GB10, a best-in-class antibody fusion protein targeting VEGF/Ang-2, exhibits promising therapeutic efficacy for neovascular eye diseases

GB10, a best-in-class antibody fusion protein targeting VEGF/Ang-2, exhibits promising therapeutic efficacy for neovascular eye diseases | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article GB10, a best-in-class antibody fusion protein targeting VEGF/Ang-2, exhibits promising therapeutic efficacy for neovascular eye diseases Xiling Wei, Yuxin Qiu, Wei Shang, Xiangling Zhang, Wenjie Yang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5135499/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Background In clinical practice, anti-vascular endothelial growth factor (VEGF) therapies have been successfully applied to patients with neovascular eye diseases. However, unmet clinical needs have not yet been fully addressed, as about 20% of patients do not response to anti-VEGF monotherapies, meanwhile, the high frequency of intravitreal (IVT) injections imposes a significant burden on patients. To overcome these challenges, we developed a novel antibody fusion protein GB10 consisting of a VEGF-Trap and an anti-angiopoietin 2 (Ang-2) variable heavy domain of heavy-chain antibody (VHH) to inhibit the pro-angiogenic pathways of VEGF and Ang-2 simultaneously for enhanced and more enduring efficacy. The activity and developability of GB10 were characterized. Methods We first explored two categorical formats for molecular construction and selected the format that demonstrated the best activity and CMC-related properties for the generation of GB10. Subsequently, we evaluated the multi-targeting capability of GB10 using bridging enzyme-linked immunosorbent assay (ELISA) and bio-layer interferometry (BLI), followed by a side-by-side comparison of the in vitro activities of GB10 and faricimab, the only marketed bispecific antibody for neovascular eye diseases, through assays such as VEGF reporter assay, human umbilical vein endothelial cells (HUVEC) proliferation, Ang-2 blocking ELISA, and Tie-2 phosphorylation. The in vivo efficacy of GB10 and faricimab was next evaluated using a non-human primate model of laser-induced choroidal neovascularization (CNV). Finally the developability of GB10 was evaluated by intraocular pharmacokinetics and stress test. Results GB10 bound VEGF and Ang-2 simultaneously with high affinity, and exhibited superior activity in vitro in inhibiting the VEGF and Ang-2 signaling pathways compared to faricimab. In vivo , GB10 demonstrated greater efficacy and durability compared to faricimab in a CNV model. GB10 also possessed a longer half-life in vitreous measured in a rabbit model. Moreover, GB10 showed excellent injectability and stability at a high-concentration of 140 mg/mL. Conclusions The superb efficacy and favorable developability profile make GB10 a potential best-in-class therapy for patients with neovascular eye diseases, warranting further evaluation in clinical settings. Neovascular eye disease VEGF Ang-2 Antibody fusion protein VEGF-Trap VHH Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Neovascular eye diseases, such as age-related macular degeneration (AMD), diabetic macular edema (DME), diabetic retinopathy (DR) and retinal vein occlusion (RVO), are major causes of severe vision impairment and blindness [ 1 ]. The progression of these diseases is often closely related to abnormal angiogenesis, in which VEGF plays a central role [ 2 ]. Currently, anti-VEGF therapies, such as Lucentis® (ranibizumab, Genentech), Eylea® (aflibercept, Regeneron) and Langmu® (conbercept, Kanghong), have become the standard treatments for neovascular eye diseases [ 3 – 7 ]. With the application of anti-VEGF therapies, the incidence of legal blindness and vision impairment caused by ocular neovascularization has been reduced, leading to lower economic and social costs and improved vision-related quality of life [ 8 ]. However, there remain several limitations and challenges for neovascular eye diseases. For example, many patients do not respond completely or suffer from a diminished response over time, which may be attributed to the limited efficacy of VEGF monotherapies resulting from solely inhibiting the VEGF pathways [ 9 ]. Approximately 20–40% of patients with AMD [ 10 ] and 15–20% of those with DR [ 11 , 12 ] do not adequately or fully respond to anti-VEGF therapies and require alternate treatment strategies for disease management. In addition to efficacy limitations, another significant challenge of anti-VEGF therapies is the durability, thereby requiring repeated intravitreal injections [ 10 , 13 , 14 ]. Frequent IVT injections can lead to high non-adherence/dropout over time, further exacerbating the illness [ 15 , 16 ]. Moreover, it increases the risk of complications, such as sustained elevation of intraocular pressure, inflammation, and hemorrhage [ 17 – 19 ]. To overcome the above challenges, novel therapeutic strategies are clearly needed. One approach is to enhance the efficacy of anti-VEGF-based therapies, including the design of multi-targeted molecules capable of inhibiting additional pathophysiological pro-angiogenic pathways. Many studies have demonstrated that angiopoietin-2 (Ang-2) plays a synergistic role with VEGF in vascular instability, leading to vascular leakage, neovascularization, and inflammation [ 20 , 21 ]. In human eyes, increased intraocular Ang-2 levels were detected in patients with DR and RVO [ 22 ]. In addition, higher levels of Ang-2 were associated with disease severity in AMD [ 23 ], suggesting a potential benefit of targeting ocular Ang-2. Therefore, the combined inhibition of VEGF and Ang-2 is expected to be more effective than VEGF inhibition alone in preventing ocular neovascularization. The other strategy is optimizing the dosage by developing high-concentration formulations that can deliver sufficient drug concentrations in a smaller volume to ensure prolonged therapeutic effects and reduced frequency of IVT administration. In recent years, there have been clinical studies that have proved the feasibility of this strategy. In the phase III HAWK and HARRIER trials [ 24 ], nAMD patients receiving 6 mg of brolucizumab achieved comparable visual improvement to those receiving 2 mg of aflibercept, but showed better improvements in retinal fluid; importantly, more than 50% of the brolucizumab 6 mg treatment arm achieved a dosing interval of 12 weeks, while the dosing interval for aflibercept 2 mg was 8 weeks. The results of the 2-year phase III PULSAR study [ 25 ] showed that the visual benefits for nAMD patients receiving 8 mg of aflibercept were similar to those receiving 2 mg of aflibercept, with 83% of patients maintaining ≥ 12-week treatment intervals. Therefore, the development of high-concentration formulations has become an important trend in the field of intravitreal injection therapy, offering patients with more convenient and effective treatment options. Currently, the use of VHH in assembling multi-targeted molecules has emerged as an important research focus in biomedicine [ 26 ]. VHH is an antibody fragment derived from camelid animals, including alpacas and llamas. Compared to single-chain variable fragments (scFv) from conventional antibodies, VHH has a smaller molecular weight and a simpler structure, which allows it to remain stable under extreme conditions (e.g., low pH), thereby enhancing the stability of the CMC process [ 27 ]. Several bispecific antibodies based on nanobodies are currently undergoing clinical evaluation for the treatment of various diseases [ 28 ]. In this study, we generated GB10, a novel multi-targeted antibody fusion protein featuring VEGF-Trap at the N-terminus and anti-Ang-2 VHH at the C-terminus, that bound and neutralized VEGF family members and Ang-2 with high potency in vitro and in vivo , and then developed it into a high-concentration formulation of 140 mg/mL. Through advancing this potentially best-in-class molecule into the clinic in the future, we hope to provide a more effective and long-term treatment option for patients with neovascular eye diseases. Methods Molecule design, expression and purification Multi-targeted molecules in two different categories were created. Category #1 was antibody fusion protein featuring VEGF-Trap at the N-terminus and anti-Ang-2 antibody at the C-terminus, and three design formats were generated. Category #2 was bispecific antibody with anti-VEGF-A antibody at the N-terminus and anti-Ang-2 antibody at the C-terminus, and two formats were made. pcDNA3.4 plasmid containing DNA fragments encoding target proteins was transfected into CHO cells by electroporation. The supernatant containing target proteins was harvested after 6 days of fed-batch cultivation and purified by Protein A affinity chromatography (AKTA, GE Healthcare). VEGF blocking assay Engineered cell lines (H-VEGF Reporter 293 Cell line, GM-C09057, Genomeditech) for VEGF signaling in assay media (DMEM, C11995500BT, Gibco) were seeded at 25,000 cells/well on a 96-well plate where human VEGF-A 165 (C083, Novoprotein) and 3-fold serially diluted samples ranging from 25 nM to 0.0004 nM had been pre-incubated. The microplate was incubated at 37 ℃, 5% CO 2 for 6 h to induce luciferase gene expression by VEGF signaling. The relative VEGF blocking activity was measured by One-Lite® Luciferase Assay System (DD1203, Vazyme) via Varioskan™ LUX (Thermo Scientific) according to the manufacturer’s protocol. Ang-2 blocking assay A recombinant human Tie-2 protein (TI2-H5255, ACROBiosystems) was used as the coating antigen in the blocking ELISA. The wells of Nunc 96-well Maxisorp plates were coated with 100 ng of Tie-2 in coating buffer (C1050, Solarbio) and incubated at 4 ℃ overnight. The next day, after blocking with blocking buffer (SW3015, Solarbio) at 37 ℃ for 2 h, the plates were washed three times with PBST (phosphate-buffered saline containing 0.05% Tween-20), then 3-fold serially diluted samples ranging from 20 nM to 0.0003 nM pre-incubated with 100 ng/mL human Ang-2 (10691-H08H, SinoBiological, His tag) were added and incubated at 37 ℃ for 1 h. After washing three times, 100 µL of HRP Anti-6X His tag® antibody (ab1187, Abcam) diluted 1:5,000 in PBST was dispensed into each well, and the plates were incubated at 37 ℃ for 30 min. Plates were washed six times and TMB Single-Component Substrate Solution (PR1200, Solarbio, 100 µL/well), and ELISA Stop Solution (C1058, Solarbio, 50 µL/well) were added sequentially. Absorbance at 450 nm was quantified via Varioskan™ LUX (Thermo Scientific). Hydrophobic Interaction Chromatography (HIC) analysis HIC analysis was performed on a 1260 Infinity II system (Agilent Technologies) with UV detection at 214 nm. Briefly, 10 µg of each sample prepared in 0.1 M phosphate buffer, pH 6.5 with 1 M ammonium sulphate was separated on a Protemix HIC Butyl-NP5 column (4.6×100 mm, 5 µm, Sepax) with a linear gradient from 44–100% of mobile phase B (mobile phase A: 0.1 M sodium dihydrogen phosphate and 1.8 M ammonium sulphate, pH 6.5; mobile phase B: 0.1 M sodium dihydrogen phosphate, pH 6.5) at a flow rate of 1.0 mL/min. Data were acquired and processed by Empower 3 software (Waters). Size Exclusion Chromatography (SEC) analysis SEC analysis was performed on a LC-2030C system (SHIMADZU) with UV detection at 280 nm. Briefly, 10 µg of sample in PBS was separated on a BioCore SEC-300 column (7.8×300 mm, 5 µm, NanoChrom) using a mobile phase consisting of 0.1 M phosphate buffered saline and 0.15 M sodium chloride, pH 7.0 at a flow rate of 0.5 mL/min. Data were acquired and processed by Empower 3 software (Waters). Generation of anti-Ang-2 VHH One alpaca was immunized with 500 µg human Ang-2 (10691-H08H, SinoBiological) emulsified with Complete Freund’s adjuvant (F5881, Sigma-Aldrich) for the first time and Incomplete Freund’s adjuvant (F5506, Sigma-Aldrich) for each subsequent immunization. Peripheral blood mononuclear cells (PBMCs) were isolated after the fifth immunization, then total RNA was extracted with TRIzol (15596018, Invitrogen) for reverse transcription (PrimeScript II kit, 6210A, TAKARA). DNA fragments encoding VHH were amplified and cloned into phagemid pCom3xss to prepare the phage VHH library. Anti-Ang-2 VHH was displayed with the assistance of helper phage M13KO7 after the VHH-phagemid was transformed into E coli. TG1 by electroporation. Human Ang-2 was used for biopanning. Approximately 2 × 10 11 plaque-forming units were added into an immuno-tube pre-coated with BSA, and incubated at 37 ℃ for 1 h. Unbound phages were collected and incubated in an immuno-tube pre-coated with human Ang-2 at 37 ℃ for 1 h for positive biopanning. The human Ang-2 bound phages were eluted using 0.1 M Glycine-HCl (pH 2.2). The Ang-2 binding affinities of candidate phages were evaluated by ELISA. The variable region sequences of positive candidates were retrieved by Sangers sequencing and recombinantly expressed in CHO cells as previously described in the Method section under “Molecule design, expression and purification”. Ang-2 and Ang-1 binding assay Nunc 96-well Maxisorp plates were coated with 1 µg/mL human Ang-2 (10691-H08H, SinoBiological), cynomolgus Ang-2 (90026-C08H, SinoBiological), mouse Ang-2 (AN2-M52H3, ACROBiosystems) and human Ang-1 (923-AN-025, R&D system) at 4°C overnight. The next day, the plates were washed with PBST, blocked for 2 h with blocking buffer and incubated with samples in 3-fold dilution series ranging from 20 nM to 0.0001 nM for 1 h at 37°C. After washing three times, 100 µL of goat anti-human IgG Fc (HRP) (ab97225, Abcam) diluted 1:10,000 in PBST was dispensed into each well, and the plates were incubated at 37 ℃ for 30 min. Finally, TMB solution and stop solution were added sequentially as previously described in the Method section under “Ang-2 blocking assay”. Absorbance at 450 nm was quantified via Varioskan™ LUX (Thermo Scientific). GB10 expression and purification GB10 belongs to Format 2 of Category #1 that comprises an N-terminal VEGF-Trap based on