Fusion protein and use thereof

Abstract

Provided in the present invention are a polypeptide capable of inhibiting VEGF signaling, a pharmaceutical composition containing the polypeptide, and the related use.

Description

Fusion proteins and uses thereof

[0001] This application claims the benefit of China Application No. 202411109729.2, filed August 13, 2024, China Application No. 202411169931.4, filed August 23, 2024, China Application No. 202510124923.6, filed January 26, 2025, and China Application No. 202510361409.4, filed March 25, 2025, which are incorporated by reference in their entirety. TECHNICAL FIELD

[0002] The present application relates to the field of biological medicine, and more specifically, the present application relates to polypeptides capable of binding VEGF, pharmaceutical compositions comprising the polypeptides, 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 regulating 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 abnormal 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] The present inventors have developed new polypeptides targeting VEGF-A, VEGF-C and VEGF-D based on extensive research, and further constructed Fc fusion proteins based on the polypeptides (e.g., Fc fusion proteins capable of simultaneously binding VEGF-A, VEGF-C and VEGF-D). Based on the polypeptides or Fc fusion proteins simultaneously targeting VEGF-A, VEGF-C and VEGF-D multi-targets, the VHH antibody targeting Ang-2 is further fused to generate fusion proteins simultaneously targeting VEGF-A, VEGF-C, VEGF-D and Ang-2. In particular, the multispecific binding molecules (fusion proteins) of the present application have better protein yield, purity, physicochemical properties and affinity compared to wild-type fusion protein molecules, and have strong VEGF-A, VEGF-C and / or VEGF-D blocking activity (e.g., blocking activity of simultaneously blocking VEGF-A, VEGF-C and VEGF-D), which can block the reporter gene activity and primary cell proliferation activity induced by 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 can be effectively applied to the prevention and / or treatment of diseases related to VEGF (e.g., VEGF-A, VEGF-C and / or VEGF-D) signaling (e.g., diseases related to abnormal angiogenesis, such as vascular eye diseases), for example, to 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 polypeptide binding VEGF, comprising a first peptide segment and / or a second peptide segment, wherein 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 respective 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 respective 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 their receptors. 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 signaling pathways 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 signaling pathways 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 a second aspect, the present application provides a polypeptide construct comprising the polypeptide of the first aspect, and an Fc peptide and an optional linker peptide derived from an immunoglobulin.

[0657] In certain embodiments, the polypeptide is covalently linked to the Fc peptide and the optional linker peptide (e.g., covalently linked by covalent bonds comprising peptide bonds, isopeptide bonds and / or disulfide bonds).

[0658] In certain embodiments, the polypeptide construct comprises the polypeptide of the first aspect, and the Fc peptide and the linker peptide, and the polypeptide is covalently linked to the Fc peptide and the linker peptide by covalent bonds comprising peptide bonds.

[0659] In certain embodiments, the polypeptide construct comprises a first peptide chain comprising: a first polypeptide, a first hinge peptide, and a first Fc peptide, wherein the polypeptide is selected from the polypeptides of the first aspect. 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.

[0660] 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 of the first aspect.

[0661] 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.

[0662] In certain embodiments, the polypeptide construct possesses one or more of the features selected from:

[0663] (i) the first polypeptide is the same as or different from the second polypeptide;

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

[0665] (iii) the first Fc peptide and the second Fc peptide are derived from the same or different immunoglobulin;

[0666] (iv) the first hinge peptide is the same as or different from the second hinge peptide;

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

[0668] (vi) the first peptide chain is the same as or different from the second peptide chain;

[0669] (vii) the first peptide chain and the second peptide chain are connected by disulfide bond formation.

[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 an 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 a human immunoglobulin (e.g., IgGl, IgG2, IgG3, or IgG4).

[0672] 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.

[0673] As will be readily understood by those skilled in the art, 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).

[0674] In certain 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).

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

[0676] In certain embodiments, the Fc peptide (e.g., the first Fc peptide and / or the second Fc peptide) is an Fc peptide derived from a human immunoglobulin (e.g., IgGl, IgG2, IgG3, or IgG4).

[0677] In certain embodiments, the Fc peptide (e.g., the first Fc peptide and / or the second Fc peptide) is an Fc peptide derived from human immunoglobulin IgGl.

[0678] 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 an Fc peptide directly derived from an immunoglobulin (e.g., a human immunoglobulin) and an Fc peptide obtained by engineering or modifying an Fc peptide derived from an immunoglobulin (e.g., a human immunoglobulin) (e.g., an Fc peptide variant).

[0679] 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).

[0680] 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.

[0681] 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.

[0682] 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).

[0683] 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 first peptide chain and / or the second peptide chain.

[0684] 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 thereof.

[0685] 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)).

[0686] 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)).

[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)).

[0688] 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).

[0689] 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)).

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

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

[0692] In certain embodiments, the polypeptide construct has cross-binding activity to human, rabbit VEGF-A, VEGF-C and / or VEGF-D.

[0693] In certain embodiments, the polypeptide construct is capable of antagonizing the binding of VEGF-A, VEGF-C, and / or VEGF-D to its receptor. In certain embodiments, the polypeptide construct is capable of simultaneously antagonizing the binding of VEGF-A, VEGF-C, and VEGF-D to its receptor.

[0694] 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.

[0695] In a third aspect, the present application provides a fusion protein comprising the polypeptide of the first aspect or the polypeptide construct of the second aspect, and a single-domain antibody or an antigen-binding fragment thereof that specifically binds to Ang-2.

[0696] Based on the disclosure provided herein, one of skill in the art will readily appreciate that the fusion protein provided herein is used in its broadest sense and is intended to encompass any bi- or multi-specific molecule comprising the VEGF-binding polypeptide or polypeptide construct of the present application and a single-domain antibody or an antigen-binding fragment thereof that specifically binds to Ang-2.

[0697] In one aspect, the fusion protein of the present application is not limited in the number of its peptide chains. Based on the disclosure provided herein, one of skill in the art will readily appreciate that the fusion protein of the present application comprising the VEGF-binding polypeptide or polypeptide construct of the present application can consist of one or more (e.g., two) molecular chains (e.g., peptide chains).

[0698] In particular, in embodiments where the fusion protein of the present application comprises the VEGF-binding polypeptide construct of the present application, the VEGF-binding polypeptide construct provided by the present application can comprise only one molecular chain (e.g., peptide chain), or two or more molecular chains (e.g., peptide chains), as described in detail herein. Based on the disclosure provided herein, one of skill in the art will readily appreciate that the fusion protein of the present application comprising the 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 construct of the present application, which can consist of one or more (e.g., two) molecular chains (e.g., peptide chains).

