Fc fusion protein simultaneously binding to VEGF and ang-2, and use thereof

Abstract

Provided in the present invention are an Fc fusion protein capable of simultaneously binding to VEGF-A, VEGF-C, VEGF-D and Ang-2, a pharmaceutical composition containing the fusion protein, and the related use.

Description

Fc fusion proteins binding to both VEGF and Ang-2 and uses thereof

[0001] This application claims the benefit of Chinese Application No. 202411109729.2, filed August 13, 2024, Chinese Application No. 202411169931.4, filed August 23, 2024, Chinese Application No. 202510124923.6, filed January 26, 2025, and Chinese Application No. 202510361437.6, filed March 25, 2025, which are incorporated by reference herein in their entirety. TECHNICAL FIELD

[0002] The present application relates to the field of biological medicine, and more specifically, the present application relates to Fc fusion proteins capable of binding to VEGF-A, VEGF-C, VEGF-D and Ang-2 simultaneously, pharmaceutical compositions containing the fusion proteins, and related uses. BACKGROUND

[0003] Vascular endothelial growth factor (VEGF) proteins and their receptors (VEGFRs) play an important role in vasculogenesis (development of the embryonic vasculature from early differentiating endothelial cells), angiogenesis (the process of forming new blood vessels from pre-existing ones), and lymphangiogenesis (the process of forming new lymphatic vessels). Platelet-derived growth factor (PDGF) proteins and their receptors (PDGFRs) are involved in the regulation of cell proliferation, survival, and migration of several cell types.

[0004] Dysfunction of the endothelial cell regulatory system is a key feature of cancer and various diseases associated with aberrant vasculogenesis, angiogenesis, and lymphangiogenesis. Angiogenesis occurs in embryonic development and the growth, repair, and regeneration of normal tissues, the female reproductive cycle, the establishment and maintenance of pregnancy, the repair of wounds and fractures. In addition to angiogenesis occurring in healthy individuals, angiogenic events are involved in many pathological processes, especially tumor growth and metastasis, and other conditions in which vascular proliferation, especially of the microvasculature, is increased, such as diabetic retinopathy, psoriasis, and rheumatoid arthritis. Inhibition of angiogenesis can be useful in preventing or lessening these pathological processes or slowing their progression.

[0005] The etiology of neovascular ophthalmic diseases is complex, and the mechanism is not fully understood. Anti-VEGF drugs have achieved remarkable clinical efficacy and have replaced laser photocoagulation (PDT) as the first-line therapy. The anti-VEGF-A drugs that have been commercially available, such as Ranibizumab, Aflibercept (Eylea) and Conbercept, can significantly improve the vision of more than half of the newly treated patients, but there are still about 15% of patients who do not respond at all and about 15% to 20% of patients who develop desensitization and loss of efficacy desensitization after anti-VEGF-A treatment. Studies have found that the compensatory increase of VEGF-C after the neutralization of VEGF-A is an important factor leading to the desensitization of anti-VEGF-A treatment in patients with wet age-related macular degeneration (wAMD). At present, in the clinical phase 2 stage, after the combination of anti-VEGF-A drugs (such as Ranibizumab, Aflibercept, etc.) and OPT-302 (anti-VEGF-C and VEGF-D), the treatment population increases, and the patients can obtain better vision improvement. However, in the treatment of eye diseases, due to the need for intravitreal injection for administration, this combined drug method requires two injections respectively, and the inconvenience of administration leads to poor patient compliance, affecting the popularization of the drug. Therefore, the development of a multi-specific molecule targeting VEGF-A, VEGF-C and VEGF-D has very important clinical value.

[0006] In addition to the VEGF family, angiopoietin (Ang) is also involved in the regulation of angiogenesis and development. Angiopoietin-2 (Angiopoietin, also known as ANGPT2 or Ang-2) belongs to the Angiopoietin family, and is abnormally released during hypoxia or inflammatory response, thereby competitively binding to tyrosine kinase receptor-2 (Tie-2), thereby blocking the role of Ang-1 in stabilizing blood vessels, thereby causing blood vessel instability and endothelial activation.

[0007] Clinical studies have found that regulating the Angiopoietin / Immunoglobulin-like loop-containing and Epidermal Growth Factor-like domain tyrosine kinase (Tie) pathway has good effects in the treatment of ocular fundus neovascular diseases. In recent years, some bispecific antibodies targeting VEGF-A and Ang-2 have been developed, such as Faricimab from Roche, which has been marketed, but the blocking activity of the bispecific antibody against VEGF-A and Ang-2 is not good enough. Therefore, there is still a need to develop new multi-specific binding molecules with stronger blocking activity. SUMMARY

[0008] Based on a large amount of research, the present inventors developed a new polypeptide targeting VEGF-A, VEGF-C and VEGF-D, and based on the polypeptide, obtained an Fc fusion protein (for example, an Fc fusion protein capable of simultaneously binding VEGF-A, VEGF-C and VEGF-D), further fused with a VHH antibody targeting Ang-2, to generate a fusion protein simultaneously targeting VEGF-A, VEGF-C, VEGF-D and Ang-2. In particular, the multispecific binding molecule (fusion protein) of the present application has better protein yield, purity, physicochemical properties and affinity compared to wild-type fusion protein molecules, and has strong VEGF-A, VEGF-C and / or VEGF-D blocking activity (for example, blocking activity of simultaneously blocking VEGF-A, VEGF-C and VEGF-D), and can block the reporter gene activity and primary cell proliferation activity induced by VEGF-A, VEGF-C and VEGF-D. Therefore, the polypeptide or fusion protein of the present application has a blocking activity more relevant to disease progression, and can be effectively applied to the prevention and / or treatment of diseases related to VEGF (for example, VEGF-A, VEGF-C and / or VEGF-D) signaling (for example, diseases related to abnormal angiogenesis, for example, vascular eye diseases), for example, it can maintain better single-dose vision improvement and longer drug efficacy time in the clinic, effectively reducing the frequency of intravitreal injection in the eye.

[0009] Therefore, in a first aspect, the present application provides a fusion protein comprising a polypeptide construct binding VEGF, and a single-domain antibody or an antigen-binding fragment thereof specifically binding Ang-2; the polypeptide construct binding VEGF comprises a polypeptide, and an Fc peptide and an optional hinge peptide derived from an immunoglobulin; wherein the polypeptide comprises a first peptide segment and / or a second peptide segment, the first peptide segment comprises or consists of immunoglobulin-like domain 2 (R2D2) of VEGFR2 (vascular endothelial growth factor receptor 2), and the second peptide segment comprises or consists of immunoglobulin-like domain 3 (R2D3) of VEGFR2;

[0010] and the polypeptide comprises a mutation (e.g., a substitution mutation) at one or more amino acid positions selected from the group consisting of: position 121, position 127, position 128, position 129, position 130, position 131, position 132, position 133, position 134, position 137, position 139, position 141, position 142, position 144, position 148, position 152, position 155, position 156, position 162, position 163, position 164, position 165, position 169, position 173, position 175, position 176, position 186, position 190, position 191, position 193, position 194, position 197, position 206, position 210, position 212, position 213, position 215, position 218, position 222, position 232, position 233, position 241, position 248, position 249, position 254, position 255, position 259, position 264, position 266, position 267, position 269, position 270, position 272, position 276, position 279, position 280, position 284, position 285, position 287, position 289, position 296, position 297, position 299, position 302, position 308, position 311, position 318, and position 323;

[0011] wherein each of the mutated amino acid positions is a natural order position relative to the amino acid sequence set forth in SEQ ID NO: 238.

[0012] As will be readily understood by those skilled in the art, the expression "each of the mutated amino acid positions is a natural order position relative to the amino acid sequence set forth in SEQ ID NO: 238" is intended to mean that the amino acid position in the polypeptide that is in a corresponding position to the amino acid position of the amino acid sequence set forth in SEQ ID NO: 238. For example, "position 121" is intended to mean the amino acid position in the polypeptide that is in a corresponding position to position 121 of SEQ ID NO: 238; "position 127" is intended to mean the amino acid position in the polypeptide that is in a corresponding position to position 127 of SEQ ID NO: 238.

[0013] As used herein, the expression "the amino acid position in the polypeptide that is in a corresponding position to position 121 of SEQ ID NO: 238" refers to the amino acid position in the sequence of the polypeptide under comparison that is in an equivalent position to the 121st amino acid residue of SEQ ID NO: 238 when the sequence of the polypeptide is optimally aligned with SEQ ID NO: 238, i.e., when the sequence of the polypeptide is aligned with SEQ ID NO: 238 to obtain the highest percentage identity.

[0014] Unless specifically indicated otherwise, or manifestly contradicted by context, the meaning of like terms in the remainder of this disclosure are defined in a manner analogous to the above.

[0015] In certain embodiments, the first peptide segment comprises or consists of amino acid residues in VEGFR2 at positions corresponding to positions 130-205 (or 130-207, or 130-210, or 130-215, or 125-205, or 125-207, or 125-210, or 125-215, or 120-205, or 120-207, or 120-210, or 120-215) of SEQ ID NO: 238. In certain embodiments, the first peptide segment comprises or consists of amino acid residues in VEGFR2 at positions corresponding to positions 120-207 of SEQ ID NO: 238.

[0016] In certain embodiments, the second peptide segment comprises or consists of amino acid residues in VEGFR2 at positions corresponding to positions 216-320 (or 216-327, or 216-335, or 211-320, or 211-327, or 211-335, or 208-320, or 208-327, or 208-335, or 206-320, or 206-327, or 206-335) of SEQ ID NO: 238. In certain embodiments, the second peptide segment comprises or consists of amino acid residues in VEGFR2 at positions corresponding to positions 208-327 of SEQ ID NO: 238.

[0017] As used herein, the expression "amino acid residues in VEGFR2 at positions corresponding to positions 130-205 of SEQ ID NO: 238" means the amino acid positions / residues in a sequence of VEGFR2 that are in equivalent positions to the amino acid residues at positions 130-205 of SEQ ID NO: 238, when the sequence of VEGFR2 is optimally aligned with SEQ ID NO: 238, i.e., when the sequence of VEGFR2 is aligned with SEQ ID NO: 238 to obtain the highest percentage identity.

[0018] Unless specifically indicated otherwise, or manifestly contradicted by context, the meaning of like terms in the remainder of this disclosure are defined in a manner analogous to the above.

[0019] In certain embodiments, the polypeptide comprises the first peptide segment.

[0020] In certain embodiments, the polypeptide comprises the second peptide segment.

[0021] In some embodiments, the polypeptide comprises the first peptide segment and the second peptide segment.

[0022] In some embodiments, the polypeptide contains mutations (e.g., substitution mutations) at one or more amino acid sites selected from the following: position 121, position 128, position 129, position 130, position 133, position 134, position 139, position 141, position 142, position 155, position 156, position 162, position 163, position 173, position 175, position 176, position 186, position 190, position 191, position 194, position 197, position 206, position 213, position 249, position 264, position 267, position 269, position 270, position 276, position 285, position 287, position 297, position 302, position 308, and position 318.

[0023] The mutated amino acid sites are the natural sequence sites relative to the amino acid sequence shown in SEQ ID NO:238.

[0024] In some embodiments, the polypeptide contains mutations (e.g., substitution mutations) at one or more amino acid sites selected from the following: position 121, position 128, position 129, position 133, position 134, position 141, position 155, position 162, position 176, position 186, position 191, position 194, position 206, position 249, position 264, position 267, position 269, position 270, position 276, position 285, position 287, position 308, and position 318;

[0025] The mutated amino acid sites are the natural sequence sites relative to the amino acid sequence shown in SEQ ID NO:238.

[0026] In some embodiments, the polypeptide contains mutations (e.g., substitution mutations) at one or more amino acid sites selected from the following: position 133, position 141, position 162, position 194, position 249, position 270, position 276, and position 308.

[0027] The mutated amino acid sites are the natural sequence sites relative to the amino acid sequence shown in SEQ ID NO:238.

[0028] In some embodiments, the polypeptide has one or more mutations selected from the following:

[0029] (1) 121N, 121C, or 121S; (2) 127P; (3) 128P; (4) 129G or 129A; (5) 130G; (6) 131P, 131L, or 131V; (7) 132H; (8) 133L, 133Y, or 133P; (9) 134E or 134Q; (10) 137H; (11) 139V; (12) 141E; (13) 142V; (14) 144M; (15) 148L; (16) 152S; (17) 155P; (18) 156D; (19) 162R, 162H, 162Y, 162T, or 162K; (20) 163T; (21) 164K; (22) 165F; (23) 169T or 169E; (24) 173N; (25) 175K or 175S; (26) 176S; (27) 186S; (28) 190S, 190N, 190A, 190V, or 190P; (29) 191L; (30) 193R or 193H; (31) 194Q or 194D; (32) 197L or 197S; (33) 206G; (34) 210R; (35) 212T; (36) 213V, 213K, or 213L; (37) 215D; (38) 218A, 218T, or 218K; (39) 222E; (40) 232E or 232Q; (41) 233P; (42) 241R; (43) 248V or 248G; (44) 249T or 249V; (45) 254E; (46) 255D or 255R; (47) 259T; (48) 264G or 264F; (49) 266N; (50) 267T, 267D, or 267N; (51) 269R; (52) 270V or 270S; (53) 272I; (54) 276V, 276S, or 276R; (55) 279R; (56) 280P; (57) 284K; (58) 285T, 285E, or 285S; (59) 287H or 287L; (60) 289S; (61) 296N; (62) 297I; (63) 299L; (64) 302S; (65) 308K, 308T, or 308S; (66) 311T; (67) 318H; (68) 323T;

[0030] wherein each of the mutated amino acid positions is relative to the natural order of the amino acid sequence set forth in SEQ ID NO: 238.

[0031] As used herein, the mutation "121N" means that the amino acid residue in the polypeptide at the position corresponding to position 121 of SEQ ID NO: 238 is replaced with N; the mutation "127P" means that the amino acid residue in the polypeptide at the position corresponding to position 127 of SEQ ID NO: 238 is replaced with P.

[0032] Unless specifically indicated otherwise or clearly contradicted by context, the meaning of similar expressions, as used herein, is defined by analogy with the above.

[0033] In certain embodiments, the polypeptide possesses one or more mutations selected from the group consisting of:

[0034] (1) 121N or 121S; (2) 128P; (3) 129G; (4) 130G; (5) 133Y or 133P; (6) 134E or 134Q; (7) 139V; (8) 141E; (9) 142V; (10) 155P; (11) 156D; (12) 162R or 162H; (13) 163T; (14) 173N; (15) 175K; (16) 176S; (17) 186S; (18) 190S, 190V or 190P; (19) 191L; (20) 194Q or 194D; (21) 197L; (22) 206G; (23) 213L; (24) 249T; (25) 264G; (26) 267T, 267D or 267N; (27) 269R; (28) 270V; (29) 276V or 276S; (30) 285T, 285E or 285S; (31) 287L; (32) V297I; (33) 302S; (34) 308K or 308T; (35) 318H;

[0035] wherein the amino acid positions of each mutation are relative to the natural order of the amino acid sequence set forth in SEQ ID NO: 238.

[0036] In certain embodiments, the polypeptide possesses one or more mutations selected from the group consisting of:

[0037] (1) 121N or 121S; (2) 128P; (3) 129G; (4) 133Y or 133P; (5) 134E or 134Q; (6) 141E; (7) 155P; (8) 162R or 162H; (9) 176S; (10) 186S; (11) 191L; (12) 194Q or 194D; (13) 206G; (14) 249T; (15) 264G; (16) 267T or 267D; (17) 269R; (18) 270V; (19) 276V or 276S; (20) 285T or 285E; (21) 287L; (22) 308K or 308T; (23) 318H;

[0038] wherein each mutated amino acid position is relative to the natural order of the amino acid sequence set forth in SEQ ID NO: 238.

[0039] In certain embodiments, the polypeptide possesses one or more mutations selected from the group consisting of:

[0040] (1) 133P; (2) 141E; (3) 162R; (4) 194D; (5) 249T; (6) 270V; (7) 276V or 276S; (8) 308K or 308T;

[0041] wherein each mutated amino acid position is relative to the natural order of the amino acid sequence set forth in SEQ ID NO: 238.

[0042] In certain embodiments, the polypeptide possesses one or more mutations selected from the group consisting of:

[0043] (1) Y121N, Y121C, or Y121S; (2) A127P; (3) S128P; (4) V129G or V129A; (5) S130G; (6) D131P, D131L, or D131V; (7) Q132H; (8) H133L, H133Y, or H133P; (9) G134E or G134Q; (10) Y137H; (11) T139V; (12) N141E; (13) K142V; (14) K144M; (15) I148L; (16) G152S; (17) S155P; (18) N156D; (19) C162R, C162H, C162Y, C162T, or C162K; (20) A163T; (21) R164K; (22) Y165F; (23) R169T or R169E; (24) D173N; (25) N175K or N175S; (26) R176S; (27) T186S; (28) Y190S, Y190N, Y190A, Y190V, or Y190P; (29) M191L; (30) S193R or S193H; (31) Y194Q or Y194D; (32) M197L or M197S; (33) D206G; (34) Q210R; (35) I212T; (36) M213V, M213K, or M213L; (37) I215D; (38) V218A, V218T, or V218K; (39) R222E; (40) H232E or H232Q; (41) G233P; (42) K241R; (43) A248V or A248G; (44) R249T or R249V; (45) V254E; (46) G255D or G255R; (47) N259T; (48) S264G or S264F; (49) K266N; (50) H267T, H267D, or H267N; (51) H269R; (52) K270V or K270S; (53) L272I; (54) D276V, D276S, or D276R; (55) T279R; (56) Q280P; (57) E284K; (58) M285T, M285E, or M285S; (59) K287H or K287L; (60) L289S; (61) G296N; (62) V297I; (63) R299L; (64) Q302S; (65) A308K, A308T, or A308S; (66) S311T; (67) N318H; (68) R323T;

[0044] wherein each of the mutated amino acid positions is relative to the natural order of the amino acid sequence set forth in SEQ ID NO: 238.

[0045] As used herein, the mutation "Y121N" means that the amino acid residue Y in the polypeptide at the position corresponding to position 121 of SEQ ID NO: 238 is replaced with N; the mutation "A127P" means that the amino acid residue A in the polypeptide at the position corresponding to position 127 of SEQ ID NO: 238 is replaced with P.

[0046] Unless specifically indicated otherwise or clearly contradicted by context, the meaning of the remaining similar expressions herein is defined in a manner similar to the above.

[0047] In certain embodiments, the polypeptide possesses one or more mutations selected from the group consisting of:

[0048] (1) Y121N or Y121S; (2) S128P; (3) V129G; (4) S130G; (5) H133Y or H133P; (6) G134E or G134Q; (7) T139V; (8) N141E; (9) K142V; (10) S155P; (11) N156D; (12) C162R or C162H; (13) A163T; (14) D173N; (15) N175K; (16) R176S; (17) T186S; (18) Y190S, Y190V or Y190P; (19) M191L; (20) Y194Q or Y194D; (21) M197L; (22) D206G; (23) M213L; (24) R249T; (25) S264G; (26) H267T, H267D or H267N; (27) H269R; (28) K270V; (29) D276V or D276S; (30) M285T, M285E or M285S; (31) K287L; (32) V297I; (33) Q302S; (34) A308K or A308T; (35) N318H;

[0049] wherein the amino acid positions of each mutation are relative to the natural order of the amino acid sequence set forth in SEQ ID NO: 238.

[0050] In certain embodiments, the polypeptide possesses one or more mutations selected from the group consisting of:

[0051] (1) Y121N or Y121S; (2) S128P; (3) V129G; (4) H133Y or H133P; (5) G134E or G134Q; (6) N141E; (7) S155P; (8) C162R or C162H; (9) R176S; (10) T186S; (11) M191L; (12) Y194Q or Y194D; (13) D206G; (14) R249T; (15) S264G; (16) H267T or H267D; (17) H269R; (18) K270V; (19) D276V or D276S; (20) M285T or M285E; (21) K287L; (22) A308K or A308T; (23) N318H;

[0052] wherein each mutated amino acid position is relative to the natural order of the amino acid sequence set forth in SEQ ID NO: 238.

[0053] In certain embodiments, the polypeptide possesses one or more mutations selected from the group consisting of:

[0054] (1) H133P; (2) N141E; (3) C162R; (4) Y194D; (5) R249T; (6) K270V; (7) D276V or D276S; (8) A308K or A308T;

[0055] wherein each mutated amino acid position is relative to the natural order of the amino acid sequence set forth in SEQ ID NO: 238.

[0056] In certain embodiments, the polypeptide possesses one or more mutations selected from the group consisting of:

[0057] (1) Y2N, Y2C, or Y2S; (2) A8P; (3) S9P; (4) V10G or V10A; (5) S11G; (6) D12P, D12L, or D12V; (7) Q13H; (8) H14L, H14Y, or H14P; (9) G15E or G15Q; (10) Y18H; (11) T20V; (12) M22E; (13) K23V; (14) K25M; (15) I29L; (16) G33S; (17) S36P; (18) N37D; (19) C43R, C43H, C43Y, C43T, or C43K; (20) A44T; (21) R45K; (22) Y46F; (23) R50T or R50E; (24) D54N; (25) N56K or N56S; (26) R57S; (27) T67S; (28) Y71S, Y71N, Y71A, Y71V, or Y71P; (29) M72L; (30) S74R or S74H; (31) Y75Q or Y75D; (32) M78L or M78S; (33) D87G; (34) Q91R; (35) I93T; (36) M94V, M94K, or M94L; (37) I96D; (38) V99A, V99T, or V99K; (39) R103E; (40) H113E or H113Q; (41) G114P; (42) K122R; (43) A129V or A129G; (44) R130T or R130V; (45) V135E; (46) G136D or G136R; (47) N140T; (48) S145G or S145F; (49) K147N; (50) H148T, H148D, or H148N; (51) H150R; (52) K151V or K151S; (53) L153I; (54) D157V, D157S, or D157R; (55) T160R; (56) Q161P; (57) E165K; (58) M166T, M166E, or M166S; (59) K168H or K168L; (60) L170S; (61) G177N; (62) V178I; (63) R180L; (64) Q183S; (65) A189K, A189T, or A189S; (66) S192T; (67) N199H; (68) R204T;

[0058] wherein each of the mutated amino acid positions is relative to the natural order of the amino acid sequence set forth in SEQ ID NO: 234.

[0059] As used herein, the mutation "Y2N" means that the amino acid residue Y in the polypeptide at the position corresponding to position 2 of SEQ ID NO: 234 is replaced with N; the mutation "A8P" means that the amino acid residue A in the polypeptide at the position corresponding to position 8 of SEQ ID NO: 234 is replaced with P.

[0060] Unless specifically indicated otherwise or clearly contradicted by context, the meaning of like terms throughout the remainder of this text are defined in a manner analogous to the above.

[0061] In certain embodiments, the polypeptide has one or more mutations selected from the following, as compared to the polypeptide set forth in SEQ ID NO: 234:

[0062] (1) Y2N or Y2S; (2) S9P; (3) V10G; (4) S11G; (5) H14P; (6) G15E or G15Q; (7) T20V; (8) M22E; (9) K23V; (10) S36P; (11) N37D; (12) C43R or C43H; (13) A44T; (14) D54N; (15) N56K; (16) R57S; (17) T67S; (18) Y71S, Y71V, or Y71P; (19) M72L; (20) Y75Q or Y75D; (21) M78L; (22) D87G; (23) M94L; (24) R130T; (25) S145G; (26) H148T, H148D, or H148N; (27) H150R; (28) K151V; (29) D157S or D157V; (30) M166T, M166E, or M166S; (31) K168L; (32) V178I; (33) Q183S; (34) A189K or A189T; (35) N199H;

[0063] wherein the amino acid positions of each mutation are relative to the natural order of the amino acid sequence set forth in SEQ ID NO: 234.

[0064] In certain embodiments, the polypeptide has one or more mutations selected from the following, as compared to the polypeptide set forth in SEQ ID NO: 234:

[0065] (1) Y2N or Y2S; (2) S9P; (3) V10G; (4) H14P; (5) G15E or G15Q; (6) M22E; (7) S36P; (8) C43R or C43H; (9) R57S; (10) T67S; (11) M72L; (12) Y75Q or Y75D; (13) D87G; (14) R130T; (15) S145G; (16) H148T or H148D; (17) H150R; (18) K151V; (19) D157S or D157V; (20) M166T or M166E; (21) K168L; (22) A189K or A189T; (23) N199H;

[0066] wherein each mutated amino acid position is in the natural order of amino acid sequence relative to the amino acid sequence set forth in SEQ ID NO: 234.