Aflibercept, and a C-terminal anti-Ang-2 nanobody (VHH-04) on a human IgG1 backbone. VHH-04 is connected to the Fc via a (G 4 S) 4 linker. GB10 expression and purification were performed as previously described in the Method section under “Molecule design, expression and purification”. VEGF and Ang-2 affinity characterization using Bio-Layer Interferometry (BLI) A Bio-Layer Interferometry (BLI) assay was performed using an Octet® R8 (Sartorius) instrument. Each sample was immobilized onto Octet ProA Biosensors (18-5010, Sartorius) until a signal of 1.0 nm was reached. Antigen solution in a 2-fold dilution series covering a concentration range from 0.5 nM to 512 nM was added to the sample-loaded sensors, and the interaction was monitored for 300 s association and 300 s dissociation. Kinetic parameters were determined using Octet® Software Version 13 (Sartorius). Bridging assay A Nunc 96-well Maxisorp plate was coated with recombinant human VEGF-A 165 (C083, Novoprotein, 200 ng/well) overnight at 4 ℃. The next day, after blocking with blocking solution, 3-fold serially diluted samples ranging from 18 nM to 0.0009 nM were added and incubated at 37°C for 1 h. After washing three times with PBST, human Ang-2 (10691-H08H, SinoBiological, 50 ng/well) was added and incubated at 37°C for 1 h. Finally, HRP Anti-6X His tag® antibody, TMB solution and stop solution were added sequentially as previously described in the Method section under “Ang-2 blocking assay”. Absorbance at 450 nm was quantified via Varioskan™ LUX (Thermo Scientific). HUVEC proliferation assay HUVEC (C-12205, Promocell) in endothelial cell growth medium (C-22010, Promocell) was seeded at 3,000 cells/well. 4-fold serially diluted samples ranging from 25 nM to 0.0004 nM and 10 ng/mL VEGF-A 165 (C083, Novoprotein) were preincubated for 1 h at room temperature. The mixtures were then immediately added to the wells of 96-well plates containing HUVEC and incubated for 3 days at 37 ℃, 5% CO 2 . Proliferation of HUVEC was measured by CellTiter-Glo® Luminescent Cell Viability Assay kit (G7570, Promega) using a Varioskan™ LUX (Thermo Scientific) according to the manufacturer’s protocol. Tie-2 phosphorylation assay Engineered cell lines (huTie2-HEK293, C2209248, Sanyou Bio) for Ang-2 signaling in assay media (DMEM, L110KJ, Basal Media) were seeded at 32,000 cells/well on a 96-well plate where 60 nM human Ang-2 (10691-H02H, SinoBiological) and serially diluted samples ranging from 2,000 nM to 10 nM had been pre-incubated. The microplate was incubated at 37 ℃, 5% CO 2 for 1.5 h to induce Tie-2 phosphorylation by Ang-2 signaling. After incubation, cells were lysed using Cell Lysis Buffer (9803S, CST), and the lysate supernatant was added to an ELISA plate for quantification of Tie-2 phosphorylation by Human Phospho-Tie-2 DuoSet IC ELISA (DYC2720-5, R&D system) on SpectraMax 190 (Molecular Devices) according to the manufacturer’s protocol. Laser-induced CNV in cynomolgus monkeys This study was conducted at WestChina-Frontier PharmaTech Co., Ltd. in accordance with the IACUC standard animal procedures along with the IACUC guidelines that was in compliance with the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals. A total of twelve cynomolgus monkeys (supplied by Hainan Jingang Biotech Co., Ltd.) at 2–6 kg body weight were used. CNV was induced by laser photocoagulation on Day − 14 in both eyes of the cynomolgus monkeys. Nine lesions were symmetrically placed in the macula of each eye using a power setting of 0.5–0.7 W, a spot size of 50 µm and a duration of 0.05 s. Based on the number of spots with fluorescein leakage of grade 4, which was considered clinically relevant [ 29 ], ten suitable animals were selected for treatment and randomly divided into 5 groups on Day 0 as follows: vehicle group, faricimab low-dose group, faricimab high-dose group, GB10 low-dose group, and GB10 high-dose group. Then intravitreal injections of 50 µL sample solution per eye were performed on both eyes (low-dose = 1 nmol/eye; high-dose = 40 nmol/eye). The therapeutic efficacy was evaluated on Day 7, 14 and 21 after dosing by fundus fluorescein angiography (FFA) and optical coherence tomography (OCT). The images taken about 5 min after the fluorescein injections were used for comparison of improvement rate using the following equation: improvement rate (%) = [FLA (Day 0 before dosing) – FLA (Day 7, 14, 21 after dosing)] ÷ [FLA (Day 0 before dosing)] × 100%, FLA, fluorescein leakage area. The thickness of the retina around the burn spot was measured to compare improvement rates using the following equation: improvement rate (%) = [RT (Day 0 before dosing) – RT (Day 7, 14, 21 after dosing)] ÷ [RT (Day 0 before dosing) – RT (Day − 14 before laser)] × 100%, RT, retinal thickness. Pharmacokinetic analysis Pharmacokinetic study was conducted at Shenzhen Kexing Biopharm Co., Ltd. in accordance with the relevant regulations of the Institutional Animal Care and Use Committee (IACUC), also followed the Guide for the Care and Use of Laboratory Animals, Eighth Edition (National Research Council, 2011). A total of 32 New Zealand white rabbits (supplied by GuangDong Medical Laboratory Animal Center) received a single intravitreal injection of GB10 at 500 µg/eye (Group 1, n = 16) and faricimab at 500 µg/eye (Group 2, n = 16). GB10 and faricimab concentrations were determined in the vitreous up to 28 days (2 h, 6 h, 8 h, 24 h, 72 h, 168 h, 14 days, 28 days) after dosing by ELISA. The pharmacokinetic parameters in the vitreous were analyzed by a noncompartmental analysis using Phoenix WinNonlin software version 1.3 (Certara). Dynamic Light Scattering (DLS) DLS experiment was carried out using a DynaPro Plate Reader III (Wyatt Technology). Briefly, 30 µL of sample solution was carefully loaded into a 384-well plate without introducing bubbles. Hydrodynamic radius ( R h) measurement was performed at 25°C and determination of aggregation onset temperature (T agg ) was then conducted in a temperature ramp of 1°C/min from 25 to 80°C. Data acquisition and analysis were carried out via the DYNAMICS software (Wyatt). Differential Scanning Fluorimetry (DSF) DSF was conducted on a nanoDSF system (Prometheus NT.48, NanoTemper). Briefly, 15 µL of sample solution was loaded into the capillaries. The samples were heated from 20 to 80 ℃ at a rate of 1 ℃/min, and fluorescence signals were collected and analyzed using the software PR ThermControl (Prometheus). Viscosity The viscosity was measured with the Viscosizer (RheoSense micro VISC). Briefly, 300 µL of sample solution was carefully transferred in the microVisc unit without introducing bubbles. The viscosity values of GB10 at 140 mg/mL were determined in “Auto mode”. Statistical Analysis Data are represented as mean ± SD and analyzed using GraphPad Prism. The differences between the groups were compared by the multiple unpaired t-test. Statistical significance was considered for P-values below 0.05. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Results Generation and screening of VEGF and Ang-2 multi-targeted molecules Based on the feasibility of combining VEGF and Ang-2 to increase effectiveness of neovascular eye disease therapy [ 22 ], we decided to generate a novel protein therapeutics with enhanced efficacy and optimal developability. As a first step, we set out to screen five different multi-targeted molecules following the schematic diagram outlined in Fig. 1 A. First, we chose aflibercept or ranibizumab, the most commonly used VEGF-targeted drugs for ocular neovascular diseases, as the backbone to construct two design categories. Category #1 was antibody fusion protein featuring aflibercept (VEGF-Trap) at the N-terminus and scFv (Format 1) or VHH (Format 2) targeting Ang-2 at the C-terminus, where the scFv and VHH utilized a published sequence called LC10 and 166H4, respectively. Besides, a single-chain antibody fusion protein (Format 3) was also explored, as its small molecular weight may enhance passage through the retina into the choroid and reach the lesion site directly, potentially increasing efficacy. Category #2 was bispecific antibody comprising ranibizumab (anti-VEGF-A antibody) at the N-terminus and the same scFv (Format 4) or VHH (Format 5) as in Category #1 at the C-terminus. In order to compare the functional activity so that we could select the most effective formats for blocking both VEGF and Ang-2, we expressed and purified the five molecules and conducted experiments of VEGF-NFAT reporter assay and Ang-2 blocking ELISA. The results, depicted in Fig. 1 B, revealed that Format 1 and Format 2 exhibited similar IC 50 that were better when compared to Format 4 and Format 5 (Fig. 1 D). This may be attributed to the higher affinity of aflibercept for VEGF-A, or a broader spectrum of activity of aflibercept, which can block not only VEGF-A but other angiogenic factors in the VEGF family as well, such as VEGF-B and PlGF [ 30 ], in comparison with ranibizumab which is specific only for VEGF-A. In contrast, Format 3 displayed the weakest performance in this assay. Additionally, the Ang-2 inhibition curves for each format was shown in Fig. 1 C where no significant differences in the IC 50 among Formats 1, 2, 4, and 5 (Fig. 1 D) were detected, suggesting similar blocking effects on Ang-2. Format 3 again exhibited the lowest activity in Ang-2 blocking assay. Taken together, these findings suggested that Formats 1 and 2 were top choice for multi-targeted blockade. Next, because certain intrinsic properties such as solubility, aggregation propensity and expression yield have direct impacts on the success of future CMC development, we initially utilized HIC to assess the solubility of Format 1 and Format 2 indirectly and rapidly. As shown in Fig. 1 E, Format 2 exhibited a shorter retention time compared to Format 1, suggesting better solubility. In addition, when Format 1 and Format 2 were transiently expressed in CHO cells, the yield of Format 2 was 288.2 mg/L compared to 147.6 mg/L of Format 1. In the DLS assay, we observed that the interaction parameter k D was − 6.61 for Format 1 and 14.7 for Format 2. The value of k D is related to the nature of intermolecular forces: a positive k D suggests significant repulsive forces between molecules, while a negative k D suggests attractive forces that could potentially lead to self-aggregation [ 31 ]. Thus, we picked Format 2 for the predicted lower tendency to form aggregates and greater stability in solution. Moreover, many current reports have stated that VHH has higher solubility, stability, and expression yield compared to scFv [ 27 ]. Therefore, in order to facilitate a smooth transition to CMC development of a multi-targeted antibody fusion protein, we chose Format 2 for further research and development. Generation and in vitro activity of anti-Ang-2 nanobody VHH-04 In order to construct a novel multi-targeted molecule of our own, we obtained VHHs targeting Ang-2 by screening an alpaca-immunized library using phage display technology. Seven unique VHH sequences binding to Ang-2 were isolated from the target-enriched library and subsequently cloned into the pcDNA3.4 vector for recombinant expression. A competition ELISA was then conducted to assess the inhibitory effects of these VHHs on Ang-2-Tie-2 binding interaction. Figure 2 A illustrated that only VHH-04 exhibited significant blocking activity against Ang-2, achieving an IC 50 of 0.11 nM. Among the angiopoietin family members, Ang-1 and Ang-2 play important roles in vascular development by interacting with the Tie-2 receptor [ 32 ]. Various approaches have convincingly demonstrated that Ang-1 is crucial for maintaining vascular integrity and that its overall activity is anti-angiogenic, whereas Ang-2 promotes vascular development [ 33 , 34 ]. Therefore, it is crucial for the Ang-2 neutralizing antibody to selectively target Ang-2 without affecting Ang-1 signaling. To confirm the specificity of VHH-04, we conducted an ELISA assay where no binding of human Ang-1 was observed (Fig. 2 B). This signified that VHH-04 was specific for Ang-2. Furthermore, to identify relevant species for non-clinical studies, we tested the cross-species reactivity of VHH-04. As shown in Fig. 2 C, VHH-04 bound to human, cynomolgus, and mouse Ang-2 with EC 50 of 0.04 nM, 0.03 nM, and 0.03 nM, respectively. Taken together, based on its blocking activity, specificity for Ang-2 and cross-species binding activity, VHH-04 was ultimately used for bispecific assembly. Construction and characterization of GB10 that targets VEGF and Ang-2 simultaneously Now that we used above anti-Ang-2 VHH antibody to construct a multi-targeted antibody-fusion protein based on Format 2 where VEGF-Trap (Aflibercept) was at the N-terminus of a human IgG1 backbone, and Ang-2-specific VHH-04 was connected via a G 4 S linker to the C-terminus of the Fc (Fig. 3 A), and we named this molecule GB10. First, in order to confirm that GB10 was capable of engaging both VEGF and Ang-2 concurrently, a bridging ELISA was performed. The result was shown in Fig. 3 B. After coating the microplate with VEGF for capturing, GB10 specifically bound to the microplate and could be detected successfully by Ang-2-His binding on the opposite end of GB10 with an EC 50 of 0.11 nM. As expected, aflibercept, a VEGF-Trap without Ang-2 binding moiety, did not have bridging activity. Secondly, we tested the interaction between GB10 and VEGF family ligands by BLI. As shown in Fig. 3 C, GB10 exhibited high affinity to VEGF-A from human, mouse, rat and rabbit. GB10 also bound human VEGF-B and PlGF with high affinity. The binding affinity of GB10 to VEGF-A from cynomolgus monkey was not tested because the amino acid sequence of VEGF-A is identical to its human counterpart. Moreover, GB10 also bound to Ang-2 with high affinity across species including human, cynomolgus monkey, mouse and rabbit by BLI. The binding affinity results are summarized in Fig. 3 D. Taken together, we designed GB10, a multi-targeted antibody-fusion protein, that was able to bind