Bispecific binding molecule binding VEGF and ANG2 and use thereof

Abstract

Provided are an antibody against a vascular endothelial growth factor (VEGF / VEGF-A) and angiopoietin-2 (ANG-2) respectively, a bispecific binding molecule (for example, an antibody) simultaneously against VEGF / VEGF-A and ANG-2, a preparation method therefor, and a pharmaceutical composition containing the antibody or molecule and an application thereof.

Description

Negative control IgG: prepared as described above; Positive control Faricimab: prepared as described above, with an initial concentration of 13.5 ug / mL and being subjected to gradient dilution at a 1:3 ratio; Positive control BI-836880: prepared as described above, with an initial concentration of 13.5 ug / mL and being subjected to gradient dilution at a 1:3 ratio; and IEX04-012: prepared as described above, with an initial concentration of 13.5 ug / mL and being subjected to gradient dilution at a 1:3 ratio. The detection results in FIG. 7 show that the bispecific binding molecule IEX04-012 inhibits the activation of VEGF-induced KDR signaling pathways, and has better inhibitory ability compared with the control antibody BI836880. Example 10. HUVEC Proliferation Inhibition Assay of Anti-VEGF A / Ang2 Bispecific Binding Molecules In this research, the inhibitory effect of the anti-VEGF A / Ang2 bispecific binding molecules on the VEGF A-induced survival and proliferation of HUVEC cells was detected by HEK293-KDR reporter assay. The experimental procedures were as those in Example 8, and the antibodies used were as follows: Panel A: IEX04-008, negative control IgG, positive controls Faricimab and BI836880, Blank (i.e., no antibody and VEGF A), and VEGF A group (i.e., only 20 ng / mL of VEGFA added) prepared as described above with initial concentrations of 20 ug / mL and being subjected to serial dilution at a 1:3 ratio; Panel B: negative control IgG, BI-anti-VEGF, IEX04-010, Blank (i.e., no antibody and VEGF A), and VEGF A group (i.e., only 20 ng / mL of VEGFA added) prepared as described above with initial concentrations of 80 ug / mL and being subjected to serial dilution at a 1:3 ratio; and Panel C: IEX04-012, negative control IgG, positive controls Faricimab and BI836880, Blank (i.e., no antibody and VEGF A), and VEGF A group (i.e., only 20 ng / mL of VEGFA added) prepared as described above with initial concentrations of 20 nM and being subjected to serial dilution at a 1:3 ratio. The detection results in FIG. 8 show that the bispecific binding molecules IEX04-008, IEX04-010 and IEX04-012 all inhibit VEGF-induced survival and proliferation of HUVEC cells, and IEX04-012 has lower IC50 and better inhibitory ability compared with the control antibodies BI836880 and Faricimab. Example 11. Ang2 Blocking Assay of Anti-VEGF A / Ang2 Bispecific Antibodies Ang2 could bind to its native receptor Tie2, and this research detected the blocking effect of the anti-VEGF A / Ang2 bispecific binding molecules on the binding between Ang2 and Tie2 by ELISA assay and FACS assay. (1) ELISA The ability of IEX04-008, IEX04-010, IEX04-012, and control antibodies BI-836880 and Faricimab to block the binding of human Ang2 to hTie2 was detected by ELISA. The hTie2 protein (Beijing Sino Biological) was resuspended and dissolved to a concentration of 2 ug / mL with PBS, and a microplate was coated with the protein and incubated overnight. The plate was blocked with 5% BSA for 1 h, and a biotinylated antigen (Recombinant Biotinylated hAngiopoietin-2 protein (R&D)) was diluted to 600 ug / mL and added to the plate at 50 uL / well. The antibodies prepared as described above (IEX04-008, IEX04-010, IEX04-012, positive control antibodies BI836880 and Faricimab, and negative control IgG) were each diluted serially at a 1:2 ratio starting from the maximum concentration of 300 nM for a total of 8 or 12 dilution gradients. The diluted antibodies were added at 50 uL / well and incubated in PBS on ice for 30 min, and the biotinylated antigen had a final concentration of 300 ng / mL. The antigen-antibody mixture obtained as described above was incubated in the microplate for 90 min and washed three times with PBS, and the supernatant was discarded. 