[0067] In certain embodiments, the polypeptide comprises one or more mutations selected from the following, as compared to the polypeptide set forth in SEQ ID NO: 234:

[0068] (1) H14P; (2) M22E; (3) C43R or C43H; (4) Y75D; (5) R130T; (6) K151V; (7) D157S or D157V; (8) A189K or A189T;

[0069] wherein each mutated amino acid position is in the natural order of amino acid sequence relative to the amino acid sequence set forth in SEQ ID NO: 234.

[0070] In certain embodiments, the polypeptide comprises the first peptide segment.

[0071] In certain embodiments, the polypeptide comprises one or more mutations (e.g., substitution mutations) at one or more amino acid positions selected from the following: position 121, position 127, position 128, position 129, position 130, position 131, position 132, position 133, position 134, position 137, position 139, position 141, position 142, position 144, position 148, position 152, position 155, position 156, position 162, position 163, position 164, position 165, position 169, position 173, position 175, position 176, position 186, position 190, position 191, position 193, position 194, position 197, and position 206.

[0072] wherein each mutated amino acid position is in the natural order of amino acid sequence relative to the amino acid sequence set forth in SEQ ID NO: 238.

[0073] In certain embodiments, the polypeptide comprises a mutation (e.g., a substitution mutation) at one or more amino acid positions selected from: position 121, position 128, position 129, position 130, position 133, position 134, position 139, position 141, position 142, position 155, position 156, position 162, position 163, position 173, position 175, position 176, position 186, position 190, position 191, position 194, position 197, position 206;

[0074] wherein each mutated amino acid position is with respect to the natural order of the amino acid sequence set forth in SEQ ID NO: 238.

[0075] In certain embodiments, the polypeptide comprises a mutation (e.g., a substitution mutation) at one or more amino acid positions selected from: position 121, position 128, position 129, position 133, position 134, position 141, position 155, position 162, position 176, position 186, position 191, position 194, position 206;

[0076] wherein each mutated amino acid position is with respect to the natural order of the amino acid sequence set forth in SEQ ID NO: 238.

[0077] In certain embodiments, the polypeptide comprises a mutation (e.g., a substitution mutation) at one or more amino acid positions selected from: position 133, position 141, position 162, position 194;

[0078] wherein each mutated amino acid position is with respect to the natural order of the amino acid sequence set forth in SEQ ID NO: 238.

[0079] In certain embodiments, the polypeptide comprises one or more mutations selected from:

[0080] (1) 121N, 121C, or 121S; (2) 127P; (3) 128P; (4) 129G or 129A; (5) 130G; (6) 131P, 131L, or 131V; (7) 132H; (8) 133L, 133Y, or 133P; (9) 134E or 134Q; (10) 137H; (11) 139V; (12) 141E; (13) 142V; (14) 144M; (15) 148L; (16) 152S; (17) 155P; (18) 156D; (19) 162R, 162H, 162Y, 162T, or 162K; (20) 163T; (21) 164K; (22) 165F; (23) 169T or 169E; (24) 173N; (25) 175K or 175S; (26) 176S; (27) 186S; (28) 190S, 190N, 190A, 190V, or 190P; (29) 191L; (30) 193R or 193H; (31) 194Q or 194D; (32) 197L or 197S; (33) 206G;

[0081] wherein each mutated amino acid position is relative to the natural order of the amino acid sequence set forth in SEQ ID NO: 238.

[0082] In certain embodiments, the polypeptide possesses one or more mutations selected from the group consisting of:

[0083] (1) 121N or 121S; (2) 128P; (3) 129G; (4) 130G; (5) 133Y or 133P; (6) 134E or 134Q; (7) 139V; (8) 141E; (9) 142V; (10) 155P; (11) 156D; (12) 162R or 162H; (13) 163T; (14) 173N; (15) 175K; (16) 176S; (17) 186S; (18) 190S, 190V, or 190P; (19) 191L; (20) 194Q or 194D; (21) 197L; (22) 206G;

[0084] wherein each mutated amino acid position is relative to the natural order of the amino acid sequence set forth in SEQ ID NO: 238.

[0085] In certain embodiments, the polypeptide possesses one or more mutations selected from the group consisting of:

[0086] (1) 121N or 121S; (2) 128P; (3) 129G; (4) 133Y or 133P; (5) 134E or 134Q; (6) 141E; (7) 155P; (8) 162R or 162H; (9) 176S; (10) 186S; (11) 191L; (12) 194Q or 194D; (13) 206G;

[0087] wherein each mutated amino acid position is relative to the natural order of the amino acid sequence set forth in SEQ ID NO: 238.

[0088] In certain embodiments, the polypeptide comprises one or more mutations selected from the group consisting of:

[0089] (1) 133P; (2) 141E; (3) 162R; (4) 194D;

[0090] wherein each mutated amino acid position is relative to the natural order of the amino acid sequence set forth in SEQ ID NO: 238.

[0091] In certain embodiments, the polypeptide comprises the second peptide segment.

[0092] In certain embodiments, the polypeptide comprises a mutation (e.g., a substitution mutation) at one or more amino acid positions selected from the group consisting of: position 210, position 212, position 213, position 215, position 218, position 222, position 232, position 233, position 241, position 248, position 249, position 254, position 255, position 259, position 264, position 266, position 267, position 269, position 270, position 272, position 276, position 279, position 280, position 284, position 285, position 287, position 289, position 296, position 297, position 299, position 302, position 308, position 311, position 318, and position 323;

[0093] wherein each mutated amino acid position is relative to the natural order of the amino acid sequence set forth in SEQ ID NO: 238.

[0094] In certain embodiments, the polypeptide comprises a mutation (e.g., a substitution mutation) at one or more amino acid positions selected from the group consisting of: position 213, position 249, position 264, position 267, position 269, position 270, position 276, position 285, position 287, position 297, position 302, position 308, position 318;

[0095] wherein each mutated amino acid position is relative to the natural order of the amino acid sequence set forth in SEQ ID NO: 238.

[0096] In certain embodiments, the polypeptide comprises a mutation (e.g., a substitution mutation) at one or more amino acid positions selected from: position 249, position 264, position 267, position 269, position 270, position 276, position 285, position 287, position 308, position 318;

[0097] wherein each mutated amino acid position is with respect to the natural order of amino acid sequence set forth in SEQ ID NO: 238.

[0098] In certain embodiments, the polypeptide comprises a mutation (e.g., a substitution mutation) at one or more amino acid positions selected from: position 249, position 270, position 276, position 308;

[0099] wherein each mutated amino acid position is with respect to the natural order of amino acid sequence set forth in SEQ ID NO: 238.

[0100] In certain embodiments, the polypeptide possesses one or more mutations selected from:

[0101] (1) 210R; (2) 212T; (3) 213V, 213K, or 213L; (4) 215D; (5) 218A, 218T, or 218K; (7) 222E; (8) 232E or 232Q; (9) 233P; (10) 241R; (11) 248V or 248G; (12) 249T or 249V; (13) 254E; (14) 255D or 255R; (15) 259T; (16) 264G or 264F; (17) 266N; (18) 267T, 267D, or 267N; (19) 269R; (20) 270V or 270S; (21) 272I; (22) 276V, 276S, or 276R; (23) 279R; (24) 280P; (25) 284K; (26) 285T, 285E, or 285S; (27) 287H or 287L; (28) 289S; (29) 296N; (30) 297I; (31) 299L; (32) 302S; (33) 308K, 308T, or 308S; (34) 311T; (35) 318H; (36) 323T;

[0102] wherein each mutated amino acid position is with respect to the natural order of amino acid sequence set forth in SEQ ID NO: 238.

[0103] In certain embodiments, the polypeptide possesses one or more mutations selected from:

[0104] (1) 213L; (2) 249T; (3) 264G; (4) 267T, 267D, or 267N; (5) 269R; (6) 270V; (7) 276V or 276S; (8) 285T, 285E, or 285S; (9) 287L; (10) V297I; (11) 302S; (12) 308K or 308T; (13) 318H;

[0105] wherein each mutated amino acid position is with respect to the natural order of the amino acid sequence set forth in SEQ ID NO: 238.

[0106] In certain embodiments, the polypeptide possesses one or more mutations selected from the group consisting of:

[0107] (1) 249T; (2) 264G; (3) 267T or 267D; (4) 269R; (5) 270V; (6) 276V or 276S; (7) 285T or 285E; (8) 287L; (9) 308K or 308T; (10) 318H;

[0108] wherein each mutated amino acid position is with respect to the natural order of the amino acid sequence set forth in SEQ ID NO: 238.

[0109] In certain embodiments, the polypeptide possesses one or more mutations selected from the group consisting of:

[0110] (1) 249T; (2) 270V; (3) 276V or 276S; (4) 308K or 308T;

[0111] wherein each mutated amino acid position is with respect to the natural order of the amino acid sequence set forth in SEQ ID NO: 238.

[0112] In certain embodiments, the polypeptide possesses one or more mutations selected from the group consisting of any row set forth in Column B or Column E of Table 1 or Table 6, wherein each mutated amino acid position is with respect to the natural order of the amino acid sequence set forth in SEQ ID NO: 238.

[0113] In certain embodiments, the polypeptide possesses one or more mutations selected from the group consisting of:

[0114] - H133L

[0115] - V129G; S130G; H133L; G152S; C162R; R164K; T186S; M191L; Y194Q; Q210R; V218A; A248V; D276V; M285T; N318H

[0116] - Y121N; V129G; S130G; H133L; G152S; C162R; T186S; Y190S; M191L; S193R; Y194Q; Q210R; V218A; D276V; M285T

[0117] - Y121N; V129G; S130G; H133L; C162R; T186S; M191L; S193R; Y194Q; Q210R; R249T; D276V; M285T; N318H

[0118] - Y121N; V129G; H133L; K144M; C162R; T186S; Y190N; M191L; S193R; V218A; R249T; D276V; M285T

[0119] - Y121N; V129A; S130G; H133L; C162R; T186S; Y190N; S193R; Q210R; V218A; R249T; D276V; N318H

[0120] - Y121N; V129G; S130G; H133L; K144M; G152S; C162R; T186S; Y190N; S193R; Y194Q; Q210R; D276V; M285T

[0121] - Y121N; V129A; S130G; H133L; G152S; C162R; T186S; Y190N; M191L; Y194Q; Q210R; A248V; R249T; D276V; M285T; N318H

[0122] - Y121N; V129G; Q132H; H133L; G152S; C162R; T186S; M191L; S193R; Y194Q; V218A; R249T; D276V; M285T; N318H

[0123] wherein each of the mutated amino acid positions is an amino acid position in the polypeptide that is in a corresponding position to the respective amino acid position of SEQ ID NO: 238. For example, the mutation "H133L" means that the amino acid residue H in the position corresponding to position 133 of SEQ ID NO: 238 is replaced with L in the polypeptide.

[0124] In certain embodiments, the polypeptide has one or more sets of mutations selected from the group consisting of:

[0125] - H133Y

[0126] - Y121N; V129G; S130G; H133Y; G152S; C162R; Y194Q; Q210R; A248V; R249T; D276V; M285T; N318H

[0127] - Y121N; V129G; S130G; H133Y; G152S; C162R; Y194Q; Q210R; A248V; R249T; D276V; M285T; N318H

[0128] - V129G; S130G; H133Y; C162R; T186S; Y190N; S193R; V218A; R249T; D276V; M285T; N318H

[0129] - Y121N; V129G; S130G; H133Y; G152S; C162R; T186S; Y190N; S193R; Q210R; V218A; A248V; D276V; M285T; N318H

[0130] - Y121N; V129G; H133Y; G152S; C162R; T186S; Y190N; M191L; Y194Q; Q210R; K241R; D276V; S311T; N318H

[0131] - Y121N; V129G; H133Y; C162R; T186S; M191L; Y194Q; R249T; D276V; M285T; N318H

[0132] - Y121N; V129G; H133Y; G152S; C162R; T186S; Y190N; S193R; V218A; R249T; D276V; M285T; S311T; N318H

[0133] - Y121N; V129G; H133Y; G152S; C162R; T186S; Y190N; S193R; Y194Q; M213V; V218A; K241R; D276V; M285T; N318H

[0134] - Y121N; V129A; S130G; H133Y; G152S; C162R; T186S; Y190N; S193R; V218A; D276V; S311T

[0135] - Y121N; V129G; H133Y; C162R; T186S; Y190N; M191L; S193R; Q210R; M213V; R249T; D276V; M285T

[0136] - Y121N; V129G; H133Y; C162R; T186S; Y190N; M191L; S193R; Q210R; M213V; R249T; D276V; M285T

[0137] - Y121N; V129G; H133Y; C162R; T186S; Y190N; M191L; S193R; Q210R; M213V; R249T; D276V; M285T

[0138] - Y121N; V129G; H133Y; C162R; T186S; Y190N; M191L; S193R; Q210R; M213V; R249T; D276V; M285T

[0139] - Y121N; V129G; H133Y; C162R; T186S; Y190N; M191L; S193R; Q210R; M213V; R249T; D276V; M285T

[0140] - Y121N; V129G; H133Y; C162R; T186S; Y190N; M191L; S193R; Q210R; M213V; R249T; D276V; M285T

[0141] - Y121N; V129G; H133Y; C162R; T186S; Y190N; M191L; S193R; Q210R; M213V; R249T; D276V; M285T

[0142] - Y121N; V129G; H133Y; C162R; T186S; Y190N; M191L; S193R; Q210R; M213V; R249T; D276V; M285T

[0143] - Y121N; V129G; H133Y; C162R; T186S; Y190N; M191L; S193R; Q210R; M213V; R249T; D276V; M285T

[0144] V129G; H133Y; C162R; T186S; Y190N; M191L; S193R; Y194Q; Q210R; A248V; R249T; D276V; M285T; S311T

[0145] wherein each of the mutated amino acid positions is an amino acid position in the polypeptide that is in a corresponding position to the respective amino acid position of SEQ ID NO: 238.

[0146] In certain embodiments, the polypeptide possesses one or more sets of mutations selected from the group consisting of:

[0147] H133P

[0148] H133P; D276S

[0149] Y194D; H133P

[0150] Y194D; H133P; D276S

[0151] Y194D; H133P; K270V

[0152] H133P; K270V; R176S

[0153] H133P; M285E; K270V

[0154] A308T; Y194D; H133P; K270V

[0155] Y194D; H133P; D276S; K270V

[0156] Y194D; H133P; D276S; A308K

[0157] Y194D; H133P; D276S; R249T

[0158] A308T; H133P; G134E; K270V

[0159] Y194D; H133P; D276S; K270V; A308K

[0160] Y194D; H133P; M285E; K270V; R176S

[0161] Y194D; H133P; D276S; R249T; C162H

[0162] H133P; C162R; Y194D; K270V; D276V; A308T

[0163] - Y121N; H133P; C162R; Y194D; K270V; D276V; A308T

[0164] - V129G; H133P; C162R; Y194D; K270V; D276V; A308T

[0165] - V129G; H133P; Y137H; C162R; Y194D; Y221N; K270V; D276V; A308T

[0166] - H133P; Y137H; C162R; Y194D; Y221N; K270V; D276S; A308T

[0167] - H133P; N141E; C162R; Y194D; K270V; D276V; A308T

[0168] - H133P; N141E; C162R; Y194D; K270V; D276V; A308K

[0169] - H133P; Y137H; C162R; Y194D; Y221N; K270V; D276V; A308T

[0170] - H133P; C162R; Y194D; K270V; D276V; A308K

[0171] - H133P; C162K; Y194D; K270V; D276V; A308T

[0172] - H133P; C162H; Y194D; K270V; D276V; A308T

[0173] - H133P; G134E; C162R; Y194D; K270V; D276V; A308T

[0174] - H133P; S155P; C162R; Y194D; K270V; D276V; A308T

[0175] - H133P; C162R; Y194D; R249T; K270V; D276V; A308T

[0176] - S128P; H133P; C162R; Y194D; K270V; D276V; A308T

[0177] -H133P; C162R; Y194D; G255R; K270V; D276V; A308T

[0178] -H133P; G134E; S155P; C162R; Y194D; G255R; K270V; D276V; A308T

[0179] -H133P; C162R; Y194D; K270V; D276S; A308T

[0180] -Y121N; H133P; C162R; Y194D; K270V; D276S; A308T

[0181] -Y137H; H133P; C162R; Y194D; Y221N; K270V; D276S; A308T

[0182] wherein each of the mutated amino acid positions is an amino acid position in the polypeptide that corresponds to the respective amino acid position of SEQ ID NO: 238.

[0183] In certain embodiments, the polypeptide possesses one or more sets of mutations selected from the group consisting of:

[0184] -N141E

[0185] -D276S; N141E

[0186] -Y194D; N141E

[0187] -S128P; N141E

[0188] -N141E; A308K

[0189] -A308T; N141E

[0190] -Y194D; D276S; N141E

[0191] -D276S; N141E; R249T

[0192] -A308T; D276S; N141E

[0193] -D276S; N141E; C162H

[0194] -A308T; Y194D; D276S; N141E

[0195] -Y194D; D276S; N141E; A308K

[0196] Y194D; S128P; D276S; N141E

[0197] A308T; Y194D; D276S; N141E; R249T

[0198] A308T; Y194D; D276S; N141E; C162H

[0199] C162R; A308T; Y194D; D276S; N141E

[0200] Y194D; D276S; N141E; C162H; A308K

[0201] H133P; N141E; C162R; Y194D; K270V; D276V; A308T

[0202] H133P; N141E; C162R; Y194D; K270V; D276V; A308K

[0203] wherein the amino acid position of each mutation is the amino acid position in the polypeptide that is in a corresponding position to the respective amino acid position of SEQ ID NO: 238.

[0204] In certain embodiments, the polypeptide possesses one or more sets of mutations selected from the group consisting of:

[0205] C162Y

[0206] wherein the amino acid position of each mutation is the amino acid position in the polypeptide that is in a corresponding position to the respective amino acid position of SEQ ID NO: 238.

[0207] In certain embodiments, the polypeptide possesses one or more sets of mutations selected from the group consisting of:

[0208] C162R

[0209] C162R; Y194D

[0210] C162R; R249T

[0211] C162R; Y194D; D276S

[0212] C162R; A308T; Y194D; D276S

[0213] C162R; Y194D; D276S; A308K

[0214] C162R; A308T; Y194D; S128P; D276S

[0215] - H133P; C162R; Y194D; K270V; D276V; A308T

[0216] - H133P; C162R; Y194D; K270V; D276V; A308T

[0217] - H133P; C162R; Y194D; K270V; D276V; A308T

[0218] - H133P; C162R; Y194D; K270V; D276V; A308T

[0219] - H133P; C162R; Y194D; K270V; D276V; A308T

[0220] - H133P; C162R; Y194D; K270V; D276V; A308T

[0221] - H133P; C162R; Y194D; K270V; D276V; A308T

[0222] - H133P; C162R; Y194D; K270V; D276V; A308T

[0223] - H133P; C162R; Y194D; K270V; D276V; A308T

[0224] - H133P; C162R; Y194D; K270V; D276V; A308T

[0225] - H133P; C162R; Y194D; K270V; D276V; A308T

[0226] - H133P; C162R; Y194D; K270V; D276V; A308T

[0227] - H133P; C162R; Y194D; K270V; D276V; A308T

[0228] - H133P; C162R; Y194D; K270V; D276V; A308T

[0229] -H133P; C162R; Y194D; G255R; K270V; D276V; A308T

[0230] -H133P; G134E; S155P; C162R; Y194D; G255R; K270V; D276V; A308T

[0231] -H133P; C162R; Y194D; K270V; D276S; A308T

[0232] -Y121N; H133P; C162R; Y194D; K270V; D276S; A308T

[0233] -Y137H; H133P; C162R; Y194D; Y221N; K270V; D276S; A308T

[0234] -V129G; S130G; H133L; G152S; C162R; R164K; T186S; M191L; Y194Q; Q210R; V218A; A248V; D276V; M285T; N318H

[0235] -V129A; S130G; H133Y; G152S; C162R; M191L; S193R; Y194Q; Q210R; R249T; D276V; M285T

[0236] -Y121N; V129G; S130G; H133Y; G152S; C162R; Y194Q; Q210R; A248V; R249T; D276V; M285T

[0237] -V129G; S130G; H133L; G152S; C162R; T186S; Y190S; M191L; S193R; Y194Q; Q210R; V218A; D276V; M285T

[0238] -V129G; S130G; H133Y; C162R; T186S; Y190N; S193R; V218A; R249T; D276V; M285T; N318H

[0239] -Y121N; V129G; S130G; H133L; C162R; T186S; M191L; S193R; Y194Q; Q210R; R249T; D276V; M285T; N318H

[0240] - Y121N; V129G; S130G; H133Y; G152S; C162R; T186S; Y190N; S193R; Q210R; V218A; A248V; D276V; M285T; N318H

[0241] - Y121N; V129G; H133L; K144M; C162R; T186S; Y190N; M191L; S193R; V218A; R249T; D276V; M285T

[0242] - Y121N; V129G; H133Y; G152S; C162R; T186S; Y190N; M191L; Y194Q; Q210R; K241R; D276V; S311T; N318H

[0243] - Y121N; V129G; H133Y; C162R; T186S; M191L; Y194Q; R249T; D276V; M285T; N318H

[0244] - Y121N; V129G; H133Y; G152S; C162R; T186S; Y190N; S193R; V218A; R249T; D276V; M285T; S311T; N318H

[0245] - Y121N; V129G; H133Y; G152S; C162R; T186S; Y190N; S193R; Y194Q; M213V; V218A; K241R; D276V; M285T; N318H

[0246] - Y121N; V129A; S130G; H133Y; G152S; C162R; T186S; Y190N; S193R; V218A; D276V; S311T

[0247] - Y121N; V129A; S130G; H133Y; G152S; C162R; T186S; Y190N; Y194Q; Q210R; A248V; D276V; M285T; S311T; N318H

[0248] - Y121N; V129G; H133Y; C162R; T186S; Y190N; M191L; S193R; Q210R; M213V; R249T; D276V; M285T

[0249] - Y121N; V129A; S130G; H133Y; G152S; C162R; Y190N; S193R; Q210R; V218A; R249T; D276V; M285T; N318H

[0250] - Y121N; V129G; H133Y; G152S; C162R; Y190N; M191L; Y194Q; A248V; R249T; D276V; M285T

[0251] - Y121N; V129G; H133Y; K144M; G152S; C162R; T186S; Y190N; Y194Q; A248V; D276V; M285T; N318H

[0252] - Y121N; V129A; S130G; H133Y; G152S; C162R; T186S; Y190N; S193R; Y194Q; Q210R; D276V; M285T

[0253] - Y121N; H133Y; C162R; M191L; S193R; Q210R; V218A; R249T; D276V; N318H

[0254] - Y121N; V129A; S130G; H133L; C162R; T186S; Y190N; S193R; Q210R; V218A; R249T; D276V; N318H

[0255] - Y121N; V129G; S130G; H133L; K144M; G152S; C162R; T186S; Y190N; S193R; Y194Q; Q210R; D276V; M285T

[0256] - Y121N; V129G; H133Y; C162R; T186S; Y190N; Y194Q; Q210R; D276V; M285T

[0257] - Y121N; V129A; S130G; H133L; G152S; C162R; T186S; Y190N; M191L; Y194Q; Q210R; A248V; R249T; D276V; M285T; N318H

[0258] - Y121N; V129G; H133Y; G152S; C162R; T186S; Y190N; M191L; S193R; Y194Q; Q210R; V218A; R249T; D276V; M285T; N318H

[0259] - Y121N; V129G; H133Y; G152S; C162R; T186S; Y190N; M191L; S193R; Y194Q; Q210R; V218A; R249T; D276V; M285T; N318H

[0260] - V129G; H133Y; C162R; T186S; Y190N; M191L; S193R; Y194Q; Q210R; A248V; R249T; D276V; M285T; S311T

[0261] wherein each of the mutated amino acid positions is an amino acid position in the polypeptide that is in a corresponding position to the corresponding amino acid position of SEQ ID NO: 238.

[0262] In certain embodiments, the polypeptide possesses one or more sets of mutations selected from the group consisting of:

[0263] - C162H

[0264] - D276S; C162H

[0265] - Y194D; C162H

[0266] - Y194D; D276S; C162H

[0267] - D276S; N141E; C162H

[0268] - A308T; Y194D; D276S; C162H

[0269] - A308T; Y194D; D276S; N141E; C162H

[0270] - Y194D; H133P; D276S; R249T; C162H

[0271] - Y194D; D276S; N141E; C162H; A308K

[0272] - H133P; C162H; Y194D; K270V; D276V; A308T

[0273] wherein each mutated amino acid position is an amino acid position in the polypeptide that is in a corresponding position to a corresponding amino acid position of SEQ ID NO: 238.

[0274] In certain embodiments, the polypeptide possesses one or more sets of mutations selected from the group consisting of:

[0275] - C162T

[0276] wherein each mutated amino acid position is an amino acid position in the polypeptide that is in a corresponding position to a corresponding amino acid position of SEQ ID NO: 238.