both VEGF and Ang-2 with high affinity at the same time. GB10 effectively blocks VEGF and Ang-2 signaling pathways in vitro To verify the functional activity of GB10 in inhibiting VEGF signaling pathway, we performed two in vitro cellular assay and compared its efficacy with faricimab, a marketed bispecific antibody targeting both VEGF-A and Ang-2. First, the neutralizing activity of GB10 and faricimab were studied on a NFAT-driven luciferase reporter assay using HEK293 cells overexpressing VEGFR. The results in Fig. 4 A demonstrated that both GB10 and faricimab reduced the signal in a dose-dependent manner. Faricimab displayed an IC 50 of 0.15 nM, while GB10 exhibited a 7.5-fold greater blocking potency with an IC 50 of 0.02 nM. Secondly, HUVEC proliferation assay was used to evaluate the impact of GB10 and faricimab on VEGF-induced angiogenesis. GB10 demonstrated approximately 2-fold greater inhibitory potency than faricimab with IC 50 ​​of 0.03 nM and 0.05 nM, respectively (Fig. 4 B). Next, an Ang-2-Tie-2 blocking ELISA assay and a Tie-2 phosphorylation assay were developed in order to evaluate the inhibitory activity of GB10 and faricimab on Ang-2 signaling pathway. Notably, when compared side-by-side in blocking ELISA (Fig. 4 C), GB10 showed a 94-fold greater activity than faricimab with IC 50 of 0.15 nM and 14.17 nM, respectively. The functional activity was further characterized using Tie-2 phosphorylation assay which measured the level of tyrosine-phosphorylation of human Tie-2 in cell lysates in the presence of GB10 or faricimab (Fig. 4 D). The results indicated that GB10 (IC 50 = 49.90 nM) was more potent in inhibiting Ang-2-induced Tie-2 phosphorylation compared to faricimab (IC 50 = 442.40 nM). Moreover, faricimab only achieved a maximum inhibition rate of approximately 70%, while GB10 was able to achieve complete inhibition. GB10 shows potent in vivo efficacy in laser-induced CVN model The superior in vitro activity prompted us to investigate how GB10 would perform against faricimab in vivo in laser-induced CNV model in cynomolgus monkeys, which is a widely used model for studying ocular neovascularization [ 35 , 36 ]. The therapeutic effect of GB10 or faricimab was evaluated by fundus fluorescein angiography (FFA) to assess the levels of vascular leakage, and by optical coherence tomography (OCT) to quantify changes in retinal thickness on Days 7, 14 and 21 after laser damage with respect to Day 0. Figure 5 A shows CNV induction, intravitreal injection and imaging schedule after dosing. The representative pictures measured by FFA was shown in Fig. 5 B and the improvement rate of the leakage area of grade 4 spots was shown in Table 1 . CNV development peaked at about 2–3 weeks after laser photocoagulation and had a tendency to resolve itself overtime, therefore, we compared the effects of the vehicle and the treatment groups at the same time points. On Day 7 after dosing, both GB10 and faricimab were effective in reducing the leakage area compared to the vehicle group. However, GB10 achieved an improvement rate exceeding 70% at low dose (1 nmol/eye) compared to ~ 47% of faricimab, and in the high-dose (40 nmol/eye) group, the CNV spot was completely resolved by GB10 compared to 79% of improvement rate by the faricimab group, suggesting a better efficacy of GB10. Similarly, on Day 14, the efficacy of GB10 persisted and remained statistically significant in the low-dose group compared to the vehicle group. In contrast, no statistically significant difference was observed in the low-dose group receiving faricimab. On Day 21, our results indicated that GB10 maintained a significant effect compared to the vehicle group in both the low-dose and high-dose groups, whereas no statistical differences were observed in any of the faricimab groups. Taken together, GB10 demonstrated sustained efficacy in the treatment of laser-induced CNV, underscoring its potential as a more potent therapeutic option than faricimab. The degree of vessel leakiness was associated with morphological changes in the retina at the site of laser injury. A distorted retinal architecture was apparent after a laser lesion, as revealed by thickening of the retina. To further assess the efficacy of GB10, we evaluated the improvement rate based on retinal thickness using OCT as an additional efficacy indicator, as illustrated in Fig. 5 C and Table 2 . OCT imaging revealed recovery in both the GB10 and faricimab treatment groups, with GB10 demonstrating a superior improvement compared to faricimab. Specifically, compared with the vehicle group, both low-dose and high-dose GB10 exhibited statistically significant increase in improvement rates at all time points. This suggests that GB10 effectively inhibited retinopathy over an extended duration, potentially facilitating more favorable conditions for vision recovery. In contrast, faricimab demonstrated statistically significant differences only on Day 7 for both low-dose and high-dose groups, but the significance did not persist in subsequent days. These data further supported that GB10 was more effective than faricimab in vivo . Table 1 Summary of improvement rate of fluorescent leakage area in grade 4 spots. Groups Time point Vehicle Low-dose (1 nmol/eye) High-dose (40 nmol/eye) Faricimab GB10 Faricimab GB10 Day 7 -15.38 ± 54.58% 46.66 ± 23.40% 70.22 ± 14.94% 78.88 ± 35.31% 100.00 ± 0.00% Day 14 8.60 ± 59.52% 57.46 ± 12.21% 71.49 ± 13.08% 84.81 ± 30.38% 93.64 ± 12.72% Day 21 35.47 ± 46.45% 67.13 ± 19.92% 92.24 ± 9.65% 79.64 ± 40.73% 100.00 ± 0.00% All numerical data were presented as mean ± standard deviation (SD). n = 4 per group. Table 2 Summary of improvement rate of retinal thickness. Groups Time point Vehicle Low-dose (1 nmol/eye) High-dose (40 nmol/eye) Faricimab GB10 Faricimab GB10 Day 7 33.34 ± 21.28% 85.43 ± 27.09% 111.91 ± 31.48% 93.47 ± 31.54% 94.26 ± 16.93% Day 14 57.72 ± 25.43% 93.57 ± 19.53% 120.75 ± 33.06% 98.89 ± 31.99% 115.02 ± 10.77% Day 21 73.95 ± 29.76% 96.68 ± 24.94% 124.73 ± 28.20% 108.85 ± 59.66% 123.51 ± 12.20% All numerical data were presented as mean ± standard deviation (SD). n = 4 per group. Intraocular pharmacokinetics of GB10 in a rabbit model Next, we evaluated the pharmacokinetic parameters of both GB10 and faricimab in vivo . We utilized New Zealand white rabbits for our ocular pharmacokinetic study due to the similarity of rabbit retinal structure to higher animals and human. Our research mainly focused on monitoring the changes in concentrations of GB10 and faricimab in vitreous samples by ELISA, and the results are presented in Fig. 6 . The data revealed differences in both T max and C max of GB10 and faricimab at the same dosage. Specifically, GB10 reached its maximum concentration (C max = 483.5 µg/mL) at 2.7 h after dosing, while faricimab reached its peak concentration (C max = 410.2 µg/mL) at 6.2 h, suggesting that GB10 may provide a more rapid therapeutic effect in clinical. Over time, the concentrations of both drugs gradually decreased and stabilized at a constant level. Further analysis showed that the half-life (T 1/2 ) of GB10 was 59.4 h, whereas the T 1/2 of faricimab was 39.6 h, suggesting that GB10 has a longer duration in the vitreous humor. The extended half-life of GB10 may present an advantage, allowing for a more sustained therapeutic effect. Additionally, the total drug exposure (AUC 0 − t ) calculated in this study was 44,867 h × µg/mL for GB10 and 46,315 h × µg/mL for faricimab. Moreover, no ocular inflammation or adverse events were observed in the intraocular PK study, indicating that GB10 demonstrated good tolerance in this experimental model (data not shown). The developability of GB10 in high-concentration formulation In the development of ocular drugs, it is crucial to consider not only the efficacy but also the frequency of dosing for patient compliance. In order to reduce the frequency of administration, GB10 was developed as a high-concentration formulation like faricimab. Given that higher protein concentration could potentially lead to an exponential increase in viscosity, which may affect the accuracy of delivered dose and cause pain at injection site, we first evaluated the viscosity of GB10 at high concentrations to ensure its injectability. As shown in Fig. 7 A, the viscosity was measured as low as 13.0 cP at 140 mg/mL, indicating favorable properties of GB10 for injection into eyes with low-gauge syringes even at high concentrations. Secondly, since there is a positive correlation between intravitreal retention time and the size of hydrodynamic radius of the drug [ 37 – 39 ], we analyzed GB10 using DLS, and the data showed that its hydrodynamic radius was 8.7 nm (Fig. 7 A), larger than monoclonal antibody with average hydrodynamic radius of 5.4 nm [ 40 ]. This result may correspond to the longer half-life of GB10 (59.4 h) in the vitreous than that of faricimab (39.6 h), which was evaluated in the previous chapter titled “Intraocular pharmacokinetics of GB10 in a rabbit model”. In addition, GB10 showed good thermal stability at 140 mg/mL, with a T agg of 56.2 ℃ and a T m of 63.0 ℃ (Fig. 7 A). Finally, we investigated the developability of GB10 in order to better understand the potential CMC risk. The developability assessment of GB10 was conducted by subjecting GB10 to a series of stress conditions, including high temperature (40°C), oxidation, pH, and repeated freeze-thaw. The stressed samples were then analyzed for their purity by SEC and in vitro activity. As illustrated in Fig. 7 B, the purity of GB10 was not compromised across a range of stress conditions confirmed by SEC. However, VEGF blocking assay revealed that oxidation and pH treatments resulted in ~ 10% reduction on Day 7 compared to T0 (Fig. 7 C). Similarly, the Ang-2 blocking activity of GB10 also decreased under high temperature, oxidation, and pH stress (Fig. 7 D). It is important to note that due to assay variations, relative activities between 70% and 130% are generally considered unaffected. Therefore, we believed that the change in the activity of GB10 under these stress conditions was acceptable. Besides, our studies demonstrated that there were no significant changes in SEC purity, VEGF blocking activity and Ang-2 blocking activity of GB10 after three and five freeze-thaw cycles (data not shown). Taken together, GB10 exhibited an excellent developability profile and could be successfully formulated at high-concentration, which is expected to improve the convenience of its clinical application. Discussion Despite major medical advances that have been achieved with VEGF inhibitors, unmet clinical needs have not yet fully addressed as there are still non-responders and a lack of long-term efficacy in anti-VEGF monotherapies. In this study, we developed GB10, a more effective and durable antibody fusion protein, to address these unmet clinical needs. One advancement in this study is that GB10 demonstrates significantly greater efficacy. First, in contrast to VEGF monotherapy, GB10 additionally targets Ang-2, another crucial growth factor involved in neoangiogenesis and vascular permeability. Previous publications have shown that retinal neovascularization typically involves complex pathological mechanisms, making the inhibition of a single-target pathway, such as VEGF, often inadequate for achieving optimal therapeutic effects [ 41 , 42 ]. Second, in contrast to faricimab, a marketed bispecific antibody only targeting VEGF-A and Ang-2, GB10 based on the framework of VEGF-Trap can target additional VEGF family members, including VEGF-B and PlGF, both of which also play important roles in angiogenesis [ 43 – 45 ]. The multi-pronged approach of GB10 has best-in-class potential in the treatment of ocular neovascular diseases. Moreover, compared to faricimab, GB10 showed greater in vitro and in vivo efficacy, which may be attributed to the molecular design. Specifically, faricimab is an asymmetric monovalent (1 + 1) heterodimeric bispecific antibody [ 22 ], and there may be concerns that its monovalence may not fully suppress the relevant signaling pathways; GB10 is designed as a symmetric homodimer (2 + 2) antibody fusion protein that enables more effective inhibition compared to monovalent antibody fragments. As we expected, in vitro efficacy studies showed that GB10 was 7.5-fold more potent in VEGF blocking assay; and the inhibition rate of GB10 reached 100% on Ang-2-Tie-2 signaling pathway, whereas that of faricimab was only 70%. Consistent with the in vitro efficacy studies, GB10 exhibited superior in vivo efficacy compared to equimolar faricimab, based on an evaluation conducted in a laser-induced cynomolgus monkey CNV model. Our results showed that GB10 significantly reduced fluorescent leakage and restored retinal thickness, and in all dosage group, the improvement rate of GB10 was superior to that of faricimab. Therefore, according to these results observed in non-human primate model which closely mimics the conditions observed in human diseases and clinical efficacy of drug treatment, we can infer that GB10 may also effectively reduce pathological angiogenesis in human eyes and improve visual function in clinical applications. The other advancement is that GB10 demonstrates a longer duration of efficacy than that of faricimab. Besides higher biological activity discussed above, the following observations may contribute to longer durability of GB10. First, PK data revealed that the half-life of GB10 (59.4 h) in the vitreous of rabbit eyes was longer than that of faricimab (39.6 h), reducing the frequency of IVT injections. Second, the stability of GB10 is a crucial factor influencing the duration of efficacy. Developability assessment demonstrated that the purity and in vitro efficacy of GB10 could not be affected under various stress conditions including elevated temperature, non-physiological pH and oxidation among others, ensuring that GB10 can sustain effective concentrations in the eyes for an extended period after injection. This good stability of GB10 is