100 uL of Avidin-HRP (Invitrogen) diluted at a 1:10000 ratio was added to each well, incubated at room temperature for 30 min, and washed six times with PBS. ATMB chromogenic solution (solarbio) was added to the plate at 100 pL / well for color development for 1 min, and the reaction was stopped by adding a stop solution (Solarbio) at 100 pL / well. The plate was read on a microplate reader at OD450 and OD620 per well. The experimental results (see FIG. 9) show that IEX04-008, IEX04-010, IEX04-012, and the control antibody BI836880 all have a complete blocking effect, and that IC50 of the bispecific binding molecules of the present invention are significantly smaller than that of the positive control antibody. (2) Flow cytometry assay (FACS) The ability of IEX04-008, IEX04-010, IEX04-012, positive control antibodies BI-836880 and Faricimab, and negative control IgG to block the binding of human Ang2-hFc to Tie2 on the cell surface was detected by FACS. The antigen hAng2-Fc protein (Beijing Sino Biological, Catalog No. 10691-H02H) was diluted to 4 ug / mL and added at 50 pL / well. The antibodies prepared as described above (IEX04-008, IEX04-010, IEX04-012, positive control antibodies BI-836880 and Faricimab, and negative control IgG) were each 2-fold diluted gradiently starting from the maximum concentration of 800 nM for a total of 12 dilution gradients. The diluted antibodies were added at 50 uL / well and incubated in PBS on ice for 30 min, the antigen hAng2-Fc protein had a final concentration of 2 ug / mL, and each antibody had a maximum final concentration of 400 nM. 293-Tie2 cells prepared as described above were adjusted to 2 x 105 cells / well and added at 100 pL / well. The cells were centrifuged at 300 g for 5 min. The supernatant was discarded. Then the cells were resuspended in an antigen-antibody mixture. The mixture was incubated on ice for 30 min. PBS was added at 100 pL / well. After centrifugation at 300 g for 5 min, the mixture was washed once with PBS. 100 pL of goat anti-human IgG-PE (Southern Biotech) diluted at a 1:200 ratio was added to each well. After a 20-min ice bath, PBS was added at 100 pL / well. The mixture was centrifuged at 300 g for 5 min, prior to one wash with PBS. The cells were resuspended with 100 pL of PBS. The cell fluorescence signal values were measured by a flow cytometer (BD Biosciences). According to MFI, the concentration-dependent curve was fitted with GraphPad. The results are shown in FIG. 10. It is shown that the bispecific binding molecules IEX04-008, IEX04-010, and IEX04-012 can all effectively block the binding of human Ang2-hFc to Tie2, and have IC50 lower than that of the positive control. Example 12. Ang2 Phosphorylation Inhibition Assay of Anti-VEGF A / Ang2 Bispecific Binding Molecules This example demonstrates the inhibitory effect of the bispecific binding molecules of the present invention on hAng2-Fc-induced Tie2 phosphorylation by using hAng2-induced phosphorylation assay. In this research, Expi293 cells overexpressing Tie2, 293-Tie2, were co-incubated with the bispecific binding molecules and a recombinant hAng2-Fc protein, and the content of phosphorylated Tie2 in the system was detected, thereby reflecting the inhibitory effect of different antibodies on the hAng2-Fc-induced Tie2 phosphorylation. The overexpressed 293-Tie2 cells prepared above were diluted to 2 x 106 cells / mL and added to a 96-well plate at 100 pL per well, the plate was centrifuged at 400 g for 5 min, and the supernatant was discarded. An experimental medium was prepared using Expi293 medium (Thermo, Catalog No. A1435102), in which test antibodies (IEX04-012 prepared above, positive controls BI-836880 and Faricimab, and negative control IgG) with maximum final concentration of 60 pg / mL were each added and then serially diluted at a 1:2 ratio; the final concentration of hAng2-Fc (Beijing Sino Biological, Catalog No. 10691-H02H) was 2.5 pg / mL. The cells were resuspended with 100 pL of the experimental medium per well and incubated at 37 °C for 15 min. The medium was removed by centrifugation. 