "essentially consisting of", and "consist of", and "mainly consisting of", "essentially consisting of", and "consist of" are subordinate concepts to "contain", "have", or "include".

[0290] A "fusion protein" is an isolated protein unrelated to other proteins, polypeptides, or molecules, either as a purified product of recombinant host cell culture or as a purified extract. Fusion protein construction techniques are based on molecular cloning techniques, and specific experimental methods can be found in laboratory manuals such as "Molecular Cloning," 2nd and 3rd editions. DNA encoding the above fusion proteins can be obtained by conventional techniques, such as total gene synthesis or splicing from Flt1 or KDR fragments. The vectors used may be plasmid vectors commonly used in molecular biology. A protein secretion signal sequence is added to the amino terminus of each fusion protein to ensure secretion of the protein from cells. The vector sequence includes a promoter for driving gene expression, protein translation initiation and termination signals, and a polyadenylic acid (PolyA) sequence. The vector also contains an antibiotic resistance gene to facilitate propagation of the plasmid in bacteria. The vector also contains a eukaryotic cell selectable gene for screening stably transfected cell lines. After constructing the plasmids for the various fusion proteins described above, cells can be transfected with the plasmid DNA to express the corresponding proteins. A variety of expression systems can be used to express these fusion proteins, including, but not limited to, mammalian cells, bacteria, yeast, insect cells, and the like. Proteins expressed from mammalian cells have glycosyl modifications. Because the amino acid sequences of the fusion proteins of the present disclosure contain glycosylatable amino acids, mammalian cells are the cells of choice for expressing these proteins. A variety of mammalian cells can be used for large-scale protein expression, including 293 cells, CHO cells, SP20 cells, NSO cells, COS cells, BHK cells, and PerC6 cells. Many other cells can also be used for the expression and production of these proteins and are therefore included in the list of cells that can be used in the present disclosure. A plasmid encoding the polypeptide can be introduced into the cell by transfection.Transfection methods include, but are not limited to, electroporation, liposome-mediated transfection, etc. After expression of the fusion protein, the concentration of the fusion protein in the cell culture medium can be measured using enzyme-linked immunosorbent assay (ELISA) or other methods. Because these fusion proteins contain an immunoglobulin Fc fragment, the expressed fusion protein can be extracted by protein A affinity chromatography. Since the basic role of the various fusion proteins disclosed herein is to block the VEGF signaling pathway, these fusion proteins can be used for angiogenesis- or VEGF-related diseases. The fusion protein DNA sequence can also be inserted into an appropriate vector and expressed in the patient's body using gene therapy or cell therapy methods. Therefore, the methods for using the fusion proteins disclosed herein can take various forms, including not only the protein itself but also the corresponding DNA sequence encoding the amino acid sequence of the fusion protein.

[0291] A "payload" refers to one or more polynucleotides or polynucleotide regions encoded by or in a viral genome, or the expression product of such polynucleotides or polynucleotide regions, e.g., a transgene, a polynucleotide encoding a polypeptide, or a regulatory polynucleotide. In some embodiments of the present disclosure, the payload encodes or expresses a target gene, such as a transgene encoding a VEGF-binding molecule. A "payload construct" is one or more polynucleotide regions encoding or comprising a payload flanked on one or both sides by inverted terminal repeat (ITR) sequences, and the payload construct serves as a template for replication in viral producer cells to produce a viral genome. A "payload construct vector" is a vector encoding or comprising a payload construct and regulatory regions used for replication and expression in bacteria and cells. A "payload construct expression vector" is a vector encoding or comprising a payload construct and further comprising one or more polynucleotide regions encoding or comprising components for viral expression in viral replicating cells.

[0292] An "AAV" vector or "rAAV vector" refers to an adeno-associated virus (AAV) vector or recombinant AAV (rAAV) vector that contains a polynucleotide sequence not derived from AAV (i.e., a polynucleotide heterologous to AAV, such as a nucleic acid sequence encoding a therapeutic genetic modification (e.g., aflibercept)), typically a sequence of interest for use in genetic transformation of a cell. Typically, the heterologous polynucleotide is flanked by at least one, and typically two, AAV inverted terminal repeats (ITRs). The term rAAV vector includes both rAAV vector particles and rAAV vector plasmids. rAAV vectors may be single-stranded (ssAAV) or self-complementary (scAAV). An "AAV virus" or "rAAV vector particle" refers to a viral particle consisting of at least one AAV capsid protein (typically consisting of all capsid proteins of wild-type AAV) and a polynucleotide rAAV vector. When a particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a genetic modification that is delivered to a mammalian cell), it is typically referred to as an "rAAV vector particle" or simply an "rAAV vector." Thus, production of an rAAV particle necessarily includes production of an rAAV vector, since such a vector is contained within the rAAV particle.

[0293] "Operatively linked" may refer to the juxtaposition of genetic elements in a relationship permitting them to operate in their intended manner. For example, a promoter can be operably linked to a coding region if the promoter serves to initiate transcription of the coding sequence. Spacer residues can be present between the promoter and the coding region so long as their functional relationship is maintained.