attributed to its molecular design strategy. Compared to other molecules in the scFv format, GB10 — in the VHH format — exhibited a low tendency to aggregate, resulting in superior conformational and colloidal stability, as indicated by the results from HIC and DLS assays. Moreover, GB10 could achieve an ultra-high concentration of 140 mg/mL, exhibiting a clear competitive advantage compared to marketed high-concentration IVT drugs, such as Vabysmo® (faricimab, Roche, 120 mg/mL) as equal molar concentration of GB10 can achieve relatively longer duration than that of faricimab. Among patients receiving faricimab, only about 45% were able to achieve a dosing interval of 4 months within the first year [ 46 ]. In contrast, GB10 at 140 mg/mL, higher than that of faricimab, is expected to significantly prolong the dosing interval, thereby increasing the proportion of patients who can achieve a dosing interval of 4 months or even longer. Overall, GB10 shows good durability, making it more convenient and effective for patients to use. Conclusions In conclusion, GB10, a novel antibody fusion protein demonstrates significant advantages including multi-targeted blockade, excellent in vitro activity and in vivo efficacy, as well as ultra-high concentration formulations. These advantages may position GB10 as a promising best-in-class therapeutic candidate for neovascular eye diseases, deserving further evaluation from bench to bedside. Declarations Declarations Ethics approval All animal studies were approved by the Institutional Animal Care and Use Committee guidelines of WestChina-Frontier PharmaTech Co., Ltd. (China) or Shenzhen Kexing Biopharm Co., Ltd. (China), a subsidiary of Kexing Biopharm Co., Ltd. Consent for publication Not applicable. Competing interests The authors declare no competing interests. Authors details 1 Drug Discovery, Centre for Research and Development, Kexing Biopharm Co., Ltd. Shenzhen 518057, China Funding This research was funded by Shenzhen Kexing Biopharm Co., Ltd. Author contributions Suofu Qin conceptualized and supervised the study. Xiling Wei performed the experiments for VHH nanobody discovery and Ang-2 functional activity assessment in vitro , and drafted the manuscript. Yuxin Qiu designed and expressed these antibody fusion proteins. Wei Shang performed cell-based functional activity studies in vitro . Xiangling Zhang conducted experiments in vivo . Wenjie Yang, Chengyong Yang and Xi Chen coordinated the developability assessment. Huiming Li and Suofu Qin edited the manuscript. Acknowledgements Not applicable. Data availability All research data generated during the current study are available upon request. References Bourne RRA, Stevens GA, White RA, Smith JL, Flaxman SR, Price H, et al. Causes of vision loss worldwide, 1990–2010: a systematic analysis. Lancet Glob Health. 2013;1:e339–49. Melincovici CS, Boşca AB, Şuşman S, Mărginean M, Mihu C, Istrate M, et al. 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Abbreviations AMD Age-related macular degeneration Ang-2 Angiopoietin 2 CNV Choroidal neovascularization DLS Dynamic light scattering DME Diabetic macular edema DR Diabetic retinopathy DSF Differential scanning fluorimetry ELISA Enzyme-linked immunosorbent assay HIC Hydrophobic interaction chromatography IVT Intravitreal RVO Retinal vein occlusion scFv Single-chain variable fragment SEC Size exclusion chromatography Tie-2 Tyrosine-protein kinase receptor VEGF Vascular endothelial growth factor VHH Variable heavy domain of heavy-chain antibody Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major revision 25 Dec, 2024 Reviewers agreed at journal 20 Oct, 2024 Reviewers invited by journal 16 Oct, 2024 Editor assigned by journal 14 Oct, 2024 First submitted to journal 22 Sep, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5135499","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":366936592,"identity":"0a1cb0d7-4371-40a3-b53a-6a6d1eb76443","order_by":0,"name":"Xiling Wei","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBklEQVRIiWNgGAWjYFACxgaGhAIJBgZmxsYHEhVAAXawGCEtBkAt7MyHDSzOMDDwMBPUAgIGQMzPliZR2UaEFoPjzW0SDwws8uSdeQwkbs6zSdzPzHzw4QwGOzldHPoMzhxsNgA6rNjwMI+B4cxtaYk9zGzJhhsYko3NDmDXYnYjsfEBUEvixmYeg2TJbYeBWnjMJB8wHEjchkvL/YcNB2BaDv+dQ4yWG4wQW+YzsyU2SDZAtWzAo8X+TCLYL4kbmJkPM0gcSzPuOQz0ywwD3H6RbD/+TPJHRV3i/P6D7T8kamxk29ubDz7sqbCTw6UFDgxQFRgQUA4C8g1EKBoFo2AUjIKRCQBvIV4nILr91wAAAABJRU5ErkJggg==","orcid":"","institution":"Kexing Biopharm Co.,LTD.","correspondingAuthor":true,"prefix":"","firstName":"Xiling","middleName":"","lastName":"Wei","suffix":""},{"id":366936593,"identity":"ab35247a-51ca-40fe-a904-a18465a317b6","order_by":1,"name":"Yuxin Qiu","email":"","orcid":"","institution":"Kexing Biopahrm Co., Ltd.","correspondingAuthor":false,"prefix":"","firstName":"Yuxin","middleName":"","lastName":"Qiu","suffix":""},{"id":366936594,"identity":"39b1d775-ff78-4513-a7d7-c0a74fd42a42","order_by":2,"name":"Wei Shang","email":"","orcid":"","institution":"Kexing Biopharm Co., Ltd.","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Shang","suffix":""},{"id":366936595,"identity":"01d19744-cc74-49b7-b297-9d9b888495fc","order_by":3,"name":"Xiangling Zhang","email":"","orcid":"","institution":"Kexing Biopharm Co., Ltd.","correspondingAuthor":false,"prefix":"","firstName":"Xiangling","middleName":"","lastName":"Zhang","suffix":""},{"id":366936596,"identity":"384fdeff-c84f-4745-86ea-f7478f70de10","order_by":4,"name":"Wenjie Yang","email":"","orcid":"","institution":"Kexing Biopharm Co., Ltd.","correspondingAuthor":false,"prefix":"","firstName":"Wenjie","middleName":"","lastName":"Yang","suffix":""},{"id":366936597,"identity":"c0691697-5d14-42eb-a047-c80ffea14f2b","order_by":5,"name":"Chengyong Yang","email":"","orcid":"","institution":"Kexing Biopharm Co., Ltd.","correspondingAuthor":false,"prefix":"","firstName":"Chengyong","middleName":"","lastName":"Yang","suffix":""},{"id":366936598,"identity":"b1e59562-4450-4558-ab34-9537571d177c","order_by":6,"name":"Xi Chen","email":"","orcid":"","institution":"Kexing Biopharm Co., Ltd.","correspondingAuthor":false,"prefix":"","firstName":"Xi","middleName":"","lastName":"Chen","suffix":""},{"id":366936599,"identity":"8e483ff0-5a7e-4357-b10d-7e28791dc63c","order_by":7,"name":"Huiming Li","email":"","orcid":"","institution":"Kexing Biopharm Co., Ltd.","correspondingAuthor":false,"prefix":"","firstName":"Huiming","middleName":"","lastName":"Li","suffix":""},{"id":366936600,"identity":"edb2392e-eb19-46ab-a70a-5b6115e877ce","order_by":8,"name":"Suofu Qin","email":"","orcid":"","institution":"Kexing Biopharm Co., Ltd.","correspondingAuthor":false,"prefix":"","firstName":"Suofu","middleName":"","lastName":"Qin","suffix":""}],"badges":[],"createdAt":"2024-09-23 06:14:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5135499/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5135499/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":69290415,"identity":"e5398c76-99c5-4a16-ad20-7414d3876369","added_by":"auto","created_at":"2024-11-18 21:38:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":472463,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDesign and selection of different molecular formats. \u003c/strong\u003e(A) Schematic overview of the structure. (B-C) The inhibitory activity of Format 1/2/3/4/5 on VEGF-A\u003csub\u003e165\u003c/sub\u003e-induced luciferase reporter gene expression in H-VEGF Reporter 293 cells (B) and on Ang-2 binding to Tie-2, as measured by ELISA (C). The data were analyzed using a four-parameter curve fitting method with GraphPad Prism and presented as mean ± SD. (D) Summary table of IC\u003csub\u003e50\u003c/sub\u003e values for each format. (E) Retention time of Format 1 and Format 2, as determined by the HIC.\u003c/p\u003e","description":"","filename":"OnlineFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5135499/v1/b7156f5a402093c0d1bd4cd3.png"},{"id":69290849,"identity":"867932ca-a6bc-450a-8166-4c82e899ad83","added_by":"auto","created_at":"2024-11-18 21:54:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":226397,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGeneration of anti-Ang-2 nanobody.\u003c/strong\u003e (A) Blocking assay of VHHs on Ang-2 binding to Tie-2 by competition ELISA. (B-C) The specific binding activity (B) and cross-species activity (C) of VHH-04 were detected by ELISA. The data were analyzed using a four-parameter curve fitting method with GraphPad Prism and presented as mean ± SD.\u003c/p\u003e","description":"","filename":"OnlineFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5135499/v1/5e5899fa27eea58c51820db2.png"},{"id":69290563,"identity":"fbd23165-2fa5-469e-aba4-a322879ac903","added_by":"auto","created_at":"2024-11-18 21:46:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":386673,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConstruction and binding affinity of GB10. \u003c/strong\u003e(A) GB10 comprises a VEGF-Trap (aflibercept) at the N-terminal end of a human IgG1 Fc and an Ang-2-specific VHH-04 connected via a G\u003csub\u003e4\u003c/sub\u003eS linker to the C-terminus (green line). (B) GB10 is capable of binding to VEGF and Ang-2 simultaneously. (C-D) Summary data table of binding kinetics of GB10 to VEGF family ligands of human/rat/mouse/rabbit (C) and Ang-2 family ligands of human/cynomolgus/mouse/rabbit (D) determined by BLI.\u003c/p\u003e","description":"","filename":"OnlineFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5135499/v1/51ad0f66568a78feebdea19a.png"},{"id":69290422,"identity":"a8eaaa25-83ab-4735-a563-4af4584863cc","added_by":"auto","created_at":"2024-11-18 21:38:47","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":359417,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIn vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e functional activity of GB10. \u003c/strong\u003e(A-B) The inhibitory potency of GB10 and faricimab was assessed by VEGF-A\u003csub\u003e165\u003c/sub\u003e-induced expression of a luciferase reporter (A) and HUVEC proliferation assay (B). (C) The blocking activity of GB10 and faricimab on Ang-2/Tie-2 binding in ELISA assay. (D) The inhibitory potency of GB10 compared with faricimab on the Ang-2 signaling pathway, as measured by a Tie-2 phosphorylation assay. The data were analyzed using a four-parameter curve fitting method with GraphPad Prism and presented as mean ± SD.\u003c/p\u003e","description":"","filename":"OnlineFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5135499/v1/5e9a3fb2a91e29172f340f09.png"},{"id":69290420,"identity":"3309de6f-aaed-4355-bc1b-ac0d96b9f5b3","added_by":"auto","created_at":"2024-11-18 21:38:47","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2852247,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEfficacy of GB10 in a laser-induced CNV model in cynomolgus monkeys. \u003c/strong\u003e(A) Schematic representation of the experimental setup including modeling, grouping, treatment, and efficacy readouts. (B) Representative pictures of FFA on Day 0 (before dosing), Day 7, 14 and 21 after intravitreal injection of vehicle, GB10 and faricimab of 1 nmol/eye and 40 nmol/eye, respectively. The improvement rates of fluorescence leakage area (%) in grade 4 spots among groups were analyzed using multiple unpaired t test and presented as mean ± SD (n = 4 per group). (C) Representative pictures of OCT on Day 0 (before dosing), Day 7, 14 and 21 after intravitreal injection of vehicle, GB10 and faricimab of 1 nmol/eye and 40 nmol/eye, respectively. The improvement rates of retinal thickness (%) were analyzed using multiple unpaired t test and presented as mean ± SD (n = 4 per group). GB10 and faricimab group were compared with vehicle group, *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001, ****\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.0001, ns, no significance.\u003c/p\u003e","description":"","filename":"OnlineFigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5135499/v1/4cef594fafb47586c3d8abf4.png"},{"id":69290565,"identity":"f6ffceb1-a4f2-4d8a-b0be-00d775e77591","added_by":"auto","created_at":"2024-11-18 21:46:47","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":178807,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIntraocular pharmacokinetics of GB10 in rabbits.\u003c/strong\u003e Time-concentration plots for GB10 and faricimab concentrations in the eyes of New Zealand white rabbits after intravitreal administration. Points represent the observed concentrations, and lines represent the estimated concentrations determined by the noncompartmental model. All data were presented as mean ± SD (n = 16 per group).\u003c/p\u003e","description":"","filename":"OnlineFigure6.png","url":"https://assets-eu.researchsquare.com/files/rs-5135499/v1/48f272f7d86c4c09b21e5ccc.png"},{"id":69290421,"identity":"c1e4f6f6-cfe3-4a4d-8ae2-ca06f12a42b9","added_by":"auto","created_at":"2024-11-18 21:38:47","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":331974,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDevelopability assessment of GB10. \u003c/strong\u003e(A) Summery data table of the \u003cem\u003eR\u003c/em\u003eh/T\u003csub\u003eagg\u003c/sub\u003e/T\u003csub\u003em\u003c/sub\u003e/viscosity of GB10 in 140 mg/mL. (B-D) Developability of GB10 upon stress. Two-time points (Day 0 and Day 7) were chosen to investigate whether high temperature (40 ℃), oxidation and pH affect the SEC purity (B), relative activity \u003cem\u003ein vitro\u003c/em\u003e on VEGF blocking (C) and Ang-2 blocking (D) of GB10.