[0699] In another aspect, the fusion proteins of the present application are not limited in the number of VEGF-binding polypeptides or polypeptide constructs 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 or polypeptide constructs and one or more single-domain antibodies or antigen-binding fragments thereof that specifically bind to Ang-2.

[0700] In another aspect, the fusion proteins of the present application are not limited in the manner in which the single-domain antibodies or antigen-binding fragments thereof that specifically bind to Ang-2 are fused to the VEGF-binding polypeptides or polypeptide constructs. In certain embodiments, the single-domain antibodies or antigen-binding fragments thereof that specifically bind to Ang-2 can be fused to the N-terminus and / or the C-terminus of the VEGF-binding polypeptides. In certain fusion protein embodiments comprising a hinge peptide, the single-domain antibodies or antigen-binding fragments thereof that specifically bind to Ang-2 can be fused between the VEGF-binding polypeptides and the hinge peptide, or, on the same side of the VEGF-binding polypeptides and the hinge peptide (e.g., on the N-terminus of the VEGF-binding polypeptides and the hinge peptide, or, on the C-terminus of the VEGF-binding polypeptides and the hinge peptide).

[0701] In certain embodiments, the single-domain antibodies or antigen-binding fragments thereof that specifically bind to Ang-2 can be fused to the N-terminus and / or the C-terminus of the VEGF-binding polypeptide constructs. In certain embodiments, the single-domain antibodies or antigen-binding fragments thereof that specifically bind to Ang-2 can be fused internally to the VEGF-binding polypeptide constructs; for example, the single-domain antibodies or antigen-binding fragments thereof that specifically bind to Ang-2 can be fused between the VEGF-binding polypeptides and the Fc peptides of the VEGF-binding polypeptide constructs; for example, the single-domain antibodies or antigen-binding fragments thereof that specifically bind to Ang-2 can be fused between the VEGF-binding polypeptides and the hinge peptides of the VEGF-binding polypeptide constructs.

[0702] In certain embodiments, the fusion proteins comprise one or more polypeptides of the first aspect and one or more single-domain antibodies or antigen-binding fragments thereof that specifically bind to Ang-2.

[0703] In certain embodiments, the fusion proteins comprise one or more polypeptides of the first aspect and one or more single-domain antibodies or antigen-binding fragments thereof that specifically bind to Ang-2.

[0704] In certain embodiments, the fusion protein comprises one or more polypeptide constructs of the second aspect, and one or more single-domain antibodies or antigen-binding fragments thereof that specifically bind to Ang-2. In certain embodiments, the fusion protein comprises the polypeptide of one or more polypeptide constructs of the second aspect, and one or more single-domain antibodies or antigen-binding fragments thereof that specifically bind to Ang-2.

[0705] In certain embodiments, the polypeptide of the first aspect or the polypeptide construct of the second aspect is covalently linked to the single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2 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).

[0706] 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 of the first aspect. In certain embodiments, the polypeptide of the first aspect 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.

[0707] In certain embodiments, the fusion protein comprises, in order from N-terminus to C-terminus: a polypeptide of the first aspect and the single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2. In certain embodiments, the fusion protein comprises, in order from N-terminus to C-terminus: the single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2 and a polypeptide of the first aspect.

[0708] In certain embodiments, the fusion protein comprises, in order from N-terminus to C-terminus: a polypeptide of the first aspect, a peptide linker, and the single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2. In certain embodiments, the fusion protein comprises, in order from N-terminus to C-terminus: the single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2, a peptide linker, and a polypeptide of the first aspect.

[0709] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2 is located at the N-terminus or C-terminus (e.g., C-terminus) of the polypeptide construct of the second aspect. In certain embodiments, the polypeptide construct of the second aspect 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.

[0710] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2 is located between the polypeptide of the polypeptide construct and the Fc peptide.

[0711] In certain embodiments, when the polypeptide construct of the second aspect 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 of the second aspect. 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 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.

[0712] In certain embodiments, when the polypeptide construct of the second aspect 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 of the second aspect. 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 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.

[0713] In certain embodiments, the fusion protein comprises a first peptide chain comprising: the first peptide chain of the polypeptide construct of the second aspect 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 of the second aspect 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 of the second aspect.

[0714] In certain embodiments, the fusion protein further comprises a second peptide chain comprising: the second peptide chain of the polypeptide construct of the second aspect 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 of the second aspect 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 of the second aspect.

[0715] In certain embodiments, the fusion protein comprises a first peptide chain comprising: the first peptide chain of the polypeptide construct of the second aspect, 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 of the second aspect, 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 of the second aspect.

[0716] In certain embodiments, the fusion protein further comprises a second peptide chain comprising: the second peptide chain of the polypeptide construct of the second aspect, 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 of the second aspect, 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 of the second aspect.

[0717] 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 of the first aspect. 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.

[0718] 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 of the first aspect. 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.

[0719] 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 single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2; 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 single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2.

[0720] 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 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; and / or, the second peptide chain 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.

[0721] 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 single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2, 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 single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2, a second hinge peptide, and a second Fc peptide.

[0722] In certain embodiments, 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.

[0723] 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 of the first aspect. 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.

[0724] 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 of the first aspect. 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.

[0725] 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 single-domain antibody or an antigen-binding fragment thereof that specifically binds to Ang-2, wherein the first polypeptide is selected from the polypeptides of the first aspect. 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 single-domain antibody or the antigen-binding fragment thereof that specifically binds to Ang-2, the second peptide linker, the first hinge peptide, and the first Fc peptide.

[0726] 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 single-domain antibody or an antigen-binding fragment thereof that specifically binds to Ang-2, wherein the second polypeptide is selected from the polypeptides of the first aspect. In certain embodiments, the fusion protein comprises, in order from N-terminus to C-terminus: the second polypeptide, the third peptide linker, the second single-domain antibody or the antigen-binding fragment thereof that specifically binds to Ang-2, the fourth peptide linker, the second hinge peptide, and the second Fc peptide.

[0727] In certain embodiments, the fusion protein comprises a first peptide chain comprising: a first polypeptide, a first peptide linker, a second peptide linker, a third peptide linker, and a first single-domain antibody or an antigen-binding fragment thereof that specifically binds to Ang-2, wherein the first polypeptide is selected from the polypeptides of the first aspect. In certain embodiments, the first peptide chain of the fusion protein comprises, in order from N-terminus to C-terminus: the first single-domain antibody or the antigen-binding fragment thereof that specifically binds to Ang-2, the first peptide linker, the first polypeptide, the second peptide linker, the first polypeptide, the third peptide linker, and the first single-domain antibody or the antigen-binding fragment thereof that specifically binds to Ang-2.

[0728] In certain embodiments, the fusion protein consists of the first peptide chain.