[0294] In the present disclosure, "VEGFR2 D2D3" can be used interchangeably with "R2D2-R2D3" and "R2D2 R2D3." "VEGFR1D2-VEGFR2D3" can be used interchangeably with "R1D2-R2D3" and "R1D2 R2D3." Example

[0295] The present disclosure will be further explained below in conjunction with examples, but these examples are not intended to limit the scope of the present disclosure. Experimental methods for which specific conditions are not specified in the examples of the present disclosure generally follow standard conditions, such as those found in Cold Spring Harbor's Antibody Technology Laboratory Manual and Molecular Cloning Manual, or conditions suggested by raw material or product manufacturers. Reagents for which specific sources are not specified are standard commercially available reagents. Example 1: VEGF binding molecules and point mutation design

[0296] In this example, three molecular formats of VEGF-binding molecules (FIG. 1) were designed, which are listed in order from N-terminus to C-terminus as follows: Molecular form 1:VEGFR1 D2+VEGFR2 D3+ VEGFR2 D2D3 + human IgG1 hinge region and CH2CH3 constant region, Molecular form 2: VEGFR2 D2D3 +VEGFR1 D2 +VEGFR2 D3 +human IgG1 hinge region and CH2CH3 constant region, Molecular Form 3: Heavy Chain: VEGFR2 D2D3 +Human IgG1 hinge region and CH2CH3 constant region +Bevacizumab VH+CH1, light chain: Bevacizumab VL+CL.

[0297] To improve the stability and ligand affinity of the fusion protein, a series of point mutations were designed in the VEGF trap scaffold molecule (i.e., VEGFR2 D2D3 domain, i.e., outlined above) in the VEGF binding molecule: 1. Five mutants were designed based on the VEGFR2 D2D3 sequence (SEQ ID NO: 69), with the cysteine ​​at position 162 (C162) mutated to A / V / S / I / L, respectively.

[0298] Wild-type VEGFR2 D2D3 sequence: [ka] 2. Six single point mutations and two combination mutations were designed based on the VEGFR2 D2D3 sequence (SEQ ID NO: 69) (Table 1).

[0299] 3. Based on the VEGFR2 D2D3 sequence (SEQ ID NO: 69), single point mutations were designed in the D2 or D3 domain (Tables 2 and 3), respectively.

[0300] [Table 1] [Table 2] [Table 3] Example 2: Plasmid construction expressing VEGF-binding molecules, protein expression and purification

[0301] The VEGF-binding molecules designed in Example 1 were synthesized using a total gene synthesis method, in which the VEGF-C antagonistic activity was mainly provided by VEGFR2 D2D3, and the VEGF-A antagonistic activity was mainly provided by the Fab of VEGFR1D2-VEGFR2D3 (Aflibercept) or Bevacizumab.

[0302] After optimizing the expression host for amino acids, the genes encoding the above nucleotide sequences were inserted into pTT5 vector, and expression vectors were designed and synthesized according to the above sequences, and sequence verification was performed.

[0303] Expression and purification of VEGF-binding molecules: Plasmids that passed validation were transfected into human 293F cells with PEI and continuously cultured. 293F cells were cultured in serum-free medium (OPM-293 CD03, Shanghai OPM Bioscience) until logarithmic growth phase and then used for cell transfection. 45 μg of the plasmid was dissolved in 10 mL of Opti-MEM® I Reduced Serum Medium (GIBCO, 31985-070) and mixed to homogeneity. 200 μg of PEI was then added and mixed homogeneously. The mixture was incubated at room temperature for 15 min and then added to 50 mL of cells. The cell culture conditions were 5% CO2, 37°C, and 125 rpm / min. On days 1 and 3 of the culture period, feeding was continued until cell viability reached less than 70%, and the cell supernatant was collected and centrifuged. The centrifugal filtered cell culture medium was loaded onto a protein A column, washed with phosphate buffer, eluted with glycine-HCl buffer (pH 2.7, 0.1 M Gly-HCl), neutralized with 1 M Tris-HCl (pH 9.0), and dialyzed against phosphate buffer to obtain the final purified protein. Detection by SDS-PAGE and SEC-HPLC confirmed that the target fusion protein was obtained.

[0304] The specific fusion protein sequence synthesized in this example is as follows: Aflibercept (PR1076) [ka] >OPT-302(HRAMD-C3) [ka] Bevacizumab Heavy chain: [ka] Light chain: [ka] >PR1010(R1D2-R2D3-linker-R2D2-R2D3-Fc)

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[0305] The inhibitory effect of VEGF-binding molecules on the activation of KDR signaling was evaluated by detecting the expression level of the VEGF-A-induced NFAT-RE Luc2P KDR cell reporter gene. The experimental steps were as follows: 3A.1 Culture medium preparation: Complete medium: High-sugar DMEM (90%) + FBS (10%), Complete medium: High-sugar DMEM (90%) + FBS (10%), 100 μg / mL hygromycin B, 250 μg / mL G-418. Assay medium: High-sugar DMEM (99%) + FBS (1%). 3A.2 Dilution of VEGF binding molecule samples: Samples to be detected were diluted in a gradient, with a maximum concentration of 300 nM, followed by a 3-fold gradient dilution. VEGFA dilution: The final VEGFA concentration was 250 ng / mL.