\u003c/p\u003e","description":"","filename":"OnlineFigure7.png","url":"https://assets-eu.researchsquare.com/files/rs-5135499/v1/c05d523f1c62381478a3a16d.png"},{"id":69291085,"identity":"b98449a3-1fc5-47c9-b69e-d8c2107d39ac","added_by":"auto","created_at":"2024-11-18 22:03:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7889174,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5135499/v1/6b9e76a3-c97c-403a-9602-b05323a0b4d2.pdf"}],"financialInterests":"","formattedTitle":"GB10, a best-in-class antibody fusion protein targeting VEGF/Ang-2, exhibits promising therapeutic efficacy for neovascular eye diseases","fulltext":[{"header":"Background","content":"\u003cp\u003eNeovascular eye diseases, such as age-related macular degeneration (AMD), diabetic macular edema (DME), diabetic retinopathy (DR) and retinal vein occlusion (RVO), are major causes of severe vision impairment and blindness [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The progression of these diseases is often closely related to abnormal angiogenesis, in which VEGF plays a central role [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Currently, anti-VEGF therapies, such as Lucentis\u0026reg; (ranibizumab, Genentech), Eylea\u0026reg; (aflibercept, Regeneron) and Langmu\u0026reg; (conbercept, Kanghong), have become the standard treatments for neovascular eye diseases [\u003cspan additionalcitationids=\"CR4 CR5 CR6\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. With the application of anti-VEGF therapies, the incidence of legal blindness and vision impairment caused by ocular neovascularization has been reduced, leading to lower economic and social costs and improved vision-related quality of life [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, there remain several limitations and challenges for neovascular eye diseases. For example, many patients do not respond completely or suffer from a diminished response over time, which may be attributed to the limited efficacy of VEGF monotherapies resulting from solely inhibiting the VEGF pathways [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Approximately 20\u0026ndash;40% of patients with AMD [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] and 15\u0026ndash;20% of those with DR [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] do not adequately or fully respond to anti-VEGF therapies and require alternate treatment strategies for disease management. In addition to efficacy limitations, another significant challenge of anti-VEGF therapies is the durability, thereby requiring repeated intravitreal injections [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Frequent IVT injections can lead to high non-adherence/dropout over time, further exacerbating the illness [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Moreover, it increases the risk of complications, such as sustained elevation of intraocular pressure, inflammation, and hemorrhage [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo overcome the above challenges, novel therapeutic strategies are clearly needed. One approach is to enhance the efficacy of anti-VEGF-based therapies, including the design of multi-targeted molecules capable of inhibiting additional pathophysiological pro-angiogenic pathways. Many studies have demonstrated that angiopoietin-2 (Ang-2) plays a synergistic role with VEGF in vascular instability, leading to vascular leakage, neovascularization, and inflammation [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In human eyes, increased intraocular Ang-2 levels were detected in patients with DR and RVO [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In addition, higher levels of Ang-2 were associated with disease severity in AMD [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], suggesting a potential benefit of targeting ocular Ang-2. Therefore, the combined inhibition of VEGF and Ang-2 is expected to be more effective than VEGF inhibition alone in preventing ocular neovascularization. The other strategy is optimizing the dosage by developing high-concentration formulations that can deliver sufficient drug concentrations in a smaller volume to ensure prolonged therapeutic effects and reduced frequency of IVT administration. In recent years, there have been clinical studies that have proved the feasibility of this strategy. In the phase III HAWK and HARRIER trials [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], nAMD patients receiving 6 mg of brolucizumab achieved comparable visual improvement to those receiving 2 mg of aflibercept, but showed better improvements in retinal fluid; importantly, more than 50% of the brolucizumab 6 mg treatment arm achieved a dosing interval of 12 weeks, while the dosing interval for aflibercept 2 mg was 8 weeks. The results of the 2-year phase III PULSAR study [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] showed that the visual benefits for nAMD patients receiving 8 mg of aflibercept were similar to those receiving 2 mg of aflibercept, with 83% of patients maintaining\u0026thinsp;\u0026ge;\u0026thinsp;12-week treatment intervals. Therefore, the development of high-concentration formulations has become an important trend in the field of intravitreal injection therapy, offering patients with more convenient and effective treatment options.\u003c/p\u003e \u003cp\u003eCurrently, the use of VHH in assembling multi-targeted molecules has emerged as an important research focus in biomedicine [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. VHH is an antibody fragment derived from camelid animals, including alpacas and llamas. Compared to single-chain variable fragments (scFv) from conventional antibodies, VHH has a smaller molecular weight and a simpler structure, which allows it to remain stable under extreme conditions (e.g., low pH), thereby enhancing the stability of the CMC process [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Several bispecific antibodies based on nanobodies are currently undergoing clinical evaluation for the treatment of various diseases [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we generated GB10, a novel multi-targeted antibody fusion protein featuring VEGF-Trap at the N-terminus and anti-Ang-2 VHH at the C-terminus, that bound and neutralized VEGF family members and Ang-2 with high potency \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e, and then developed it into a high-concentration formulation of 140 mg/mL. Through advancing this potentially best-in-class molecule into the clinic in the future, we hope to provide a more effective and long-term treatment option for patients with neovascular eye diseases.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMolecule design, expression and purification\u003c/h2\u003e \u003cp\u003eMulti-targeted molecules in two different categories were created. Category #1 was antibody fusion protein featuring VEGF-Trap at the N-terminus and anti-Ang-2 antibody at the C-terminus, and three design formats were generated. Category #2 was bispecific antibody with anti-VEGF-A antibody at the N-terminus and anti-Ang-2 antibody at the C-terminus, and two formats were made.\u003c/p\u003e \u003cp\u003epcDNA3.4 plasmid containing DNA fragments encoding target proteins was transfected into CHO cells by electroporation. The supernatant containing target proteins was harvested after 6 days of fed-batch cultivation and purified by Protein A affinity chromatography (AKTA, GE Healthcare).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eVEGF blocking assay\u003c/h3\u003e\n\u003cp\u003eEngineered cell lines (H-VEGF Reporter 293 Cell line, GM-C09057, Genomeditech) for VEGF signaling in assay media (DMEM, C11995500BT, Gibco) were seeded at 25,000 cells/well on a 96-well plate where human VEGF-A\u003csub\u003e165\u003c/sub\u003e (C083, Novoprotein) and 3-fold serially diluted samples ranging from 25 nM to 0.0004 nM had been pre-incubated. The microplate was incubated at 37 ℃, 5% CO\u003csub\u003e2\u003c/sub\u003e for 6 h to induce luciferase gene expression by VEGF signaling. The relative VEGF blocking activity was measured by One-Lite\u0026reg; Luciferase Assay System (DD1203, Vazyme) via Varioskan\u0026trade; LUX (Thermo Scientific) according to the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e\n\u003ch3\u003eAng-2 blocking assay\u003c/h3\u003e\n\u003cp\u003eA recombinant human Tie-2 protein (TI2-H5255, ACROBiosystems) was used as the coating antigen in the blocking ELISA. The wells of Nunc 96-well Maxisorp plates were coated with 100 ng of Tie-2 in coating buffer (C1050, Solarbio) and incubated at 4 ℃ overnight. The next day, after blocking with blocking buffer (SW3015, Solarbio) at 37 ℃ for 2 h, the plates were washed three times with PBST (phosphate-buffered saline containing 0.05% Tween-20), then 3-fold serially diluted samples ranging from 20 nM to 0.0003 nM pre-incubated with 100 ng/mL human Ang-2 (10691-H08H, SinoBiological, His tag) were added and incubated at 37 ℃ for 1 h. After washing three times, 100 \u0026micro;L of HRP Anti-6X His tag\u0026reg; antibody (ab1187, Abcam) diluted 1:5,000 in PBST was dispensed into each well, and the plates were incubated at 37 ℃ for 30 min. Plates were washed six times and TMB Single-Component Substrate Solution (PR1200, Solarbio, 100 \u0026micro;L/well), and ELISA Stop Solution (C1058, Solarbio, 50 \u0026micro;L/well) were added sequentially. Absorbance at 450 nm was quantified via Varioskan\u0026trade; LUX (Thermo Scientific).\u003c/p\u003e\n\u003ch3\u003eHydrophobic Interaction Chromatography (HIC) analysis\u003c/h3\u003e\n\u003cp\u003eHIC analysis was performed on a 1260 Infinity II system (Agilent Technologies) with UV detection at 214 nm. Briefly, 10 \u0026micro;g of each sample prepared in 0.1 M phosphate buffer, pH 6.5 with 1 M ammonium sulphate was separated on a Protemix HIC Butyl-NP5 column (4.6\u0026times;100 mm, 5 \u0026micro;m, Sepax) with a linear gradient from 44\u0026ndash;100% of mobile phase B (mobile phase A: 0.1 M sodium dihydrogen phosphate and 1.8 M ammonium sulphate, pH 6.5; mobile phase B: 0.1 M sodium dihydrogen phosphate, pH 6.5) at a flow rate of 1.0 mL/min. Data were acquired and processed by Empower 3 software (Waters).\u003c/p\u003e\n\u003ch3\u003eSize Exclusion Chromatography (SEC) analysis\u003c/h3\u003e\n\u003cp\u003eSEC analysis was performed on a LC-2030C system (SHIMADZU) with UV detection at 280 nm. Briefly, 10 \u0026micro;g of sample in PBS was separated on a BioCore SEC-300 column (7.8\u0026times;300 mm, 5 \u0026micro;m, NanoChrom) using a mobile phase consisting of 0.1 M phosphate buffered saline and 0.15 M sodium chloride, pH 7.0 at a flow rate of 0.5 mL/min. Data were acquired and processed by Empower 3 software (Waters).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eGeneration of anti-Ang-2 VHH\u003c/h2\u003e \u003cp\u003eOne alpaca was immunized with 500 \u0026micro;g human Ang-2 (10691-H08H, SinoBiological) emulsified with Complete Freund\u0026rsquo;s adjuvant (F5881, Sigma-Aldrich) for the first time and Incomplete Freund\u0026rsquo;s adjuvant (F5506, Sigma-Aldrich) for each subsequent immunization.\u003c/p\u003e \u003cp\u003ePeripheral blood mononuclear cells (PBMCs) were isolated after the fifth immunization, then total RNA was extracted with TRIzol (15596018, Invitrogen) for reverse transcription (PrimeScript II kit, 6210A, TAKARA). DNA fragments encoding VHH were amplified and cloned into phagemid pCom3xss to prepare the phage VHH library. Anti-Ang-2 VHH was displayed with the assistance of helper phage M13KO7 after the VHH-phagemid was transformed into \u003cem\u003eE coli.\u003c/em\u003e TG1 by electroporation.\u003c/p\u003e \u003cp\u003eHuman Ang-2 was used for biopanning. Approximately 2 \u0026times; 10\u003csup\u003e11\u003c/sup\u003e plaque-forming units were added into an immuno-tube pre-coated with BSA, and incubated at 37 ℃ for 1 h. Unbound phages were collected and incubated in an immuno-tube pre-coated with human Ang-2 at 37 ℃ for 1 h for positive biopanning. The human Ang-2 bound phages were eluted using 0.1 M Glycine-HCl (pH 2.2). The Ang-2 binding affinities of candidate phages were evaluated by ELISA.\u003c/p\u003e \u003cp\u003eThe variable region sequences of positive candidates were retrieved by Sangers sequencing and recombinantly expressed in CHO cells as previously described in the Method section under \u0026ldquo;Molecule design, expression and purification\u0026rdquo;.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAng-2 and Ang-1 binding assay\u003c/h3\u003e\n\u003cp\u003eNunc 96-well Maxisorp plates were coated with 1 \u0026micro;g/mL human Ang-2 (10691-H08H, SinoBiological), cynomolgus Ang-2 (90026-C08H, SinoBiological), mouse Ang-2 (AN2-M52H3, ACROBiosystems) and human Ang-1 (923-AN-025, R\u0026amp;D system) at 4\u0026deg;C overnight. The next day, the plates were washed with PBST, blocked for 2 h with blocking buffer and incubated with samples in 3-fold dilution series ranging from 20 nM to 0.0001 nM for 1 h at 37\u0026deg;C. After washing three times, 100 \u0026micro;L of goat anti-human IgG Fc (HRP) (ab97225, Abcam) diluted 1:10,000 in PBST was dispensed into each well, and the plates were incubated at 37 ℃ for 30 min. Finally, TMB solution and stop solution were added sequentially as previously described in the Method section under \u0026ldquo;Ang-2 blocking assay\u0026rdquo;. Absorbance at 450 nm was quantified via Varioskan\u0026trade; LUX (Thermo Scientific).\u003c/p\u003e\n\u003ch3\u003eGB10 expression and purification\u003c/h3\u003e\n\u003cp\u003eGB10 belongs to Format 2 of Category #1 that comprises an N-terminal VEGF-Trap based on Aflibercept, and a C-terminal anti-Ang-2 nanobody (VHH-04) on a human IgG1 backbone. VHH-04 is connected to the Fc via a (G\u003csub\u003e4\u003c/sub\u003eS)\u003csub\u003e4\u003c/sub\u003e linker. GB10 expression and purification were performed as previously described in the Method section under \u0026ldquo;Molecule design, expression and purification\u0026rdquo;.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eVEGF and Ang-2 affinity characterization using Bio-Layer Interferometry (BLI)\u003c/h2\u003e \u003cp\u003eA Bio-Layer Interferometry (BLI) assay was performed using an Octet\u0026reg; R8 (Sartorius) instrument. Each sample was immobilized onto Octet ProA Biosensors (18-5010, Sartorius) until a signal of 1.0 nm was reached. Antigen solution in a 2-fold dilution series covering a concentration range from 0.5 nM to 512 nM was added to the sample-loaded sensors, and the interaction was monitored for 300 s association and 300 s dissociation. Kinetic parameters were determined using Octet\u0026reg; Software Version 13 (Sartorius).