[0277] In certain embodiments, the polypeptide possesses one or more sets of mutations selected from the group consisting of:

[0278] - C162K

[0279] - H133P; C162K; Y194D; K270V; D276V; A308T

[0280] wherein each mutated amino acid position is an amino acid position in the polypeptide that is in a corresponding position to a corresponding amino acid position of SEQ ID NO: 238.

[0281] In certain embodiments, the polypeptide possesses one or more sets of mutations selected from the group consisting of:

[0282] - Y194Q

[0283] - V129G; S130G; H133L; G152S; C162R; R164K; T186S; M191L; Y194Q; Q210R; V218A; A248V; D276V; M285T; N318H

[0284] - V129A; S130G; H133Y; G152S; C162R; M191L; S193R; Y194Q; Q210R; R249T; D276V; M285T

[0285] - Y121N; V129G; S130G; H133Y; G152S; C162R; Y194Q; Q210R; A248V; R249T; D276V; M285T

[0286] - V129G; S130G; H133L; G152S; C162R; T186S; Y190S; M191L; S193R; Y194Q; Q210R; V218A; D276V; M285T

[0287] - Y121N; V129G; S130G; H133L; C162R; T186S; M191L; S193R; Y194Q; Q210R; R249T; D276V; M285T; N318H

[0288] - Y121N; V129G; H133Y; G152S; C162R; T186S; Y190N; M191L; Y194Q; Q210R; K241R; D276V; S311T; N318H

[0289] - Y121N; V129G; H133Y; C162R; T186S; M191L; Y194Q; R249T; D276V; M285T; N318H

[0290] - Y121N; V129G; H133Y; G152S; C162R; T186S; Y190N; S193R; Y194Q; M213V; V218A; K241R; D276V; M285T; N318H

[0291] - Y121N; V129A; S130G; H133Y; G152S; C162R; T186S; Y190N; Y194Q; Q210R; A248V; D276V; M285T; S311T; N318H

[0292] - Y121N; V129G; H133Y; G152S; C162R; Y190N; M191L; Y194Q; A248V; R249T; D276V; M285T

[0293] - Y121N; V129G; H133Y; K144M; G152S; C162R; T186S; Y190N; Y194Q; A248V; D276V; M285T; N318H

[0294] - Y121N; V129A; S130G; H133Y; G152S; C162R; T186S; Y190N; S193R; Y194Q; Q210R; D276V; M285T

[0295] - Y121N; V129G; S130G; H133L; K144M; G152S; C162R; T186S; Y190N; S193R; Y194Q; Q210R; D276V; M285T

[0296] - Y121N; V129G; H133Y; C162R; T186S; Y190N; Y194Q; Q210R; D276V; M285T

[0297] - Y121N; V129A; S130G; H133L; G152S; C162R; T186S; Y190N; M191L; Y194Q; Q210R; A248V; R249T; D276V; M285T; N318H

[0298] - Y121N; V129G; Q132H; H133L; G152S; C162R; T186S; M191L; S193R; Y194Q; V218A; R249T; D276V; M285T; N318H

[0299] - Y121N; V129G; H133Y; G152S; C162R; T186S; Y190N; M191L; S193R; Y194Q; Q210R; V218A; R249T; D276V; M285T; N318H

[0300] - V129G; H133Y; C162R; T186S; Y190N; M191L; S193R; Y194Q; Q210R; A248V; R249T; D276V; M285T; S311T

[0301] wherein each of the mutated amino acid positions is an amino acid position in the polypeptide that is in a corresponding position to the corresponding amino acid position of SEQ ID NO: 238.

[0302] In certain embodiments, the polypeptide possesses one or more sets of mutations selected from the group consisting of:

[0303] - Y194D

[0304] - Y194D; D276S

[0305] - Y194D; R249T

[0306] - Y194D; C162H

[0307] - Y194D; N141E

[0308] - Y194D; D276V

[0309] - Y194D; S128P

[0310] - Y194D; A308K

[0311] - Y194D; S155P

[0312] - Y194D; D206G

[0313] - A308T; Y194D

[0314] - C162R; Y194D

[0315] - Y194D; H133P

[0316] - Y194D; R176S

[0317] - Y194D; K270V

[0318] - Y194D; D276S; R249T

[0319] - Y194D; D276S; A308K

[0320] - Y194D; D276S; N141E

[0321] - Y194D; D276S; C162H

[0322] - Y194D; S128P; D276S

[0323] - A308T; Y194D; D276S

[0324] - Y194D; D276S; D206G

[0325] - C162R; Y194D; D276S

[0326] - Y194D; D276S; S155P

[0327] - Y194D; H133P; D276S

[0328] - Y194D; D276S; H269R

[0329] - Y194D; D276S; G134E

[0330] - Y194D; D276S; Y121S

[0331] - Y194D; D276S; H267D

[0332] - Y194D; D276S; K287L

[0333] - Y194D; D276S; K270V

[0334] - A308T; Y194D; R176S

[0335] Y194D; H133P; D276S; K270V

[0336] Y194D; G134E; K270V

[0337] A308T; Y194D; H133P; K270V

[0338] Y194D; H133P; D276S; K270V

[0339] Y194D; H133P; D276S; A308K

[0340] A308T; Y194D; S128P; D276S

[0341] A308T; Y194D; D276S; N141E

[0342] Y194D; H133P; D276S; R249T

[0343] Y194D; D276S; H267T; A308K

[0344] A308T; Y194D; D276S; D206G

[0345] Y194D; S128P; D276S; A308K

[0346] Y194D; D276S; N141E; A308K

[0347] A308T; Y194D; D276S; R249T

[0348] Y194D; S128P; D276S; N141E

[0349] Y194D; D276S; D206G; A308K

[0350] A308T; Y194D; D276S; C162H

[0351] C162R; A308T; Y194D; D276S

[0352] Y194D; D276S; G134Q; A308K

[0353] C162R; Y194D; D276S; A308K

[0354] Y194D; H133P; D276S; K270V; A308K

[0355] A308T; Y194D; D276S; N141 E; R249T

[0356] C162R; A308T; Y194D; S128P; D276S

[0357] Y194D; S128P; D276S; D206G; A308K

[0358] Y194D; H133P; M285E; K270V; R176S

[0359] A308T; Y194D; D276S; N141 E; C162H

[0360] C162R; A308T; Y194D; D276S; N141 E

[0361] Y194D; H133P; D276S; R249T; C162H

[0362] Y194D; D276S; N141 E; C162H; A308K

[0363] H133P; C162R; Y194D; K270V; D276V; A308T

[0364] Y121 N; H133P; C162R; Y194D; K270V; D276V; A308T

[0365] V129G; H133P; C162R; Y194D; K270V; D276V; A308T

[0366] V129G; H133P; Y137H; C162R; Y194D; Y221 N; K270V; D276V; A308T

[0367] H133P; Y137H; C162R; Y194D; Y221 N; K270V; D276S; A308T

[0368] H133P; N141 E; C162R; Y194D; K270V; D276V; A308T

[0369] H133P; N141 E; C162R; Y194D; K270V; D276V; A308K

[0370] H133P; Y137H; C162R; Y194D; Y221 N; K270V; D276V; A308T

[0371] - H133P; C162R; Y194D; K270V; D276V; A308T

[0372] - H133P; C162K; Y194D; K270V; D276V; A308T

[0373] - H133P; C162H; Y194D; K270V; D276V; A308T

[0374] - H133P; G134E; C162R; Y194D; K270V; D276V; A308T

[0375] - H133P; S155P; C162R; Y194D; K270V; D276V; A308T

[0376] - H133P; C162R; Y194D; R249T; K270V; D276V; A308T

[0377] - S128P; H133P; C162R; Y194D; K270V; D276V; A308T

[0378] - H133P; C162R; Y194D; G255R; K270V; D276V; A308T

[0379] - H133P; G134E; S155P; C162R; Y194D; G255R; K270V; D276V; A308T

[0380] - H133P; C162R; Y194D; K270V; D276S; A308T

[0381] - Y121N; H133P; C162R; Y194D; K270V; D276S; A308T

[0382] - Y137H; H133P; C162R; Y194D; Y221N; K270V; D276S; A308T

[0383] wherein each of the mutated amino acid positions is an amino acid position in the polypeptide that is in a corresponding position to the corresponding amino acid position of SEQ ID NO: 238.

[0384] In certain embodiments, the polypeptide possesses one or more sets of mutations selected from the group consisting of:

[0385] - R249V

[0386] wherein each mutated amino acid position is an amino acid position in the polypeptide that is in a corresponding position to the corresponding amino acid position of SEQ ID NO: 238.

[0387] In certain embodiments, the polypeptide possesses one or more sets of mutations selected from the group consisting of:

[0388] - R249T

[0389] - D276S; R249T

[0390] - Y194D; R249T

[0391] - C162R; R249T

[0392] - A308T; R249T

[0393] - Y194D; D276S; R249T

[0394] - D276S; N141E; R249T

[0395] - Y194D; H133P; D276S; R249T

[0396] - A308T; Y194D; D276S; R249T

[0397] - A308T; Y194D; D276S; N141E; R249T

[0398] - Y194D; H133P; D276S; R249T; C162H

[0399] - H133P; C162R; Y194D; R249T; K270V; D276V; A308T

[0400] - V129A; S130G; H133Y; G152S; C162R; M191L; S193R; Y194Q; Q210R; R249T; D276V; M285T

[0401] - Y121N; V129G; S130G; H133Y; G152S; C162R; Y194Q; Q210R; A248V; R249T; D276V; M285T

[0402] - V129G; S130G; H133Y; C162R; T186S; Y190N; S193R; V218A; R249T; D276V; M285T; N318H

[0403] - Y121N; V129G; S130G; H133L; C162R; T186S; M191L; S193R; Y194Q; Q210R; R249T; D276V; M285T; N318H

[0404] - Y121N; V129G; H133L; K144M; C162R; T186S; Y190N; M191L; S193R; V218A; R249T; D276V; M285T

[0405] - Y121N; V129G; H133Y; C162R; T186S; M191L; Y194Q; R249T; D276V; M285T; N318H

[0406] - Y121N; V129G; H133Y; G152S; C162R; T186S; Y190N; S193R; V218A; R249T; D276V; M285T; S311T; N318H

[0407] - Y121N; V129G; H133Y; C162R; T186S; Y190N; M191L; S193R; Q210R; M213V; R249T; D276V; M285T

[0408] - Y121N; V129A; S130G; H133Y; G152S; C162R; Y190N; S193R; Q210R; V218A; R249T; D276V; M285T; N318H

[0409] - Y121N; V129G; H133Y; G152S; C162R; Y190N; M191L; Y194Q; A248V; R249T; D276V; M285T

[0410] - Y121N; H133Y; C162R; M191L; S193R; Q210R; V218A; R249T; D276V; N318H

[0411] - Y121N; V129A; S130G; H133L; C162R; T186S; Y190N; S193R; Q210R; V218A; R249T; D276V; N318H

[0412] - Y121N; V129G; H133Y; G152S; C162R; T186S; Y190N; M191L; S193R; Y194Q; Q210R; V218A; R249T; D276V; M285T; N318H

[0413] - Y121N; V129G; H133Y; G152S; C162R; T186S; Y190N; M191L; S193R; Y194Q; Q210R; V218A; R249T; D276V; M285T; N318H

[0414] - Y121N; V129G; H133Y; G152S; C162R; T186S; Y190N; M191L; S193R; Y194Q; Q210R; V218A; R249T; D276V; M285T; N318H

[0415] - V129G; H133Y; C162R; T186S; Y190N; M191L; S193R; Y194Q; Q210R; A248V; R249T; D276V; M285T; S311T

[0416] wherein each mutated amino acid position is an amino acid position in the polypeptide that is in a corresponding position to the corresponding amino acid position of SEQ ID NO: 238.

[0417] In certain embodiments, the polypeptide possesses one or more sets of mutations selected from the group consisting of:

[0418] - K270S

[0419] wherein each mutated amino acid position is an amino acid position in the polypeptide that is in a corresponding position to the corresponding amino acid position of SEQ ID NO: 238.

[0420] In certain embodiments, the polypeptide possesses one or more sets of mutations selected from the group consisting of:

[0421] - K270V

[0422] - Y194D; K270V

[0423] - K270V; A308K

[0424] - Y194D; D276S; K270V

[0425] - Y194D; H133P; K270V

[0426] - Y194D; G134E; K270V

[0427] - S128P; K270V; A308K

[0428] - H133P; K270V; R176S

[0429] - H133P; M285E; K270V

[0430] - A308T; Y194D; H133P; K270V

[0431] - Y194D; H133P; D276S; K270V

[0432] - A308T; H133P; G134E; K270V

[0433] - A308T; G134E; K270V; R176S

[0434] - M285T; G134E; K270V; R176S

[0435] - Y194D; H133P; D276S; K270V; A308K

[0436] - S128P; D276S; K270V; A308K; S264G

[0437] - Y194D; H133P; M285E; K270V; R176S

[0438] - H133P; C162R; Y194D; K270V; D276V; A308T

[0439] - Y121N; H133P; C162R; Y194D; K270V; D276V; A308T

[0440] - V129G; H133P; C162R; Y194D; K270V; D276V; A308T

[0441] - V129G; H133P; Y137H; C162R; Y194D; Y221N; K270V; D276V; A308T

[0442] - H133P; Y137H; C162R; Y194D; Y221N; K270V; D276S; A308T

[0443] - H133P; N141E; C162R; Y194D; K270V; D276V; A308T

[0444] -H133P; N141E; C162R; Y194D; K270V; D276V; A308K

[0445] -H133P; Y137H; C162R; Y194D; Y221N; K270V; D276V; A308T

[0446] -H133P; C162R; Y194D; K270V; D276V; A308K

[0447] -H133P; C162K; Y194D; K270V; D276V; A308T

[0448] -H133P; C162H; Y194D; K270V; D276V; A308T

[0449] -H133P; G134E; C162R; Y194D; K270V; D276V; A308T

[0450] -H133P; S155P; C162R; Y194D; K270V; D276V; A308T

[0451] -H133P; C162R; Y194D; R249T; K270V; D276V; A308T

[0452] -S128P; H133P; C162R; Y194D; K270V; D276V; A308T

[0453] -H133P; C162R; Y194D; G255R; K270V; D276V; A308T

[0454] -H133P; G134E; S155P; C162R; Y194D; G255R; K270V; D276V; A308T

[0455] -H133P; C162R; Y194D; K270V; D276S; A308T

[0456] -Y121N; H133P; C162R; Y194D; K270V; D276S; A308T

[0457] -Y137H; H133P; C162R; Y194D; Y221N; K270V; D276S; A308T

[0458] wherein each mutated amino acid position is an amino acid position in the polypeptide that is in a corresponding position to a corresponding amino acid position of SEQ ID NO: 238.

[0459] In certain embodiments, the polypeptide possesses one or more sets of mutations selected from the group consisting of:

[0460] -D276R

[0461] wherein each mutated amino acid position is an amino acid position in the polypeptide that is in a corresponding position to a corresponding amino acid position of SEQ ID NO: 238.

[0462] In certain embodiments, the polypeptide possesses one or more sets of mutations selected from the group consisting of:

[0463] -D276S

[0464] -Y194D; D276S

[0465] -D276S; N141E

[0466] -D276S; R249T

[0467] -D276S; D206G

[0468] -S128P; D276S

[0469] -D276S; A308K

[0470] -D276S; S155P

[0471] -A308T; D276S

[0472] -D276S; C162H

[0473] -H133P; D276S

[0474] -D276S; H269R

[0475] -D276S; R176S

[0476] -D276S; G134E

[0477] -D276S; Y121S

[0478] -D276S; M285T

[0479] -Y194D; D276S; R249T

[0480] -Y194D; D276S; A308K

[0481] Y194D; D276S; N141E

[0482] Y194D; D276S; C162H

[0483] Y194D; S128P; D276S

[0484] A308T; Y194D; D276S

[0485] Y194D; D276S; D206G

[0486] C162R; Y194D; D276S

[0487] Y194D; D276S; S155P

[0488] Y194D; H133P; D276S

[0489] Y194D; D276S; H269R

[0490] Y194D; D276S; G134E

[0491] Y194D; D276S; Y121S

[0492] Y194D; D276S; H267D

[0493] D276S; N141E; R249T

[0494] Y194D; D276S; K287L

[0495] A308T; D276S; N141E

[0496] Y194D; D276S; K270V

[0497] S128P; D276S; A308K

[0498] D276S; N141E; C162H

[0499] Y194D; H133P; D276S; K270V

[0500] Y194D; H133P; D276S; A308K

[0501] A308T; Y194D; S128P; D276S

[0502] A308T; Y194D; D276S; N141E

[0503] Y194D; H133P; D276S; R249T

[0504] Y194D; D276S; H267T; A308K

[0505] A308T; Y194D; D276S; D206G

[0506] Y194D; S128P; D276S; A308K

[0507] Y194D; D276S; N141E; A308K

[0508] A308T; Y194D; D276S; R249T

[0509] Y194D; S128P; D276S; N141E

[0510] Y194D; D276S; D206G; A308K

[0511] A308T; Y194D; D276S; C162H

[0512] C162R; A308T; Y194D; D276S

[0513] Y194D; D276S; G134Q; A308K

[0514] C162R; Y194D; D276S; A308K

[0515] Y194D; H133P; D276S; K270V; A308K

[0516] S128P; D276S; K270V; A308K; S264G

[0517] A308T; Y194D; D276S; N141E; R249T

[0518] C162R; A308T; Y194D; S128P; D276S

[0519] Y194D; S128P; D276S; D206G; A308K

[0520] A308T; Y194D; D276S; N141E; C162H

[0521] C162R; A308T; Y194D; D276S; N141E

[0522] Y194D; H133P; D276S; R249T; C162H

[0523] Y194D; D276S; N141E; C162H; A308K

[0524] H133P; Y137H; C162R; Y194D; Y221N; K270V; D276S; A308T

[0525] H133P; C162R; Y194D; K270V; D276S; A308T

[0526] Y121N; H133P; C162R; Y194D; K270V; D276S; A308T

[0527] Y137H; H133P; C162R; Y194D; Y221N; K270V; D276S; A308T

[0528] wherein each of the mutated amino acid positions is an amino acid position in the polypeptide that is in a corresponding position to the respective amino acid position of SEQ ID NO: 238.

[0529] In certain embodiments, the polypeptide possesses one or more sets of mutations selected from the group consisting of:

[0530] D276V

[0531] Y194D; D276V

[0532] H133P; C162R; Y194D; K270V; D276V; A308T

[0533] Y121N; H133P; C162R; Y194D; K270V; D276V; A308T

[0534] V129G; H133P; C162R; Y194D; K270V; D276V; A308T

[0535] V129G; H133P; Y137H; C162R; Y194D; Y221N; K270V; D276V; A308T

[0536] H133P; N141E; C162R; Y194D; K270V; D276V; A308T

[0537] H133P; N141E; C162R; Y194D; K270V; D276V; A308K

[0538] - H133P; Y137H; C162R; Y194D; Y221N; K270V; D276V; A308T

[0539] - H133P; C162R; Y194D; K270V; D276V; A308K

[0540] - H133P; C162K; Y194D; K270V; D276V; A308T

[0541] - H133P; C162H; Y194D; K270V; D276V; A308T

[0542] - H133P; G134E; C162R; Y194D; K270V; D276V; A308T

[0543] - H133P; S155P; C162R; Y194D; K270V; D276V; A308T

[0544] - H133P; C162R; Y194D; R249T; K270V; D276V; A308T

[0545] - S128P; H133P; C162R; Y194D; K270V; D276V; A308T

[0546] - H133P; C162R; Y194D; G255R; K270V; D276V; A308T

[0547] - H133P; G134E; S155P; C162R; Y194D; G255R; K270V; D276V; A308T

[0548] - V129G; S130G; H133L; G152S; C162R; R164K; T186S; M191L; Y194Q; Q210R; V218A; A248V; D276V; M285T; N318H

[0549] - V129A; S130G; H133Y; G152S; C162R; M191L; S193R; Y194Q; Q210R; R249T; D276V; M285T

[0550] - Y121N; V129G; S130G; H133Y; G152S; C162R; Y194Q; Q210R; A248V; R249T; D276V; M285T

[0551] - V129G; S130G; H133L; G152S; C162R; T186S; Y190S; M191L; S193R; Y194Q; Q210R; V218A; D276V; M285T

[0552] - V129G; S130G; H133Y; C162R; T186S; Y190N; S193R; V218A; R249T; D276V; M285T; N318H

[0553] - Y121N; V129G; S130G; H133L; C162R; T186S; M191L; S193R; Y194Q; Q210R; R249T; D276V; M285T; N318H

[0554] - Y121N; V129G; S130G; H133Y; G152S; C162R; T186S; Y190N; S193R; Q210R; V218A; A248V; D276V; M285T; N318H

[0555] - Y121N; V129G; H133L; K144M; C162R; T186S; Y190N; M191L; S193R; V218A; R249T; D276V; M285T

[0556] - Y121N; V129G; H133Y; G152S; C162R; T186S; Y190N; M191L; Y194Q; Q210R; K241R; D276V; S311T; N318H

[0557] - Y121N; V129G; H133Y; C162R; T186S; M191L; Y194Q; R249T; D276V; M285T; N318H

[0558] - Y121N; V129G; H133Y; G152S; C162R; T186S; Y190N; S193R; V218A; R249T; D276V; M285T; S311T; N318H

[0559] - Y121N; V129G; H133Y; G152S; C162R; T186S; Y190N; S193R; Y194Q; M213V; V218A; K241R; D276V; M285T; N318H

[0560] - Y121N; V129A; S130G; H133Y; G152S; C162R; T186S; Y190N; S193R; Q210R; V218A; R249T; D276V; M285T; N318H

[0561] - Y121N; V129G; H133Y; G152S; C162R; Y190N; M191L; Y194Q; A248V; R249T; D276V; M285T

[0562] - Y121N; V129G; H133Y; G152S; C162R; Y190N; M191L; Y194Q; A248V; R249T; D276V; M285T

[0563] - Y121N; V129G; H133Y; G152S; C162R; Y190N; M191L; Y194Q; A248V; R249T; D276V; M285T

[0564] - Y121N; V129G; H133Y; G152S; C162R; Y190N; M191L; Y194Q; A248V; R249T; D276V; M285T

[0565] - Y121N; V129G; H133Y; G152S; C162R; Y190N; M191L; Y194Q; A248V; R249T; D276V; M285T

[0566] - Y121N; V129G; H133Y; G152S; C162R; Y190N; M191L; Y194Q; A248V; R249T; D276V; M285T

[0567] - Y121N; V129G; H133Y; G152S; C162R; Y190N; M191L; Y194Q; A248V; R249T; D276V; M285T

[0568] - Y121N; V129G; H133Y; G152S; C162R; Y190N; M191L; Y194Q; A248V; R249T; D276V; M285T

[0569] - Y121N; V129G; S130G; H133L; K144M; G152S; C162R; T186S; Y190N; S193R; Y194Q; Q210R; D276V; M285T

[0570] - Y121N; V129G; H133Y; C162R; T186S; Y190N; Y194Q; Q210R; D276V; M285T

[0571] - Y121N; V129A; S130G; H133L; G152S; C162R; T186S; Y190N; M191L; Y194Q; Q210R; A248V; R249T; D276V; M285T; N318H

[0572] - Y121N; V129G; Q132H; H133L; G152S; C162R; T186S; M191L; S193R; Y194Q; V218A; R249T; D276V; M285T; N318H

[0573] - Y121N; V129G; H133Y; G152S; C162R; T186S; Y190N; M191L; S193R; Y194Q; Q210R; V218A; R249T; D276V; M285T; N318H

[0574] - V129G; H133Y; C162R; T186S; Y190N; M191L; S193R; Y194Q; Q210R; A248V; R249T; D276V; M285T; S311T

[0575] wherein each of the mutated amino acid positions is an amino acid position in the polypeptide that is in a corresponding position to the corresponding amino acid position of SEQ ID NO: 238.