[0306] 3A.3 Preparation of reporter gene cell line: Glo-Response™ NFAT-RE-luc2P / KDR HEK293 cells. After digestion, wash the cells with 5 mL of PBS, then resuspend the cells in assay medium and culture at a density of 8 x 10 cells. 5 50 μL / well was added to a 96-well cell culture plate, i.e., the cell number was 4 × 10 4 / Well.

[0307] 3A.4 Treatment with VEGF-binding molecules: Different concentrations of VEGF-binding molecules were added to cell wells at 50 μL / well and cultured at 37°C in a 5% CO2 incubator for 6 hours. Each concentration was replicated three times, and controls with and without VEGF-A were also set up.

[0308] 3A.5 Color Development: Bright-Glo Color Development Solution was equilibrated to room temperature. 50 μL of Bright-Glo Color Development Solution was added to the 96-well plate after incubation, and the plate was shaken at room temperature for 10 minutes to ensure uniform mixing. The plate was then left to stand for 2 minutes. The plate was then placed in a multifunction plate reader, and the chemiluminescence readings were taken.

[0309] 3A.6 Data Processing:

number

number

[0310] To evaluate the binding activity of the VEGF-binding molecule to VEGF-C, this example used a BAF3-VEGFR2 / R3 cell proliferation assay. BAF3-VEGFR2 / R3 is a BA-F3 cell line that overexpresses human VEGFR2 / R3, and cell proliferation depends on the exogenously added growth factor VEGF-C. The basic experimental steps are as follows: 3B.1 Cell culture: The above cell lines were cultured at 1 × 10 to 1.5 × 10 cells / ml in RPMI 1640 medium containing 10% FBS (supplemented with 10 ng / mL mouse IL-3, 1 μg / mL puromycin, and 250 μg / mL G418). 5 Culture the cells at 1 × 10 cells / mL in a 37°C, 5% CO2 incubator. 6 The cells were plated at a cell density not exceeding 100 cells / mL.

[0311] 3B.2 Cell proliferation experiment: Cells in the logarithmic growth phase were taken, washed three times with PBS, and centrifuged at 800 r / min for 3 min. The cell density was adjusted with RPMI1640 (FBS: 2%, 143 ng / mL VEFG-C) and then cultured at 2 × 10 4 50 μL of VEGF-C solution was added to a 96-well plate at 50 μL per well. Subsequently, 50 μL of gradient-diluted VEGF-binding molecules (maximum concentration 300 nM, 3-fold dilution) was added. Control groups were also set up, with no VEGF-C added and with VEGF-C alone. After 3 days of culture, 30 μL of cell titer-glo was added, mixed evenly, and cells were counted.

[0312] 3B.3 Data Processing:

number

number

[0313] In this example, the binding activity of VEGF-A and VEGF-C to VEGF-binding molecules was evaluated by the above two in vitro experiments, and the specific data are shown in Table 4: [Table 4-1] [Table 4-2]

[0314] The in vitro activity of the VEGF binding molecules of the present disclosure against VEGF-A and VEGF-C is comparable to the positive controls Aflibercept and OPT-302.

[0315] [Table 5]

[0316] "-" indicates that no activity was detected.

[0317] [Table 6]

[0318] As shown in Tables 5 and 6, PR1160 is a VEGFR2 D2D3(C162V)-Fc fusion protein and has a certain in vitro VEGF-A inhibitory activity, but the activity is weaker than that of the positive control Aflibercept, and the inhibitory activities of the other fusion proteins against VEGF-A and VEGF-C are equivalent to those of the two positive control molecules, respectively. As shown in Table 7, the inhibitory activities of the VEGF-binding molecules PR1145, PR1174, PR1163, and PR1186 against VEGF-A and VEGF-C are equivalent to those of the two positive control molecules, and are stronger than the inhibitory activities of PR1010 and bevazicumab against VEGF-A.

[0319] [Table 7-1] [Table 7-2] Example 4: Thermostability analysis of different VEGF binding molecules

[0320] The thermal stability of a fusion protein is an important criterion for evaluating the superiority or inferiority of a molecule, and the development of a fusion protein molecule with relatively excellent thermal stability has positive implications for process production, drug delivery, etc. In this example, an accelerated thermal stability evaluation was performed on candidate molecules.

[0321] The specific experimental steps are as follows: The candidate molecule was diluted to 1 mg / mL and divided into three 1.5 mL EP tubes, 100 μL per tube. One of these was taken as a time point 0 sample and frozen for storage. The remaining samples were placed in a temperature and humidity incubator at 40°C and 75% humidity. One sample was taken every two weeks and four weeks and frozen for storage. After collecting the three time points (0, 2, and 4 weeks), the samples were thawed at 4°C and centrifuged at 12,000 rpm / min for 10 minutes to remove the precipitate. The supernatant was then subjected to HPLC analysis. The HPLC used in this example was a Waters ACQUITY Arc column with an XBridge BEH 200Å SEC 3.5 μm column, a flow rate of 0.5 mL / min, and a 30 μL sample draw. The final sample HPLC spectrum was obtained and is shown in the table below.