100 pL of NP-40 lysis buffer containing 1% protease and a 30 phosphatase inhibitor was added, and the plate was left to stand on ice for 30 min. The plate was centrifuged at 2000 g, and the protein supernatant was collected and stored in a refrigerator at -80 °C. The concentration of pTie2 was detected according to the instruction of a phosphorylated Tie2 ELISA kit (R&D, DYC2720E), and a microplate was coated with a capture antibody at a concentration of 4 ug / mL and incubated at 4 °C overnight. The plate was washed three times with PBST and blocked with 5% BSA for 1 h. 100 uL of the sample to be detected and a control pTie2 (R&D, DYC2720E) were added to make a standard curve, and the mixture was incubated at room temperature for 2 h (if the concentration of pTie2 in the sample was too high and exceeded the ELISA detection range, the mixture obtained could be subjected to 2- to 3-fold dilution). The plate was washed three times with PBST, 100 uL of anti-pTyr antibody conjugated with HRP (R&D, Catalog No. DYC2720E) was added, and the mixture was incubated at room temperature for 2 h. The plate was washed 6 times with PBST, 100 uL of TMB was added for color development, and after 15 min, 100 uL of stop buffer was added to stop the reaction. OD450-OD620 of each well was measured using a spectrophotometer. The experimental results are shown in FIG. 11. The antibody IEX04-012 of the present invention can effectively inhibit hAng2-Fc-induced 293-Tie2 phosphorylation in vitro, and IC50 is superior to that of the positive control. Example 13. Inhibition Assay of Ang2 Vascular Endothelial Cell Leakage by Anti-VEGF A / Ang2 Bispecific Binding Molecules This research identified the effect and function of anti-VEGF A / Ang2 bispecific binding molecules on vascular endothelial cell leakage by HUVEC-Tie2 leakage assay. HUVEC-Tie2 cells overexpressing Tie2 were obtained by transfecting HUVEC cells (Allcells, Catalog No. H-001-CN) with lentivirus. 300 uL of EGM-2 medium was plated on the bottom layer of a mini-well 96-well insert culture dish, the cells were digested with accutase (Sigma) and HUVEC-Tie2 was obtained. The cells were resuspended to 1 x 107 cells / mL using the EGM-2 medium and plated at 100 uL / well on the top layer of the culture dish. The lower chamber medium (EGM-2 medium) was replaced every 24 h, and after 24 h, the lower chamber medium was replaced by an experimental medium, wherein the experimental medium contained the following components: Blank: EGM-2 medium (Lonza, Catalog No. CC-5035); VEGF A group: EGM-2 Medium + 20 ng / mL VEGF (R&D, Catalog No. 293-VE); IgG group (VEGF A + IgG): EGM-2 medium + 20 ng / mL VEGF + 10 ug / mL IgG; Ang1 group (VEGF A + Ang1): EGM-2 medium + 20 ng / mL VEGF (R&D, Catalog No. 293-VE) + 200 ng / mL Ang1 (R&D, Catalog No. 923-AN); IEX04-012 group (VEGF A + IEX04-012): EGM-2 medium + 20 ng / mL VEGF + 10 ug / mL IEX04-012; BI-836880 group (VEGF A + BI-836880): EGM-2 medium + 20 ng / mL VEGF + 10 ug / mL BI-836880; and Faricimab group (VEGF A + Faricimab): EGM-2 medium + 20 ng / mL VEGF + 10 ug / mL Faricimab. The experimental medium described above was placed and incubated at 37 °C with 5% CO2. After 24 h, 1 uL of FITC-Dextran (Sigma, Catalog No. FD2000S-1G) (4 mg / mL) was added to the experimental medium per well. The mixture was placed at 37 °C with 5% CO2. After 30 min, the lower chamber medium was taken out, diluted at a 1:10 ratio with PBS, and detected by a multimode microplate reader at an exciting wavelength of 488 nm and an emitting wavelength of 535 nm. The experimental results are shown in FIG. 12, showing that the antibody IEX04-012 of the present invention can effectively reduce the VEGF-induced vascular endothelial cell permeability. Example 14. Laser-Induced Choroidal Neovascularization Pharmacodynamic Test This assay adopted a rhesus monkey laser-induced choroidal neovascularization model to determine the neovascularization resistance of the bispecific binding molecule IEX04-012 of the present invention. Rhesus monkey: Species: a rhesus monkey; grade: a normal grade; weight: 3.30-4.20 kg of body weight when purchased, and 3.354.35 kg of body weight during molding; source: Sichuan Hengshu Bio-Technology; production license number: SCXK (Sichuan) 2019-029; laboratory animal quality certification number: No.0023356. In this test, laser photocoagulation was performed around the macular fovea of the fundus of the rhesus monkey to induce the choroidal neovascularization in the fundus and establish an animal model similar to the human choroidal neovascularization. Before and after 20 days of photocoagulation, fundus fluorescence angiography was performed to judge the molding condition. 