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eBridging assay\u003c/h2\u003e \u003cp\u003eA Nunc 96-well Maxisorp plate was coated with recombinant human VEGF-A\u003csub\u003e165\u003c/sub\u003e (C083, Novoprotein, 200 ng/well) overnight at 4 ℃. The next day, after blocking with blocking solution, 3-fold serially diluted samples ranging from 18 nM to 0.0009 nM were added and incubated at 37\u0026deg;C for 1 h. After washing three times with PBST, human Ang-2 (10691-H08H, SinoBiological, 50 ng/well) was added and incubated at 37\u0026deg;C for 1 h. Finally, HRP Anti-6X His tag\u0026reg; antibody, TMB solution and stop solution were added sequentially as previously described in the Method section under \u0026ldquo;Ang-2 blocking assay\u0026rdquo;. Absorbance at 450 nm was quantified via Varioskan\u0026trade; LUX (Thermo Scientific).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eHUVEC proliferation assay\u003c/h2\u003e \u003cp\u003eHUVEC (C-12205, Promocell) in endothelial cell growth medium (C-22010, Promocell) was seeded at 3,000 cells/well. 4-fold serially diluted samples ranging from 25 nM to 0.0004 nM and 10 ng/mL VEGF-A\u003csub\u003e165\u003c/sub\u003e (C083, Novoprotein) were preincubated for 1 h at room temperature. The mixtures were then immediately added to the wells of 96-well plates containing HUVEC and incubated for 3 days at 37 ℃, 5% CO\u003csub\u003e2\u003c/sub\u003e. Proliferation of HUVEC was measured by CellTiter-Glo\u0026reg; Luminescent Cell Viability Assay kit (G7570, Promega) using a Varioskan\u0026trade; LUX (Thermo Scientific) according to the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eTie-2 phosphorylation assay\u003c/h2\u003e \u003cp\u003eEngineered cell lines (huTie2-HEK293, C2209248, Sanyou Bio) for Ang-2 signaling in assay media (DMEM, L110KJ, Basal Media) were seeded at 32,000 cells/well on a 96-well plate where 60 nM human Ang-2 (10691-H02H, SinoBiological) and serially diluted samples ranging from 2,000 nM to 10 nM had been pre-incubated. The microplate was incubated at 37 ℃, 5% CO\u003csub\u003e2\u003c/sub\u003e for 1.5 h to induce Tie-2 phosphorylation by Ang-2 signaling. After incubation, cells were lysed using Cell Lysis Buffer (9803S, CST), and the lysate supernatant was added to an ELISA plate for quantification of Tie-2 phosphorylation by Human Phospho-Tie-2 DuoSet IC ELISA (DYC2720-5, R\u0026amp;D system) on SpectraMax 190 (Molecular Devices) according to the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eLaser-induced CNV in cynomolgus monkeys\u003c/h2\u003e \u003cp\u003e This study was conducted at WestChina-Frontier PharmaTech Co., Ltd. in accordance with the IACUC standard animal procedures along with the IACUC guidelines that was in compliance with the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals. A total of twelve cynomolgus monkeys (supplied by Hainan Jingang Biotech Co., Ltd.) at 2\u0026ndash;6 kg body weight were used. CNV was induced by laser photocoagulation on Day \u0026minus;\u0026thinsp;14 in both eyes of the cynomolgus monkeys. Nine lesions were symmetrically placed in the macula of each eye using a power setting of 0.5\u0026ndash;0.7 W, a spot size of 50 \u0026micro;m and a duration of 0.05 s. Based on the number of spots with fluorescein leakage of grade 4, which was considered clinically relevant [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], ten suitable animals were selected for treatment and randomly divided into 5 groups on Day 0 as follows: vehicle group, faricimab low-dose group, faricimab high-dose group, GB10 low-dose group, and GB10 high-dose group. Then intravitreal injections of 50 \u0026micro;L sample solution per eye were performed on both eyes (low-dose\u0026thinsp;=\u0026thinsp;1 nmol/eye; high-dose\u0026thinsp;=\u0026thinsp;40 nmol/eye). The therapeutic efficacy was evaluated on Day 7, 14 and 21 after dosing by fundus fluorescein angiography (FFA) and optical coherence tomography (OCT). The images taken about 5 min after the fluorescein injections were used for comparison of improvement rate using the following equation: improvement rate (%) = [FLA (Day 0 before dosing) \u0026ndash; FLA (Day 7, 14, 21 after dosing)] \u0026divide; [FLA (Day 0 before dosing)] \u0026times; 100%, FLA, fluorescein leakage area. The thickness of the retina around the burn spot was measured to compare improvement rates using the following equation: improvement rate (%) = [RT (Day 0 before dosing) \u0026ndash; RT (Day 7, 14, 21 after dosing)] \u0026divide; [RT (Day 0 before dosing) \u0026ndash; RT (Day \u0026minus;\u0026thinsp;14 before laser)] \u0026times; 100%, RT, retinal thickness.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003ePharmacokinetic analysis\u003c/h2\u003e \u003cp\u003e Pharmacokinetic study was conducted at Shenzhen Kexing Biopharm Co., Ltd. in accordance with the relevant regulations of the Institutional Animal Care and Use Committee (IACUC), also followed the Guide for the Care and Use of Laboratory Animals, Eighth Edition (National Research Council, 2011). A total of 32 New Zealand white rabbits (supplied by GuangDong Medical Laboratory Animal Center) received a single intravitreal injection of GB10 at 500 \u0026micro;g/eye (Group 1, n\u0026thinsp;=\u0026thinsp;16) and faricimab at 500 \u0026micro;g/eye (Group 2, n\u0026thinsp;=\u0026thinsp;16). GB10 and faricimab concentrations were determined in the vitreous up to 28 days (2 h, 6 h, 8 h, 24 h, 72 h, 168 h, 14 days, 28 days) after dosing by ELISA. The pharmacokinetic parameters in the vitreous were analyzed by a noncompartmental analysis using Phoenix WinNonlin software version 1.3 (Certara).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eDynamic Light Scattering (DLS)\u003c/h2\u003e \u003cp\u003eDLS experiment was carried out using a DynaPro Plate Reader III (Wyatt Technology). Briefly, 30 \u0026micro;L of sample solution was carefully loaded into a 384-well plate without introducing bubbles. Hydrodynamic radius (\u003cem\u003eR\u003c/em\u003eh) measurement was performed at 25\u0026deg;C and determination of aggregation onset temperature (T\u003csub\u003eagg\u003c/sub\u003e) was then conducted in a temperature ramp of 1\u0026deg;C/min from 25 to 80\u0026deg;C. Data acquisition and analysis were carried out via the DYNAMICS software (Wyatt).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eDifferential Scanning Fluorimetry (DSF)\u003c/h2\u003e \u003cp\u003eDSF was conducted on a nanoDSF system (Prometheus NT.48, NanoTemper). Briefly, 15 \u0026micro;L of sample solution was loaded into the capillaries. The samples were heated from 20 to 80 ℃ at a rate of 1 ℃/min, and fluorescence signals were collected and analyzed using the software PR ThermControl (Prometheus).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eViscosity\u003c/h2\u003e \u003cp\u003eThe viscosity was measured with the Viscosizer (RheoSense micro VISC). Briefly, 300 \u0026micro;L of sample solution was carefully transferred in the microVisc unit without introducing bubbles. The viscosity values of GB10 at 140 mg/mL were determined in \u0026ldquo;Auto mode\u0026rdquo;.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eData are represented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD and analyzed using GraphPad Prism. The differences between the groups were compared by the multiple unpaired t-test. Statistical significance was considered for P-values below 0.05. *\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ***\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; ****\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eGeneration and screening of VEGF and Ang-2 multi-targeted molecules\u003c/h2\u003e \u003cp\u003eBased on the feasibility of combining VEGF and Ang-2 to increase effectiveness of neovascular eye disease therapy [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], we decided to generate a novel protein therapeutics with enhanced efficacy and optimal developability. As a first step, we set out to screen five different multi-targeted molecules following the schematic diagram outlined in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFirst, we chose aflibercept or ranibizumab, the most commonly used VEGF-targeted drugs for ocular neovascular diseases, as the backbone to construct two design categories. Category #1 was antibody fusion protein featuring aflibercept (VEGF-Trap) at the N-terminus and scFv (Format 1) or VHH (Format 2) targeting Ang-2 at the C-terminus, where the scFv and VHH utilized a published sequence called LC10 and 166H4, respectively. Besides, a single-chain antibody fusion protein (Format 3) was also explored, as its small molecular weight may enhance passage through the retina into the choroid and reach the lesion site directly, potentially increasing efficacy. Category #2 was bispecific antibody comprising ranibizumab (anti-VEGF-A antibody) at the N-terminus and the same scFv (Format 4) or VHH (Format 5) as in Category #1 at the C-terminus. In order to compare the functional activity so that we could select the most effective formats for blocking both VEGF and Ang-2, we expressed and purified the five molecules and conducted experiments of VEGF-NFAT reporter assay and Ang-2 blocking ELISA. The results, depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, revealed that Format 1 and Format 2 exhibited similar IC\u003csub\u003e50\u003c/sub\u003e that were better when compared to Format 4 and Format 5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). This may be attributed to the higher affinity of aflibercept for VEGF-A, or a broader spectrum of activity of aflibercept, which can block not only VEGF-A but other angiogenic factors in the VEGF family as well, such as VEGF-B and PlGF [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], in comparison with ranibizumab which is specific only for VEGF-A. In contrast, Format 3 displayed the weakest performance in this assay. Additionally, the Ang-2 inhibition curves for each format was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC where no significant differences in the IC\u003csub\u003e50\u003c/sub\u003e among Formats 1, 2, 4, and 5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD) were detected, suggesting similar blocking effects on Ang-2. Format 3 again exhibited the lowest activity in Ang-2 blocking assay. Taken together, these findings suggested that Formats 1 and 2 were top choice for multi-targeted blockade.\u003c/p\u003e \u003cp\u003eNext, because certain intrinsic properties such as solubility, aggregation propensity and expression yield have direct impacts on the success of future CMC development, we initially utilized HIC to assess the solubility of Format 1 and Format 2 indirectly and rapidly. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, Format 2 exhibited a shorter retention time compared to Format 1, suggesting better solubility. In addition, when Format 1 and Format 2 were transiently expressed in CHO cells, the yield of Format 2 was 288.2 mg/L compared to 147.6 mg/L of Format 1. In the DLS assay, we observed that the interaction parameter \u003cem\u003ek\u003c/em\u003e\u003csub\u003eD\u003c/sub\u003e was \u0026minus;\u0026thinsp;6.61 for Format 1 and 14.7 for Format 2. The value of \u003cem\u003ek\u003c/em\u003e\u003csub\u003eD\u003c/sub\u003e is related to the nature of intermolecular forces: a positive \u003cem\u003ek\u003c/em\u003e\u003csub\u003eD\u003c/sub\u003e suggests significant repulsive forces between molecules, while a negative \u003cem\u003ek\u003c/em\u003e\u003csub\u003eD\u003c/sub\u003e suggests attractive forces that could potentially lead to self-aggregation [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Thus, we picked Format 2 for the predicted lower tendency to form aggregates and greater stability in solution. Moreover, many current reports have stated that VHH has higher solubility, stability, and expression yield compared to scFv [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Therefore, in order to facilitate a smooth transition to CMC development of a multi-targeted antibody fusion protein, we chose Format 2 for further research and development.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGeneration and\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e \u003cb\u003eactivity of anti-Ang-2 nanobody VHH-04\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn order to construct a novel multi-targeted molecule of our own, we obtained VHHs targeting Ang-2 by screening an alpaca-immunized library using phage display technology.\u003c/p\u003e \u003cp\u003eSeven unique VHH sequences binding to Ang-2 were isolated from the target-enriched library and subsequently cloned into the pcDNA3.4 vector for recombinant expression. A competition ELISA was then conducted to assess the inhibitory effects of these VHHs on Ang-2-Tie-2 binding interaction. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA illustrated that only VHH-04 exhibited significant blocking activity against Ang-2, achieving an IC\u003csub\u003e50\u003c/sub\u003e of 0.11 nM.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAmong the angiopoietin family members, Ang-1 and Ang-2 play important roles in vascular development by interacting with the Tie-2 receptor [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Various approaches have convincingly demonstrated that Ang-1 is crucial for maintaining vascular integrity and that its overall activity is anti-angiogenic, whereas Ang-2 promotes vascular development [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Therefore, it is crucial for the Ang-2 neutralizing antibody to selectively target Ang-2 without affecting Ang-1 signaling. To confirm the specificity of VHH-04, we conducted an ELISA assay where no binding of human Ang-1 was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). This signified that VHH-04 was specific for Ang-2. Furthermore, to identify relevant species for non-clinical studies, we tested the cross-species reactivity of VHH-04. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, VHH-04 bound to human, cynomolgus, and mouse Ang-2 with EC\u003csub\u003e50\u003c/sub\u003e of 0.04 nM, 0.03 nM, and 0.03 nM, respectively.\u003c/p\u003e \u003cp\u003eTaken together, based on its blocking activity, specificity for Ang-2 and cross-species binding activity, VHH-04 was ultimately used for bispecific assembly.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eConstruction and characterization of GB10 that targets VEGF and Ang-2 simultaneously\u003c/h2\u003e \u003cp\u003eNow that we used above anti-Ang-2 VHH antibody to construct a multi-targeted antibody-fusion protein based on Format 2 where VEGF-Trap (Aflibercept) was at the N-terminus of a human IgG1 backbone, and Ang-2-specific VHH-04 was connected via a G\u003csub\u003e4\u003c/sub\u003eS linker to the C-terminus of the Fc (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), and we named this molecule GB10.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFirst, in order to confirm that GB10 was capable of engaging both VEGF and Ang-2 concurrently, a bridging ELISA was performed. The result was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB. After coating the microplate with VEGF for capturing, GB10 specifically bound to the microplate and could be detected successfully by Ang-2-His binding on the opposite end of GB10 with an EC\u003csub\u003e50\u003c/sub\u003e of 0.11 nM. As expected, aflibercept, a VEGF-Trap without Ang-2 binding moiety, did not have bridging activity. Secondly, we tested the interaction between GB10 and VEGF family ligands by BLI. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, GB10 exhibited high affinity to VEGF-A from human, mouse, rat and rabbit. GB10 also bound human VEGF-B and PlGF with high affinity. The binding affinity of GB10 to VEGF-A from cynomolgus monkey was not tested because the amino acid sequence of VEGF-A is identical to its human counterpart. Moreover, GB10 also bound to Ang-2 with high affinity across species including human, cynomolgus monkey, mouse and rabbit by BLI. The binding affinity results are summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD. Taken together, we designed GB10, a multi-targeted antibody-fusion protein, that was able to bind both VEGF and Ang-2 with high affinity at the same time.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGB10 effectively blocks VEGF and Ang-2 signaling pathways\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo verify the functional activity of GB10 in inhibiting VEGF signaling pathway, we performed two \u003cem\u003ein vitro\u003c/em\u003e cellular assay and compared its efficacy with faricimab, a marketed bispecific antibody targeting both VEGF-A and Ang-2. First, the neutralizing activity of GB10 and faricimab were studied on a NFAT-driven luciferase reporter assay using HEK293 cells overexpressing VEGFR. The results in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA demonstrated that both GB10 and faricimab reduced the signal in a dose-dependent manner. Faricimab displayed an IC\u003csub\u003e50\u003c/sub\u003e of 0.15 nM, while GB10 exhibited a 7.5-fold greater blocking potency with an IC\u003csub\u003e50\u003c/sub\u003e of 0.02 nM. Secondly, HUVEC proliferation assay was used to evaluate the impact of GB10 and faricimab on VEGF-induced angiogenesis. GB10 demonstrated approximately 2-fold greater inhibitory potency than faricimab with IC\u003csub\u003e50\u003c/sub\u003e ​​of 0.03 nM and 0.05 nM, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Next, an Ang-2-Tie-2 blocking ELISA assay and a Tie-2 phosphorylation assay were developed in order to evaluate the inhibitory activity of GB10 and faricimab on Ang-2 signaling pathway. Notably, when compared side-by-side in blocking ELISA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), GB10 showed a 94-fold greater activity than faricimab with IC\u003csub\u003e50\u003c/sub\u003e of 0.15 nM and 14.17 nM, respectively. The functional activity was further characterized using Tie-2 phosphorylation assay which measured the level of tyrosine-phosphorylation of human Tie-2 in cell lysates in the presence of GB10 or faricimab (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). The results indicated that GB10 (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;49.90 nM) was more potent in inhibiting Ang-2-induced Tie-2 phosphorylation compared to faricimab (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;442.40 nM). Moreover, faricimab only achieved a maximum inhibition rate of approximately 70%, while GB10 was able to achieve complete inhibition.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eGB10 shows potent\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e \u003cb\u003eefficacy in laser-induced CVN model\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe superior \u003cem\u003ein vitro\u003c/em\u003e activity prompted us to investigate how GB10 would perform against faricimab \u003cem\u003ein vivo\u003c/em\u003e in laser-induced CNV model in cynomolgus monkeys, which is a widely used model for studying ocular neovascularization [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The therapeutic effect of GB10 or faricimab was evaluated by fundus fluorescein angiography (FFA) to assess the levels of vascular leakage, and by optical coherence tomography (OCT) to quantify changes in retinal thickness on Days 7, 14 and 21 after laser damage with respect to Day 0. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA shows CNV induction, intravitreal injection and imaging schedule after dosing.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe representative pictures measured by FFA was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and the improvement rate of the leakage area of grade 4 spots was shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. CNV development peaked at about 2\u0026ndash;3 weeks after laser photocoagulation and had a tendency to resolve itself overtime, therefore, we compared the effects of the vehicle and the treatment groups at the same time points. On Day 7 after dosing, both GB10 and faricimab were effective in reducing the leakage area compared to the vehicle group. However, GB10 achieved an improvement rate exceeding 70% at low dose (1 nmol/eye) compared to ~\u0026thinsp;47% of faricimab, and in the high-dose (40 nmol/eye) group, the CNV spot was completely resolved by GB10 compared to 79% of improvement rate by the faricimab group, suggesting a better efficacy of GB10. Similarly, on Day 14, the efficacy of GB10 persisted and remained statistically significant in the low-dose group compared to the vehicle group. In contrast, no statistically significant difference was observed in the low-dose group receiving faricimab. On Day 21, our results indicated that GB10 maintained a significant effect compared to the vehicle group in both the low-dose and high-dose groups, whereas no statistical differences were observed in any of the faricimab groups. Taken together, GB10 demonstrated sustained efficacy in the treatment of laser-induced CNV, underscoring its potential as a more potent therapeutic option than faricimab.\u003c/p\u003e \u003cp\u003eThe degree of vessel leakiness was associated with morphological changes in the retina at the site of laser injury. A distorted retinal architecture was apparent after a laser lesion, as revealed by thickening of the retina. To further assess the efficacy of GB10, we evaluated the improvement rate based on retinal thickness using OCT as an additional efficacy indicator, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. OCT imaging revealed recovery in both the GB10 and faricimab treatment groups, with GB10 demonstrating a superior improvement compared to faricimab. Specifically, compared with the vehicle group, both low-dose and high-dose GB10 exhibited statistically significant increase in improvement rates at all time points. This suggests that GB10 effectively inhibited retinopathy over an extended duration, potentially facilitating more favorable conditions for vision recovery. In contrast, faricimab demonstrated statistically significant differences only on Day 7 for both low-dose and high-dose groups, but the significance did not persist in subsequent days. These data further supported that GB10 was more effective than faricimab \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSummary of improvement rate of fluorescent leakage area in grade 4 spots.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c6\" namest=\"c2\"\u003e \u003cp\u003eGroups\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTime point\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eVehicle\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eLow-dose (1 nmol/eye)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eHigh-dose (40 nmol/eye)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFaricimab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGB10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFaricimab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGB10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDay 7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-15.38\u0026thinsp;\u0026plusmn;\u0026thinsp;54.58%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e46.66\u0026thinsp;\u0026plusmn;\u0026thinsp;23.40%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e70.22\u0026thinsp;\u0026plusmn;\u0026thinsp;14.94%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e78.88\u0026thinsp;\u0026plusmn;\u0026thinsp;35.31%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e100.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDay 14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.60\u0026thinsp;\u0026plusmn;\u0026thinsp;59.52%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e57.46\u0026thinsp;\u0026plusmn;\u0026thinsp;12.21%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e71.49\u0026thinsp;\u0026plusmn;\u0026thinsp;13.08%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e84.81\u0026thinsp;\u0026plusmn;\u0026thinsp;30.38%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e93.64\u0026thinsp;\u0026plusmn;\u0026thinsp;12.72%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDay 21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e35.47\u0026thinsp;\u0026plusmn;\u0026thinsp;46.45%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e67.13\u0026thinsp;\u0026plusmn;\u0026thinsp;19.92%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e92.24\u0026thinsp;\u0026plusmn;\u0026thinsp;9.65%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e79.64\u0026thinsp;\u0026plusmn;\u0026thinsp;40.73%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e100.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAll numerical data were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). n\u0026thinsp;=\u0026thinsp;4 per group.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSummary of improvement rate of retinal thickness.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c6\" namest=\"c2\"\u003e \u003cp\u003eGroups\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTime point\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eVehicle\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eLow-dose (1 nmol/eye)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eHigh-dose (40 nmol/eye)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFaricimab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGB10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFaricimab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGB10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDay 7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e33.34\u0026thinsp;\u0026plusmn;\u0026thinsp;21.28%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e85.43\u0026thinsp;\u0026plusmn;\u0026thinsp;27.09%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e111.91\u0026thinsp;\u0026plusmn;\u0026thinsp;31.48%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e93.47\u0026thinsp;\u0026plusmn;\u0026thinsp;31.54%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e94.26\u0026thinsp;\u0026plusmn;\u0026thinsp;16.93%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDay 14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e57.72\u0026thinsp;\u0026plusmn;\u0026thinsp;25.43%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e93.57\u0026thinsp;\u0026plusmn;\u0026thinsp;19.53%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e120.75\u0026thinsp;\u0026plusmn;\u0026thinsp;33.06%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e98.89\u0026thinsp;\u0026plusmn;\u0026thinsp;31.99%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e115.02\u0026thinsp;\u0026plusmn;\u0026thinsp;10.77%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDay 21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e73.95\u0026thinsp;\u0026plusmn;\u0026thinsp;29.76%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e96.68\u0026thinsp;\u0026plusmn;\u0026thinsp;24.94%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e124.73\u0026thinsp;\u0026plusmn;\u0026thinsp;28.20%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e108.85\u0026thinsp;\u0026plusmn;\u0026thinsp;59.66%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e123.51\u0026thinsp;\u0026plusmn;\u0026thinsp;12.20%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAll numerical data were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). n\u0026thinsp;=\u0026thinsp;4 per group.