[0729] In certain embodiments, the fusion protein possesses one or more of the following features:

[0730] (i) the first polypeptide is the same as or different from the second polypeptide;

[0731] (ii) the first hinge peptide and the second hinge peptide are derived from the same or different immunoglobulin;

[0732] (iii) the first Fc peptide and the second Fc peptide are derived from the same or different immunoglobulin;

[0733] (iv) the first hinge peptide is the same as or different from the second hinge peptide;

[0734] (v) the first Fc peptide and the second Fc peptide are the same or different;

[0735] (vi) the first peptide chain and the second peptide chain are the same or different;

[0736] (vii) the first peptide chain and the second peptide chain are connected by a disulfide bond formation;

[0737] (viii) the first single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2 and the second single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2 are the same or different;

[0738] (ix) the first peptide linker, the second peptide linker, the third peptide linker, and the fourth peptide linker are the same or different.

[0739] 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).

[0740] 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).

[0741] 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.

[0742] As will be readily understood by one skilled in the art, 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).

[0743] In certain 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).

[0744] In certain embodiments, the Fc peptide (e.g., the first Fc peptide and / or the second Fc peptide) is an Fc peptide derived from an immunoglobulin (e.g., IgGl, IgG2, IgG3, or IgG4).

[0745] In certain embodiments, the Fc peptide (e.g., the first Fc peptide and / or the second Fc peptide) is an Fc peptide derived from a human immunoglobulin (e.g., IgGl, IgG2, IgG3, or IgG4).

[0746] In certain embodiments, the Fc peptide (e.g., the first Fc peptide and / or the second Fc peptide) is an Fc peptide derived from a human immunoglobulin IgGl.

[0747] As those skilled in the art will readily appreciate, the Fc peptide derived from an immunoglobulin (e.g., a human immunoglobulin) includes both an Fc peptide directly derived from an immunoglobulin (e.g., a human immunoglobulin) and an Fc peptide (e.g., an Fc peptide variant) obtained by engineering or modifying an Fc peptide derived from an immunoglobulin (e.g., a human immunoglobulin).

[0748] 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).

[0749] 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.

[0750] 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 (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 Kabat definition.

[0751] 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 (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 (e.g., 1, 2, 3, 4 or 5) amino acid substitutions, deletions, or additions compared to any one of the CDRs in (i).

[0752] 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);

[0753] 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 (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 set forth in SEQ ID NO: 248, or (iii) a sequence having one or several (e.g., 1, 2, 3, 4 or 5) amino acid substitutions, deletions, or additions compared to (i).

[0754] 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 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 set forth in any one of SEQ ID NOs: 243-247, 255, 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).

[0755] 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 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 set forth in any one of SEQ ID NOs: 243-247, and / or the second peptide chain comprises: (i) an amino acid sequence as set forth in any one of SEQ ID NOs: 243-247 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 set forth in any one of SEQ ID NOs: 243-247.

[0756] In certain embodiments, the fusion protein comprises the first peptide chain and optionally the 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.

[0757] In certain embodiments, the fusion protein comprises the first peptide chain and optionally the 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.

[0758] In certain embodiments, the fusion protein comprises the first peptide chain and optionally the 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.

[0759] 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.

[0760] 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.

[0761] In some embodiments, the fusion protein consists of one peptide chain. In some embodiments, the fusion protein comprises an amino acid sequence as set forth in SEQ ID NO: 255.

[0762] In a fourth aspect, the present application provides an isolated nucleic acid molecule encoding the polypeptide of the first aspect, the polypeptide construct of the second aspect, or the fusion protein of the third aspect.

[0763] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding the polypeptide of the first aspect.

[0764] In some embodiments, the isolated nucleic acid molecule encodes the polypeptide construct of the second aspect, which comprises a first polypeptide and a second polypeptide; the isolated nucleic acid molecule comprises a first nucleotide sequence encoding the first polypeptide and a second nucleotide sequence encoding the second polypeptide.

[0765] In some embodiments, the isolated nucleic acid molecule encodes the fusion protein of the third aspect, which comprises a first polypeptide and a second polypeptide; the isolated nucleic acid molecule comprises a first nucleotide sequence encoding the first polypeptide and a second nucleotide sequence encoding the second polypeptide.

[0766] In some embodiments, the first nucleotide sequence, the second nucleotide sequence are the same or different from each other.

[0767] In some embodiments, the first nucleotide sequence, the second nucleotide sequence are present on the same or different isolated nucleic acid molecules.

[0768] In a fifth aspect, the present application provides a vector comprising the isolated nucleic acid molecule of the fourth aspect. In some embodiments, the vector is a cloning vector or an expression vector.

[0769] In some embodiments, the vector comprises a nucleotide sequence encoding the polypeptide of the first aspect.

[0770] In certain embodiments, the vector encodes a polypeptide construct of the second aspect, the polypeptide construct 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.

[0771] In certain embodiments, the vector encodes a fusion protein of the third 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.

[0772] In certain embodiments, the first nucleotide sequence, the second nucleotide sequence are the same or different from each other.

[0773] In certain embodiments, the first nucleotide sequence, the second nucleotide sequence are present on the same or different vector molecules.

[0774] In a sixth aspect, the present application provides a host cell comprising the isolated nucleic acid molecule of the fourth aspect or the vector of the fifth aspect.

[0775] 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).

[0776] The polypeptide, polypeptide construct, or 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 polypeptide, polypeptide construct, or 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 polypeptide, polypeptide construct, or fusion protein of the present application is expressed.

[0777] In a seventh aspect, the present application provides a method of preparing the polypeptide of the first aspect, the polypeptide construct of the second aspect, or the fusion protein of the third aspect, comprising, culturing the host cell of the sixth aspect under conditions that allow expression of the polypeptide, the polypeptide construct, or the fusion protein, and recovering the polypeptide, the polypeptide construct, or the fusion protein from the culture of the cultured host cell.

[0778] In an eighth aspect, the present application provides a conjugate comprising the polypeptide of the first aspect, the polypeptide construct of the second aspect, or the fusion protein of the third aspect, and a therapeutic agent linked to the polypeptide, the polypeptide construct, or the fusion protein.

[0779] 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).

[0780] In a ninth aspect, the present application provides a pharmaceutical composition comprising the polypeptide of the first aspect, the polypeptide construct of the second aspect, the fusion protein of the third aspect, the isolated nucleic acid molecule of the fourth aspect, the vector of the fifth aspect, the host cell of the sixth aspect, or the conjugate of the eighth aspect, and a pharmaceutically acceptable carrier and / or excipient.

[0781] In certain embodiments, the pharmaceutical composition further comprises an additional pharmaceutically active agent.

[0782] 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).