[0576] In certain embodiments, the polypeptide possesses one or more sets of mutations selected from the group consisting of:

[0577] - A308T

[0578] - A308T; D276S

[0579] - A308T; Y194D

[0580] - A308T; N141E

[0581] - A308T; R249T

[0582] - A308T; Y194D; D276S

[0583] A308T; D276S; N141E

[0584] A308T; Y194D; R176S

[0585] A308T; Y194D; H133P; K270V

[0586] A308T; Y194D; S128P; D276S

[0587] A308T; Y194D; D276S; N141E

[0588] A308T; Y194D; D276S; D206G

[0589] A308T; Y194D; D276S; R249T

[0590] A308T; H133P; G134E; K270V

[0591] A308T; Y194D; D276S; C162H

[0592] C162R; A308T; Y194D; D276S

[0593] A308T; G134E; K270V; R176S

[0594] A308T; Y194D; D276S; N141E; R249T

[0595] C162R; A308T; Y194D; S128P; D276S

[0596] A308T; Y194D; D276S; N141E; C162H

[0597] C162R; A308T; Y194D; D276S; N141E

[0598] H133P; C162R; Y194D; K270V; D276V; A308T

[0599] Y121N; H133P; C162R; Y194D; K270V; D276V; A308T

[0600] V129G; H133P; C162R; Y194D; K270V; D276V; A308T

[0601] V129G; H133P; Y137H; C162R; Y194D; Y221N; K270V; D276V; A308T

[0602] H133P; Y137H; C162R; Y194D; Y221N; K270V; D276S; A308T

[0603] H133P; N141E; C162R; Y194D; K270V; D276V; A308T

[0604] H133P; Y137H; C162R; Y194D; Y221N; K270V; D276V; A308T

[0605] H133P; C162R; Y194D; K270V; D276V; A308K

[0606] H133P; C162K; Y194D; K270V; D276V; A308T

[0607] H133P; C162H; Y194D; K270V; D276V; A308T

[0608] H133P; G134E; C162R; Y194D; K270V; D276V; A308T

[0609] H133P; S155P; C162R; Y194D; K270V; D276V; A308T

[0610] H133P; C162R; Y194D; R249T; K270V; D276V; A308T

[0611] S128P; H133P; C162R; Y194D; K270V; D276V; A308T

[0612] H133P; C162R; Y194D; G255R; K270V; D276V; A308T

[0613] H133P; G134E; S155P; C162R; Y194D; G255R; K270V; D276V; A308T

[0614] H133P; C162R; Y194D; K270V; D276S; A308T

[0615] Y194D; D276S; A308K

[0616] Y194D; D276S; A308K

[0617] wherein each of the mutated amino acid positions is an amino acid position in the polypeptide that is in a corresponding position to the respective amino acid position of SEQ ID NO: 238.

[0618] In certain embodiments, the polypeptide possesses one or more sets of mutations selected from the group consisting of:

[0619] A308K

[0620] D276S; A308K

[0621] Y194D; A308K

[0622] N141E; A308K

[0623] K270V; A308K

[0624] Y194D; D276S; A308K

[0625] S128P; D276S; A308K

[0626] S128P; K270V; A308K

[0627] Y194D; H133P; D276S; A308K

[0628] Y194D; D276S; H267T; A308K

[0629] Y194D; S128P; D276S; A308K

[0630] Y194D; D276S; N141E; A308K

[0631] Y194D; D276S; D206G; A308K

[0632] Y194D; D276S; G134Q; A308K

[0633] C162R; Y194D; D276S; A308K

[0634] Y194D; H133P; D276S; K270V; A308K

[0635] - Y194D; D276S; K270V; A308K; S264G

[0636] - Y194D; D276S; N141E; C162H; A308K

[0637] - Y194D; D276S; N141E; C162H; A308K

[0638] - H133P; N141E; C162R; Y194D; K270V; D276V; A308K

[0639] - H133P; C162R; Y194D; K270V; D276V; A308K

[0640] wherein each of the mutated amino acid positions is an amino acid position in the polypeptide that is in a corresponding position to the corresponding amino acid position of SEQ ID NO: 238.

[0641] In certain embodiments, the second peptide segment is directly linked or optionally linked via a peptide linker to the N-terminus or C-terminus (e.g., C-terminus) of the first peptide segment.

[0642] In certain embodiments, the second peptide segment is directly linked (e.g., not linked via a peptide linker) to the N-terminus or C-terminus (e.g., C-terminus) of the first peptide segment.

[0643] In certain embodiments, the immunoglobulin-like domain 2 and the immunoglobulin-like domain 3 are derived from the same or different VEGFR2.

[0644] In certain embodiments, the immunoglobulin-like domain 2 and the immunoglobulin-like domain 3 are derived from the same species of VEGFR2 (e.g., the immunoglobulin-like domain 2 and the immunoglobulin-like domain 3 are derived from the same or different human VEGFR2).

[0645] In certain embodiments, the immunoglobulin-like domain 2 and the immunoglobulin-like domain 3 are derived from the same VEGFR2 (e.g., human VEGFR2).

[0646] In certain embodiments, the polypeptide further comprises a signal peptide and / or a tag peptide (e.g., a purification tag peptide, a detection tag peptide).

[0647] In certain embodiments, the polypeptide comprises the signal peptide and / or the tag peptide at its N-terminus and / or C-terminus (e.g., N-terminus).

[0648] In certain embodiments, the polypeptide consists of the first peptide segment and the second peptide segment.

[0649] In certain embodiments, the polypeptide is not naturally occurring.

[0650] In certain embodiments, the polypeptide possesses binding activity to human VEGF-A, VEGF-C and / or VEGF-D.

[0651] In certain embodiments, the polypeptide possesses binding activity to human VEGF-A, VEGF-C and VEGF-D.

[0652] In certain embodiments, the polypeptide possesses cross-binding activity to human, rabbit VEGF-A, VEGF-C and / or VEGF-D.

[0653] In certain embodiments, the polypeptide is capable of antagonizing the binding of VEGF-A, VEGF-C and / or VEGF-D to its receptor. In certain embodiments, the polypeptide is capable of simultaneously antagonizing the binding of VEGF-A, VEGF-C and VEGF-D to their receptors.

[0654] In certain embodiments, the polypeptide is capable of inhibiting or blocking the activation of the signaling pathway mediated by VEGF-A, VEGF-C and / or VEGF-D. In certain embodiments, the polypeptide is capable of simultaneously inhibiting or blocking the activation of the signaling pathway mediated by VEGF-A, VEGF-C and VEGF-D.

[0655] In certain embodiments, the polypeptide comprises an amino acid sequence as set forth in positions 1-208 of any one of SEQ ID NOs: 2-29, 31-108, 110-228.

[0656] In certain embodiments, in the polypeptide construct, the polypeptide is covalently linked to the Fc peptide and the optional hinge peptide (e.g., covalently linked by covalent bonds comprising peptide bonds, isopeptide bonds and / or disulfide bonds).

[0657] In certain embodiments, the polypeptide construct comprises the polypeptide, the Fc peptide and the hinge peptide, and, in the polypeptide construct, the polypeptide is covalently linked to the Fc peptide and the hinge peptide by covalent bonds comprising peptide bonds.

[0658] In certain embodiments, the polypeptide construct comprises a first peptide chain, the first peptide chain comprising: a first polypeptide, a first hinge peptide, and a first Fc peptide, wherein the first polypeptide is selected from the polypeptides as described above. In certain embodiments, the first peptide chain comprises, in order from N-terminus to C-terminus: the first polypeptide, the first hinge peptide, and the first Fc peptide.

[0659] In certain embodiments, the polypeptide construct further comprises a second peptide chain comprising a second hinge peptide and a second Fc peptide. In certain embodiments, the second peptide chain further comprises a second polypeptide capable of binding VEGF (e.g., VEGF-A, VEGF-C, and / or VEGF-D). In certain embodiments, the second polypeptide is selected from the polypeptides described above.

[0660] In certain embodiments, the second peptide chain comprises, in order from N-terminus to C-terminus: the second polypeptide, the second hinge peptide, and the second Fc peptide.

[0661] In certain embodiments, the polypeptide construct possesses one or more features selected from the group consisting of:

[0662] (i) the first polypeptide is the same as or different from the second polypeptide;

[0663] (ii) the first hinge peptide and the second hinge peptide are derived from the same or different immunoglobulin;

[0664] (iii) the first Fc peptide and the second Fc peptide are derived from the same or different immunoglobulin;

[0665] (iv) the first hinge peptide and the second hinge peptide are the same or different;

[0666] (v) the first Fc peptide and the second Fc peptide are the same or different;

[0667] (vi) the first peptide chain is the same as or different from the second peptide chain;

[0668] (vii) the first peptide chain and the second peptide chain are connected by disulfide bond formation.

[0669] In certain embodiments, the hinge peptide (e.g., the first hinge peptide and / or the second hinge peptide) is a hinge peptide derived from an immunoglobulin (e.g., IgGl, IgG2, IgG3, or IgG4).

[0670] In certain embodiments, the hinge peptide (e.g., the first hinge peptide and / or the second hinge peptide) is a hinge peptide derived from a human immunoglobulin (e.g., IgGl, IgG2, IgG3, or IgG4).

[0671] In certain embodiments, the hinge peptide (e.g., the first hinge peptide and / or the second hinge peptide) is a hinge peptide derived from human IgGl.

[0672] As will be readily understood by those skilled in the art, the Fc peptide derived from an immunoglobulin (e.g., a human immunoglobulin) includes both a Fc peptide directly derived from an immunoglobulin (e.g., a human immunoglobulin) and a Fc peptide obtained by engineering or modifying a Fc peptide derived from an immunoglobulin (e.g., a human immunoglobulin) (e.g., a Fc peptide variant).

[0673] In certain embodiments, the Fc peptide (e.g., the first Fc peptide and / or the second Fc peptide) has (i) an amino acid sequence as set forth in SEQ ID NO: 233, 241, or 242, or (ii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to (i), or (iii) a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions or additions) compared to (i).

[0674] In certain embodiments, the Fc peptide (e.g., the first Fc peptide and / or the second Fc peptide) is a Fc peptide derived from an immunoglobulin (e.g., IgG1, IgG2, IgG3, or IgG4).

[0675] In certain embodiments, the Fc peptide (e.g., the first Fc peptide and / or the second Fc peptide) is a Fc peptide derived from a human immunoglobulin (e.g., IgG1, IgG2, IgG3, or IgG4).

[0676] In certain embodiments, the Fc peptide (e.g., the first Fc peptide and / or the second Fc peptide) is a Fc peptide derived from a human immunoglobulin IgG1.

[0677] As will be readily understood by those skilled in the art, the Fc peptide derived from an immunoglobulin (e.g., a human immunoglobulin) includes both a Fc peptide directly derived from an immunoglobulin (e.g., a human immunoglobulin) and a Fc peptide obtained by engineering or modifying a Fc peptide derived from an immunoglobulin (e.g., a human immunoglobulin) (e.g., a Fc peptide variant).

[0678] In certain embodiments, the Fc peptide (e.g., the first Fc peptide and / or the second Fc peptide) comprises (i) an amino acid sequence as set forth in any one of SEQ ID NOs: 236, 239-240, or (ii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to (i), or (iii) a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to (i).

[0679] In certain embodiments, the polypeptide construct comprises: (i) an amino acid sequence as set forth in any one of SEQ ID NOs: 2-29, 31-108, 110-228 or (ii) an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100% sequence identity compared to the amino acid sequence as set forth in any one of SEQ ID NOs: 2-29, 31-108, 110-228.

[0680] In certain embodiments, the polypeptide construct comprises the first peptide chain and, optionally, the second peptide chain; wherein the first peptide chain comprises: (i) an amino acid sequence as set forth in any one of SEQ ID NOs: 2-29, 31-108, 110-228 or (ii) an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100% sequence identity compared to the amino acid sequence as set forth in any one of SEQ ID NOs: 2-29, 31-108, 110-228, and / or the second peptide chain comprises: (i) an amino acid sequence as set forth in any one of SEQ ID NOs: 2-29, 31-108, 110-228 or (ii) an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100% sequence identity compared to the amino acid sequence as set forth in any one of SEQ ID NOs: 2-29, 31-108, 110-228.

[0681] In certain embodiments, the polypeptide construct further comprises a signal peptide and / or a tag peptide (e.g., a purification tag peptide, a detection tag peptide).

[0682] In certain embodiments, the polypeptide construct comprises the signal peptide and / or the tag peptide at the N-terminus and / or the C-terminus of the polypeptide construct.

[0683] In certain embodiments, the polypeptide construct comprises the first peptide chain and optionally the second peptide chain; and the first peptide chain and / or the second peptide chain comprises the signal peptide and / or the tag peptide at the N-terminus and / or the C-terminus of the first peptide chain and / or the second peptide chain.

[0684] In certain embodiments, the polypeptide construct has good thermal stability (e.g., better than or comparable to that of T002 (a construct with an amino acid sequence as set forth in SEQ ID NO: 1)).

[0685] In certain embodiments, the polypeptide construct has good hydrophobicity (e.g., better than or comparable to that of T002 (a construct with an amino acid sequence as set forth in SEQ ID NO: 1)).

[0686] In certain embodiments, the polypeptide construct has good protein yield (e.g., better than or comparable to that of T002 (a construct with an amino acid sequence as set forth in SEQ ID NO: 1)).

[0687] In certain embodiments, the polypeptide construct has good protein yield (e.g., better than or comparable to that of T002 (a construct with an amino acid sequence as set forth in SEQ ID NO: 1)) when expressed in a eukaryotic cell (e.g., a CHO cell line, such as a CHO-S cell line).

[0688] In certain embodiments, the polypeptide construct has good protein purity (e.g., better than or comparable to that of T002 (a construct with an amino acid sequence as set forth in SEQ ID NO: 1)).

[0689] In certain embodiments, the polypeptide construct has binding activity to human VEGF-A, VEGF-C and / or VEGF-D.

[0690] In certain embodiments, the polypeptide construct has binding activity to human VEGF-A, VEGF-C and VEGF-D.

[0691] In certain embodiments, the polypeptide construct has cross-binding activity to human, rabbit VEGF-A, VEGF-C and / or VEGF-D.

[0692] In certain embodiments, the polypeptide construct is capable of antagonizing the binding of VEGF-A, VEGF-C, and / or VEGF-D to their receptors. In certain embodiments, the polypeptide construct is capable of simultaneously antagonizing the binding of VEGF-A, VEGF-C, and VEGF-D to their receptors.

[0693] In certain embodiments, the polypeptide construct is capable of inhibiting or blocking the activation of a signaling pathway mediated by VEGF-A, VEGF-C, and / or VEGF-D. In certain embodiments, the polypeptide construct is capable of simultaneously inhibiting or blocking the activation of a signaling pathway mediated by VEGF-A, VEGF-C, and VEGF-D.

[0694] Based on the disclosure provided herein, one of ordinary skill in the art will readily understand that the fusion proteins provided herein are used in their broadest sense and are intended to include any bi- or multi-specific molecule comprising a VEGF-binding polypeptide construct of the present application and a single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2.

[0695] In one aspect, the fusion proteins of the present application are not limited in the number of peptide chains. For example, as described in detail herein, the VEGF-binding polypeptide constructs provided herein can comprise only one molecular chain (e.g., peptide chain), or two or more molecular chains (e.g., peptide chains). Based on the disclosure provided herein, one of ordinary skill in the art will readily understand that the fusion proteins of the present application comprising a VEGF-binding polypeptide construct of the present application can have the same number of molecular chains (e.g., peptide chains) as the VEGF-binding polypeptide constructs of the present application, which can consist of one or more (e.g., two) molecular chains (e.g., peptide chains).

[0696] In another aspect, the fusion proteins of the present application are not limited in the number of VEGF-binding polypeptides and single-domain antibodies or antigen-binding fragments thereof that specifically bind to Ang-2 that they comprise. For example, the fusion proteins of the present application can comprise one or more VEGF-binding polypeptides and one or more single-domain antibodies or antigen-binding fragments thereof that specifically bind to Ang-2.

[0697] In another aspect, the fusion proteins of the present application are not limited in the manner of fusion of the single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2 with the polypeptide construct that binds to VEGF. In certain embodiments, the single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2 can be fused to the N-terminus and / or C-terminus of the polypeptide construct that binds to VEGF. In certain embodiments, the single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2 can be fused to the interior of the polypeptide construct that binds to VEGF; for example, the single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2 can be fused to the polypeptide that binds to VEGF and the Fc peptide of the polypeptide construct that binds to VEGF; for example, the single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2 can be fused to the polypeptide that binds to VEGF and the hinge peptide of the polypeptide construct that binds to VEGF.

[0698] In certain embodiments, the fusion proteins comprise one or more of the polypeptide constructs, and one or more of the single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2.

[0699] In certain embodiments, the fusion proteins comprise one or more of the polypeptides of the polypeptide constructs, and one or more of the single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2.

[0700] In certain embodiments, the polypeptide construct and the single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2 are covalently linked via a covalent bond comprising a peptide bond (e.g., covalently linked via a covalent bond comprising a peptide bond, an isopeptide bond, and / or a disulfide bond).

[0701] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2 is located at the N-terminus and / or C-terminus (e.g., C-terminus) of the polypeptide construct. In certain embodiments, the polypeptide construct is directly linked or optionally linked via a peptide linker to the single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2.

[0702] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2 is located between the polypeptide and the Fc peptide of the polypeptide construct.

[0703] In certain embodiments, when the polypeptide construct does not comprise a hinge peptide, the single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2 is located between the polypeptide and the Fc peptide of the polypeptide construct. In certain embodiments, the polypeptide and the single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2 are directly linked or optionally linked by a peptide linker, and the single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2 and the Fc peptide are directly linked or optionally linked by a peptide linker.

[0704] In certain embodiments, when the polypeptide construct does not comprise a hinge peptide, the single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2 is located between the polypeptide and the Fc peptide of the polypeptide construct. In certain embodiments, the polypeptide and the single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2 are directly linked or optionally linked by a peptide linker, and the single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2 and the Fc peptide are directly linked or optionally linked by a peptide linker.

[0705] In certain embodiments, the fusion protein comprises a first peptide chain comprising: a first polypeptide, a first hinge peptide, a first Fc peptide, and a first single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2, wherein the first polypeptide is selected from the polypeptides above. In certain embodiments, the first peptide chain of the fusion protein comprises, in order from N-terminus to C-terminus: (i) the first polypeptide, first hinge peptide, first Fc peptide, and first single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2; (ii) first single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2, the first polypeptide, first hinge peptide, and first Fc peptide; or (iii) the first polypeptide, first single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2, first hinge peptide, and first Fc peptide.

[0706] In certain embodiments, the fusion protein further comprises a second peptide chain comprising: (i) a second polypeptide, a second hinge peptide, a second Fc peptide, and a second single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2, wherein the second polypeptide is selected from the polypeptides above. In certain embodiments, the second peptide chain of the fusion protein comprises, in order from N-terminus to C-terminus: (i) the second polypeptide, second hinge peptide, second Fc peptide, and second single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2; (ii) second single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2, the second polypeptide, second hinge peptide, and second Fc peptide; or (iii) the second polypeptide, second single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2, second hinge peptide, and second Fc peptide.

[0707] In certain embodiments, the fusion protein comprises a first peptide chain and optionally a second peptide chain, wherein the first peptide chain comprises, in order from N-terminus to C-terminus: the first polypeptide, a first hinge peptide, a first Fc peptide, and a first Ang-2 binding single domain antibody or antigen binding fragment thereof; and / or the second peptide chain comprises, in order from N-terminus to C-terminus: the second polypeptide, a second hinge peptide, a second Fc peptide, and a second Ang-2 binding single domain antibody or antigen binding fragment thereof.

[0708] In certain embodiments, the fusion protein comprises a first peptide chain and optionally a second peptide chain, wherein the first peptide chain comprises, in order from N-terminus to C-terminus: a first Ang-2 binding single domain antibody or antigen binding fragment thereof, the first polypeptide, a first hinge peptide, and a first Fc peptide; and / or the second peptide chain comprises, in order from N-terminus to C-terminus: a second Ang-2 binding single domain antibody or antigen binding fragment thereof, the second polypeptide, a second hinge peptide, and a second Fc peptide.

[0709] In certain embodiments, the fusion protein comprises a first peptide chain and optionally a second peptide chain, wherein the first peptide chain comprises, in order from N-terminus to C-terminus: the first polypeptide, a first Ang-2 binding single domain antibody or antigen binding fragment thereof, a first hinge peptide, and a first Fc peptide; and / or the second peptide chain comprises, in order from N-terminus to C-terminus: the second polypeptide, a second Ang-2 binding single domain antibody or antigen binding fragment thereof, a second hinge peptide, and a second Fc peptide.

[0710] In certain embodiments, the adjacent domains of the first peptide chain and / or the second peptide chain of the fusion protein are each optionally linked by a linker (e.g., a peptide linker) or directly linked.

[0711] In certain embodiments, when the first or second Ang-2 binding single domain antibody or antigen binding fragment thereof is fused to the first or second Fc peptide, the first or second polypeptide, or the first or second hinge peptide, the fusion can additionally be through one or more peptide linkers.

[0712] In certain embodiments, the fusion protein comprises a first peptide chain comprising: the first peptide chain of the polypeptide construct and a first single-domain antibody or antigen binding fragment thereof that specifically binds to Ang-2. In certain embodiments, the first peptide chain of the fusion protein comprises, in order from N-terminus to C-terminus: the first peptide chain of the polypeptide construct and a first single-domain antibody or antigen binding fragment thereof that specifically binds to Ang-2. In certain embodiments, the first peptide chain of the fusion protein comprises, in order from N-terminus to C-terminus: a first single-domain antibody or antigen binding fragment thereof that specifically binds to Ang-2 and the first peptide chain of the polypeptide construct.

[0713] In certain embodiments, the fusion protein further comprises a second peptide chain comprising: the second peptide chain of the polypeptide construct and a second single-domain antibody or antigen binding fragment thereof that specifically binds to Ang-2. In certain embodiments, the second peptide chain of the fusion protein comprises, in order from N-terminus to C-terminus: the second peptide chain of the polypeptide construct and a second single-domain antibody or antigen binding fragment thereof that specifically binds to Ang-2. In certain embodiments, the second peptide chain of the fusion protein comprises, in order from N-terminus to C-terminus: a second single-domain antibody or antigen binding fragment thereof that specifically binds to Ang-2 and the second peptide chain of the polypeptide construct.

[0714] In certain embodiments, the fusion protein comprises a first peptide chain comprising: the first peptide chain of the polypeptide construct, a first peptide linker and a first single-domain antibody or antigen binding fragment thereof that specifically binds to Ang-2. In certain embodiments, the first peptide chain of the fusion protein comprises, in order from N-terminus to C-terminus: the first peptide chain of the polypeptide construct, a first peptide linker and a first single-domain antibody or antigen binding fragment thereof that specifically binds to Ang-2. In certain embodiments, the first peptide chain of the fusion protein comprises, in order from N-terminus to C-terminus: a first single-domain antibody or antigen binding fragment thereof that specifically binds to Ang-2, a first peptide linker and the first peptide chain of the polypeptide construct.

[0715] In certain embodiments, the fusion protein further comprises a second peptide chain comprising: the second peptide chain of the polypeptide construct, a second peptide linker and a second single-domain antibody or antigen binding fragment thereof that specifically binds to Ang-2. In certain embodiments, the second peptide chain of the fusion protein comprises, in order from N-terminus to C-terminus: the second peptide chain of the polypeptide construct, a second peptide linker and a second single-domain antibody or antigen binding fragment thereof that specifically binds to Ang-2. In certain embodiments, the second peptide chain of the fusion protein comprises, in order from N-terminus to C-terminus: a second single-domain antibody or antigen binding fragment thereof that specifically binds to Ang-2, a second peptide linker and the second peptide chain of the polypeptide construct.

[0716] In certain embodiments, the fusion protein comprises a first peptide chain comprising: a first polypeptide, a first hinge peptide, a first Fc peptide, and a first single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2, wherein the first polypeptide is selected from the polypeptides described above. In certain embodiments, the first peptide chain of the fusion protein comprises, in order from N-terminus to C-terminus: the first polypeptide, a first hinge peptide, a first Fc peptide, and a first single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2. In certain embodiments, the first peptide chain of the fusion protein comprises, in order from N-terminus to C-terminus: a first single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2, the first polypeptide, a first hinge peptide, and a first Fc peptide. In certain embodiments, the first peptide chain of the fusion protein comprises, in order from N-terminus to C-terminus: the first polypeptide, a first single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2, a first hinge peptide, and a first Fc peptide.

[0717] In certain embodiments, the fusion protein further comprises a second peptide chain comprising: a second polypeptide, a second hinge peptide, a second Fc peptide, and a second single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2, wherein the second polypeptide is selected from the polypeptides described above. In certain embodiments, the second peptide chain of the fusion protein comprises, in order from N-terminus to C-terminus: the second polypeptide, a second hinge peptide, a second Fc peptide, and a second single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2. In certain embodiments, the second peptide chain of the fusion protein comprises, in order from N-terminus to C-terminus: a second single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2, the second polypeptide, a second hinge peptide, and a second Fc peptide. In certain embodiments, the second peptide chain of the fusion protein comprises, in order from N-terminus to C-terminus: the second polypeptide, a second single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2, a second hinge peptide, and a second Fc peptide.