[0322] The parent molecule PR1010 in molecular form 1 had an initial purity of 90%, but after 2 weeks at 40°C, the purity decreased to below 30%, suggesting the need for urgent improvement in the stability of the molecule. After introducing the VEGFR2 C162V mutation into PR1010, the purity of the PR1094 molecule in HEK 293F cells further increased to above 95%, and the SEC purity remained high at around 90% after 4 weeks at 40°C.

[0323] As shown by the HPLC spectrum results, after the C162V mutation, the thermostability of the molecules was significantly improved, whether it was the VEGFR2 D2D3-Fc fusion protein (PR1160 compared with PR1088) or molecular form 1 (PR1094 compared with RP1010).

[0324] In addition, point mutations such as N274F (PR1113), N274I (PR1114), D276Q (PR1118), Q280E (PR1123), E284H (PR1125), Y221S / R222K (PR1135), and L313R (PR1136) can also significantly improve the stability of the fusion protein by stabilizing the VEGFR2 D2D3 domain.

[0325] To further investigate the effect of C162V / I-based combinatorial mutations on protein expression and purification, we next expressed combinatorially mutated molecules using CHO-K1: in most cases, combinatorial mutations could further improve the stability of the molecules (e.g., PR1145 / PR1149 over PR1094).

[0326] The results are shown in Tables 8-1 and 8-2, where PR1088 to PR1136 (Table 8-1) were obtained for HEK 293F expression and PR1094 to PR1179 (Table 8-2) were obtained for CHO-K1 expression.

[0327] [Table 8-1] [Table 8-2] [Table 9-1] [Table 9-2] [Table 9-3] Example 5: SPR technology measures affinity between VEGF binding molecules and different ligands

[0328] Surface plasmon resonance (SPR) was used to detect the affinity between different VEGF-binding molecules and VEGF-A / B / C proteins. (VEGF-binding molecules were captured on a Protein A chip.) The coupling level was set to 100 RU, and detection was performed using a Biacore instrument (T200, GE Healthcare, BIAC-B20-03). The running buffer was HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20). Diluted antibodies (6.25, 12.5, 25, 50, and 100 nM) were flowed through the experimental and control channels (100 nM VEGF-A / B / C) at a flow rate of 30 μL / min for 3 min, followed by 5 min of dissociation. The regeneration buffer, 10 mM glycine pH 1.5 (GE Healthcare, BR-1003-54), was then run at a flow rate of 30 μL / min for 30 seconds. Data were analyzed using Biacore 8K evaluation software.

[0329] The affinity constants of the VEGF-binding molecules for human VEGF-A, VEGF-B, and VEGF-C proteins were calculated using the Multi-Cycle method. The calculated kinetic parameters ka, kd, and KD are shown in the table below.

[0330] [Table 10] Example 6: Evaluation of the in vivo stability of VEGF binding molecules

[0331] To determine the in vivo stability of the VEGF-binding molecule, the changes in the molecule concentration in the ocular tissue after injecting the molecule into the vitreous were measured. The specific experiments were as follows: Fourteen New Zealand rabbits, half male and half female, weighing 1.5-2.5 kg, were purchased and divided into four groups: a high-dose PR1145 group, a low-dose PR1145 group, a high-dose PR1163 group, and a blank control group. All procedures were performed bilaterally. For the 12 animals in the treatment group, vitreous and aqueous humor samples were collected 3 h, 3 d, 7 d, 14 d, and 28 d after administration to the vitreous of both eyes. The samples were then analyzed for target protein content using enzyme-linked immunosorbent assay (ELISA). The experiment was completed by Chenghe Tuochuang Biotechnology Co., Ltd. The doses were listed in Table 10.

[0332] [Table 11]

[0333] Figure 4 shows the content of VEGF-binding molecules in rabbits measured 3 hours, 3 days, 7 days, 14 days, and 28 days after intravitreal injection. The results show that the PR1145 high-dose group, PR1145 low-dose group, and PR1163 high-dose group reached maximum drug concentrations in their bodies 3 days after injection, and PR1145 and PR1163 could be detected in their bodies for 14 days, indicating that the VEGF-binding molecules have relatively good in vivo stability. Example 7: In vivo efficacy evaluation of VEGF-binding molecules

[0334] To evaluate the inhibitory effect of VEGF-binding molecules on choroidal neovascularization, we conducted efficacy testing using a laser-induced choroidal neovascularization (CNV) model. This model is a classic model that is currently widely adopted internationally for testing anti-choroidal neovascularization drugs. Specific experiments were performed as follows: Forty-two rats, half male and half female, aged 8 to 10 weeks, were purchased and divided into seven groups: a high-dose PR1145 group, a low-dose PR1145 group, control group 1 (Aflibercept), control group 2 (OPT302), and a negative control group (PBS). The dosing schedule is shown in Table 11. Six animals in each group underwent laser CNV model construction in both eyes. One week later, 4 μL of the protein molecule was injected into both eyes. Two weeks later, fundus angiography was performed to assess efficacy. PR1145 (3.75 mg / mL) and Aflibercept (2.5 mg / mL) were administered at equimolar doses, with the molar ratio of Aflibercept (2.5 mg / mL) to OPT302 (10 mg / mL) being 1:4.