[0783] In a tenth aspect, the present application provides use of the polypeptide of the first aspect, the polypeptide construct of the second aspect, the fusion protein of the third aspect, the isolated nucleic acid molecule of the fourth aspect, the vector of the fifth aspect, the host cell of the sixth aspect, the conjugate of the eighth aspect, or the pharmaceutical composition of the ninth aspect for the manufacture of a medicament for the prevention and / or treatment of a disease associated with VEGF (e.g., VEGF-A, VEGF-C, and / or VEGF-D) signaling in a subject.

[0784] 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.

[0785] 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.

[0786] 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.

[0787] In certain embodiments, the subject is a mammal, e.g., a human or a rabbit.

[0788] In certain embodiments, the polypeptide, polypeptide construct, 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 with) another pharmaceutically active agent.

[0789] 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).

[0790] In an eleventh 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 polypeptide of the first aspect, the polypeptide construct of the second aspect, the fusion protein of the third aspect, the isolated nucleic acid molecule of the fourth aspect, the vector of the fifth aspect, the host cell of the sixth aspect, the conjugate of the eighth aspect, or the pharmaceutical composition of the ninth aspect.

[0791] 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.

[0792] 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.

[0793] 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.

[0794] In certain embodiments, the subject is a mammal, e.g., a human or a rabbit.

[0795] In certain embodiments, the polypeptide, polypeptide construct, fusion protein, isolated nucleic acid molecule, vector, host cell, conjugate, or pharmaceutical composition is used alone or is administered in combination with (e.g., concurrently or sequentially) another pharmaceutically active agent.

[0796] 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).

[0797] The polypeptide or polypeptide construct or 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 injectable 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 polypeptide or polypeptide construct or 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 polypeptide or polypeptide construct or 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 injection solutions can be prepared as sterile lyophilized powders for reconstitution with a suitable solvent (e.g., 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) prior to use.

[0798] The polypeptide or polypeptide construct or 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, intraleaflet, intracisternal, inguinal, intravesical, local (e.g., powder, salve, or drops), or nasal routes. However, for many therapeutic uses, the preferred route of administration / means 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 of administration and / or means of administration will vary depending on the intended purpose. In certain embodiments, the polypeptide or polypeptide construct or fusion protein or conjugate or pharmaceutical composition of the present application is administered by intravenous injection or bolus.

[0799] Definitions of Terms

[0800] In the present application, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by a person of ordinary skill in the art. Also, the virology, biochemistry, immunology laboratory operation steps used herein are conventional steps widely used in the corresponding field. At the same time, in order to better understand the present application, the definitions and explanations of related terms are provided as follows.

[0801] When the terms "for example", "for instance", "such as", "including", "containing" or variations thereof are used herein, these terms shall not be considered as limiting, but shall be interpreted as meaning "but not limited to" or "without limitation".

[0802] Unless otherwise indicated herein or clearly contradicted by context, the terms "a" and "an" and "the" and similar referents in the context of describing the application (especially in the context of the following claims) are to be interpreted in an inclusive rather than an exclusive sense.

[0803] 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).

[0804] Those skilled in the art understand that VEGFR2 can include VEGFR2 of various species of origin, and there can be differences between the amino acid sequences of VEGFR2 of different species of origin. In the present application, when describing VEGFR2, it not only includes VEGFR2 as shown in SEQ ID NO: 238, but also includes VEGFR2 of various species of origin whose amino acid sequences are different from SEQ ID NO: 238.

[0805] 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.

[0806] 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).

[0807] 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.

[0808] 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.

[0809] 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.

[0810] Herein, unless the context clearly indicates otherwise, when referring to the term "single-domain antibody", this includes not only the intact single-domain antibody, but also antigen-binding fragments of the single-domain antibody.

[0811] 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 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.

[0812] 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.

[0813] 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.

[0814] 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 the dimerization between two Fc peptides contained by the two heavy chains.

[0815] 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 = number of identical overlapping positions / total number of positions x 100%). In certain embodiments, the two sequences are the same length.

[0816] 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.

[0817] 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. In addition, a vector can contain a replication origin.

[0818] 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.

[0819] 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.

[0820] Unless otherwise indicated herein, or otherwise clear from context, "A, B, and / or C" or similar statements are to be understood to mean "A, B, C, or any combination thereof," for example, it can be construed to mean any one selected from the group consisting of A, B, C, A and B, A and C, B and C, A and B and C.

[0821] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is compatible, in a pharmacological and / or physiological sense, 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 acids), 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.

[0822] As used herein, the term "prevent" refers to an approach taken to stop or delay the occurrence of a disease or disorder or symptoms thereof in a subject. As used herein, the term "treat" refers to an approach taken to obtain a beneficial or desired clinical result. For the purposes of the present 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.

[0823] 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.

[0824] 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.

[0825] Advantages of the Invention

[0826] 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

[0827] Figure 1A: Results of activity assays for VEGF Trap fusion protein mutants to inhibit hVEGF-A-induced HUVEC primary cell proliferation.

[0828] Figure IB: Results of activity assays for VEGF Trap fusion protein mutants to inhibit hVEGF-C-induced HUVEC primary cell proliferation.

[0829] Figure 1C: Results of activity assays for VEGF Trap fusion protein mutants to inhibit hVEGF-D-induced HUVEC primary cell proliferation.

[0830] Figures 2A-2V: Results of activity assays for VEGF Trap AI molecules to inhibit hVEGF-A-induced HEK293-hVEGFR2-hVEGFR3-Luc reporter cell activation.

[0831] Figures 3A-3W: Results of activity assays for VEGF Trap AI molecules to inhibit hVEGF-C-induced HEK293-hVEGFR2-hVEGFR3-Luc reporter cell activation.

[0832] Figures 4A-4X: Results of activity assays for VEGF Trap AI molecules to inhibit hVEGF-D-induced HEK293-hVEGFR2-hVEGFR3-Luc reporter cell activation.

[0833] Figures 5A-5B: Results of activity assays for VEGF Trap AI molecules to inhibit hVEGF-A-induced HUVEC primary cell proliferation.

[0834] Figures 6A-6B: Results of activity assays for VEGF Trap AI molecules to inhibit hVEGF-C-induced HUVEC primary cell proliferation.

[0835] Figures 7A-7B: Results of activity assays for VEGF Trap AI molecules to inhibit hVEGF-D-induced HUVEC primary cell proliferation.

[0836] Figures 8A-8C: Results of activity assays for AI-A1 molecules to inhibit hVEGF-A, hVEGF-C, hVEGF-D-induced HUVEC primary cell proliferation.

[0837] 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.

[0838] Figures 10A to 10C: Results of activity assay of AI-A6 molecule to inhibit hVEGF-A, hVEGF-C, hVEGF-D-induced HUVEC primary cell proliferation.