[0718] In certain embodiments, the fusion protein comprises a first peptide chain comprising: a first polypeptide, a first hinge peptide, a first Fc peptide, a first peptide linker, and a first single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2, wherein the first polypeptide is selected from the polypeptides described above. In certain embodiments, the first peptide chain of the fusion protein comprises, in order from N-terminus to C-terminus: the first polypeptide, a first hinge peptide, a first Fc peptide, a first peptide linker, and a first single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2. In certain embodiments, the first peptide chain of the fusion protein comprises, in order from N-terminus to C-terminus: a first single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2, a first peptide linker, the first polypeptide, a first hinge peptide, and a first Fc peptide. In certain embodiments, the first peptide chain of the fusion protein comprises, in order from N-terminus to C-terminus: the first polypeptide, a first peptide linker, a first single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2, a first hinge peptide, and a first Fc peptide. In certain embodiments, the first peptide chain of the fusion protein comprises, in order from N-terminus to C-terminus: the first polypeptide, a first single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2, a first peptide linker, a first hinge peptide, and a first Fc peptide.

[0719] In certain embodiments, the fusion protein further comprises a second peptide chain comprising: a second polypeptide, a second hinge peptide, a second Fc peptide, a second peptide linker, and a second single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2, wherein the second polypeptide is selected from the polypeptides described above. In certain embodiments, the second peptide chain of the fusion protein comprises, in order from N-terminus to C-terminus: the second polypeptide, a second hinge peptide, a second Fc peptide, a second peptide linker, and a second single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2. In certain embodiments, the second peptide chain of the fusion protein comprises, in order from N-terminus to C-terminus: a second single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2, a second peptide linker, the second polypeptide, a second hinge peptide, and a second Fc peptide. In certain embodiments, the second peptide chain of the fusion protein comprises, in order from N-terminus to C-terminus: the second polypeptide, a second peptide linker, a second single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2, a second hinge peptide, and a second Fc peptide. In certain embodiments, the second peptide chain of the fusion protein comprises, in order from N-terminus to C-terminus: the second polypeptide, a second single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2, a second peptide linker, a second hinge peptide, and a second Fc peptide.

[0720] In certain embodiments, the fusion protein comprises a first peptide chain comprising: a first polypeptide, a first hinge peptide, a first Fc peptide, a first peptide linker, a second peptide linker, and a first Ang-2 binding nanobody or antigen binding fragment thereof, wherein the first polypeptide is selected from the polypeptides described above. In certain embodiments, the first peptide chain of the fusion protein comprises, in order from N-terminus to C-terminus: the first polypeptide, the first peptide linker, the first Ang-2 binding nanobody or antigen binding fragment thereof, the second peptide linker, the first hinge peptide, and the first Fc peptide.

[0721] In certain embodiments, the fusion protein further comprises a second peptide chain comprising: a second polypeptide, a second hinge peptide, a second Fc peptide, a third peptide linker, a fourth peptide linker, and a second Ang-2 binding nanobody or antigen binding fragment thereof, wherein the second polypeptide is selected from the polypeptides described above. In certain embodiments, the fusion protein comprises, in order from N-terminus to C-terminus: the second polypeptide, the third peptide linker, the second Ang-2 binding nanobody or antigen binding fragment thereof, the fourth peptide linker, the second hinge peptide, and the second Fc peptide.

[0722] In certain embodiments, the fusion protein has one or more of the following features:

[0723] (i) the first polypeptide is the same as or different from the second polypeptide;

[0724] (ii) the first hinge peptide and the second hinge peptide are derived from the same or different immunoglobulin;

[0725] (iii) the first Fc peptide and the second Fc peptide are derived from the same or different immunoglobulin;

[0726] (iv) the first hinge peptide is the same as or different from the second hinge peptide;

[0727] (v) the first Fc peptide is the same as or different from the second Fc peptide;

[0728] (vi) the first peptide chain is the same as or different from the second peptide chain;

[0729] (vii) the first peptide chain and the second peptide chain are connected by disulfide bond formation between them;

[0730] (viii) the first Ang-2 binding nanobody or antigen binding fragment thereof and the second Ang-2 binding nanobody or antigen binding fragment thereof are the same or different;

[0731] (ix) the first peptide linker, the second peptide linker, the third peptide linker, and the fourth peptide linker are the same or different.

[0732] In some embodiments, the hinge peptide (e.g., the first hinge peptide and / or the second hinge peptide) is a hinge peptide derived from an immunoglobulin (e.g., IgGl, IgG2, IgG3, or IgG4).

[0733] In some embodiments, the hinge peptide (e.g., the first hinge peptide and / or the second hinge peptide) is a hinge peptide derived from a human immunoglobulin (e.g., IgGl, IgG2, IgG3, or IgG4).

[0734] In some embodiments, the hinge peptide (e.g., the first hinge peptide and / or the second hinge peptide) is a hinge peptide derived from human IgGl.

[0735] As those skilled in the art will readily appreciate, the hinge peptide derived from an immunoglobulin (e.g., a human immunoglobulin) includes both a hinge peptide directly derived from an immunoglobulin (e.g., a human immunoglobulin) and a hinge peptide obtained by engineering or modifying a hinge peptide derived from an immunoglobulin (e.g., a human immunoglobulin) (e.g., an artificially designed hinge peptide).

[0736] In some embodiments, the hinge peptide (e.g., the first hinge peptide and / or the second hinge peptide) has (i) an amino acid sequence as set forth in SEQ ID NO: 233, 241, or 242, or (ii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to (i), or (iii) a sequence having one or several amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) compared to (i).

[0737] In some embodiments, the Fc peptide (e.g., the first Fc peptide and / or the second Fc peptide) is a Fc peptide derived from an immunoglobulin (e.g., IgGl, IgG2, IgG3, or IgG4).

[0738] In some embodiments, the Fc peptide (e.g., the first Fc peptide and / or the second Fc peptide) is a Fc peptide derived from a human immunoglobulin (e.g., IgGl, IgG2, IgG3, or IgG4).

[0739] In some embodiments, the Fc peptide (e.g., the first Fc peptide and / or the second Fc peptide) is a Fc peptide derived from human immunoglobulin IgGl.

[0740] As will be readily understood by those skilled in the art, the Fc peptide derived from an immunoglobulin (e.g., human immunoglobulin) includes both the Fc peptide directly derived from an immunoglobulin (e.g., human immunoglobulin) and the Fc peptide (e.g., Fc peptide variant) obtained by modification or alteration of the Fc peptide derived from an immunoglobulin (e.g., human immunoglobulin).

[0741] In certain embodiments, the Fc peptide (e.g., the first Fc peptide and / or the second Fc peptide) comprises (i) an amino acid sequence as set forth in any one of SEQ ID NOs: 236, 239-240, or (ii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to (i), or (iii) a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to (i).

[0742] In certain embodiments, the peptide linker (e.g., the first peptide linker, the second peptide linker, the third peptide linker, the fourth peptide linker) is selected from a peptide linker comprising one or more glycine (G) and / or serine (S), e.g., a flexible peptide comprising Gn, (G4S)n, or (G5S)n, n being an integer not less than 0, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In certain embodiments, the peptide linker (e.g., the first peptide linker, the second peptide linker, the third peptide linker, the fourth peptide linker) is selected from a peptide linker comprising one or more glycine (G) and / or serine (S), e.g., a flexible peptide comprising (G4S)n or (G5S)n, n being an integer not less than 0, e.g., 1, 2, 3 or 4. In certain embodiments, the peptide linker (e.g., the first peptide linker, the second peptide linker, the third peptide linker, the fourth peptide linker) has a sequence as set forth in any one of SEQ ID NOs: 252-254.

[0743] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2 (e.g., the first single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2 and / or the second single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2) comprises a CDR1, a CDR2, and a CDR3 comprised in a VHH as set forth in (i) SEQ ID NO: 248 or 256; or (ii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to any one of the CDRs in (i), or (iii) a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions or additions) compared to any one of the CDRs in (i). In certain embodiments, the CDRs are according to the Rabat definition.

[0744] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2 (e.g., the first single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2 and / or the second single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2) comprises: (i) a CDR1 as set forth in SEQ ID NO: 249; a CDR2 as set forth in SEQ ID NO: 250; and, a CDR3 as set forth in SEQ ID NO: 251 or 257; or (ii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to any one of the CDRs in (i), or (iii) a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions or additions) compared to any one of the CDRs in (i).

[0745] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2 (e.g., the first single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2 and / or the second single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2) comprises a heavy chain framework region derived from a human immunoglobulin (e.g., a heavy chain framework region comprised in an amino acid sequence encoded by a human heavy chain germline antibody gene);

[0746] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2 (e.g., the first single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2 and / or the second single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2) comprises: (i) a sequence as set forth in SEQ ID NO: 248 or 256, or (ii) an amino acid sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100% sequence identity compared to the amino acid sequence set forth in SEQ ID NO: 248 or 256, or (iii) a sequence with one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to (i).

[0747] In certain embodiments, the fusion protein comprises: (i) a sequence as set forth in any one of SEQ ID NOs: 243-247, 255, or (ii) an amino acid sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100% sequence identity compared to the amino acid sequence set forth in any one of SEQ ID NOs: 243-247, 255, or (iii) a sequence with one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to (i).

[0748] In certain embodiments, the fusion protein comprises the first peptide chain and, optionally, the second peptide chain; wherein the first peptide chain comprises: (i) an amino acid sequence as set forth in any one of SEQ ID NOs: 243-247, 255 or (ii) an amino acid sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100% sequence identity compared to the amino acid sequence set forth in any one of SEQ ID NOs: 243-247, 255, and / or the second peptide chain comprises: (i) an amino acid sequence as set forth in any one of SEQ ID NOs: 243-247, 255 or (ii) an amino acid sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100% sequence identity compared to the amino acid sequence set forth in any one of SEQ ID NOs: 243-247, 255.

[0749] In some embodiments, the fusion protein comprises a first peptide chain and optionally a second peptide chain, wherein the first peptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 243, and / or the second peptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 243.

[0750] In some embodiments, the fusion protein comprises a first peptide chain and optionally a second peptide chain, wherein the first peptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 244, and / or the second peptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 244.

[0751] In some embodiments, the fusion protein comprises a first peptide chain and optionally a second peptide chain, wherein the first peptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 245, and / or the second peptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 245.

[0752] In some embodiments, the fusion protein comprises a first peptide chain and optionally a second peptide chain, wherein the first peptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 246, and / or the second peptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 246.

[0753] In some embodiments, the fusion protein comprises a first peptide chain and optionally a second peptide chain, wherein the first peptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 247, and / or the second peptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 247.

[0754] In some embodiments, the fusion protein comprises a first peptide chain and optionally a second peptide chain, wherein the first peptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 255, and / or the second peptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 255.

[0755] In a second aspect, the present application provides an isolated nucleic acid molecule encoding the fusion protein of the first aspect.

[0756] In some embodiments, the isolated nucleic acid molecule encodes the fusion protein of the first aspect, the fusion protein comprising a first polypeptide and a second polypeptide; the isolated nucleic acid molecule comprising a first nucleotide sequence encoding the first polypeptide and a second nucleotide sequence encoding the second polypeptide.

[0757] In some embodiments, the first nucleotide sequence, the second nucleotide sequence are the same or different from each other.

[0758] In certain embodiments, the first nucleotide sequence, the second nucleotide sequence are present on the same or different isolated nucleic acid molecules.

[0759] In a third aspect, the present application provides a vector comprising the isolated nucleic acid molecule of the second aspect. In certain embodiments, the vector is a cloning vector or an expression vector.

[0760] In certain embodiments, the vector encodes the fusion protein of the first aspect, the fusion protein comprising a first polypeptide and a second polypeptide; the vector comprising a first nucleotide sequence encoding the first polypeptide and a second nucleotide sequence encoding the second polypeptide.

[0761] In certain embodiments, the first nucleotide sequence, the second nucleotide sequence are the same or different from each other.

[0762] In certain embodiments, the first nucleotide sequence, the second nucleotide sequence are present on the same or different vector molecules.

[0763] In a fourth aspect, the present application provides a host cell comprising the isolated nucleic acid molecule of the second aspect or the vector of the third aspect.

[0764] Such host cells include, but are not limited to, prokaryotic cells such as bacterial cells (e.g., E. coli cells), and eukaryotic cells such as fungal cells (e.g., yeast cells), insect cells, plant cells and animal cells (e.g., mammalian cells, such as mouse cells, human cells, etc.). In certain embodiments, the host cell is a microorganism. In certain embodiments, the host cell is a eukaryotic cell (e.g., a CHO cell line, such as a CHO-S cell line).

[0765] The fusion protein of the present application can be prepared in various methods known in the art, for example, by genetic engineering recombination techniques. For example, a DNA molecule encoding the fusion protein of the present application is obtained by chemical synthesis or PCR amplification. The resulting DNA molecule is inserted into an expression vector, which is then transfected into a host cell. Then, the transfected host cell is cultured under specific conditions, and the fusion protein of the present application is expressed.

[0766] In a fifth aspect, the present application provides a method of preparing the fusion protein of the first aspect, comprising culturing the host cell of the fourth aspect under conditions permitting expression of the fusion protein, and recovering the fusion protein from the culture of the host cell.

[0767] In a sixth aspect, the present application provides a conjugate comprising the fusion protein of the first aspect, and a therapeutic agent linked to the fusion protein.

[0768] In certain embodiments, the therapeutic agent is selected from a VEGF antagonist (e.g., a VEGF-A antagonist, a VEGF-C antagonist, a VEGF-D antagonist, an antagonist that simultaneously antagonizes VEGF-A, VEGF-C, and VEGF-D).

[0769] In a seventh aspect, the present application provides a pharmaceutical composition comprising the fusion protein of the first aspect, the isolated nucleic acid molecule of the second aspect, the vector of the third aspect, the host cell of the fourth aspect, or the conjugate of the sixth aspect, and a pharmaceutically acceptable carrier and / or excipient.

[0770] In certain embodiments, the pharmaceutical composition further comprises an additional pharmaceutically active agent.

[0771] In certain embodiments, the additional pharmaceutically active agent is selected from a VEGF antagonist (e.g., a VEGF-A antagonist, a VEGF-C antagonist, a VEGF-D antagonist, an antagonist that simultaneously antagonizes VEGF-A, VEGF-C, and VEGF-D).

[0772] In an eighth aspect, the present application provides use of the fusion protein of the first aspect, the isolated nucleic acid molecule of the second aspect, the vector of the third aspect, the host cell of the fourth aspect, the conjugate of the sixth aspect, or the pharmaceutical composition of the seventh aspect for the manufacture of a medicament for preventing and / or treating a disease associated with VEGF (e.g., VEGF-A, VEGF-C, and / or VEGF-D) signaling in a subject.

[0773] In certain embodiments, the prevention and / or treatment of the disease associated with VEGF (e.g., VEGF-A, VEGF-C, and / or VEGF-D) signaling would benefit from antagonism of VEGF signaling.

[0774] In certain embodiments, the disease associated with VEGF (e.g., VEGF-A, VEGF-C, and / or VEGF-D) signaling is selected from a disease associated with abnormal angiogenesis.

[0775] In certain embodiments, the disease associated with VEGF (e.g., VEGF-A, VEGF-C, and / or VEGF-D) signaling is selected from the group consisting of: a vascular- derived ocular disease (e.g., macular degeneration, macular edema (e.g., wet age-related macular degeneration, macular edema following retinal vein occlusion, diabetic macular edema), retinal vein occlusion (e.g., central retinal vein occlusion, branch retinal vein occlusion), choroidal neovascular glaucoma, iridal neovascular glaucoma, neovascular glaucoma, post-surgical fibrosis of glaucoma, proliferative vitreoretinopathy, optic disc neovascularization, corneal neovascularization, retinal neovascularization, vitreous neovascularization, pannus, pterygium, chorioretinopathy, retinopathy of prematurity, vascular retinopathy, diabetic retinopathy), a cancer (e.g., lung cancer, kidney cancer, uterine cancer, prostate cancer, bladder cancer, ovarian cancer, colon cancer, breast cancer, leukemia, lymphoma, myeloma, sarcoma, glioma, pancreatic cancer), an autoimmune disease (e.g., psoriasis, rheumatoid arthritis, psoriatic arthritis), and any combination thereof.

[0776] In certain embodiments, the subject is a mammal, e.g., a human or a rabbit.

[0777] In certain embodiments, the fusion protein, the isolated nucleic acid molecule, the vector, the host cell, the conjugate, or the pharmaceutical composition is used alone or in combination with (e.g., concurrently or sequentially) another pharmaceutically active agent.

[0778] In certain embodiments, the additional pharmaceutically active agent is selected from the group consisting of a VEGF antagonist (e.g., a VEGF-A antagonist, a VEGF-C antagonist, a VEGF-D antagonist, an antagonist that simultaneously antagonizes VEGF-A, VEGF-C, and VEGF-D).

[0779] In a ninth aspect, the present application provides a method for preventing and / or treating a disease associated with VEGF (e.g., VEGF-A, VEGF-C, and / or VEGF-D) signaling in a subject, comprising: administering to a subject in need thereof an effective amount of the fusion protein of the first aspect, the isolated nucleic acid molecule of the second aspect, the vector of the third aspect, the host cell of the fourth aspect, the conjugate of the sixth aspect, or the pharmaceutical composition of the seventh aspect.

[0780] In certain embodiments, the prevention and / or treatment of the disease associated with VEGF (e.g., VEGF-A, VEGF-C, and / or VEGF-D) signaling would benefit from antagonism of VEGF signaling.

[0781] In certain embodiments, the disease associated with VEGF (e.g., VEGF-A, VEGF-C, and / or VEGF-D) signaling is selected from the group consisting of diseases associated with abnormal angiogenesis.

[0782] In certain embodiments, the disease associated with VEGF (e.g., VEGF-A, VEGF-C, and / or VEGF-D) signaling is selected from the group consisting of: vascular- derived ocular diseases (e.g., macular degeneration, macular edema (e.g., wet age-related macular degeneration, macular edema following retinal vein occlusion, diabetic macular edema), retinal vein occlusion (e.g., central retinal vein occlusion, branch retinal vein occlusion), choroidal neovascular glaucoma, iridal neovascular glaucoma, neovascular glaucoma, post-surgical fibrosis of glaucoma, proliferative vitreoretinopathy, optic disc neovascularization, corneal neovascularization, retinal neovascularization, vitreous neovascularization, pannus, pterygium, chorioretinopathy, retinopathy of prematurity, vascular retinopathy, diabetic retinopathy), cancer (e.g., lung cancer, kidney cancer, uterine cancer, prostate cancer, bladder cancer, ovarian cancer, colon cancer, breast cancer, leukemia, lymphoma, myeloma, sarcoma, glioma, pancreatic cancer), autoimmune diseases (e.g., psoriasis, rheumatoid arthritis, psoriatic arthritis), and any combination thereof.

[0783] In certain embodiments, the subject is a mammal, e.g., a human or a rabbit.

[0784] In certain embodiments, the fusion protein, isolated nucleic acid molecule, vector, host cell, conjugate, or pharmaceutical composition is used alone or in combination with (e.g., concurrently or sequentially) another pharmaceutically active agent.

[0785] In certain embodiments, the additional pharmaceutically active agent is selected from the group consisting of VEGF antagonists (e.g., VEGF-A antagonists, VEGF-C antagonists, VEGF-D antagonists, antagonists that simultaneously antagonize VEGF-A, VEGF-C, and VEGF-D).

[0786] The fusion protein or conjugate or pharmaceutical composition of the present application can be formulated into any dosage form known in the medical arts, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, sterile powders for injection, and concentrated solutions for injection), inhalants, sprays, and the like. The preferred dosage form depends on the intended mode of administration and therapeutic use. The fusion protein or conjugate or pharmaceutical composition of the present application should be sterile and stable under the conditions of manufacture and storage. A preferred dosage form is an injection. Such injections can be sterile injection solutions. For example, sterile injection solutions can be prepared by incorporating the fusion protein or conjugate or pharmaceutical composition of the present application in the required amount in an appropriate solvent with one or more of the other ingredients, as described below, as desired, followed by filtered sterilization. Furthermore, sterile solutions can be prepared by vacuum or freeze drying (lyophilization), which yields a powder of the fusion protein or conjugate or pharmaceutical composition of the present application plus any additional desired ingredient(s), followed by reconstitution into a solution or dispersion for convenient storage and administration. Such lyophilized powders can be dispensed on a carrier, for example, water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% NaCl), dextrose solution (e.g., 5% dextrose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffered solution (e.g., phosphate buffered saline), Ringer's solution, and any combination thereof.

[0787] The fusion protein or conjugate or pharmaceutical composition of the present application can be administered by any suitable method known in the art, including, but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, endocytic reticulum, inguinal, intravesical, local (e.g., powder, salve, or drops), or nasal routes. However, for many therapeutic uses, the preferred route of administration is parenteral administration (e.g., intravenous injection or bolus, subcutaneous injection, intraperitoneal injection, intramuscular injection). The skilled artisan will appreciate that the route and / or mode of administration will vary depending upon the desired goal. In certain embodiments, the fusion protein or conjugate or pharmaceutical composition of the present application is administered by intravenous injection or bolus.

[0788] Definitions of Terms

[0789] In the present application, the scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art, unless otherwise indicated. Also, the laboratory procedures of virology, biochemistry, immunology used herein are conventional procedures well known in the art. In addition, the following definitions and explanations of terms are provided for better understanding of the present application.

[0790] When the terms "for example", "for instance", "such as", "including", "containing", or variations thereof, are used herein, these terms are not to be interpreted in an exclusive or exhaustive sense, i.e., there can be additional items or components that are neither recited nor implied by the foregoing terms.

[0791] The terms "a" and "an" and "the" and similar referents are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context, in describing the invention (particularly in the context of the following claims).

[0792] As used herein, the term "VEGFR2" (vascular endothelial cell growth factor receptor 2), also known as VEGFR-2, Flk-1, KDR, is a receptor protein expressed on vascular endothelial cells, which plays an important role in processes such as angiogenesis, which mainly activates a series of signal pathways by binding with vascular endothelial cell growth factors (such as VEGF-A), thereby promoting the proliferation, migration, survival and vascularization of vascular endothelial cells. An exemplary amino acid sequence of VEGFR2 can be obtained from public databases (e.g., GenBank database, UniProt database).

[0793] Those skilled in the art understand that VEGFR2 can include VEGFR2 of various different species origins, and there can be differences between the amino acid sequences of VEGFR2 of different species origins. In the present application, when VEGFR2 is described, it not only includes VEGFR2 as shown in SEQ ID NO: 238, but also includes VEGFR2 of various species origins whose amino acid sequences are different from SEQ ID NO: 238.

[0794] As understood by one of skill in the art, VEGFR2 can comprise a plurality of immunoglobulin-like domains (or Ig-like domains), for example, naturally occurring VEGFR2 typically comprises seven immunoglobulin-like domains, designated D1, D2, D3, D4, D5, D6, D7, or R2D1, R2D2, R2D3, R2D4, R2D5, R2D6, R2D7, respectively, which domains play important roles in the functioning of VEGFR2. Descriptions of the various domains of VEGF receptor proteins can be found in US6100071A, which is incorporated by reference herein in its entirety. In certain embodiments, the immunoglobulin-like domain 2 of VEGFR2 (R2D2) comprises or consists of the amino acid residues in VEGFR2 corresponding to positions 130-205 (or 130-207, or 130-210, or 130-215, or 125-205, or 125-207, or 125-210, or 125-215, or 120-205, or 120-207, or 120-210, or 120-215) of SEQ ID NO: 238. In certain embodiments, the immunoglobulin-like domain 3 of VEGFR2 (R2D3) comprises or consists of the amino acid residues in VEGFR2 corresponding to positions 216-320 (or 216-327, or 216-335, or 211-320, or 211-327, or 211-335, or 208-320, or 208-327, or 208-335, or 206-320, or 206-327, or 206-335) of SEQ ID NO: 238.

[0795] In the present context, the term "polypeptide construct" is used in its broadest sense. Generally, the polypeptide construct is generally intended to mean a construct comprising one or more polypeptide or protein components, wherein the one or more polypeptide or protein components can each independently have different origins or different biological activities or functions, and are linked by covalent and / or non-covalent means (e.g., covalent linkage by covalent bonds comprising peptide bonds, isopeptide bonds and / or disulfide bonds, and / or non-covalent linkage by hydrogen bonds). The polypeptide construct of the present application is not limited in the number of molecular chains (e.g., peptide chains) it comprises, for example, the polypeptide construct of the present application can comprise only one molecular chain (e.g., peptide chain), or two or more molecular chains (e.g., peptide chains) covalently and / or non-covalently linked (e.g., covalent linkage by covalent bonds comprising peptide bonds, isopeptide bonds and / or disulfide bonds, and / or non-covalent linkage by hydrogen bonds) between the two or more molecular chains (e.g., peptide chains). Likewise, it is readily understood by those skilled in the art that in embodiments comprising multiple polypeptide or protein components, the multiple polypeptide or protein components comprised by the polypeptide construct of the present application can be located in whole or in part in the same molecular chain (e.g., peptide chain), or each in a different molecular chain (e.g., peptide chain).