[0335] [Table 12]

[0336] Fundus photographs of the rats were taken before model construction, and no abnormalities were observed in the fundus of any rat. As shown in Figure 5A, 7 days after model construction, clear laser spot and sodium fluorescein leakage were observed in the PBS group, indicating successful establishment of the model. All rats were administered 7 days after CNV model construction, and fluorescein fundus angiography (FFA) was performed 14 days after administration. Increased fluorescent spot and sodium fluorescein leakage were observed in the PBS group, indicating persistent progression and a worsening trend of laser-induced choroidal neovascularization within 21 days after model construction. On day 14 after administration, the inhibition of choroidal neovascularization by VEGF-binding molecules was measured by FFA. The results are shown in Figure 5B. Compared with the PBS group, the high-dose PR1145 group, the low-dose PR1145 group, Aflibercept, and OPT302 all significantly improved laser-induced choroidal neovascularization sodium fluorescein leakage. The improvement rate with PR1145 (3.75 mg / mL) was 15.8%, which was higher than the improvement rate with Aflibercept (2.5 mg / mL) (12.5%). PR1145 (3.75 mg / mL) and OPT302 (10 mg / mL) had significant improvement effects, but the dose of PR1145 was significantly reduced. Example 8: Construction of gene expression cassettes for AAV delivery

[0337] The expression vector provided in this example comprises a nucleic acid molecule that expresses a VEGF-binding molecule, the structure of which is shown in FIG. 2, and which comprises, from the 5' end to the 3' end, a CMV enhancer, a promoter, a 5'UTR, a Kozak sequence (gccacc), a VEGFR-binding molecule coding sequence, a 3'UTR, a WPRE, and an SV40 polyA, wherein the nucleic acid sequence of the CMV enhancer is shown in SEQ ID NO: 74, the nucleic acid sequence of the CMV promoter is shown in SEQ ID NO: 75, and the 5'UTR is shown in SEQ ID NO: 76. ozak sequence (gccacc), the coding sequence of the VEGF-binding molecule is set forth in any one of SEQ ID NOs: 78 to 86 (wherein SEQ ID NOs: 78 to 80 correspond to codon-optimized sequences 1, 2, and 3 of PR1145, SEQ ID NOs: 81 to 83 correspond to codon-optimized sequences 1, 2, and 3 of PR1174, and SEQ ID NOs: 84 to 86 correspond to codon-optimized sequences 1, 2, and 3 of PR1163), the 3'UTR sequence is set forth in SEQ ID NO: 87, the WPRE sequence is set forth in SEQ ID NO: 88, and the SV40 polyA sequence is set forth in SEQ ID NO: 89.

[0338] CMV enhancer sequence: [ka] CMV promoter sequence: [ka] 5'UTR sequence [ka] Kozak sequence: gccacc, Codon-optimized sequence 1 of PR1145 [ka] Codon-optimized sequence 2 of PR1145 [ka] Codon-optimized sequence 3 of PR1145 [ka] Codon-optimized sequence 1 of PR1174 [ka] Codon-optimized sequence 2 of PR1174 [ka] Codon-optimized sequence 3 of PR1174 [ka] Codon-optimized sequence 1 of PR1163 [ka] [ka] Codon-optimized sequence 2 of PR1163 [ka] [ka] Codon-optimized sequence 3 of PR1163 [ka] [ka] 3'-UTR sequence: [ka] WPRE sequence: [ka] SV40 polyA sequence: [ka] Capsid protein sequence: [ka] Example 9: Construction of expression vectors

[0339] An expression cassette was constructed, and an AAV packaging plasmid expressing the VEGF-binding molecule protein gene was constructed through conventional molecular biological procedures such as enzyme digestion, ligation, transformation, and cloning screening and identification (see Figure 3). The plasmid contained, in order, a CMV enhancer, promoter, 5'UTR, Kozak sequence (gccacc), VEGFR-binding molecule, 3'UTR, WPRE, and SV40 polyA. The expression cassette had inverted terminal repeats (ITRs) on both sides, with EcoRV and BSMI enzyme digestion sites. Example 10: Preparation and purification of AAV virus

[0340] The amino acid sequence of the AAV capsid used to deliver the gene expression cassette in this example is set forth in SEQ ID NO: 77. Referring to the method for packaging and purifying recombinant AAV viruses reported by Martin Lock et al., the AAV Rep and Cap protein expression plasmids, auxiliary plasmids, and the expression vector of Example 9 were co-transfected and packaged into HEK293 cells using PEI to prepare recombinant adeno-associated viruses. After 48 hours of transfection, the cells and culture supernatant were collected, and the AAV viruses were purified using iogisol ultragradient centrifugation (see Martin Lock, et al. Rapid, Simple, and Versatile Manufacturing of Recombinant Adeno-Associated Viral Vectors at Scale. HUMAN GENE THERAPY, 2010. 21: 1259-1271). The recombinant adeno-associated virus vectors were obtained, and the viral titers were measured using real-time fluorescent quantitative PCR. Example 11: Intraocular expression experiment

[0341] The following experiment was conducted to measure the expression levels and changes of pharmacophore proteins in four tissues of rabbit eyes (vitreous, aqueous humor, retina, and choroid) after injection of AAV-1174 (PR1174 (nucleic acid sequence is SEQ ID NO: 81)) and ADVM-022 (rAAV2.7m8-aflibercept in WO2017218974). Thirty-two New Zealand rabbits, aged 2 to 5 months, were purchased and divided into four groups (eight rabbits per group). The vitreous of both eyes was injected according to the doses listed in Table 12 (two high and two low doses were injected in each group). On day 58 after injection, the vitreous, aqueous humor, retina, and choroid tissues of both eyes were collected. At each time point, two rabbits and four eyes were sampled for each treatment group. The samples were returned to our laboratory and the pharmacophore protein content was measured using enzyme-linked immunosorbent assay.