[0839] Figures 11A to 11C: Results of activity assay of AI-A1, AI-A3, AI-A4, AI-A5 molecules to inhibit hVEGF-A, hVEGF-C, hVEGF-D-induced HEK293-hVEGFR2-hVEGFR3-Luc reporter cell activation.

[0840] Figures 12A to 12C: Results of activity assay of AI-A6 molecule to inhibit hVEGF-A, hVEGF-C, hVEGF-D-induced HEK293-hVEGFR2-hVEGFR3-Luc reporter cell activation.

[0841] Figure 13: Results of activity assay of AI-A1, AI-A3, AI-A4, AI-A5 molecules to inhibit hAng-2-induced HEK293-hTie2-βgal reporter cell activation.

[0842] Figure 14: Results of activity assay of AI-A6 molecule to inhibit hAng-2-induced HEK293-hTie2-βgal reporter cell activation.

[0843] Figure 15: Improvement rate of leakage area of T002-AI2-93, T002-AI2-103 molecules in DL-α-AAA-induced in vivo RNV model.

[0844] Figure 16: Improvement rate of leakage area of AI-A1, AI-A6 molecules in DL-α-AAA-induced in vivo RNV model. DETAILED DESCRIPTION

[0845] 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).

[0846] 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, NY, 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.

[0847] Example 1: Preparation of Antigen Protein

[0848] 1.1 Transient expression of antigens

[0849] 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 with C-terminal fusion of 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 was performed using 293E cells (Gibco) in FreeStyle 293 Expression Medium (Thermo Fisher, 12338018). TM 6 The cells were seeded at 0.3-0.4 x 10 6 cells / mL in 500 mL cell culture flasks 24 hours before transfection and incubated at 37°C, 5% CO2, 120 rpm. The cells were transfected when the cell density reached 0.8-1.2 x 10 TM cells / mL. For transfection, 150 μL of 293fectin (life, 12347-500) was first added to 3 mL of Opti-MEM (Gibco, 31985070) and mixed well, and incubated at room temperature for 5 minutes. Meanwhile, 100 μg of pcDNA3.4 (Invitrogen) expression plasmids containing the coding sequences of hVEGF-A, hVEGF-C, and hVEGF-D antigens, respectively, were diluted to 3 mL with Opti-MEM. 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 well. The cell culture flasks were incubated at 37°C, 5% CO2, 120 rpm for 7 days. On the 3rd and 5th days after transfection, 7.5 mL of Peptone (BD, 211677) was added.

[0850] 1.2 Purification of antigens

[0851] ​hFc tag protein purification: The cell culture fluid was centrifuged at 2000g for 20 min, and the supernatant was collected and filtered with a 0.22-micron filter. The Prism A (Cytiva) affinity chromatography (protein purification liquid chromatography system: Cytiva, AKTA avant 25) was used for elution with 20 mM citric acid-sodium citrate (pH 3.2), and then the pH was adjusted to neutral with 1M Tris base. The purified sample was subjected to SDS-PAGE with 4-20% gradient gel to determine the protein purity.

[0852] His tag protein purification: The supernatant was collected and filtered with a 0.22-micron filter. The Ni column affinity chromatography was used, and the sample was loaded into a Ni chromatography column that had 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, elution was performed under the following conditions: (1) elution was performed with 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), sample collection was performed according to the ultraviolet absorption peak peak type, and after the ultraviolet absorption peak was stable and did not fluctuate, the second step of elution was performed; (2) elution was performed with 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), sample collection was performed according to the ultraviolet absorption peak peak type, and after the ultraviolet absorption peak was stable and did not fluctuate, the third step of elution was performed; (3) elution was performed with 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), sample collection was performed according to the ultraviolet absorption peak peak type, and after the ultraviolet absorption peak was stable and did not fluctuate, the collection was stopped. The purified sample was subjected to SDS-PAGE with 4-20% gradient gel for detection.

[0853] mFc tag protein purification: The supernatant was collected and filtered with a 0.22-micron filter, and the Prism A (GE) affinity chromatography was used for elution with 20 mM citric acid-sodium citrate, pH 3.0, and the pH was adjusted to neutral with 1M Tris base. The purified sample was subjected to SDS-PAGE with 4-20% gradient gel to determine the protein purity.

[0854] Example 2: Random mutation library

[0855] 2.1 Construction of random mutation library

[0856] 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.

[0857] 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℃.

[0858] 2.2 Random mutation library panning

[0859] 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.

[0860] 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.

[0861] The bacteria liquid after 1-4 rounds of screening 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.

[0862] 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.

[0863] Example 3: Combination mutation library

[0864] 3.1 Construction of combination mutation library

[0865] 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.

[0866] 3.2 Screening of the combinatorial mutation library

[0867] The screening method of the combinatorial mutation library was as described in the 2.2 section of Example 2.

[0868] 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).

[0869] Table 1: Combinatorial mutant molecules

[0870] Example 4: Preparation of VEGF Trap fusion protein

[0871] 4.1 Synthesis of VEGF Trap fusion protein plasmid

[0872] 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 the expression of the VEGF Trap fusion protein mutant.

[0873] 4.2 Transient expression of VEGF Trap fusion protein

[0874] Twenty-four hours before transfection, 2.4-2.6 x 10 6 The cell culture bottles were placed in a shaker at 37°C, 8% CO2, and 120 rpm for culture. 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 diluted into 4 mL of OptiPRO SFM (Thermo Fisher, 12309050) and incubated at room temperature for 30 s. Meanwhile, 100 μg of pcDNA3.4 expression plasmid containing the gene of interest was diluted into 4 mL of OptiPRO SFM and incubated at room temperature for 30 s. The diluted transfection reagent and plasmid were mixed well and incubated at room temperature for 1-2 min. Then, the mixture was added into the cells and mixed well. The culture flask was incubated at 37 °C, 8% CO2, 120 rpm for 18-22 h. TM CHO Reagent (Thermo Fisher, A29130) was added into 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 into 4 mL of OptiPRO SFM and incubated at room temperature for 30 s. The diluted transfection reagent and plasmid were mixed well and incubated at room temperature for 1-2 min. Then, the mixture was added into the cells and mixed well. The culture flask was incubated at 37 °C, 8% CO2, 120 rpm for 18-22 h. TM CHO Reagent (Thermo Fisher, A29130) was added into 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 into 4 mL of OptiPRO SFM and incubated at room temperature for 30 s. The diluted transfection reagent and plasmid were mixed well and incubated at room temperature for 1-2 min. Then, the mixture was added into the cells and mixed well. The culture flask was incubated at 37 °C, 8% CO2, 120 rpm for 18-22 h. TM CHO Reagent (Thermo Fisher, A29130) was added into 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 into 4 mL of OptiPRO SFM and incubated at room temperature for 30 s. The diluted transfection reagent and plasmid were mixed well and incubated at room temperature for 1-2 min. Then, the mixture was added into the cells and mixed well. The culture flask was incubated at 37 °C, 8% CO2, 120 rpm for 18-22 h. TM CHO Reagent (Thermo Fisher, A29130) was added into 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 into 4 mL of OptiPRO SFM and incubated at room temperature for 30 s. The diluted transfection reagent and plasmid were mixed well and incubated at room temperature for 1-2 min. Then, the mixture was added into the cells and mixed well. The culture flask was incubated at 37 °C, 8% CO2, 120 rpm for 18-22 h.