20 rhesus monkeys (half male and half female) which were successfully molded were selected to be divided into 5 groups, namely a model control group, an IEX04-012 low-dose group, an IEX04-012 high-dose group, an Eylea group, and a Faricimab group, wherein each group contained 4 monkeys, half male and female. On day 21 after photocoagulation, each group of monkeys was administered individually according to the doses in the table. IEX04-012, Eylea (Bayer), or Faricimab (all dissolved in 0.9% sodium chloride injectable solution) was administered by intravitreal injection at both eyes, and the model control was given an equal volume of 0.9% sodium chloride injectable solution. Color fundus photography, fundus fluorescence angiography (leak spot statistics and leak area measurements) (Robin J Goody, Wenzheng Hu, Afshin Shafiee et al., Optimization of laser-induced choroidal neovascularization in African green monkeys. Experimental Eye Research, Exp Eye Res. 2011 92(6):464-72), and optical coherence tomography (OCT, Wang Q, Lin X, Xiang W et al., Assessment of laser induction of Bruch's membrane disruption in monkey by spectral-domain optical coherence tomography. British Journal of Ophthalmology, 2015, 99(1):119-24) were performed on each group of animals on day 7, 14, 21, and 28 after the administration to observe the inhibition effect of the test sample on the choroidal neovascularization. The animals were euthanized on day 29 after the administration, and then both eyes were taken out for histological examination with immunohistochemical (HE) staining. Experimental design table Group Dose Administration volume Concentration Route of administration Control / / X mg / ml IEX04-012 Low dose 10 ug / eye 50 gL / eye 0.2mg / ml Intravitreal administration IEX04-012 High dose 30 gg / eye 50 gL / eye 0.6mg / ml Intravitreal administration Eylea 10 gg / eye 50 gL / eye 0.2mg / ml Intravitreal administration Faricimab 30 gg / eye 50 gL / eye 0.6mg / ml Intravitreal administration The results in FIGs. 13-15 show that the bispecific binding molecule of the present invention showed significant anti-neovascularization after 28 days of administration, and the statistics of the number of grade four leakage spots (FIG. 13A) and the number of grade three to four leakage spots (FIG. 13B) showed that the treated animals of the IEX04-012 group had significantly fewer high-leakage spots than those of the control and positive control administration groups. The OCT results showed a significant decrease in retinal thickness in the animals of the IEX04-012 treatment group, suggesting that the retinal edema degree is reduced and the effect is superior to that of the control (FIG. 14). The fundus fluorescence angiographic results show that the fundus leakage area of the IEX04-012 treatment group was significantly reduced, and the effect was superior to those of positive controls Eylea and Faricimab (FIG. 15), indicating that the antibody of the present invention can significantly inhibit the leakage caused by neovascularization. In conclusion, it is verified that the antibody of the present invention in combination with the anti-VEGF inhibitor has a significant inhibitory effect on the laser-induced fundus neovascularization, and has the function of protecting the integrity of blood vessels. 