[0796] As used herein, the term "single-domain antibody" (sdAb) has the meaning generally understood by those skilled in the art, and refers to an antibody fragment composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region, VHH), typically derived from the variable region of a heavy chain antibody (e.g., a camelid antibody or a shark antibody). Single-domain antibodies are also known as nanobodies, and the two terms are used interchangeably. Typically, a nanobody consists of 4 framework regions and 3 complementarity determining regions, with the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Nanobodies can be truncated at the N- or C-terminus to include only part of FR1 and / or FR4, or to lack one or both of those framework regions, as long as antigen binding and specificity are substantially maintained.

[0797] As used herein, the term "antigen-binding fragment" of a single-domain antibody refers to a polypeptide comprising a fragment of a single-domain antibody that retains the ability to bind specifically to the same antigen to which the single-domain antibody binds, and / or competes with the single-domain antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion". See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nded. Raven Press, N.Y. (1989)), which is incorporated by reference in its entirety for all purposes. Antigen-binding fragments of the nanobodies of the application can be generated by recombinant DNA techniques or by enzymatic or chemical cleavage of the nanobodies of the application. In some embodiments, the "antigen-binding fragment" of the single-domain antibody can be truncated at the N- or C-terminus compared to the full-length single-domain antibody to include only part of FR1 and / or FR4, or lack one or both of those framework regions, as long as it essentially retains antigen-binding and specificity.

[0798] Antigen-binding fragments of single-domain antibodies can be obtained from a given single-domain antibody, such as the nanobodies provided herein, using conventional techniques known to those of skill in the art, such as recombinant DNA techniques or enzymatic or chemical cleavage, and screened for specificity in the same manner as for the intact nanobodies.

[0799] Herein, unless the context clearly indicates otherwise, when referring to the term "single-domain antibody", this includes not only intact single-domain antibodies, but also antigen-binding fragments of single-domain antibodies.

[0800] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. There are three CDRs in a nanobody, designated CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991) and / or the Chothia-Conway numbering system (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)), which are incorporated by reference in their entirety for all purposes. thEd. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given Nanobody, one of skill in the art will readily identify the CDRs defined by each numbering system. Moreover, the correspondence between the different numbering systems is well known to one of skill in the art (see, e.g., Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). In the present application, the CDRs contained by the single-domain antibody or antigen-binding fragment thereof of the application can be determined according to the various numbering systems known in the art. In certain embodiments, the CDRs contained by the single-domain antibody or antigen-binding fragment thereof of the application are preferably determined by the Kabat, Chothia, or IMGT numbering system.

[0801] As used herein, the term "framework region" or "FR" residues refer to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.

[0802] As used herein, the term "Fc peptide", "Fc", "Fc segment", "Fc fragment", or "Fc region" means an antibody fragment formed by the second constant region (CH2), the third constant region (CH3) of an immunoglobulin heavy chain. The Fc fragment of an antibody has a variety of different functions, but is not involved in the binding of antigens. For example, the Fc peptide can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system.

[0803] As used herein, the term "hinge peptide" or "hinge region" means the hinge peptide of an immunoglobulin heavy chain, which is located at the C-terminus of the CH1 region of an immunoglobulin heavy chain for connecting the CH2 region of an immunoglobulin heavy chain. The hinge peptide usually contains cysteines, which can be used to form disulfide bonds between two heavy chains containing the hinge peptide of an immunoglobulin to mediate dimerization between two Fc peptides contained by the two heavy chains.

[0804] As used herein, the term "identity" is used in reference to the match between sequences of two polypeptides or two nucleic acids. To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity = # of identical positions / total # of positions x 100%). In certain embodiments, the two sequences are the same length.

[0805] Determination of percent identity between two sequences can also be accomplished using a mathematical algorithm. One non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5877. Such an algorithm is incorporated in the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403.

[0806] As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When the vector is capable of directing the expression of a polynucleotide inserted into it, the vector is referred to as an expression vector. A vector can be introduced into a host cell by transformation, transduction or transfection, and directs the expression of elements of genetic material it carries in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1 -derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papova viruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector can contain a replication origin.

[0807] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to, prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0808] The writing of the twenty conventional amino acids referred to herein follows the conventional usage. See, e.g., Immunology - A Synthesis (2 nd Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present application, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also in the present application, amino acids are generally represented by the one-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

[0809] Unless otherwise indicated herein or contradicted by context, "A, B, and / or C" or similar expressions shall be understood as "A, B, C, or any combination thereof," e.g., as any one selected from A, B, C, A and B, A and C, B and C, A and B and C.

[0810] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is compatible, in pharmacological and / or physiological terms, with the subject and active ingredient, and is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19 thed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to, pH adjusting agents, surfactants, adjuvants, ionic strength enhancers, diluents, agents to maintain osmotic pressure, agents to retard absorption, preservatives, stabilizers. For example, pH adjusting agents include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic or non-ionic surfactants, such as Tween-80. Adjuvants include, but are not limited to, inorganic adjuvants (such as aluminum hydroxide, vanadium), biological adjuvants (such as Mycobacterium tuberculosis, BCG, Corynebacterium parvum, Bordetella pertussis, Gram-negative bacterial endotoxins, B subunit of cholera toxin, muramyl dipeptide, cytokines), synthetic adjuvants (such as double-stranded polynucleotides, uridylic acid), oil adjuvants (such as Freund's complete adjuvant, peanut oil emulsion), and nano-adjuvants, etc. Ionic strength enhancers include, but are not limited to, sodium chloride. Agents to maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents to retard absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol), etc. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thiomersal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning commonly understood by those skilled in the art as capable of stabilizing the desired activity of the active ingredients in the pharmaceutical, including, but not limited to, sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried lactoserum, albumin or casein) or their degradation products (such as lactalbumin hydrolysate), etc. In certain exemplary embodiments, the pharmaceutically acceptable carrier or excipient comprises a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquids are selected from the group consisting of water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% NaCl), dextrose solution (e.g., 5% dextrose), surfactant-containing solutions (e.g., 0.01% polysorbate 20), pH-buffered solutions (e.g., phosphate-buffered solutions), Ringer's solution, and any combination thereof.

[0811] As used herein, the term "prevention" refers to a method carried out in order to stop or delay the occurrence of a disease or disorder or symptoms in a subject. As used herein, the term "treatment" refers to a method carried out in order to obtain a beneficial or desired clinical result. For the purposes of this application, a beneficial or desired clinical result includes, but is not limited to, alleviation of symptoms, diminishment of extent of disease, stabilization (i.e., not worsening) of the state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Moreover, "treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0812] As used herein, the term "subject" refers to a mammal, for example, a human. In certain embodiments, the subject (e.g., human) has a disease associated with abnormal angiogenesis or is at risk of having such a disease.

[0813] As used herein, the term "effective amount" refers to an amount that is sufficient to achieve or at least partially achieve a desired effect. For example, an effective amount for preventing a disease (e.g., an autoimmune disease) refers to an amount that is sufficient to prevent, stop, or delay the occurrence of the disease; an effective amount for treating a disease refers to an amount that is sufficient to cure or at least partially stop the disease and its complications in a patient already having the disease. Determining such effective amounts is well within the capabilities of those skilled in the art. For example, an amount effective for therapeutic use will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient such as age, body weight, and sex, the mode of administration of a drug, and other therapies being administered concurrently, etc.

[0814] Advantages of the Invention

[0815] The polypeptides or Fc fusion proteins targeting VEGF provided in the present application are capable of simultaneously binding to VEGF-A, VEGF-C and VEGF-D. In particular, the fusion proteins provided in the present application have better protein yield, purity, physicochemical properties and affinity compared to wild-type fusion protein molecules, and have stronger VEGF-A, VEGF-C and / or VEGF-D blocking activity (e.g., blocking activity of simultaneously blocking VEGF-A, VEGF-C and VEGF-D). Therefore, the polypeptides or fusion proteins of the present application have blocking activity more relevant to disease progression, and are expected to be effectively applied to the prevention and / or treatment of diseases associated with VEGF (e.g., VEGF-A, VEGF-C and / or VEGF-D) signaling (e.g., diseases associated with abnormal angiogenesis, for example, vascular-derived eye diseases). BRIEF DESCRIPTION OF DRAWINGS

[0816] Figure 1A: Results of activity assays for VEGF Trap fusion protein mutants to inhibit hVEGF-A-induced HUVEC primary cell proliferation.

[0817] Figure IB: Results of activity assays for VEGF Trap fusion protein mutants to inhibit hVEGF-C-induced HUVEC primary cell proliferation.

[0818] Figure 1C: Results of activity assays for VEGF Trap fusion protein mutants to inhibit hVEGF-D-induced HUVEC primary cell proliferation.

[0819] Figures 2A-2V: Results of activity assays for VEGF Trap AI molecules to inhibit hVEGF-A-induced HEK293-hVEGFR2-hVEGFR3-Luc reporter cell activation.

[0820] Figures 3A-3W: Results of activity assays for VEGF Trap AI molecules to inhibit hVEGF-C-induced HEK293-hVEGFR2-hVEGFR3-Luc reporter cell activation.

[0821] Figures 4A-4X: Results of activity assays for VEGF Trap AI molecules to inhibit hVEGF-D-induced HEK293-hVEGFR2-hVEGFR3-Luc reporter cell activation.

[0822] Figures 5A-5B: Results of activity assays for VEGF Trap AI molecules to inhibit hVEGF-A-induced HUVEC primary cell proliferation.

[0823] Figures 6A-6B: Results of activity assays for VEGF Trap AI molecules to inhibit hVEGF-C-induced HUVEC primary cell proliferation.

[0824] Figures 7A-7B: Results of activity assays for VEGF Trap AI molecules to inhibit hVEGF-D-induced HUVEC primary cell proliferation.

[0825] Figures 8A-8C: Results of activity assays for AI-A1 molecules to inhibit hVEGF-A, hVEGF-C, hVEGF-D-induced HUVEC primary cell proliferation.

[0826] Figures 9A-9C: Results of activity assays for AI-A3, AI-A4, AI-A5 molecules to inhibit hVEGF-A, hVEGF-C, hVEGF-D-induced HUVEC primary cell proliferation.

[0827] Figure 10A to Figure 10C: The results of the activity assay of AI-A1, AI-A3, AI-A4, AI-A5 molecules inhibiting the activation of HEK293-hVEGFR2-hVEGFR3-Luc reporter cells induced by hVEGF-A, hVEGF-C, hVEGF-D.

[0828] Figure 11A to Figure 11C: The results of the activity assay of AI-A18 molecule inhibiting the activation of HEK293-hVEGFR2-hVEGFR3-Luc reporter cells induced by hVEGF-A, hVEGF-C, hVEGF-D.

[0829] Figure 12: The results of the activity assay of AI-A1, AI-A3, AI-A4, AI-A5 molecules inhibiting the activation of HEK293-hTie2-βgal reporter cells induced by hAng-2.

[0830] Figure 13: The results of the activity assay of AI-A18 molecule inhibiting the activation of HEK293-hTie2-βgal reporter cells induced by hAng-2.

[0831] Figure 14: The improvement rate of leakage area of AI-A1 molecule in DL-α-AAA induced in vivo RNV model. DETAILED DESCRIPTION

[0832] The present application will now be described with reference to the following examples which are intended to illustrate the present application (but not to limit the present application).

[0833] Unless otherwise indicated, the molecular biology experimental methods and immunoassay procedures employed in the present application were performed according to the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nded., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, and F. M. Ausubel et al., Short Protocols in Molecular Biology, 3rded., John Wiley & Sons, Inc., 1995. Those skilled in the art know that the examples describe the present application by way of illustration, and are not intended to limit the scope of the present application claimed.

[0834] Example 1: Preparation of Antigen Protein

[0835] 1.1 Transient Expression of Antigen

[0836] FreeStyle 293E cells (Gibco) were used to express the antigen protein in FreeStyle 293E Expression Medium (Gibco) according to the manufacturer's instructions. TM293 expression medium (Thermo Fisher, 12338018) was used for transient expression of hVEGF-A (human VEGF-A, NCBI, P15692-4), hVEGF-C (human VEGF-C, NCBI, P49767), hVEGF-D (human VEGF-D, NCBI, O43915) antigens fused with hIgG1 Fc (containing hinge peptide (SEQ ID NO: 242) and Fc segment (SEQ ID NO: 239) of human IgG1), His (SEQ ID NO: 235), mIgG1 Fc (containing hinge peptide (SEQ ID NO: 241) and Fc segment (SEQ ID NO: 236) of mouse IgG1), and Avi (B) (SEQ ID NO: 237) tags at C-terminus, respectively. 24 hours before transfection, 0.3-0.4 x 10 6 cells / mL of 293E cells were inoculated in 500 mL cell culture flasks, and the flasks were incubated at 37°C, 5% CO2, and 120 rpm in a shaker. The cell density was measured before transfection, and when it reached 0.8-1.2 x 10 6 cells / mL, transfection was performed. For transfection, 150 μL of 293fectin TM (life, 12347-500) was added to 3 mL of Opti-MEM (Gibco, 31985070) and mixed, and incubated at room temperature for 5 minutes. Meanwhile, 100 μg of pcDNA3.4 (Invitrogen) expression plasmids each containing the coding sequence of hVEGF-A, hVEGF-C, and hVEGF-D antigens were diluted with Opti-MEM to 3 mL. The diluted transfection reagent and plasmids were mixed well and incubated at room temperature for 15 minutes, and then the mixture was added to the cells and mixed. The flasks were incubated at 37°C, 5% CO2, and 120 rpm in a shaker for 7 days. On the 3rd and 5th days after transfection of the cells, 7.5 mL of Peptone (BD, 211677) was added.

[0837] 1.2 Antigen purification

[0838] hFc-tagged protein purification: The cell culture solution was centrifuged at 2000 g for 20 min, and the supernatant was collected and filtered with a 0.22-μm filter. The Prism A (Cytiva) affinity chromatography (protein purification liquid chromatography system: Cytiva, AKTA avant 25) was performed using 20 mM citric acid-sodium citrate (pH 3.2) for elution, and then the pH was adjusted to neutral using 1 M Tris base. The purified sample was subjected to SDS-PAGE using a 4-20% gradient gel to determine the protein purity.

[0839] His-tag protein purification: collect the supernatant, filter the supernatant with 0.22 micron filter membrane. Use Ni column affinity chromatography, load into the Ni chromatography column which has been equilibrated with 20 mM PB-500 mM NaCl, pH 7.4 buffer, after equilibration with 20 mM PB-500 mM NaCl, pH 7.4 buffer, elute, elution conditions are: (1) use A liquid (20 mM PB-500 mM NaCl, pH 7.4) + 4% B liquid (20 mM PB-500 mM NaCl-500 mM imidazole, pH 7.4) elution, according to the ultraviolet absorption peak peak type sample collection, after the ultraviolet absorption peak is stable and not fluctuation, the second step elution; (2) use A liquid (20 mM PB-500 mM NaCl, pH 7.4) + 20% B liquid (20 mM PB-500 mM NaCl-500 mM imidazole, pH 7.4) elution, according to the ultraviolet absorption peak peak type sample collection, after the ultraviolet absorption peak is stable and not fluctuation, the third step elution; (3) use A liquid (20 mM PB-500 mM NaCl, pH 7.4) + 60% B liquid (20 mM PB-500 mM NaCl-500 mM imidazole, pH 7.4) elution, according to the ultraviolet absorption peak peak type sample collection, after the ultraviolet absorption peak is stable and not fluctuation, stop collection. The purified sample is detected by SDS-PAGE with 4-20% gradient gel.

[0840] mFc-tag protein purification: collect the supernatant, filter the supernatant with 0.22 micron filter membrane, through Prism A (GE) affinity chromatography, elute with 20 mM citric acid-citric acid sodium, pH 3.0, adjust the pH to neutral with 1 M Tris base. The purified sample is detected by SDS-PAGE with 4-20% gradient gel to determine the protein purity.

[0841] Example 2: Random mutation library

[0842] 2.1 Construction of random mutation library

[0843] VEGF Trap was constructed by fusing the second domain of human VEGFR2 (R2D2, amino acids 120-207 of SEQ ID NO: 238) directly to the third domain of human VEGFR2 (R2D3, amino acids 208-327 of SEQ ID NO: 238). To express the tandem R2D2 and R2D3 domains, a DNA fragment (DNA synthesis was commissioned to Shengong Biotechnology Co., Ltd., Changchun) encoding a fusion protein of wild-type VEGF Trap (human R2D2 and R2D3 domains) and wild-type human IgG1 hinge peptide (SEQ ID NO: 242) and Fc segment (SEQ ID NO: 239) was synthesized, the amino acid sequence of the fusion protein is shown as SEQ ID NO: 1, which is named as T002.

[0844] Using T002 molecule as a template, 3 rounds of PCR mutation were performed with reference to Takara PrimeScript TM II 1 st Strand cDNA Synthesis Kit (Agilent 200550), pCANTAB5E was selected as the phage display vector, and after enzyme digestion and purification recovery of the fragments and the vector, random mutation library mass ligation and electrotransformation were performed to complete library construction, and the bacterial solution was stored at -80℃.

[0845] 2.2 Random mutation library panning

[0846] An appropriate amount of -80℃ mutant library bacterial solution was inoculated into 880 mL of 2xYT-AG medium (containing 100 μg / mL ampicillin and 2% glucose), and cultured at 37℃, 200 rpm until OD600=0.5-0.6. The cell density was 100 times that of the helper phage, and the infection was 1.5 h. The bacterial cells were collected by centrifugation, resuspended in 440 mL of 2xYT-AK medium (containing 100 μg / mL ampicillin and 70 μg / mL kanamycin), and cultured at 30℃, 200 rpm overnight. The culture supernatant was collected by centrifugation at 10000g, 4℃ for 20 min, and recombinant phage was prepared by PEG / NaCl precipitation method.

[0847] Biotinylated hVEGF-A-Avi(B)-His or hVEGF-C-Avi(B)-His was used as antigen, and 5% Milk-PBS (M-PBS) was used for blocking; then 10 13The first round of starting phage for antibody antigen binding, PBST (0.05% Tween 20, pH 7.4) was used to wash off the unbound phage, glycine-HCl (Glycine-HCl, pH 2.2) was used to elute the phage, the eluted phage was re-infected into TG1, the eluted product was amplified, and the phage was purified by PEG / NaCl precipitation for the next round of screening. A total of 4 rounds of conventional enrichment screening of phage library were carried out, and the titer of the eluted product was determined.

[0848] The bacteria liquid after 1-4 rounds of panning was diluted and plated, and cultured overnight; single colonies were picked and cultured in 96-well deep well plates containing 0.5 mL / well 2xYT-AG medium overnight; then the overnight culture was transferred to 96-well deep well plates containing 0.5 mL / well 2xYT-AG (the content of glucose was 0.05%) medium at a volume ratio of 1:10, and cultured to OD600=0.6-0.8, then 1 mM IPTG was added at a final concentration of 1 mM, and induced at 30°C overnight, the next day the supernatant was centrifuged and transferred to a clean 96-well deep well plate, and 3% bovine serum albumin (BSA) was added at a final concentration to obtain a single clone induction supernatant sample. The 96-well enzyme-labeled plate was coated with hVEGF-A-hIgG1Fc-Avi(B) or hVEGF-C-hIgG1Fc-Avi(B) as antigen, after blocking, 50 μL of single clone induction supernatant sample was added to each well, and incubated at 25°C for 1 h; then 250 μL of PBST (0.05% Tween 20, pH 7.4) was added to each well, shaken for 5-10 s, the solution was discarded, and the operation was repeated 3-5 times; then 50 μL of anti-His-HRP antibody (Proteintech, HRP-66005, 1:20000) PBS diluent was added to each well, and incubated at 25°C for 1 h; then 250 μL of PBST (0.05% Tween 20, pH 7.4) was added to each well, shaken for 5-10 s, the solution was discarded, and the operation was repeated 5 times; 50 μL of TMB color developing liquid was added to each well, and color developed for 5 min, then 50 μL of 1M H2SO4 was added to each well to stop the color development; the OD450 absorbance value was measured using an enzyme-labeled instrument.

[0849] According to the ELISA data of 5208 single clone phage, 132 positive clones with binding activity were selected for sequencing. Finally, 9 positive clones with different sequences were selected for further experiments.

[0850] Example 3: Combination mutation library

[0851] 3.1 Construction of combination mutation library

[0852] According to the sequence characteristics of the 9 positive clones obtained from the random mutation library and DS (Discovery Studio) rational design analysis, 26 sites were finally selected for the construction of a combinatorial library. The amino acid distribution of each site was determined according to the sequence distribution of the 9 positive clones, and the wild-type amino acid distribution was introduced. Using T002 as a template, primers were designed, and an overlap PCR library was constructed. pCANTAB5E was selected as the phage display vector, and the fragments and vectors were subjected to enzyme digestion, purification and recovery treatment. Then, combinatorial mutation library mass ligation and electroporation were performed to complete the library construction. The bacterial solution was stored at -80°C.

[0853] 3.2 Screening of the combinatorial mutation library

[0854] The screening method of the combinatorial mutation library was as described in section 2.2 of Example 2.

[0855] According to the ELISA data of the 2232 single clone phages, 189 positive clones with binding activity were selected for sequencing. Finally, 27 positive clones with different sequences were obtained for further experiments. The amino acid sequences of the 27 positive clones are shown as SEQ ID NOs: 2-28, and the mutation site information is shown in Table 1. In Table 1, each mutation site of each mutation contained in each mutant is described by the sequence of VEGFR2 (SEQ ID NO: 238).

[0856] Table 1: Combinatorial mutant molecules

[0857] Example 4: Preparation of VEGF Trap fusion protein

[0858] 4.1 Synthesis of VEGF Trap fusion protein plasmid

[0859] The DNA sequence encoding the VEGF Trap fusion protein mutant obtained by screening the combinatorial mutation library of Example 3 was synthesized. The DNA fragment was cloned into the multiple cloning site downstream of the CMV promoter on the expression vector pcDNA3.4 for expression of the VEGF Trap fusion protein mutant.

[0860] 4.2 Transient expression of VEGF Trap fusion protein

[0861] 24 hours before transfection, 2.4-2.6 x 10 6 The cell culture bottles were incubated in a 37°C, 8% CO2, 120 rpm shaker. The cell density was determined before transfection, and the cell density reached 6-7 x 10 6When the cell density reached 6 x 105cells / mL, the cell density was adjusted to 6 x 105cells / mL 6 cells / mL and transfected. First, 320 μL of ExpiFectamine (Thermo Fisher, A29130) was added to 4 mL of OptiPRO SFM (Thermo Fisher, 12309050) and mixed well, and incubated at room temperature for 30 s. Meanwhile, 100 μg of pcDNA3.4 expression plasmid containing the gene of interest was diluted with 4 mL of OptiPRO SFM and mixed well, and incubated at room temperature for 30 s. Then, the diluted transfection reagent and plasmid were mixed well and incubated at room temperature for 1-2 min, and then the mixture was added to the cells and mixed well. The culture bottle was placed in a 37°C, 8% CO2, 120 rpm shaker for 18-22 h. Then, 600 μL of CHO-S enhancer and 16 mL of ExpiCHO Feed (Thermo Fisher, A29130) were added. Then, the culture bottle was placed in a 32°C, 5% CO2, 120 rpm shaker for 6 days. On the 5th day after cell transfection, 16 mL of Feed was added. TM CHO Reagent (Thermo Fisher, A29130) was added to 4 mL of OptiPRO SFM (Thermo Fisher, 12309050) and mixed well, and incubated at room temperature for 30 s. Meanwhile, 100 μg of pcDNA3.4 expression plasmid containing the gene of interest was diluted with 4 mL of OptiPRO SFM and mixed well, and incubated at room temperature for 30 s. Then, the diluted transfection reagent and plasmid were mixed well and incubated at room temperature for 1-2 min, and then the mixture was added to the cells and mixed well. The culture bottle was placed in a 37°C, 8% CO2, 120 rpm shaker for 18-22 h. Then, 600 μL of CHO-S enhancer and 16 mL of ExpiCHO Feed (Thermo Fisher, A29130) were added. Then, the culture bottle was placed in a 32°C, 5% CO2, 120 rpm shaker for 6 days. On the 5th day after cell transfection, 16 mL of Feed was added. TM CHO Reagent (Thermo Fisher, A29130) was added to 4 mL of OptiPRO SFM (Thermo Fisher, 12309050) and mixed well, and incubated at room temperature for 30 s. Meanwhile, 100 μg of pcDNA3.4 expression plasmid containing the gene of interest was diluted with 4 mL of OptiPRO SFM and mixed well, and incubated at room temperature for 30 s. Then, the diluted transfection reagent and plasmid were mixed well and incubated at room temperature for 1-2 min, and then the mixture was added to the cells and mixed well. The culture bottle was placed in a 37°C, 8% CO2, 120 rpm shaker for 18-22 h. Then, 600 μL of CHO-S enhancer and 16 mL of ExpiCHO Feed (Thermo Fisher, A29130) were added. Then, the culture bottle was placed in a 32°C, 5% CO2, 120 rpm shaker for 6 days. On the 5th day after cell transfection, 16 mL of Feed was added. TM CHO Reagent (Thermo Fisher, A29130) was added to 4 mL of OptiPRO SFM (Thermo Fisher, 12309050) and mixed well, and incubated at room temperature for 30 s. Meanwhile, 100 μg of pcDNA3.4 expression plasmid containing the gene of interest was diluted with 4 mL of OptiPRO SFM and mixed well, and incubated at room temperature for 30 s. Then, the diluted transfection reagent and plasmid were mixed well and incubated at room temperature for 1-2 min, and then the mixture was added to the cells and mixed well. The culture bottle was placed in a 37°C, 8% CO2, 120 rpm shaker for 18-22 h. Then, 600 μL of CHO-S enhancer and 16 mL of ExpiCHO Feed (Thermo Fisher, A29130) were added. Then, the culture bottle was placed in a 32°C, 5% CO2, 120 rpm shaker for 6 days. On the 5th day after cell transfection, 16 mL of Feed was added. TM CHO Reagent (Thermo Fisher, A29130) was added to 4 mL of OptiPRO SFM (Thermo Fisher, 12309050) and mixed well, and incubated at room temperature for 30 s. Meanwhile, 100 μg of pcDNA3.4 expression plasmid containing the gene of interest was diluted with 4 mL of OptiPRO SFM and mixed well, and incubated at room temperature for 30 s. Then, the diluted transfection reagent and plasmid were mixed well and incubated at room temperature for 1-2 min, and then the mixture was added to the cells and mixed well. The culture bottle was placed in a 37°C, 8% CO2, 120 rpm shaker for 18-22 h. Then, 600 μL of CHO-S enhancer and 16 mL of ExpiCHO Feed (Thermo Fisher, A29130) were added. Then, the culture bottle was placed in a 32°C, 5% CO2, 120 rpm shaker for 6 days. On the 5th day after cell transfection, 16 mL of Feed was added.