[0875] 4.3 Purification of VEGF Trap fusion protein

[0876] The cell culture solution was centrifuged at 2000 g for 20 min, and the supernatant was collected and filtered through a 0.22-μm 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.

[0877] Example 5: Purity, physicochemical properties, and affinity of VEGF Trap fusion protein

[0878] 5.1 Determination of protein purity by size exclusion chromatography (SEC-HPLC)

[0879] 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.

[0880] Table 2: VEGF Trap fusion protein expression and purification detection results

[0881] 5.2 VEGF Trap fusion protein thermal stability and hydrophobicity detection

[0882] 5.2.1 Protein solubility temperature characterization detection

[0883] 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.

[0884] 5.2.2 Hydrophobic chromatography HIC detection

[0885] 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.

[0886] The results show that the VEGF Trap fusion protein mutants have good hydrophobicity and better thermal stability than T002.

[0887] Table 3: VEGF Trap fusion protein hydrophobicity and thermal stability detection results

[0888] 5.3 VEGF Trap fusion protein and VEGF-A, VEGF-C, VEGF-D antigen affinity determination

[0889] Using Sensor Chip Protein A biosensor chip (Cytiva, 29-1275-56), the VEGF Trap fusion protein was immobilized on the chip as the ligand, 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. Kinetic fitting was performed using Biacore 8K Evaluation (Cytiva), and the affinity constants are shown in Table 4. The controls used were Eylea (derived from CN103349781B, with an amino acid sequence as SEQ ID NO: 229), OPT-302 (derived from US9745558B2, with an amino acid sequence as SEQ ID NO: 230).

[0890] The results show that the VEGF Trap fusion protein mutants have good affinity with hVEGF-A, hVEGF-C, and hVEGF-D.

[0891] Table 4: VEGF Trap fusion protein mutant and hVEGF-A, hVEGF-C, hVEGF-D antigen affinity detection results

[0892] Example 6: Activity assay for inhibiting HUVEC primary cell proliferation induced by VEGF-A, VEGF-C, VEGF-D

[0893] 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.

[0894] 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.

[0895] The results show that T002-P44910 presents a dose-dependent inhibition of HUVEC primary cell proliferation.

[0896] Table 5: Activity assay results for VEGF Trap fusion protein inhibiting HUVEC primary cell proliferation induced by hVEGF-A, hVEGF-C, and hVEGF-D

[0897] Note: “NA” indicates no inhibitory activity, and “ / ” indicates not detected.

[0898] Example 7: Construction of VEGF Trap AI molecules and combination mutants

[0899] 7.1 Design and identification of VEGF Trap AI molecules and combination mutants

[0900] 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 the mutation site of each mutation contained in each mutant is described by the sequence of VEGFR2 (SEQ ID NO: 238). The T002 mutant designed based on AI above 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.

[0901] Table 6: VEGF Trap AI molecules

[0902] Table 7: Expression and purification detection results of VEGF Trap AI molecules

[0903] Table 8: Hydrophobicity and thermal stability detection results of VEGF Trap AI molecules

[0904] Example 8: Assay of VEGF Trap AI molecules for inhibiting VEGF-induced activation of HEK293-hVEGFR2-hVEGFR3-Luc reporter cells

[0905] 8.1 Preparation of HEK293-VEGFR2-VEGFR3 overexpression cell line

[0906] The human VEGFR2 (KDR) protein coding gene (UniProt, Gene ID: CCDS3497.1) was ligated to the pLVX-IRES-Puro (OriGene, A3005) plasmid to construct a 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 a lentivirus plasmid of hVEGFR3 protein.

[0907] The lentivirus was packaged using a three-plasmid system (envelope plasmid pCMV-VSVG, packaging plasmid pCMV-dr8.91 and the above-mentioned pLVX plasmid), then concentrated, and finally obtained a lentivirus solution. The above-mentioned two lentivirus solutions were used to infect HEK293-NFAT-RE-luc2p cells (Promega, R8510, a luciferase reporter cell line with NFAT response element). After infection, the infected cells were subjected to pressure screening by 400 μg / mL geneticin (GENETICIN, Gibco), 0.5 μg / mL puromycin (PUROMYCIN, Invitrogen), and a 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.

[0908] 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

[0909] 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.

[0910] 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)].

[0911] 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.

[0912] 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

[0913] Note: “ / ” means not detected; “NA” means no inhibitory activity.

[0914] 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

[0915] Example 9: Activity assay of VEGF Trap AI molecule to inhibit HUVEC primary cell proliferation induced by VEGF-A, VEGF-C, VEGF-D

[0916] Activity assay of VEGF Trap AI molecule to inhibit hVEGF-A, hVEGF-C, hVEGF-D induced HUVEC primary cell proliferation 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.

[0917] Table 11: Activity assay results of VEGF Trap AI molecule to inhibit hVEGF-A, hVEGF-C, hVEGF-D induced HUVEC primary cell proliferation

[0918] Note: " / " means not detected; "NA" means no inhibitory activity.

[0919] Example 10: Design and identification of quadruple blocking fusion protein molecule

[0920] The above VEGF Trap AI molecule, hinge peptide, Fc peptide and anti-Ang-2 VHH (the sequence of VHH is shown in SEQ ID NO: 248, and the sequences of CDR1-3 are shown in SEQ ID NO: 249-251) are combined to obtain a quadruple blocking fusion protein molecule that can simultaneously block VEGF-A, VEGF-C, VEGF-D and Ang-2.

[0921] 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, the sequence of which is shown in SEQ ID NO: 243.

[0922] 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), a Fc peptide (SEQ ID NO: 239), a G4S linker (SEQ ID NO: 252), a VHH against Ang-2, the sequence of which is shown in SEQ ID NO: 244.

[0923] 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), a Fc peptide (SEQ ID NO: 239), a G4S linker (SEQ ID NO: 252), a VHH against Ang-2, the sequence of which is shown in SEQ ID NO: 245.