[0342] [Table 13]

[0343] Figure 6 shows the results of intraocular expression. At D58, both the high-dose AAV-1174 group and the low-dose AAV-1174 group were able to stably express PR1174 protein in the aqueous humor, vitreous, retina, and choroid of rabbits, and the protein expression level was higher than that of ADVM-022. Example 12: In vivo efficacy experiments

[0344] The laser-induced choroidal neovascularization model is a classic model for testing antichoroidal neovascularization drugs that is currently commonly used internationally. This model was used to verify the efficacy of AAV-1174 to test its inhibitory effect on choroidal neovascularization.

[0345] Thirty-six rats were purchased, half male and half female, aged 8 to 10 weeks, and divided into six groups, each consisting of six rats. Each group was administered intravitreal injection according to the doses listed in Table 13. AAV-1174 (a pharmacophore protein expressing PR1174 (the nucleic acid sequence of which is SEQ ID NO: 81)) was administered, and laser model construction was performed on both eyes three weeks after administration. Fundus angiography was performed one week after model construction to detect the efficacy of the drug.

[0346] [Table 14] [Table 15]

[0347] Fundus images of the rats were taken before model construction, and no abnormalities were observed in the fundus of any rat. All rats underwent CNV model construction 21 days after administration, and FFA detection was performed 28 days after administration. In the PBS group, clear laser spot and sodium fluorescein leakage were observed 7 days after model construction, indicating successful model establishment. On 28 days after administration, FFA detection of fluorescent spots was performed, and a grade 3 spot leakage rate was detected. As shown in Table 14 and Figure 7, the difference between the AAV-1174 (high dose) group and the PBS group was statistically significant, demonstrating that AAV-1174 had a significant inhibitory effect on laser-induced choroidal neovascularization sodium fluorescein leakage.

Claims

1. A VEGF binding molecule comprising: a first VEGF binding domain; and and a second VEGF binding domain, Among them, the first VEGF-binding domain comprises immunoglobulin-like domain 2 of VEGFR2 (R2D2) and immunoglobulin-like domain 3 of VEGFR2 (R2D3); The second VEGF binding domain comprises a VEGF Trap or comprises an anti-VEGF antibody or an antigen-binding fragment thereof. VEGF binding molecule.

2. The VEGF Trap comprises the immunoglobulin-like domain 2 (R1D2) of VEGFR1 and / or the immunoglobulin-like domain 3 (R2D3) of VEGFR2; The VEGF binding molecule of claim 1.

3. the first VEGF-binding domain comprises amino acid mutations at any one or more positions selected from positions 162, 221, 222, 274, 276, 277, 280, 284, 288, 289, and 313, the amino acid mutation sites being naturally occurring sites with respect to the amino acid sequence set forth in SEQ ID NO:1; Preferably, the first VEGF binding domain comprises: -162nd place, - 162nd and 221st place, - 162nd and 222nd place, - 162nd and 274th place, - 162nd and 276th place, - 162nd and 277th place, - 162nd and 289th place, - 162nd and 313th place, - 221st and 222nd place, - 277th and 289th place, - 162nd, 221st and 222nd places, - 162nd, 277th and 289th place, - 162nd, 221st, 222nd and 274th place, and comprising an amino acid mutation at a position selected from any one or more of the following groups: More preferably, the first VEGF binding domain comprises: -162nd place, - 162nd, 221st, 222nd and 274th place, containing an amino acid mutation at a position selected from any one of the following groups:

3. A VEGF binding molecule according to claim 1 or 2.

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

5. the first VEGF-binding domain comprises amino acid mutations at any one or more positions selected from positions 135, 197, 199, 213, 215, 219, 257, 275, 277, 278, 283, 289, 313, and 314, the sites of the amino acid mutations being naturally occurring sites with respect to 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. A VEGF binding molecule according to any one of claims 1 to 3.

6. The first VEGF binding domain comprises: -135Y, -197Y, - 199Q or 199T, -213N, - 215F or 215H, -219I, -257Q or 257T, - 275L or 275E, -277R, - 278D, 278S or 278E, -283T, -289Y, -313Y, - 314S or 314Q, and Preferably, the first VEGF binding domain comprises the amino acid mutation 283T. The VEGF binding molecule of claim 5.

7. The first VEGF-binding domain comprises an amino acid mutation at any one or more positions selected from 162, 221, 222, 274, 276, 277, 280, 284, 288, 289, and 313; and comprising an amino acid mutation at any one or more positions selected from among positions 135, 197, 199, 213, 215, 219, 257, 275, 277, 278, 283, 289, 313, and 314; Preferably, the first VEGF binding domain comprises: - 162nd and 275th place, - 162nd and 283rd place, - 162nd and 313th place, - 162nd, 274th and 283rd place, - 162nd, 221st, 222nd and 283rd place, - 162nd, 221st, 222nd, 274th and 283rd positions, and comprising an amino acid mutation at a position selected from the group consisting of: More preferably, the first VEGF binding domain comprises: - 162nd and 283rd place, - 162nd, 221st, 222nd, 274th and 283rd positions, containing an amino acid mutation at a position selected from any one of the following groups: A VEGF binding molecule according to any one of claims 1 to 6.