29 days after the administration, rhesus monkeys were anesthetized with pentobarbital sodium according to the body weight (at about 30 mg / kg by intravenous injection, and the dose could be adjusted according to health conditions of the animals) and euthanized by bleeding from abdominal aorta or femoral artery. The rhesus monkeys were roughly observed, and bilateral eyeballs were taken. Some animal eyes were fixed in a modified Davidson's fixative solution and embedded in paraffin, and laser-molded areas were selected for conventional HE staining such as CD31 IHC staining to perform the histopathological examination. In the pathological section of the antibody group of the present invention, the retinopathy area was significantly reduced, the retinal edema was alleviated, and the tissue hyperplasia of the laser injury area was reduced compared with the anti-VEGF single treatment. The results show better retina morphological improvement (see FIG. 16), the inhibition of the retinochoroidal neovascularization, and the enhancement of the blood vessel integrity function (FIG. 17). SEQUENCE LISTING SEQID NO Antibody name LA42F8 (VEGFAVHH) 1 HCDR1 GFNLDYYPIG 2 HCDR2 CISSVGSTNYADSVKG 3 HCDR3 DPLCSALILPPPFLS 4 VH QLQLVESGGGLVQPGGSLRLSCAASGFNLDYYPIGWFRQA PGKEREGVSCISSVGSTNYADSVKGRFTISRDNAKNTVYLQ MNSLKPEDTAVYYCAADPLCSALILPPPFLSWGQGTQVTVS S Antibody name LA42F8.5 1 HCDR1 GFNLDYYPIG 2 HCDR2 CISSVGSTNYADSVKG 3 HCDR3 DPLCSALILPPPFLS 5 VH EVQLLESGGGLVQPGGSLRLSCAASGFNLDYYPIGWFRQA PGKGLEGVSCISSVGSTNYADSVKGRFTISRDNSKNTVYLQ MNSLRAEDTAVYYCAADPLCSALILPPPFLSWGQGTQVTVS S Antibody name LA46E11 (VEGFAVHH) 6 HCDR1 GSIFSINAMG 7 HCDR2 TMIGGSSTFYADSVNG 8 HCDR3 DVTGRSGTSIWNKRDDY 9 VH QVQLEESGGGLVQPGGSLRLSCVASGSIFSINAMGWHRQAP GSQRELVATMIGGSSTFYADSVNGRFTISRDNAKNTVYLQM NSLKLEDTAVYYCYADVTGRSGTSIWNKRDDYWGQGTLV TVSS Antibody name LA46E11.8 (VEGFAVHH) 6 HCDR1 GSIFSINAMG 10 HCDR2 TMIGGSSTFYAESVQG 8 HCDR3 DVTGRSGTSIWNKRDDY 11 VH EVQLLESGGGLVQPGGSLRLSCAASGSIFSINAMGWHRQAP GKQRELVATMIGGSSTFYAESVQGRFTISRDNSKNTVYLQM NSLRAEDTAVYYCYADVTGRSGTSIWNKRDDYWGQGTQV TVSS Antibody name LA24C11 (Ang2VHH) 16 HCDR1 GFALDYYAIG 17 HCDR2 CISSGDGSTYYADSVKG 18 HCDR3 DSRGDDVACEGLRRNEYDY 19 VH QVQLVESGGGLVQPGGSLRLSCAASGFALDYYAIGWFRQA PGKEREGVSCISSGDGSTYYADSVKGRFTISRDNAKNTVYL QMNSLKPEDTAVYYCATDSRGDDVACEGLRRNEYDYWGQ GTQVTVSS Antibody name LA24C11.10 (Ang2 VHH) 16 HCDR1 GFALDYYAIG 20 HCDR2 CISSGEGSTYYADSVKG 18 HCDR3 DSRGDDVACEGLRRNEYDY 21 VH EVQLLESGGGLVQPGGSLRLSCAASGFALDYYAIGWFRQAPGK GLEGVSCISSGEGSTYYADSVKGRFTISRDNSKNTVYLQMNSLR AEDTA VYYCATDSRGDD VA CEGLRRNEYDYWGQGTL VTVSS Antibody name IEX04-008 LA46E11.8 (VHH) (underlined) + linker (bold) + LA46E11.8 (VHH) (underlined) + LA24C11.10 (italic) 22 Full length EVQLLESGGGLVQPGGSLRLSCAASGSIFSINAMGWHRQAP GKQRELVATMIGGSSTFYAESVQGRFTISRDNSKNTVYLQM NSLRAEDTAVYYCYADVTGRSGTSIWNKRDDYWGQGTQV tvssggggsggggsevqllesggglvqpggslrlscaasg SIFSINAMGWHRQAPGKQRELVATMIGGSSTFYAESVQGRF TISRDNSKNTVYLQMNSLRAEDTAVYYCYADVTGRSGTSI WNKRDDYWGQGTQVTVSSGGGGSGGGGSEVQLLESGGG L VQPGGSLRLSCAASGFALD YYAIG WFRQAPGKGLEG VSCISSG EGSTYYADSVKGRFIISRDNSKNTVYLQMNSLRAEDIAVYYCAT DSRGDD VA CEGLRRNEYD YWGQGIL VTVSS Antibody name IEX04-010 VEGFR extracellular domain + Fc + linker (underlined) + LA24C11.10 (italic) 24 Full length SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKK FPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGH LYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTART ELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLS TLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSEVQL LESGGGLVQPGGSLRLSCAASGFALDYYAIGWFRQAPGKGLEG VSCISSGEGSIYYADSVKGRFIISRDNSKNTVYLQMNSLRAEDIA VYYCAIDSRGDD VA CEGLRRNEYDYWGQGIL VTVSS * Name Aflibercept (VEGFR extracellular domain (underlined) + Fc (italic + bold), wherein FLT1 domain 2 is underlined, and KDR domain 3 is underlined and bold) 25 Full length SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKK FPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGH LYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTAR TELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMK KFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVH EKOKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM1SRTPEV TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP1EKT1SKA KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSD1AVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPG 26 VEGFR extracellular domain SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKK FPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGH LYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTART ELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLS TLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK 27 Fc DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPG \ Antibody name IEX04-012 Ranibizumab scFv containing disulfide bond mutations + linker (bold) + LA24C11.10 (italic) 28 Full length DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPG KAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQQYSTVPWTFGCGTKVEIKGGGGSGGGGSGGGGS GGGGSEVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGM NWVRQAPGKCLEWVGWINTYTGEPTYAADFKRRFTFSLD TSKSTAYLQMNSLRAEDTAVYYCAKYPYYYGTSHWYFDV WGQGTLVTVSSGGGGSGGGGSGGGGSEVQLLESGGGLVQ PGGSLRLSCAASGFALDYYAIGWFRQAPGKGLEGVSCISSGEGS TYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCATDSR GDD VA CEGLRRNEYD YWGQGTL VTVSS * Ranibizumab scFv containing disulfide bond mutations 29 scFv DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPG KAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQQYSTVPWTFGCGTKVEIKGGGGSGGGGSGGGGS GGGGSEVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGM NWVRQAPGKCLEWVGWINTYTGEPTYAADFKRRFTFSLD TSKSTAYLQMNSLRAEDTAVYYCAKYPYYYGTSHWYFDV WGQGTLVTVSS 30 VL DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPG KAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQQYSTVPWTFGCGTKVEIK 31 LCDR1 QDISNYLN 32 LCDR2 FTSSLHS 33 LCDR3 QQYSTVPWT 34 VH EVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQ APGKCLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTA YLQMNSLRAEDTAVYYCAKYPYYYGTSHWYFDVWGQGT LVTVSS 35 HCDR1 GYDFTHY 36 HCDR2 NTYTGE 37 HCDR3 YPYYYGTSHWYFDV 23 Linker (GGGGS)n, wherein n = 1, 2, 3, or 4 Positive control antibody BI836880 (VEGFA terminal) 12 VH DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQA PGKEREFVVAISKGGYKYDAVSLEGRFTISRDNAKNTVYLQ INSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVT VSS Positive control antibody Faricimab 13 HC 1 EVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQ APGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTA YLQMNSLRAEDTAVYYCAKYPYYYGTSHWYFDVWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAA GGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLAQDWL NGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCR DELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNA YTQKSLSLSPGK 40 LC1 DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPG KAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQL KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC 14 HC2 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVR QAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSIS TAYMELSRLRSDDTAVYYCARSPNPYYYDSSGYYYPGAFDI WGQGTMVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLN NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKT HTCPPCPAPEAAGGPSVFLFPPKPKDTLMASRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV SVLTVLAQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQP REPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFS CSVMHEALHNAYTQKSLSLSPGK 41 LC2 SYVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQ APVLVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEA DYYCQVWDSSSDHWVFGGGTKLTVLSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSC Positive control antibody BI836880 (VEGFA / HSA / Ang2) 15 VH DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQA PGKEREFVVAISKGGYKYDAVSLEGRFTISRDNAKNTVYLQ INSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVT VSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFT FSSFGMSWVRQAPGRGLEWVSSISGSGSDTLYADSVKGRFT ISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLV TVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAVSGI TLDDYAIGWFRQAPGKEREGVSAIRSSGGSTYYADSVKGRF TISSDNSKNTVYLQMNSLRPEDTAVYYCAAVPAGRLRYGE QWYPIYEYDAWGQGTLVTVSS Negative control IgG 38 Heavy chain EVRLLESGGGLVQPGGSLRLSCAASGFTFSNYAMGWVRQA PGKGLEWVSAISGSGGSTYYADSVKGRFTTSRDDSKNALY LQMNSLRAEDTAVYYCARGGPGWYAADVWGQGTTVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK 42 Light Chain DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPG KAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQQADLPAFAFGGGTKVEIKRTVAAPSVFIFPPSDEQLK SGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC Positive control antibody BI-anti-VEGF 39 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQA PGKEREFVVAISKGGYKYDAVSLEGRFTISRDNAKNTVYLQ INSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVT VSS