[0924] 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, a G4S linker (SEQ ID NO: 252), a hinge peptide (SEQ ID NO: 233), a Fc peptide (SEQ ID NO: 239), the sequence of which is shown in SEQ ID NO: 246.

[0925] 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, a hinge peptide (SEQ ID NO: 242), a Fc peptide (SEQ ID NO: 239), the sequence of which is shown in SEQ ID NO: 247.

[0926] The structure of the AI-A6 molecule from N-terminus to C-terminus is anti-Ang-2 VHH, G10 linker (SEQ ID NO: 254), VEGFR2 D2D3 domain of T002-AI2-93 (amino acid sequence shown in positions 1-208 of SEQ ID NO: 209), G4S linker (SEQ ID NO: 252), VEGFR2 D2D3 domain of T002-AI2-93 (amino acid sequence shown in positions 1-208 of SEQ ID NO: 209), G10 linker (SEQ ID NO: 254), anti-Ang-2 VHH, and the sequence is shown in SEQ ID NO: 255.

[0927] The fusion protein was constructed with reference to Examples 4 and 5, and after gene synthesis, vector construction and extraction, the CHO-S cells were transiently transfected, and the Prism A (Cytiva) affinity chromatography was used to obtain the fusion protein. The expression and purification, hydrophobicity and thermal stability detection results of the fusion protein are shown in Tables 12 and 13. The results show that the quadruple blocking fusion protein molecule has good yield and purity, and has good hydrophobicity and thermal stability.

[0928] Table 12: Expression and purification detection results of quadruple blocking fusion protein

[0929] Table 13: Hydrophobicity and thermal stability detection results of quadruple blocking fusion protein

[0930] Example 11: Activity determination of quadruple blocking fusion protein for inhibiting HUVEC primary cell proliferation induced by VEGF-A, VEGF-C and VEGF-D

[0931] The activity determination of the quadruple blocking fusion protein molecule for inhibiting HUVEC primary cell proliferation induced by hVEGF-A, hVEGF-C and hVEGF-D was carried out with reference to Example 6, and the results are shown in Figures 8A-8C, 9A-9C, 10A-10C and Tables 14-16. The results show that the quadruple blocking fusion protein molecule presents dose-dependent inhibition of HUVEC primary cell proliferation.

[0932] Table 14: Activity determination results of AI-A1 for inhibiting HUVEC primary cell proliferation induced by hVEGF-A, hVEGF-C and hVEGF-D

[0933] Note: "NA" means no inhibitory activity, " / " means not detected, "Not fitted" means unable to fit.

[0934] Table 15: Results of activity assay of AI-A3, AI-A4, AI-A5 to inhibit hVEGF-A, hVEGF-C, hVEGF-D induced HUVEC primary cell proliferation

[0935] Note: "NA" means no inhibitory activity, " / " means not tested, "Not fitted" means not fitted.

[0936] Table 16: Results of activity assay of AI-A6 to inhibit hVEGF-A, hVEGF-C, hVEGF-D induced HUVEC primary cell proliferation

[0937] Note: "NA" means no inhibitory activity, " / " means not tested, "Not fitted" means not fitted.

[0938] Example 12: Activity assay of quadruple-blockade fusion protein to inhibit VEGF-A, VEGF-C, VEGF-D induced HEK293-hVEGFR2-hVEGFR3-Luc reporter cell activation

[0939] The activity assay of quadruple-blockade fusion protein molecules to inhibit hVEGF-A, hVEGF-C, hVEGF-D induced HEK293-hVEGFR2-hVEGFR3-Luc reporter cell activation was performed according to Example 8, and the positive control antibody Faricimab was derived from Roche, batch number B1535B01. The results are shown in Figures 11A-11C, 12A-12C and Tables 17-18. The results show that the quadruple-blockade fusion protein molecules exhibit dose-dependent blockade of hVEGF-A, hVEGF-C and hVEGF-D induced HEK293-hVEGFR2-hVEGFR3-Luc cell signaling pathway activation.

[0940] Table 17: Results of activity assay of quadruple-blockade fusion protein to inhibit hVEGF-A, hVEGF-C, hVEGF-D induced HEK293-hVEGFR2-hVEGFR3-Luc reporter cell activation

[0941] Note: "NA" means no inhibitory activity, " / " means not tested.

[0942] Table 18: Results of activity assay of AI-A6 to inhibit hVEGF-A, hVEGF-C, hVEGF-D induced HEK293-hVEGFR2-hVEGFR3-Luc reporter cell activation

[0943] Note: "NA" means no inhibitory activity, " / " means not detected.

[0944] Example 13: Activity assay for inhibiting hAng-2-induced activation of HEK293-hTie2-βgal reporter cells

[0945] HEK293-hTie2-βgal cells [Discover X, 93-1024C1, a ProLink TM (PK) tagged RTK, enzyme receptor (EA) tagged SH2 domain and untagged co-receptor, activation of the RTK-PK generates functional enzyme to hydrolyze a substrate to produce a chemiluminescent signal] were purchased and subjected to stress culture by 400 μg / mL GENETICIN (Gibco) and 200 μg / mL Hygromycin B (Invitrogen) after purchase. 1 x 10 4 cells / well were seeded in 96-well full white plates (CORNING, 3917) using DMEM (Gibco, C12430500BT) experimental medium containing 1% FBS (Gibco, 10091-148).

[0946] hAng-2 (SinoBio, Cat No: 10691-H02H) was diluted in experimental medium to a final concentration of 1 μg / ml, 25 μl / well was added as the control group. On the basis of the control group, AI-A1, AI-A3, AI-A4, AI-A5, AI-A6, positive control Faricimab and negative control hIgG1 isotype were diluted in experimental medium to a maximum concentration of 1500 nM, 5-fold dilution, 8 or 9 concentration gradients, and 25 μl / well was added as the sample group. At the same time, the blank group was set up by adding the same volume of experimental medium. Incubate in a 37°C, 5% CO2 incubator for 24 hours. Add 100 μL / well of the Luciferase Assay System (Promage, E4740), avoid light, room temperature for 1 h, and use the SpectraMax iD5 multifunctional microplate reader (Molecular Devices) to detect the luminescence value. Reporter gene inhibition rate (%) = 100% x [1- (RLU sample group-RLU blank group) / (RLU control group-RLU blank group)].

[0947] ​The results are shown in FIGS. 13 and 14 and Tables 19-20, which show that AI-A1, AI-A3, AI-A4, AI-A5, and AI-A6 all exhibited dose-dependent blocking of hAng-2-induced activation of the signaling pathway of HEK293-hTie2-βgal reporter cells, and had stronger blocking activity than the marketed Faricimab.