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

8. A VEGF binding molecule according to any one of claims 7.

9. A VEGF binding molecule comprising: at least one VEGF binding domain, the VEGF binding domain comprising immunoglobulin-like domain 2 of VEGFR2 (R2D2) and immunoglobulin-like domain 3 of VEGFR2 (R2D3); wherein the VEGF-binding domain comprises amino acid mutations at one or more positions selected from positions 162, 221, 222, 274, 276, 277, 280, 284, 288, 289, and 313, and the amino acid mutation sites are natural sites relative to the amino acid sequence shown in SEQ ID NO:1; VEGF binding molecule.

10. The VEGF binding domain is -162nd place, - 162nd and 221st place, - 162nd and 222nd place, - 162nd and 274th place, - 162nd and 276th place, - 162nd and 277th place, - 162nd and 289th place, - 162nd and 313th place, - 221st and 222nd place, - 277th and 289th place, - 162nd, 221st and 222nd places, - 162nd, 277th and 289th place, - 162nd, 221st, 222nd and 274th place, and comprising an amino acid mutation at a position selected from any one or more of the following groups: Preferably, the VEGF binding domain comprises: -162nd place, - 162nd, 221st, 222nd and 274th place, containing an amino acid mutation at a position selected from any one of the following groups:

10. The VEGF binding molecule of claim 9.

11. The VEGF binding domain is - 162A, 162V, 162S, 162I or 162L, - 221S or 221N, -222K, - 274F or 274I, -276Q, -277R, -280E, -284H, -288Y, -289Y, -313R, and Preferably, the VEGF binding domain comprises: -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, -162V / 221S / 222K / 274F, and More preferably, the VEGF binding domain comprises -162V, -162V / 221S / 222K / 274F, The amino acid mutations are selected from any one or more of the following groups:

11. A VEGF binding molecule according to claim 9 or 10.

12. the VEGF-binding domain comprises amino acid mutations at any one or more positions selected from positions 135, 197, 199, 213, 215, 219, 257, 275, 277, 278, 283, 289, 313, and 314, the amino acid mutation sites being naturally occurring sites with respect to the amino acid sequence set forth in SEQ ID NO:1; Preferably, the VEGF binding domain comprises an amino acid mutation at position 283.

11. A VEGF binding molecule according to claim 9 or 10.

13. The VEGF binding domain is -135Y, -197Y, - 199Q or 199T, -213N, - 215F or 215H, -219I, -257Q or 257T, - 275L or 275E, -277R, - 278D, 278S or 278E, -283T, -289Y, -313Y, - 314S or 314Q, and Preferably, the VEGF binding domain comprises the amino acid mutation 283T.

13. The VEGF binding molecule of claim 12.

14. The VEGF-binding domain comprises an amino acid mutation at any one or more positions selected from the group consisting of 162, 221, 222, 274, 276, 277, 280, 284, 288, 289, and 313; and comprising an amino acid mutation at any one or more positions selected from among positions 135, 197, 199, 213, 215, 219, 257, 275, 277, 278, 283, 289, 313, and 314; Preferably, the VEGF binding domain comprises: - 162nd and 275th place, - 162nd and 283rd place, - 162nd and 313th place, - 162nd, 274th and 283rd place, - 162nd, 221st, 222nd and 283rd place, - 162nd, 221st, 222nd, 274th and 283rd positions, and comprising an amino acid mutation at a position selected from any one or more of the following groups: Preferably, the VEGF binding domain comprises: - 162nd and 283rd place, - 162nd, 221st, 222nd, 274th and 283rd positions, containing an amino acid mutation at a position selected from any one or more of the following groups: A VEGF binding molecule according to any one of claims 9 to 13.

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

15. The VEGF binding molecule of claim 14.

16. Further comprising an Fc, preferably the Fc is a human IgG1, human IgG2, human IgG3 or human IgG4 Fc; A VEGF binding molecule according to any one of claims 1 to 15.

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

18. The VEGF binding molecule comprises, from the N-terminus to the C-terminus: R2D2-R2D3-Fc, R1D2-R2D3-R2D2-R2D3-Fc, R1D2-R2D3-linker-R2D2-R2D3-Fc, R2D2-R2D3-R1D2-R2D3-Fc, R2D2-R2D3-linker-R1D2-R2D3-Fc, Any one selected from 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, the VH and VL are the VH and VL of bevacizumab, or the VH and VL of ranibizumab; Preferably, the linker is a peptide linker, More preferably, the linker is selected from: (GS)a(GGS)b(GGGS)c(GGGGS)d(GGGGG)e, in which a, b, c, d, e are each independently an integer equal to or greater than 0; or The linker is (EAAAK) ３ , (EAAAR) ３ , (EGGGK) ３ , (EGGGR) ３ , (DAAAR) ３ , (DAAAK) ３ , (DGGGR) ３ Or (DGGGK) ３ or The linker is ｘ S) ｙ wherein x is selected from an integer of 1 to 5, and y is selected from an integer of 1 to 6; A VEGF binding molecule according to any one of claims 1 to 17.