[0862] 4.3 Purification of VEGF Trap fusion protein

[0863] The cell culture solution was centrifuged at 2000 g for 20 min, and the supernatant was collected and filtered through a 0.22 micron filter. The supernatant was subjected to PrismA (Cytiva) affinity chromatography (protein purification liquid chromatography system: Cytiva, AKTA avant 25) and eluted with 20 mM citric acid-sodium citrate (pH 3.2), and then the pH was adjusted to neutral with 1 M Tris base. The purity of the purified sample was determined by SDS-PAGE using 4-20% gradient gel.

[0864] Example 5: Purity, physicochemical properties, and affinity of VEGF Trap fusion protein

[0865] 5.1 Determination of protein purity by size exclusion chromatography (SEC-HPLC)

[0866] The purified protein sample was diluted to 1 mg / ml with the mobile phase, eluted with a high performance liquid chromatograph (Waters 2489) and a chromatographic column TSK_GEL3000SWxl (TOSOH) 7.8 x 300 mm, 5 μm under the condition of a mobile phase of 50 mmol / L PB-300 mmol / L NaCl (pH 7.0), a flow rate of 1 ml / min, an injection volume of 10 μl, a detection wavelength of 214 nm, and an elution time of 16 min. Then, peak cutting integration analysis was performed on the chromatogram. The results are shown in Table 2. The results show that the yield and / or purity of the VEGF Trap fusion protein mutant is better than T002.

[0867] Table 2: VEGF Trap fusion protein expression and purification detection results

[0868] 5.2 Thermal stability and hydrophobicity detection of VEGF Trap fusion protein

[0869] 5.2.1 Protein solubility temperature characterization detection

[0870] The purified protein sample was diluted to 0.5-2 mg / ml with PBS (pH 7.4) buffer, and the fluorescent dye (Protein Thermal Shift Dye Kit TM ) was diluted to 8x, mixed with the corresponding volume of PBS (pH 7.4) buffer at a dye / buffer volume ratio of 1 / 2. The sample was mixed with the dye mixture in a 96-well plate, 12.5 μl of sample was added per well, 7.5 μl of dye mixture was added per well, and the bubbles were removed by centrifugation at 4000 r / min for 1 min. The sample was slowly heated using a fluorescence quantitative PCR instrument (Thermo Fisher Scientific, 7500 Real-Time PCR System), and the change in protein conformation during heating was monitored by fluorescence dye or endogenous protein fluorescence signal to calculate the thermal denaturation key temperature Tm value. Protein Thermal Shift Software 1.4 was used for data analysis. The results are shown in Table 3.

[0871] 5.2.2 Hydrophobic chromatography HIC detection

[0872] The purified protein sample was diluted to 1 mg / ml with ultrapure water, eluted with a high performance liquid chromatograph (Waters 2489) and a chromatographic column MabPac TMHIC-10 (Thermo) 4.6 x 100 mm, 5 μm, gradient elution under the condition of mobile phase A: 0.1 M potassium phosphate buffer-1 M ammonium sulfate (pH 6.8) and mobile phase B: 0.1 M potassium phosphate buffer (pH 6.8) (mobile phase A is replaced by mobile phase B after 20 min, and mobile phase B is replaced by mobile phase A from 28 min to the end), flow rate 1 ml / min, injection volume 10 μl, detection wavelength 280 nm, elution time 40 min. Then the chromatogram is integrated and analyzed. The results are shown in Table 3.

[0873] The results show that the VEGF Trap fusion protein mutants have good hydrophobicity and better thermal stability than T002.

[0874] Table 3: VEGF Trap fusion protein hydrophobicity and thermal stability detection results

[0875] 5.3 VEGF Trap fusion protein and VEGF-A, VEGF-C, VEGF-D antigen affinity determination

[0876] Sensor Chip Protein A biosensor chip (Cytiva, 29-1275-56) was used to immobilize VEGF Trap fusion protein as ligand on the chip, and different concentrations of antigens hVEGF-A-Avi(B)-His, hVEGF-C-Avi(B)-His, hVEGF-D-Avi(B)-His were flowed through the chip at a flow rate of 30 μL / min, the antigens combined with the receptor molecules, the binding time was 240 s, and the dissociation time was 1800 s or 600 s. Biacore 8K Evaluation (Cytiva) was used for kinetic fitting, and the affinity constant was obtained as shown in Table 4. The controls used were Eylea (derived from CN103349781B, the amino acid sequence is SEQ ID NO: 229), OPT-302 (derived from US9745558B2, the amino acid sequence is SEQ ID NO: 230).

[0877] The results show that the VEGF Trap fusion protein mutants have good affinity with hVEGF-A, hVEGF-C, hVEGF-D.

[0878] Table 4: VEGF Trap fusion protein mutant and hVEGF-A, hVEGF-C, hVEGF-D antigen affinity detection results

[0879] Example 6: Activity assay for inhibiting HUVEC primary cell proliferation induced by VEGF-A, VEGF-C, VEGF-D

[0880] HUVEC cells (Nanjing Keygen, CBP60340) were adjusted to a density of 1 x 10 5 cells / mL, 50 μL / well were inoculated in a 96-well full-white plate (CORNING, 3917), i.e. 5 x 10 3 cells / well.

[0881] The experimental medium was diluted to a final concentration of 0.25 μg / ml for hVEGF-A (Sino bio, 11066-HNAH), 0.1 μg / ml for hVEGF-C (Acrobiosystems, VEC-H4225), and 3 μg / ml for hVEGF-D (Sino bio, 10557-H08H), and 25 μl / well was added as the control group. On the basis of the control group, the experimental medium was diluted to a maximum concentration of 500 nM for T002-P44910, T002, positive control Eylea, positive control OPT-302, and negative control hIgG1 isotype (non-relevant isotype negative control antibody), 4-fold dilution, 9 concentration gradients, and 25 μl / well was added as the sample group when detecting hVEGF-A, hVEGF-C, and hVEGF-D. A blank group was also set up by adding an equal volume of experimental medium. Incubation was carried out in a 37°C, 5% CO2 incubator for 96 hours. Cell Counting-Lite (Nanjing Nvzhan, DD1101-02) 100 μL / well was added, protected from light, and incubated at room temperature for 5 min. The light emission value was detected by a SpectraMax iD5 multifunctional enzyme labeler (Molecular Devices). The reporter gene inhibition rate (%) = 100% x [1-(RLU sample group-RLU blank group) / (RLU control group-RLU blank group)]. The results are shown in FIGS. 1A-1C and Table 5.

[0882] The results show that T002-P44910 presents a dose-dependent inhibition of HUVEC primary cell proliferation.

[0883] Table 5: Activity assay results for VEGF Trap fusion protein inhibiting HUVEC primary cell proliferation induced by hVEGF-A, hVEGF-C, and hVEGF-D

[0884] Note: “NA” indicates no inhibitory activity, and “ / ” indicates not detected.

[0885] Example 7: Construction of VEGF Trap AI molecules and combination mutants

[0886] 7.1 Design and identification of VEGF Trap AI molecules and combination mutants

[0887] By AI-assisted drug discovery, the process of mutual recognition and binding between VEGF and VEGFR molecules is simulated, and the generated new molecules are sorted by calculation indexes such as affinity and drugability, beneficial residues are selected for mutation, and at the same time, combination mutation is performed on these beneficial residues; the mutant is obtained by mutating the 1-208 amino acids in the wild type T002 sequence shown in SEQ ID NO: 1, and the mutation site information of each mutant is shown in Table 6, wherein each mutation site contained in each mutant is described by the sequence of VEGFR2 (SEQ ID NO: 238). The T002 mutant obtained based on AI design is constructed into Fc fusion protein according to Example 4 and Example 5, and the sequence is shown as SEQ ID NO: 29-228, wherein the hinge peptide as shown in SEQ ID NO: 233 or SEQ ID NO: 242 and the Fc segment as shown in SEQ ID NO: 239 or SEQ ID NO: 240 are used, after gene synthesis, vector construction and extraction, the CHO-S cells are transiently transfected, and then PrismA (Cytiva) affinity chromatography is used to obtain the mutant, and the expression and purification, hydrophobicity and thermal stability detection results are shown in Table 7 and Table 8. The results show that the yield and / or purity of the VEGF Trap AI molecule is better than that of T002, and it has good hydrophobicity and thermal stability better than T002.

[0888] Table 6: VEGF Trap AI molecules

[0889] Table 7: Expression and purification detection results of VEGF Trap AI molecules

[0890] Table 8: Hydrophobicity and thermal stability detection results of VEGF Trap AI molecules

[0891] Example 8: Activity assay of VEGF Trap AI molecules for inhibiting VEGF-induced activation of HEK293-hVEGFR2-hVEGFR3-Luc reporter cells

[0892] 8.1 Preparation of HEK293-VEGFR2-VEGFR3 overexpression cell line

[0893] The human VEGFR2 (KDR) protein coding gene (UniProt, Gene ID: CCDS3497.1) was ligated to the pLVX-IRES-Puro (OriGene, A3005) plasmid to construct the lentivirus plasmid of hVEGFR2 protein. The human VEGFR3 protein coding gene (UniProt, Gene ID: CCDS4457.1) was ligated to the pLVX-IRES-Neo (OriGene, A3220) plasmid to construct the lentivirus plasmid of hVEGFR3 protein.

[0894] The lentivirus was packaged using the three-plasmid system (envelope plasmid pCMV-VSVG, packaging plasmid pCMV-dr8.91 and the above-mentioned pLVX plasmid), then concentrated, and finally obtained the lentivirus solution. The HEK293-NFAT-RE-luc2p cells (Promega, R8510, a luciferase reporter cell line with NFAT response element) were infected with the above-mentioned two kinds of lentivirus solutions. After infection, the infected cells were subjected to pressure screening by 400 μg / mL geneticin (Gibco) and 0.5 μg / mL puromycin (Invitrogen), and the resistant pool was obtained. The expression of hVEGFR2 and hVEGFR3 proteins on the cell surface was determined by FACS. The identified positive pool cells were subjected to monoclonalization by limited dilution method, and the cell strain HEK293-hVEGFR2-hVEGFR3-Luc overexpressing hVEGFR2 and hVEGFR3 was obtained after expansion and identification.

[0895] 8.2 Activity determination of VEGF Trap AI molecule in inhibiting the activation of HEK293-hVEGFR2-hVEGFR3-Luc reporter cells induced by VEGF-A, VEGF-C and VEGF-D

[0896] The HEK293-hVEGFR2-hVEGFR3-Luc cells were adjusted to a density of 6×10 5 cells / mL, 50 μL / well were inoculated in a 96-well full white plate (CORNING, 3917), i.e. 3×10 4 cells / well.

[0897] The experimental medium was diluted with hVEGF-A (SinoBio, item number: 11066-HNAH) at a final concentration of 1 ng / ml; hVEGF-C-Avi(B)-His at a final concentration of 50 ng / ml; and hVEGF-D-Avi(B)-His at a final concentration of 500 ng / ml, 25 μl / well was added respectively, as a control group. On the basis of the control group, the experimental medium was gradiently diluted with T002-P44910, T002, positive control Eylea, positive control OPT-302, BP002 and BP003 (derived from patent CN116162148A, sequences as shown in SEQ ID NO: 231 and 232) and negative control hIgG1 isotype, the highest concentration was 200 nM, 8-fold dilution, 9 concentration gradients, and the sample addition amount was 25 μl / well when detecting hVEGF-A, hVEGF-C and hVEGF-D, as a sample group. At the same time, a blank group was set up by adding an equal volume of experimental medium. Incubate in a 37°C, 5% CO2 incubator for 4 hours. Add Bright-Lite (Norgen, DD1204-02) 100 μL / well, avoid light, room temperature for 3 min, and detect the luminescence value by SpectraMax iD5 multifunctional microplate reader (Molecular Devices). Reporter gene inhibition rate (%) = 100% x [1-(RLU sample group-RLU blank group) / (RLU control group-RLU blank group)].

[0898] As shown in FIGS. 2A to 2V, FIGS. 3A to 3W, FIGS. 4A to 4X and Tables 9-10, the results showed that the VEGF Trap molecules all presented dose-dependent blocking of hVEGF-A, hVEGF-C and hVEGF-D induced activation of HEK293-hVEGFR2-hVEGFR3-Luc cell signaling pathway.

[0899] Table 9: Activity determination results of VEGF Trap AI molecules inhibiting hVEGF-A, hVEGF-C and hVEGF-D induced activation of HEK293-hVEGFR2-hVEGFR3-Luc reporter cells

[0900] Note: “ / ” indicates not detected; “NA” indicates no inhibitory activity.

[0901] Table 10: Activity determination results of VEGF Trap AI molecules inhibiting hVEGF-A, hVEGF-C and hVEGF-D induced activation of HEK293-hVEGFR2-hVEGFR3-Luc reporter cells

[0902] Example 9: Activity assay of VEGF Trap AI molecule to inhibit HUVEC primary cell proliferation induced by VEGF-A, VEGF-C, VEGF-D

[0903] Activity assay of VEGF Trap AI molecule to inhibit HUVEC primary cell proliferation induced by hVEGF-A, hVEGF-C, hVEGF-D was performed according to Example 6, and the results are shown in FIG. 5A-5B, FIG. 6A-6B, FIG. 7A-7B and Table 11. The results show that the VEGF Trap AI molecule presents dose-dependent inhibition of HUVEC primary cell proliferation.

[0904] Table 11: Activity assay results of VEGF Trap AI molecule to inhibit HUVEC primary cell proliferation induced by hVEGF-A, hVEGF-C, hVEGF-D

[0905] Note: " / " means not detected; "NA" means no inhibitory activity.

[0906] Example 10: Design and identification of quadruple blocking fusion protein molecule

[0907] The above VEGF Trap AI molecule, hinge peptide, Fc peptide and anti-Ang-2 VHH or VHH2 (VHH sequence as shown in SEQ ID NO: 248, CDR1-3 sequence as shown in SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251; VHH2 sequence as shown in SEQ ID NO: 256, CDR1-3 sequence as shown in SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 257) are combined to obtain a quadruple blocking fusion protein molecule that can simultaneously block VEGF-A, VEGF-C, VEGF-D and Ang-2.

[0908] The structure of AI-A1 molecule from N-terminal to C-terminal is in turn VEGFR2 D2D3 domain of T002-AI2-93 (amino acid sequence shown in SEQ ID NO: 209, 1-208), hinge peptide (SEQ ID NO: 233), Fc peptide (SEQ ID NO: 240), G4S linker (SEQ ID NO: 252), anti-Ang-2 VHH (SEQ ID NO: 248), the sequence of which is shown in SEQ ID NO: 243.

[0909] The structure of the AI-A2 molecule from N-terminus to C-terminus is in turn VEGFR2 D2D3 domain of T002-AI2-101 (amino acid sequence shown in SEQ ID NO: 217, 1-208), a hinge peptide (SEQ ID NO: 233), an Fc peptide (SEQ ID NO: 239), a G4S linker (SEQ ID NO: 252), a VHH against Ang-2 (SEQ ID NO: 248), the sequence of which is shown in SEQ ID NO: 244.

[0910] The structure of the AI-A3 molecule from N-terminus to C-terminus is in turn VEGFR2 D2D3 domain of T002-AI2-103 (amino acid sequence shown in SEQ ID NO: 219, 1-208), a hinge peptide (SEQ ID NO: 233), an Fc peptide (SEQ ID NO: 239), a G4S linker (SEQ ID NO: 252), a VHH against Ang-2 (SEQ ID NO: 248), the sequence of which is shown in SEQ ID NO: 245.

[0911] The structure of the AI-A4 molecule from N-terminus to C-terminus is in turn VEGFR2 D2D3 domain of T002-AI2-93 (amino acid sequence shown in SEQ ID NO: 209, 1-208), a G5S linker (SEQ ID NO: 253), a VHH against Ang-2 (SEQ ID NO: 248), a G4S linker (SEQ ID NO: 252), a hinge peptide (SEQ ID NO: 233), an Fc peptide (SEQ ID NO: 239), the sequence of which is shown in SEQ ID NO: 246.

[0912] The structure of the AI-A5 molecule from N-terminus to C-terminus is in turn VEGFR2 D2D3 domain of T002-AI2-93 (amino acid sequence shown in SEQ ID NO: 209, 1-208), a G5S linker (SEQ ID NO: 253), a VHH against Ang-2 (SEQ ID NO: 248), a hinge peptide (SEQ ID NO: 242), an Fc peptide (SEQ ID NO: 239), the sequence of which is shown in SEQ ID NO: 247.

[0913] The structure of the AI-A18 molecule from N-terminus to C-terminus is in turn VEGFR2 D2D3 domain of T002-AI2-93 (amino acid sequence shown in SEQ ID NO: 209, 1-208), hinge peptide (SEQ ID NO: 233), Fc peptide (SEQ ID NO: 240), G4S linker (SEQ ID NO: 252), VHH2 of anti-Ang-2 (SEQ ID NO: 256), sequence shown in SEQ ID NO: 255.

[0914] The Fc fusion proteins were constructed according to the embodiments 4 and 5, and after gene synthesis, vector construction and extraction, the CHO-S cells were transiently transfected, and then subjected to PrismA (Cytiva) affinity chromatography. The expression and purification, hydrophobicity and thermal stability detection results of the fusion proteins are shown in Tables 12 and 13. The results show that the quadruple blocking fusion protein molecules have good yield and purity, and also have good hydrophobicity and thermal stability.

[0915] Table 12: Expression and purification detection results of quadruple blocking fusion proteins

[0916] Table 13: Hydrophobicity and thermal stability detection results of quadruple blocking fusion proteins

[0917] Example 11: Activity determination of quadruple blocking fusion proteins in inhibiting HUVEC primary cell proliferation induced by VEGF-A, VEGF-C and VEGF-D

[0918] The activity determination of the quadruple blocking fusion protein molecules in inhibiting HUVEC primary cell proliferation induced by hVEGF-A, hVEGF-C and hVEGF-D was carried out according to the embodiment 6, and the results are shown in Figures 8A-8C, 9A-9C, and Tables 14 and 15. The results show that the quadruple blocking fusion protein molecules exhibit dose-dependent inhibition of HUVEC primary cell proliferation.

[0919] Table 14: Activity determination results of AI-A1 in inhibiting HUVEC primary cell proliferation induced by hVEGF-A, hVEGF-C and hVEGF-D

[0920] Note: “NA” represents no inhibitory activity, “ / ” represents not detected, and “Not fitted” represents unable to fit.

[0921] Table 15: Activity determination results of AI-A3, AI-A4 and AI-A5 in inhibiting HUVEC primary cell proliferation induced by hVEGF-A, hVEGF-C and hVEGF-D

[0922] Note: "NA" means no inhibitory activity, " / " means not tested, "Not fitted" means not fitted.

[0923] Example 12: Activity assay of quadruple blockade fusion protein to inhibit VEGF-A, VEGF-C, VEGF-D induced activation of HEK293-hVEGFR2-hVEGFR3-Luc reporter cells

[0924] Activity assay of quadruple blockade fusion protein molecules to inhibit hVEGF-A, hVEGF-C, hVEGF-D induced activation of HEK293-hVEGFR2-hVEGFR3-Luc reporter cells was performed as described in Reference Example 8, with Faricimab as positive control antibody (Roche, lot B1535B01). The results are shown in Figures 10A-10C, 11A-11C, and Tables 16 and 17. The results show that the quadruple blockade fusion protein molecules exhibited dose-dependent blockade of hVEGF-A, hVEGF-C, and hVEGF-D induced activation of the signaling pathway in HEK293-hVEGFR2-hVEGFR3-Luc cells.

[0925] Table 16: Activity assay results of quadruple blockade fusion protein to inhibit hVEGF-A, hVEGF-C, hVEGF-D induced activation of HEK293-hVEGFR2-hVEGFR3-Luc reporter cells

[0926] Note: "NA" means no inhibitory activity, " / " means not tested.

[0927] Table 17: Activity assay results of quadruple blockade fusion protein AI-A18 to inhibit hVEGF-A, hVEGF-C, hVEGF-D induced activation of HEK293-hVEGFR2-hVEGFR3-Luc reporter cells

[0928] Note: "NA" means no inhibitory activity, " / " means not tested.

[0929] Example 13: Activity assay to inhibit hAng-2 induced activation of HEK293-hTie2-βgal reporter cells

[0930] HEK293-hTie2-βgal cells [Discover X, 93-1024C1, a cell line that can simultaneously express ProLink TM(PK) tagged RTK, enzyme acceptor (EA) tagged SH2 domain and untagged co-receptor, activation of the RTK-PK generates functional enzyme to hydrolyze the substrate to produce chemiluminescent signal], cells were purchased and cultured under pressure by 400 μg / mL GENETICIN (Gibco) and 200 μg / mL Hygromycin B (Invitrogen). 1 x 10 4

[0931] The experimental medium was diluted with hAng-2 (SinoBio, Item No.: 10691-H02H) to a final concentration of 1 μg / ml, and 25 μl / well was added as a control group. On the basis of the control group, the experimental medium was gradiently diluted with AI-A1, AI-A3, AI-A4, AI-A5, AI-A18, positive control Faricimab and negative control hIgG1 isotype, with the highest concentration being 1500 nM, 5-fold dilution, 8 or 9 concentration gradients, and the sample was added in an amount of 25 μl / well when detecting hAng-2, as a sample group. At the same time, a blank group was set up by adding an equal volume of experimental medium. Incubate in a 37°C, 5% CO2 incubator for 24 hours. Add The Assay System (Promage, E4740) 100 μL / well, avoid light, room temperature for 1 h, and the SpectraMax iD5 multifunctional enzyme marker (Molecular Devices) detects the luminescence value. Reporter gene inhibition rate (%) = 100% x [1-(RLU sample group-RLU blank group) / (RLU control group-RLU blank group)].

[0932] The results are shown in Figures 12 and 13 and Tables 18 and 19, and the results show that AI-A1, AI-A3, AI-A4, AI-A5, AI-A18 all present a dose-dependent blocking of hAng-2-induced activation of the HEK293-hTie2-βgal reporter cell signaling pathway, and have stronger blocking activity than the marketed Faricimab.

[0933] Table 18: Results of activity determination of quadruple blocking fusion protein for inhibiting hAng-2-induced activation of HEK293-hTie2-βgal reporter cells

[0934] Note: "NA" means no inhibitory activity, " / " means not detected.​

[0935] Table 19: Results of the activity assay of AI-A18 to inhibit hAng-2-induced activation of HEK293-hTie2-βgal reporter cells

[0936] Example 14: DL-α-AAA-induced retinal neovascularization model in Dutch rabbits

[0937] In this experiment, the anti-neovascularization effect of the test product of the present application was determined using a DL-α-aminoadipic acid (DL-α-AAA)-induced retinal neovascularization (RNV) model in Dutch rabbits.