[0948] Table 19: Results of activity determination of quadruple blocking fusion proteins in inhibiting hAng-2-induced activation of HEK293-hTie2-βgal reporter cells

[0949] Note: "NA" indicates no inhibitory activity, and " / " indicates not detected.

[0950] Table 20: Results of activity determination of AI-A6 in inhibiting hAng-2-induced activation of HEK293-hTie2-βgal reporter cells

[0951] Note: "NA" indicates no inhibitory activity, and " / " indicates not detected.

[0952] Example 14: DL-α-AAA-induced rabbit model of retinal neovascularization

[0953] In this experiment, a DL-α-aminoadipic acid (DL-α-AAA)-induced rabbit model of retinal neovascularization (RNV) was used to determine the anti-neovascularization effect of the molecules of the application.

[0954] In this experiment, 80 μL of DL-α-AAA solution was injected into the vitreous cavity of both eyes of the rabbits in a single injection to induce retinal neovascularization in the fundus, and an animal model similar to human fundus neovascularization was established. 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. At the same time, a Zeiss Fovea 300 fundus camera (Carl Zeiss Meditec, Inc., Germany) was used to collect images of the fundus of the animals. 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. At the same time, a Zeiss Fovea 300 fundus camera (Carl Zeiss Meditec, Inc., Germany) was used to collect images of the fundus of the animals. HRA+OCT, Fluorescein Angiography (FFA) examination was performed on the animals, and FFA early and late images of the temporal, central and nasal nerve fiber layer were collected. The early image was taken within about 1.5 min, and the late image was taken within about 1.5-3 min. The field of view was 102°. Whether RNV was generated and leaked was determined according to whether there was fluorescence leakage in the fundus of the animal. The area of fluorescence leakage was measured using Heidelberg Eye Explorer software (SPECTRALIS Software Version 6.12), and the reduction in the area of fluorescence leakage and the improvement rate of the area of fluorescence leakage were calculated:

[0955] Reduction in the area of fluorescence leakage = area of fluorescence leakage before administration - area of fluorescence leakage after administration;

[0956] Improvement rate of the area of fluorescence leakage = reduction in the area of fluorescence leakage / area of fluorescence leakage before administration x 100%.

[0957] According to the area of retinal leakage detected by FFA, the animals were evenly divided into groups. The details of the grouping and administration are shown in Tables 21 and 22. Each group had 6-7 rabbit eyes, and each eye was injected with the drug in the vitreous body. The fundus without red light images and fundus fluorescence angiography of the animals were collected at different time points after administration, and the inhibition of choroidal neovascularization by the test product was observed.

[0958] Table 21: Administration scheme

[0959] Table 22: Administration scheme

[0960] The results of fundus without red light images and fundus fluorescence angiography are shown in Figures 15 and 16. The results of Figure 15 show that T002-AI2-93 and T002-AI2-103 showed significant anti-neovascularization effects after 14 days of administration, and the inhibition of vascular leakage could last for 84 days after administration, which was better than that of the and groups, proving that the molecules T002-AI2-93 and T002-AI2-103 of the application have obvious inhibitory effects on neovascularization. The results of Figure 16 show that AI-A1 and AI-A6 showed significant anti-neovascularization effects after 14 days of administration, and the inhibition of vascular leakage could last for 84 days after administration, which was better than that of the and groups, proving that the molecules AI-A1 and AI-A6 of the application have obvious inhibitory effects on neovascularization.

[0961] While the specific embodiments of the application have been described in detail, those skilled in the art will appreciate that various modifications and alterations to the details can be made within the scope of the application as disclosed in the above teachings and that such modifications and alterations are intended to fall within the scope of the application. The scope of the application is therefore indicated by the appended claims, and any equivalents thereof.

[0962] Sequence information

Claims

1. A polypeptide that binds VEGF, comprising a first peptide segment and / or a second peptide segment, wherein, the first peptide segment comprises immunoglobulin-like domain 2 (R2D2) of VEGFR2 (vascular endothelial growth factor receptor 2), and the second peptide segment comprises 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.

2. The polypeptide 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.

3. The polypeptide 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.

4. The polypeptide 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.

5. The polypeptide 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.

6. The polypeptide 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.

7. The polypeptide of any one of claims 1-6, comprising an amino acid sequence as set forth in positions 1-208 of any one of SEQ ID NOs: 2-29, 31-108, 110-228.

8. An isolated nucleic acid molecule encoding the polypeptide of any one of claims 1-7.

9. A vector comprising the isolated nucleic acid molecule of claim 8; preferably, the vector is a cloning vector or an expression vector.

10. A host cell comprising the isolated nucleic acid molecule of claim 8 or the vector of claim 9.

11. A method of making the polypeptide of any one of claims 1-7, comprising culturing the host cell of claim 10 under conditions that allow expression of the polypeptide, and recovering the polypeptide from the cultured host cell culture.

12. A conjugate comprising the polypeptide of any one of claims 1-7 and a therapeutic agent linked to the polypeptide.

13. A pharmaceutical composition comprising the polypeptide of any one of claims 1-7, the isolated nucleic acid molecule of claim 8, the vector of claim 9, the host cell of claim 10, or the conjugate of claim 12, and a pharmaceutically acceptable carrier and / or excipient; Preferably, the pharmaceutical composition further comprises an additional pharmaceutically active agent.

14. Use of the polypeptide of any one of claims 1-7, the isolated nucleic acid molecule of claim 8, the vector of claim 9, the host cell of claim 10, the conjugate of claim 12, or the pharmaceutical composition of claim 13, for the manufacture of a medicament for preventing and / or treating 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, iridoneovascular 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, psoriasis), and any combination thereof; Preferably, the subject is a mammal, e.g., a human or a rabbit. Preferably, the polypeptide, isolated nucleic acid molecule, vector, host cell, conjugate or pharmaceutical composition is used alone or in combination with (e.g. simultaneously or sequentially) another pharmaceutically active agent.

15. 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 polypeptide of any one of claims 1-7, the isolated nucleic acid molecule of claim 8, the vector of claim 9, the host cell of claim 10, the conjugate of claim 12 or the pharmaceutical composition of claim 13; Preferably, the prevention and / or treatment of a disease associated with VEGF signaling would benefit from antagonism of VEGF signaling; Preferably, the disease associated with VEGF signaling is selected from a disease associated with abnormal angiogenesis; Preferably, the disease associated with VEGF signaling is selected from: 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 in 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; Preferably, the subject is a mammal, e.g. a human or a rabbit; Preferably, the polypeptide, isolated nucleic acid molecule, vector, host cell, conjugate or pharmaceutical composition is used alone or in combination with (e.g. simultaneously or sequentially) another pharmaceutically active agent.

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