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

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

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

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

23. further comprising a promoter and / or enhancer operably linked to the polynucleotide, Preferably, the promoter is a CMV promoter, Preferably, the enhancer is a CMV enhancer.

23. The isolated nucleic acid according to any one of claims 21 to 22.

24. Further comprising any one or any combination of a Kozak sequence, a 5'-UTR and a 3'-UTR, a WPRE sequence, a polyA sequence, and an intron; Preferably, the Kozak sequence is located between an intron and a polynucleotide encoding a VEGF binding molecule; Preferably, the polyA sequence is an sv40 polyA sequence.

24. The isolated nucleic acid according to any one of claims 21 to 23.

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

26. A vector comprising: The vector comprises the isolated nucleic acid of any one of claims 21 to 25, preferably the vector is selected from any one or combination of a plasmid, a lentiviral vector, an adenovirus, an adeno-associated virus (AAV) vector, a recombinant adeno-associated virus (rAAV) vector, or a retroviral vector, More preferably, the vector is a recombinant adeno-associated viral vector. vector.

27. A recombinant adeno-associated viral vector, comprising: (i) a polynucleotide encoding a VEGF binding molecule as defined in any one of claims 21 to 25; and (ii) an enhancer, preferably a CMV enhancer; and (iii) a promoter, preferably a CMV promoter; (iv) a 5'-UTR and a 3'-UTR; (v) a Kozak sequence; and (vi) a WPRE sequence; and (vii) an sv40 polyA sequence; and Any one or any combination of (ii) to (vii) is operably linked to (i); Recombinant adeno-associated virus vectors.

28. A recombinant adeno-associated virus particle, comprising: An isolated nucleic acid according to any one of claims 21 to 25, or A recombinant adeno-associated virus vector comprising the recombinant adeno-associated virus vector of claim 27 and a capsid protein, Preferably, the capsid protein is selected from any serotype of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11, AAV12, AAVrh8, AAVrhlO, AAV-DJ, AAV-DJ8, or a variant thereof; More preferably, the capsid protein comprises an amino acid sequence as set forth in SEQ ID NO: 77, or an amino acid sequence having at least 70% sequence identity to SEQ ID NO:

77. Recombinant adeno-associated virus particles.

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

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

31. Preferably, the disease associated with abnormal angiogenesis is a vascular eye disease or cancer; More preferably, the vascular 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, diabetic macular edema), retinal vein occlusion (e.g., central retinal vein occlusion, branch retinal vein occlusion), choroidal neovascularization, iris neovascularization, neovascular glaucoma, postoperative fibrosis of 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 cancers include small cell lung cancer, renal cancer, uterine cancer, prostate cancer, bladder cancer, ovarian cancer, colon cancer, breast cancer, leukemia, lymphoma, myeloma, sarcoma (e.g., fibrosarcoma, myxosarcoma, liposarcoma, lymphangioendothelial sarcoma, angiosarcoma, endothelial sarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, lymphangiosarcoma, synovium, mesothelioma, leiomyosarcoma, rhabdomyosarcoma), glioma, pancreatic cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland cancer, and the like. selected from the group consisting of adenocarcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchopulmonary carcinoma, choriocarcinoma, renal cell carcinoma, hepatocarcinoma, cholangiocarcinoma, seminoma, embryonal carcinoma, cervical carcinoma, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, astrocytoma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, medulloblastoma, craniopharyngioma, oligodendroglioma, meningioma, melanoma, neutrophiloma, and retinoblastoma; Use of a VEGF-binding molecule according to any one of claims 1 to 20, an isolated nucleic acid according to any one of claims 21 to 25, or a vector according to any one of claims 26 to 27, or a recombinant adeno-associated viral particle according to claim 28, in the preparation of a medicament for treating a disease associated with abnormal angiogenesis.

32. 29. A rAAV production system for producing recombinant adeno-associated viral particles according to claim 28, wherein the production system comprises: (a) a nucleic acid sequence encoding the amino acid sequence of an 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 to 27; (c) an auxiliary packaging element having sufficient AAV rep and accessory functions 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 the AAV capsid; Among them, the AAV capsid is selected from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11, AAV12, AAVrh8, AAVrhlO, AAV-DJ, AAV-DJ8, or mutants thereof, and is preferably AAV2; rAAV production system.

33. the auxiliary packaging elements are provided by a packaging cell; Preferably, the packaging cell comprises a plasmid selected from any one of pHelper, pRC9, and pGOI, or a combination thereof; 33. The rAAV production system of claim 32.

34. 29. A method for producing a recombinant adeno-associated virus particle according to claim 28, comprising: 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 by a packaging cell, The AAV capsid is selected from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11, AAV12, AAVrh8, AAVrhlO, AAV-DJ, AAV-DJ8, or mutants thereof, and is preferably AAV2; The packaging cell comprises a plasmid selected from any one of pHelper, pRC9, and pGOI, or a combination thereof; method.

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