Claims

1. A bispecific binding molecule binding to VEGF A and Ang2, comprising a first target-binding region specifically binding to VEGF A and a second target-binding region specifically binding to Ang2,wherein the first target-binding region is an anti-VEGF A scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH of the anti-VEGF A scFv comprises an HCDR1, an HCDR2, and an HCDR3, wherein the HCDR1 comprises an amino acid sequence set forth in SEQ ID NO: 35; the HCDR2 comprises an amino acid sequence set forth in SEQ ID NO: 36; and the HCDR3 comprises an amino acid sequence set forth in SEQ ID NO: 37; and the VL of the anti-VEGF A scFv comprises an LCDR1, an LCDR2, and an LCDR3, wherein the LCDR1 comprises an amino acid sequence set forth in SEQ ID NO: 31; the LCDR2 comprises an amino acid sequence set forth in SEQ ID NO: 32; and the LCDR3 comprises an amino acid sequence set forth in SEQ ID NO: 33;and wherein the second target-binding region is an anti-Ang2 VHH antibody comprising 3 CDRs, HCDR1, HCDR2, and HCDR3 as follows, whereinthe HCDR1 comprises an amino acid sequence set forth in SEQ ID NO: 16;the HCDR2 comprises an amino acid sequence set forth in SEQ ID NO: 17 or 20; andthe HCDR3 comprises an amino acid sequence set forth in SEQ ID NO: 18.