[0938] In this experiment, a single injection of 80 μL of DL-α-AAA solution into the vitreous cavity of both eyes was used to induce retinal neovascularization in the fundus and to establish an animal model similar to human fundus neovascularization. At 16 weeks after injection, a Heidelberg HRA+OCT (Heidelberg Engineering, Spectralis HRA+OCT, Germany) was used to collect red-free images of the fundus of the animals, with a field of view of 55° and an examination area of the temporal, central, and nasal nerve fiber layers, to observe the morphology of RNV. HRA+OCT (Heidelberg Engineering, Spectralis HRA+OCT, Germany) was used to collect red-free images of the fundus of the animals, with a field of view of 55° and an examination area of the temporal, central, and nasal nerve fiber layers, to observe the morphology of RNV. HRA+OCT (Heidelberg Engineering, Spectralis HRA+OCT, Germany) was used to collect red-free images of the fundus of the animals, with a field of view of 55° and an examination area of the temporal, central, and nasal nerve fiber layers, to observe the morphology of RNV.

[0939] Fluorescence leakage area reduction = fluorescence leakage area before administration - fluorescence leakage area after administration

[0940] Fluorescence leakage area improvement rate = fluorescence leakage area reduction / fluorescence leakage area before administration x 100%.

[0941] The retinal leakage area detected by FFA was relatively evenly grouped, and the grouping and administration details are shown in Table 20. Each group of 6-7 rabbit eyes was injected with each group of drugs in the vitreous body. The fundus without red light image and fundus fluorescence angiography of each group of animals were collected at different time points after administration to observe the inhibition of test product on choroidal neovascularization.

[0942] Table 20: Administration scheme

[0943] The fundus without red light image and fundus fluorescence angiography examination results are shown in Figure 14, and the results show that AI-A1 shows significant anti-angiogenesis effect after 14 days of administration, and the inhibition of vascular leakage can last for 84 days after administration, and the inhibition of vascular leakage is better than and with significant difference.

[0944] Although the specific embodiments of the present application have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details according to all the teachings disclosed, and these changes are within the scope of protection of the present application. The entire scope of the present application is given by the appended claims and any equivalents thereof.

[0945] Sequence information

Claims

A fusion protein comprising a polypeptide construct that binds VEGF, and a single-domain antibody or an antigen-binding fragment thereof that specifically binds Ang-2; the polypeptide construct that binds VEGF comprises a polypeptide, and an Fc peptide and optionally a hinge peptide derived from an immunoglobulin; wherein the polypeptide comprises a first peptide segment comprising immunoglobulin-like domain 2 (R2D2) of VEGFR2 (vascular endothelial cell growth factor receptor 2), and / or a second peptide segment comprising immunoglobulin-like domain 3 (R2D3) of VEGFR2; and the polypeptide comprises a mutation at one or more amino acid positions selected from the group consisting of positions 121, 127, 128, 129, 130, 131, 132, 133, 134, 137, 139, 141, 142, 144, 148, 152, 155, 156, 162, 163, 164, 165, 169, 173, 175, 176, 186, 190, 191, 193, 194, 197, 206, 210, 212, 213, 215, 218, 222, 232, 233, 241, 248, 249, 254, 255, 259, 264, 266, 267, 269, 270, 272, 276, 279, 280, 284, 285, 287, 289, 296, 297, 299, 302, 308, 311, 318, and 323; wherein each mutated amino acid position is a natural order position relative to the amino acid sequence set forth in SEQ ID NO:

238. The fusion protein of claim 1, wherein, the polypeptide comprises a mutation at one or more amino acid positions selected from the group consisting of positions 121, 128, 129, 130, 133, 134, 139, 141, 142, 155, 156, 162, 163, 173, 175, 176, 186, 190, 191, 194, 197, 206, 213, 249, 264, 267, 269, 270, 276, 285, 287, 297, 302, 308, 318; wherein each mutated amino acid position is a natural order position relative to the amino acid sequence set forth in SEQ ID NO:

238. the polypeptide comprises a mutation at one or more amino acid positions selected from the group consisting of positions 121, 128, 129, 130, 133, 134, 139, 141, 142, 155, 156, 162, 163, 173, 175, 176, 186, 190, 191, 194, 197, 206, 213, 249, 264, 267, 269, 270, 276, 285, 287, 297, 302, 308, 318; wherein each mutated amino acid position is a natural order position relative to the amino acid sequence set forth in SEQ ID NO:

238. Preferably, the polypeptide comprises a mutation at one or more amino acid positions selected from: position 121, position 128, position 129, position 133, position 134, position 141, position 155, position 162, position 176, position 186, position 191, position 194, position 206, position 249, position 264, position 267, position 269, position 270, position 276, position 285, position 287, position 308, position 318; wherein each mutated amino acid position is a natural order position relative to the amino acid sequence set forth in SEQ ID NO: 238; Preferably, the polypeptide comprises a mutation at one or more amino acid positions selected from: position 133, position 141, position 162, position 194, position 249, position 270, position 276, position 308; wherein each mutated amino acid position is a natural order position relative to the amino acid sequence set forth in SEQ ID NO:

238. The fusion protein of claim 1 or 2, wherein, the polypeptide has one or more mutations selected from: the polypeptide has one or more mutations selected from: the polypeptide has one or more mutations selected from: the polypeptide has one or more mutations selected from: the polypeptide has one or more mutations selected from: the polypeptide has one or more mutations selected from: the polypeptide has one or more mutations selected from: the polypeptide has one or more mutations selected from: the polypeptide has one or more mutations selected from: the polypeptide has one or more mutations selected from: the polypeptide has one or more mutations selected from: the polypeptide has one or more mutations selected from: the polypeptide has one or more mutations selected from: the polypeptide has one or more mutations selected from: the polypeptide has one or more mutations selected from: the polypeptide has one or more mutations selected from: the polypeptide has one or more mutations selected from: the polypeptide has one or more mutations selected from: the polypeptide has one or more mutations selected from: the polypeptide has one or more mutations selected from: the polypeptide has one or more mutations selected from: the polypeptide has one or more mutations selected from: the polypeptide has one or more mutations selected from: the polypeptide has one or more mutations selected from: the polypeptide has one or more mutations selected from: the polypeptide has one or more mutations selected (1) 121N, 121C, or 121S; (2) 127P; (3) 128P; (4) 129G or 129A; (5) 130G; (6) 131P, 131L, or 131V; (7) 132H; (8) 133L, 133Y, or 133P; (9) 134E or 134Q; (10) 137H; (11) 139V; (12) 141E; (13) 142V; (14) 144M; (15) 148L; (16) 152S; (17) 155P; (18) 156D; (19) 162R, 162H, 162Y, 162T, or 162K; (20) 163T; (21) 164K; (22) 165F; (23) 169T or 169E; (24) 173N; (25) 175K or 175S; (26) 176S; (27) 186S; (28) 190S, 190N, 190A, 190V, or 190P; (29) 191L; (30) 193R or 193H; (31) 194Q or 194D; (32) 197L or 197S; (33) 206G; (34) 210R; (35) 212T; (36) 213V, 213K, or 213L; (37) 215D; (38) 218A, 218T, or 218K; (39) 222E; (40) 232E or 232Q; (41) 233P; (42) 241R; (43) 248V or 248G; (44) 249T or 249V; (45) 254E; (46) 255D or 255R; (47) 259T; (48) 264G or 264F; (49) 266N; (50) 267T, 267D, or 267N; (51) 269R; (52) 270V or 270S; (53) 272I; (54) 276V, 276S, or 276R; (55) 279R; (56) 280P; (57) 284K; (58) 285T, 285E, or 285S; (59) 287H or 287L; (60) 289S; (61) 296N; (62) 297I; (63) 299L; (64) 302S; (65) 308K, 308T, or 308S; (66) 311T; (67) 318H; (68) 323T; wherein each of the mutated amino acid positions is relative to the natural order of the amino acid sequence set forth in SEQ ID NO:

238. The fusion protein of any one of claims 1-3, wherein, the polypeptide has one or more mutations selected from the group consisting of: (1) 121N or 121S; (2) 128P; (3) 129G; (4) 130G; (5) 133Y or 133P; (6) 134E or 134Q; (7) 139V; (8) 141E; (9) 142V; (10) 155P; (11) 156D; (12) 162R or 162H; (13) 163T; (14) 173N; (15) 175K; (16) 176S; (17) 186S; (18) 190S, 190V or 190P; (19) 191L; (20) 194Q or 194D; (21) 197L; (22) 206G; (23) 213L; (24) 249T; (25) 264G; (26) 267T, 267D or 267N; (27) 269R; (28) 270V; (29) 276V or 276S; (30) 285T, 285E or 285S; (31) 287L; (32) 297I; (33) 302S; (34) 308K or 308T; (35) 318H; wherein each mutated amino acid position is relative to the natural order of the amino acid sequence set forth in SEQ ID NO: 238; Preferably, the polypeptide has one or more mutations selected from the group consisting of: (1) 121N or 121S; (2) 128P; (3) 129G; (4) 133Y or 133P; (5) 134E or 134Q; (6) 141E; (7) 155P; (8) 162R or 162H; (9) 176S; (10) 186S; (11) 191L; (12) 194Q or 194D; (13) 206G; (14) 249T; (15) 264G; (16) 267T or 267D; (17) 269R; (18) 270V; (19) 276V or 276S; (20) 285T or 285E; (21) 287L; (22) 308K or 308T; (23) 318H; wherein each mutated amino acid position is relative to the natural order of the amino acid sequence set forth in SEQ ID NO: 238; Preferably, the polypeptide has one or more mutations selected from the group consisting of: (1) 133P; (2) 141E; (3) 162R; (4) 194D; (5) 249T; (6) 270V; (7) 276V or 276S; (8) 308K or 308T; wherein each mutated amino acid position is relative to the natural order of the amino acid sequence set forth in SEQ ID NO:

238. The fusion protein of any one of claims 1-4, wherein, the polypeptide has one or more mutations selected from the group consisting of the mutations set forth in any row of column B or column E of Table 1 or Table 6, wherein each mutated amino acid position is relative to the natural order of the amino acid sequence set forth in SEQ ID NO:

238. The fusion protein of any one of claims 1-5, wherein, the second peptide segment is directly linked or optionally linked via a peptide linker to the N-terminus or C-terminus (e.g., C-terminus) of the first peptide segment; Preferably, the second peptide segment is directly linked to the N- or C-terminus (e.g., C-terminus) of the first peptide segment. The fusion protein of any one of claims 1-6, wherein, The polypeptide comprises an amino acid sequence as set forth in positions 1-208 of any one of SEQ ID NOs: 2-29, 31-108, 110-228. The fusion protein of any one of claims 1-7, wherein, The polypeptide construct comprises a first peptide chain, which comprises: a first polypeptide, a first hinge peptide, and a first Fc peptide, wherein the first polypeptide is selected from the polypeptides defined in any one of claims 1-8; preferably, the first peptide chain comprises, in order from N-terminus to C-terminus: the first polypeptide, the first hinge peptide, and the first Fc peptide; Preferably, the polypeptide construct further comprises a second peptide chain, which comprises a second hinge peptide and a second Fc peptide; preferably, the second peptide chain further comprises a second polypeptide capable of binding to VEGF (e.g., VEGF-A, VEGF-C, and / or VEGF-D); preferably, the second polypeptide is selected from the polypeptides defined in any one of claims 1-8; Preferably, the second peptide chain comprises, in order from N-terminus to C-terminus: the second polypeptide, the second hinge peptide, and the second Fc peptide; Preferably, the polypeptide construct has one or more of the following features selected from: (i) the first polypeptide is the same as or different from the second polypeptide; (ii) the first hinge peptide and the second hinge peptide are derived from the same or different immunoglobulin; (iii) the first Fc peptide and the second Fc peptide are derived from the same or different immunoglobulin; (iv) the first hinge peptide is the same as or different from the second hinge peptide; (v) the first Fc peptide is the same as or different from the second Fc peptide; (vi) the first peptide chain is the same as or different from the second peptide chain; (vii) the first peptide chain and the second peptide chain are connected via disulfide bond formation. The fusion protein of any one of claims 1-8, wherein, The polypeptide construct comprises: (i) an amino acid sequence as set forth in any one of SEQ ID NOs: 2-29, 31-108, 110-228 or (ii) an amino acid sequence having at least 90% sequence identity compared to the amino acid sequence as set forth in any one of SEQ ID NOs: 2-29, 31-108, 110-228; Preferably, the polypeptide construct comprises the first peptide chain and optionally the second peptide chain; wherein the first peptide chain comprises: (i) an amino acid sequence as set forth in any one of SEQ ID NOs: 2-29, 31-108, 110-228 or (ii) an amino acid sequence having at least 90% sequence identity compared to the amino acid sequence as set forth in any one of SEQ ID NOs: 2-29, 31-108, 110-228, and / or the second peptide chain comprises: (i) an amino acid sequence as set forth in any one of SEQ ID NOs: 2-29, 31-108, 110-228 or (ii) an amino acid sequence having at least 90% sequence identity compared to the amino acid sequence as set forth in any one of SEQ ID NOs: 2-29, 31-108, 110-228. The fusion protein of any one of claims 1-9, wherein, The fusion protein comprises one or more of the polypeptides of the polypeptide construct, and one or more single-domain antibodies or antigen-binding fragments thereof that specifically bind to Ang-2. The fusion protein of any one of claims 1-10, wherein, The single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2 is located at the N-terminus and / or C-terminus (e.g., C-terminus) of the polypeptide construct; Preferably, the single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2 and the polypeptide construct are directly linked or optionally linked via a peptide linker. The fusion protein of any one of claims 1-10, wherein, The single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2 is located between the polypeptide and the Fc peptide of the polypeptide construct; Preferably, when the polypeptide construct does not comprise a hinge peptide, the single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2 is located between the polypeptide and the Fc peptide of the polypeptide construct; preferably, the polypeptide and the single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2 are directly linked or optionally linked via a peptide linker, and the single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2 and the Fc peptide are directly linked or optionally linked via a peptide linker; Preferably, when the polypeptide construct comprises a hinge peptide, the single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2 is located between the polypeptide and the hinge peptide of the polypeptide construct; preferably, the polypeptide and the single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2 are directly linked or optionally linked via a peptide linker, and the single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2 and the hinge peptide are directly linked or optionally linked via a peptide linker. The fusion protein of any one of claims 1-12, wherein: The fusion protein comprises a first peptide chain, the first peptide chain comprising: a first polypeptide, a first hinge peptide, a first Fc peptide, and a first single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2, wherein the first polypeptide is selected from the polypeptides defined in any one of claims 1-7; Preferably, the first peptide chain of the fusion protein comprises, in order from N-terminus to C-terminus: (i) the first polypeptide, the first hinge peptide, the first Fc peptide, and the first Ang-2 binding Nanobody or antigen-binding fragment thereof; (ii) the first Ang-2 binding Nanobody or antigen-binding fragment thereof, the first polypeptide, the first hinge peptide, and the first Fc peptide; or (iii) the first polypeptide, the first Ang-2 binding Nanobody or antigen-binding fragment thereof, the first hinge peptide, and the first Fc peptide; Preferably, the fusion protein further comprises a second peptide chain comprising: a second polypeptide, a second hinge peptide, a second Fc peptide, and a second Ang-2 binding Nanobody or antigen-binding fragment thereof, wherein the second polypeptide is selected from the polypeptides as defined in any one of claims 1-7; Preferably, the fusion protein second peptide chain comprises, in order from N-terminus to C-terminus: (i) the second polypeptide, the second hinge peptide, the second Fc peptide, and the second Ang-2 binding Nanobody or antigen-binding fragment thereof; (ii) the second Ang-2 binding Nanobody or antigen-binding fragment thereof, the second polypeptide, the second hinge peptide, and the second Fc peptide; or (iii) the second polypeptide, the second Ang-2 binding Nanobody or antigen-binding fragment thereof, the second hinge peptide, and the second Fc peptide; Preferably, the adjacent domains of the first peptide chain and / or the second peptide chain of the fusion protein are each optionally linked by a linker (e.g., a peptide linker) or directly linked. The fusion protein of any one of claims 6, 11-13, wherein the peptide linker is selected from a peptide linker comprising one or more glycine (G) and / or serine (S), e.g., a flexible peptide comprising Gn, (G4S)n, or (G5S)n, n being an integer not less than 0, e.g., n being 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The fusion protein of claim 13 or 14, having one or more features selected from: (i) the first polypeptide is the same or different from the second polypeptide; (ii) the first hinge peptide and the second hinge peptide are derived from the same or different immunoglobulin; (iii) the first Fc peptide and the second Fc peptide are derived from the same or different immunoglobulin; (iv) the first hinge peptide is the same or different from the second hinge peptide; (v) the first Fc peptide is the same or different from the second Fc peptide; (vi) the first peptide chain is the same or different from the second peptide chain; (vii) the first peptide chain and the second peptide chain are linked by disulfide bond formation; (viii) the first Ang-2 binding Nanobody or antigen-binding fragment thereof and the second Ang-2 binding Nanobody or antigen-binding fragment thereof are the same or different. The fusion protein of any one of claims 8, 13-15, wherein, The hinge peptide (e.g., the first hinge peptide and / or the second hinge peptide) is a hinge peptide derived from an immunoglobulin (e.g., IgG1, IgG2, IgG3, or IgG4); Preferably, the hinge peptide (e.g., the first hinge peptide and / or the second hinge peptide) is a hinge peptide derived from a human immunoglobulin (e.g., IgGl, IgG2, IgG3, or IgG4). The fusion protein of any one of claims 8, 13-16, wherein, the Fc peptide (e.g., the first Fc peptide and / or the second Fc peptide) is a Fc peptide derived from an immunoglobulin (e.g., IgGl, IgG2, IgG3, or IgG4); Preferably, the Fc peptide (e.g., the first Fc peptide and / or the second Fc peptide) is a Fc peptide derived from a human immunoglobulin (e.g., IgGl, IgG2, IgG3, or IgG4). Preferably, the Fc peptide (e.g., the first Fc peptide and / or the second Fc peptide) is a Fc peptide derived from a human immunoglobulin IgGl. The fusion protein of any one of claims 1-17, wherein, the single domain antibody or antigen binding fragment thereof that specifically binds to Ang-2 (e.g., the first single domain antibody or antigen binding fragment thereof that specifically binds to Ang-2 and / or the second single domain antibody or antigen binding fragment thereof that specifically binds to Ang-2) comprises a CDR1, a CDR2, and a CDR3 comprised in a VHH as set forth in SEQ ID NO: 248 or 256; Preferably, the single domain antibody or antigen binding fragment thereof that specifically binds to Ang-2 (e.g., the first single domain antibody or antigen binding fragment thereof that specifically binds to Ang-2 and / or the second single domain antibody or antigen binding fragment thereof that specifically binds to Ang-2) comprises a CDR1 as set forth in SEQ ID NO: 249; a CDR2 as set forth in SEQ ID NO: 250; and, a CDR3 as set forth in SEQ ID NO: 251 or 257; Preferably, the single domain antibody or antigen binding fragment thereof that specifically binds to Ang-2 (e.g., the first single domain antibody or antigen binding fragment thereof that specifically binds to Ang-2 and / or the second single domain antibody or antigen binding fragment thereof that specifically binds to Ang-2) comprises a heavy chain framework region derived from a human immunoglobulin (e.g., a heavy chain framework region comprised in an amino acid sequence encoded by a human heavy chain germline antibody gene); Preferably, the single domain antibody or antigen binding fragment thereof that specifically binds to Ang-2 (e.g., the first single domain antibody or antigen binding fragment thereof that specifically binds to Ang-2 and / or the second single domain antibody or antigen binding fragment thereof that specifically binds to Ang-2) comprises a CDR1, a CDR2, and a CDR3 comprised in a VHH as set forth in SEQ ID NO: 248 or 256. The fusion protein of any one of claims 1-18, comprising a sequence as set forth in any one of SEQ ID NOs: 243-247, 255; Preferably, the fusion protein comprises a first peptide chain and, optionally, a second peptide chain; wherein, the first peptide chain comprises a sequence as set forth in any one of SEQ ID NOs: 243-247, 255, and / or, the second peptide chain comprises a sequence as set forth in any one of SEQ ID NOs: 243-247, 255. An isolated nucleic acid molecule encoding the fusion protein of any one of claims 1-19. a vector comprising the isolated nucleic acid molecule of claim 20; preferably, the vector is a cloning vector or an expression vector. a host cell comprising the isolated nucleic acid molecule of claim 20 or the vector of claim 21. a method of making the fusion protein of any one of claims 1-19, comprising culturing the host cell of claim 22 under conditions that allow expression of the fusion protein, and recovering the fusion protein from the cultured host cell culture. a conjugate comprising the fusion protein of any one of claims 1-19 and a therapeutic agent linked to the fusion protein. a pharmaceutical composition comprising the fusion protein of any one of claims 1-19, the isolated nucleic acid molecule of claim 20, the vector of claim 21, the host cell of claim 22, or the conjugate of claim 24, and a pharmaceutically acceptable carrier and / or excipient; preferably, the pharmaceutical composition further comprises an additional pharmaceutically active agent. use of the fusion protein of any one of claims 1-19, the isolated nucleic acid molecule of claim 20, the vector of claim 21, the host cell of claim 22, the conjugate of claim 24, or the pharmaceutical composition of claim 25 for the manufacture of a medicament for the prevention and / or treatment of a disease associated with VEGF signaling in a subject; preferably, the prevention and / or treatment of the disease associated with VEGF signaling would benefit from antagonism of VEGF signaling; preferably, the disease associated with VEGF signaling is selected from the group consisting of diseases associated with abnormal angiogenesis; preferably, the disease associated with VEGF signaling is selected from the group consisting of: vascular origin eye diseases (e.g., macular degeneration, macular edema (e.g., wet age-related macular degeneration, macular edema following retinal vein occlusion, diabetic macular edema), retinal vein occlusion (e.g., central retinal vein occlusion, branch retinal vein occlusion), choroidal neovascular glaucoma, iridal neovascular glaucoma, neovascular glaucoma, post-surgical fibrosis in glaucoma, proliferative vitreoretinopathy, optic disc neovascularization, corneal neovascularization, retinal neovascularization, vitreous neovascularization, pannus, pterygium, chorioretinopathy, retinopathy of prematurity, vascular retinopathy, diabetic retinopathy), cancer (e.g., lung cancer, kidney cancer, uterine cancer, prostate cancer, bladder cancer, ovarian cancer, colon cancer, breast cancer, leukemia, lymphoma, myeloma, sarcoma, glioma, pancreatic cancer), autoimmune diseases (e.g., psoriasis, rheumatoid arthritis, psoriatic arthritis), and any combination thereof; preferably, the subject is a mammal, e.g., a human or a rabbit; preferably, the fusion protein, the isolated nucleic acid molecule, the vector, the host cell, the conjugate, or the pharmaceutical composition is used alone or in combination (e.g., concurrently or sequentially) with an additional pharmaceutically active agent. A method for preventing and / or treating a disease associated with VEGF signaling in a subject, comprising: administering to a subject in need thereof an effective amount of the fusion protein of any one of claims 1-19, the isolated nucleic acid molecule of claim 20, the vector of claim 21, the host cell of claim 22, the conjugate of claim 24, or the pharmaceutical composition of claim 25; Preferably, the prevention and / or treatment of a disease related to VEGF signaling would benefit from an antagonism of VEGF signaling; Preferably, the disease related to VEGF signaling is selected from a disease associated with abnormal angiogenesis; Preferably, the disease related to VEGF signaling is selected from: vascular origin eye diseases (e.g., macular degeneration, macular edema (e.g., wet age-related macular degeneration, macular edema following retinal vein occlusion, diabetic macular edema), retinal vein occlusion (e.g., central retinal vein occlusion, branch retinal vein occlusion), choroidal neovascular glaucoma, iridal neovascular glaucoma, neovascular glaucoma, post-surgical fibrosis in glaucoma, proliferative vitreoretinopathy, optic disc neovascularization, corneal neovascularization, retinal neovascularization, vitreous neovascularization, pannus, pterygium, chorioretinopathy, retinopathy of prematurity, vascular retinopathy, diabetic retinopathy), cancer (e.g., lung cancer, kidney cancer, uterine cancer, prostate cancer, bladder cancer, ovarian cancer, colon cancer, breast cancer, leukemia, lymphoma, myeloma, sarcoma, glioma, pancreatic cancer), autoimmune diseases (e.g., psoriasis, rheumatoid arthritis, psoriatic arthritis), and any combination thereof; Preferably, the subject is a mammal, such as a human or a rabbit; Preferably, the fusion protein, the isolated nucleic acid molecule, the vector, the host cell, the conjugate, or the pharmaceutical composition is used alone or in combination with (e.g., concurrently or sequentially with) another pharmaceutically active agent.

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