2. The bispecific binding molecule according to claim 1, wherein the bispecific binding molecule is a bispecific antibody.

3. The bispecific binding molecule according to claim 1 or 2, having the following structure:VL of the anti-VEGF A scFv-linker-VH of the anti-VEGF A scFv-linker-anti-Ang2 VHH orVH of the anti-VEGF A scFv-linker-VL of the anti-VEGF A scFv-linker-anti-Ang2 VHH.

4. The bispecific binding molecule according to any one of claims 1-3, wherein the anti-Ang2 VHH comprises an amino acid sequence set forth in SEQ ID NO: 21 or 19, or comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 21 or 19.

5. The bispecific binding molecule according to any one of claims 1-4, whereinthe VH of the anti-VEGF A scFv comprises an amino acid sequence set forth in SEQ ID NO: 34, or comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 34; andthe VL of the anti-VEGF A scFv comprises an amino acid sequence set forth in SEQ ID NO: 30, or comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 30.2022287100   24 Aug 20266. The bispecific binding molecule according to any one of claims 3-5, wherein the linker comprises an amino acid sequence set forth in SEQ ID NO: 23.

7. The bispecific binding molecule according to any one of claims 1-6, wherein the bispecific binding molecule comprises an amino acid sequence set forth in SEQ ID NO: 28, or comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 28.

8. The bispecific binding molecule according to any one of claims 1-7, wherein the bispecific binding molecule is a bispecific antibody comprising an amino acid sequence set forth in SEQ ID NO: 28.

9. A nucleic acid molecule encoding the bispecific binding molecule according to any one of claims 1-8.

10. An expression vector comprising the nucleic acid molecule according to claim 9.

11. A host cell comprising the nucleic acid molecule according to claim 9 or the expression vector according to claim 10.

12. The host cell according to claim 11, wherein the host cell is:(a) prokaryotic, optionally wherein the prokaryotic host cell is an E.coli cell, such as TG1; or(b) eukaryotic, optionally wherein the eukaryotic host cell is a CHO cell or a 293 cell, such as a 293F or Expi-293 cell.

13. A method for preparing the bispecific binding molecule according to any one of claims 1-8, comprising culturing the host cell according to claim 11 or 12 under conditions suitable for expressing the bispecific binding molecule, and optionally recovering the bispecific binding molecule from the host cell or the host cell medium.

14. An immunoconjugate comprising the bispecific binding molecule according to any one of claims 1-8.

15. A pharmaceutical composition or formulation comprising the bispecific binding molecule according to any one of claims 1-8, and optionally one or more additional therapeutic agents, and further optionally a pharmaceutical supplementary material.

16. A method for preventing or treating an ocular disease in a subject, comprising administering to the subject an effective amount of the bispecific binding molecule according to any one of claims 18, the immunoconjugate according to claim 14, or the pharmaceutical composition or formulation according to claim 15.

17. The method according to claim 16, wherein the ocular disease is an ocular disease associated with angiogenesis, such as an ocular disease associated with corneal neovascularization.2022287100   24 Aug 202618. The method according to claim 16 or 17, wherein the method further comprises administering to the subject one or more therapies, such as therapeutic modalities and / or additional therapeutic agents.

19. Use of the bispecific binding molecule according to any one of claims 1-8, the immunoconjugate according to claim 14, or the pharmaceutical composition or formulation according to claim 15 in the manufacture of a medicament for the treatment or prevention of an ocular disease.

Images