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eIntraocular pharmacokinetics of GB10 in a rabbit model\u003c/h2\u003e \u003cp\u003eNext, we evaluated the pharmacokinetic parameters of both GB10 and faricimab \u003cem\u003ein vivo\u003c/em\u003e. We utilized New Zealand white rabbits for our ocular pharmacokinetic study due to the similarity of rabbit retinal structure to higher animals and human. Our research mainly focused on monitoring the changes in concentrations of GB10 and faricimab in vitreous samples by ELISA, and the results are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. The data revealed differences in both T\u003csub\u003emax\u003c/sub\u003e and C\u003csub\u003emax\u003c/sub\u003e of GB10 and faricimab at the same dosage. Specifically, GB10 reached its maximum concentration (C\u003csub\u003emax\u003c/sub\u003e = 483.5 \u0026micro;g/mL) at 2.7 h after dosing, while faricimab reached its peak concentration (C\u003csub\u003emax\u003c/sub\u003e = 410.2 \u0026micro;g/mL) at 6.2 h, suggesting that GB10 may provide a more rapid therapeutic effect in clinical. Over time, the concentrations of both drugs gradually decreased and stabilized at a constant level. Further analysis showed that the half-life (T\u003csub\u003e1/2\u003c/sub\u003e) of GB10 was 59.4 h, whereas the T\u003csub\u003e1/2\u003c/sub\u003e of faricimab was 39.6 h, suggesting that GB10 has a longer duration in the vitreous humor. The extended half-life of GB10 may present an advantage, allowing for a more sustained therapeutic effect. Additionally, the total drug exposure (AUC\u003csub\u003e0\u0026thinsp;\u0026minus;\u0026thinsp;t\u003c/sub\u003e) calculated in this study was 44,867 h\u0026thinsp;\u0026times;\u0026thinsp;\u0026micro;g/mL for GB10 and 46,315 h\u0026thinsp;\u0026times;\u0026thinsp;\u0026micro;g/mL for faricimab. Moreover, no ocular inflammation or adverse events were observed in the intraocular PK study, indicating that GB10 demonstrated good tolerance in this experimental model (data not shown).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eThe developability of GB10 in high-concentration formulation\u003c/h2\u003e \u003cp\u003eIn the development of ocular drugs, it is crucial to consider not only the efficacy but also the frequency of dosing for patient compliance. In order to reduce the frequency of administration, GB10 was developed as a high-concentration formulation like faricimab.\u003c/p\u003e \u003cp\u003eGiven that higher protein concentration could potentially lead to an exponential increase in viscosity, which may affect the accuracy of delivered dose and cause pain at injection site, we first evaluated the viscosity of GB10 at high concentrations to ensure its injectability. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, the viscosity was measured as low as 13.0 cP at 140 mg/mL, indicating favorable properties of GB10 for injection into eyes with low-gauge syringes even at high concentrations. Secondly, since there is a positive correlation between intravitreal retention time and the size of hydrodynamic radius of the drug [\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], we analyzed GB10 using DLS, and the data showed that its hydrodynamic radius was 8.7 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA), larger than monoclonal antibody with average hydrodynamic radius of 5.4 nm [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. This result may correspond to the longer half-life of GB10 (59.4 h) in the vitreous than that of faricimab (39.6 h), which was evaluated in the previous chapter titled \u0026ldquo;Intraocular pharmacokinetics of GB10 in a rabbit model\u0026rdquo;. In addition, GB10 showed good thermal stability at 140 mg/mL, with a T\u003csub\u003eagg\u003c/sub\u003e of 56.2 ℃ and a T\u003csub\u003em\u003c/sub\u003e of 63.0 ℃ (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFinally, we investigated the developability of GB10 in order to better understand the potential CMC risk. The developability assessment of GB10 was conducted by subjecting GB10 to a series of stress conditions, including high temperature (40\u0026deg;C), oxidation, pH, and repeated freeze-thaw. The stressed samples were then analyzed for their purity by SEC and \u003cem\u003ein vitro\u003c/em\u003e activity. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, the purity of GB10 was not compromised across a range of stress conditions confirmed by SEC. However, VEGF blocking assay revealed that oxidation and pH treatments resulted in ~\u0026thinsp;10% reduction on Day 7 compared to T0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Similarly, the Ang-2 blocking activity of GB10 also decreased under high temperature, oxidation, and pH stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). It is important to note that due to assay variations, relative activities between 70% and 130% are generally considered unaffected. Therefore, we believed that the change in the activity of GB10 under these stress conditions was acceptable. Besides, our studies demonstrated that there were no significant changes in SEC purity, VEGF blocking activity and Ang-2 blocking activity of GB10 after three and five freeze-thaw cycles (data not shown). Taken together, GB10 exhibited an excellent developability profile and could be successfully formulated at high-concentration, which is expected to improve the convenience of its clinical application.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eDespite major medical advances that have been achieved with VEGF inhibitors, unmet clinical needs have not yet fully addressed as there are still non-responders and a lack of long-term efficacy in anti-VEGF monotherapies. In this study, we developed GB10, a more effective and durable antibody fusion protein, to address these unmet clinical needs.\u003c/p\u003e \u003cp\u003eOne advancement in this study is that GB10 demonstrates significantly greater efficacy. First, in contrast to VEGF monotherapy, GB10 additionally targets Ang-2, another crucial growth factor involved in neoangiogenesis and vascular permeability. Previous publications have shown that retinal neovascularization typically involves complex pathological mechanisms, making the inhibition of a single-target pathway, such as VEGF, often inadequate for achieving optimal therapeutic effects [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Second, in contrast to faricimab, a marketed bispecific antibody only targeting VEGF-A and Ang-2, GB10 based on the framework of VEGF-Trap can target additional VEGF family members, including VEGF-B and PlGF, both of which also play important roles in angiogenesis [\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The multi-pronged approach of GB10 has best-in-class potential in the treatment of ocular neovascular diseases. Moreover, compared to faricimab, GB10 showed greater \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e efficacy, which may be attributed to the molecular design. Specifically, faricimab is an asymmetric monovalent (1\u0026thinsp;+\u0026thinsp;1) heterodimeric bispecific antibody [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], and there may be concerns that its monovalence may not fully suppress the relevant signaling pathways; GB10 is designed as a symmetric homodimer (2\u0026thinsp;+\u0026thinsp;2) antibody fusion protein that enables more effective inhibition compared to monovalent antibody fragments. As we expected, \u003cem\u003ein vitro\u003c/em\u003e efficacy studies showed that GB10 was 7.5-fold more potent in VEGF blocking assay; and the inhibition rate of GB10 reached 100% on Ang-2-Tie-2 signaling pathway, whereas that of faricimab was only 70%. Consistent with the \u003cem\u003ein vitro\u003c/em\u003e efficacy studies, GB10 exhibited superior \u003cem\u003ein vivo\u003c/em\u003e efficacy compared to equimolar faricimab, based on an evaluation conducted in a laser-induced cynomolgus monkey CNV model. Our results showed that GB10 significantly reduced fluorescent leakage and restored retinal thickness, and in all dosage group, the improvement rate of GB10 was superior to that of faricimab. Therefore, according to these results observed in non-human primate model which closely mimics the conditions observed in human diseases and clinical efficacy of drug treatment, we can infer that GB10 may also effectively reduce pathological angiogenesis in human eyes and improve visual function in clinical applications.\u003c/p\u003e \u003cp\u003eThe other advancement is that GB10 demonstrates a longer duration of efficacy than that of faricimab. Besides higher biological activity discussed above, the following observations may contribute to longer durability of GB10. First, PK data revealed that the half-life of GB10 (59.4 h) in the vitreous of rabbit eyes was longer than that of faricimab (39.6 h), reducing the frequency of IVT injections. Second, the stability of GB10 is a crucial factor influencing the duration of efficacy. Developability assessment demonstrated that the purity and in vitro efficacy of GB10 could not be affected under various stress conditions including elevated temperature, non-physiological pH and oxidation among others, ensuring that GB10 can sustain effective concentrations in the eyes for an extended period after injection. This good stability of GB10 is attributed to its molecular design strategy. Compared to other molecules in the scFv format, GB10 \u0026mdash; in the VHH format \u0026mdash; exhibited a low tendency to aggregate, resulting in superior conformational and colloidal stability, as indicated by the results from HIC and DLS assays. Moreover, GB10 could achieve an ultra-high concentration of 140 mg/mL, exhibiting a clear competitive advantage compared to marketed high-concentration IVT drugs, such as Vabysmo\u0026reg; (faricimab, Roche, 120 mg/mL) as equal molar concentration of GB10 can achieve relatively longer duration than that of faricimab. Among patients receiving faricimab, only about 45% were able to achieve a dosing interval of 4 months within the first year [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. In contrast, GB10 at 140 mg/mL, higher than that of faricimab, is expected to significantly prolong the dosing interval, thereby increasing the proportion of patients who can achieve a dosing interval of 4 months or even longer. Overall, GB10 shows good durability, making it more convenient and effective for patients to use.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, GB10, a novel antibody fusion protein demonstrates significant advantages including multi-targeted blockade, excellent \u003cem\u003ein vitro\u003c/em\u003e activity and \u003cem\u003ein vivo\u003c/em\u003e efficacy, as well as ultra-high concentration formulations. These advantages may position GB10 as a promising best-in-class therapeutic candidate for neovascular eye diseases, deserving further evaluation from bench to bedside.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eDeclarations\u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eEthics approval\u003c/strong\u003e \u003cp\u003e All animal studies were approved by the Institutional Animal Care and Use Committee guidelines of WestChina-Frontier PharmaTech Co., Ltd. (China) or Shenzhen Kexing Biopharm Co., Ltd. (China), a subsidiary of Kexing Biopharm Co., Ltd.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eAuthors details\u003c/h2\u003e \u003cp\u003e \u003csup\u003e1\u003c/sup\u003e Drug Discovery, Centre for Research and Development, Kexing Biopharm Co., Ltd. Shenzhen 518057, China\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research was funded by Shenzhen Kexing Biopharm Co., Ltd.\u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003eSuofu Qin conceptualized and supervised the study. Xiling Wei performed the experiments for VHH nanobody discovery and Ang-2 functional activity assessment \u003cem\u003ein vitro\u003c/em\u003e, and drafted the manuscript. Yuxin Qiu designed and expressed these antibody fusion proteins. Wei Shang performed cell-based functional activity studies \u003cem\u003ein vitro\u003c/em\u003e. Xiangling Zhang conducted experiments \u003cem\u003ein vivo\u003c/em\u003e. Wenjie Yang, Chengyong Yang and Xi Chen coordinated the developability assessment. Huiming Li and Suofu Qin edited the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eAll research data generated during the current study are available upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBourne RRA, Stevens GA, White RA, Smith JL, Flaxman SR, Price H, et al. 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Invest Ophthalmol Vis Sci. 2021;62:428\u0026ndash;428.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Abbreviations","content":" \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eAMD\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eAge-related macular degeneration\u003c/div\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eAng-2\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eAngiopoietin 2\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eCNV\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eChoroidal neovascularization\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eDLS\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eDynamic light scattering\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eDME\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eDiabetic macular edema\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eDR\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eDiabetic retinopathy\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eDSF\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eDifferential scanning fluorimetry\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eELISA\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eEnzyme-linked immunosorbent assay\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eHIC\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eHydrophobic interaction chromatography\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eIVT\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eIntravitreal\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eRVO\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eRetinal vein occlusion\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003escFv\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eSingle-chain variable fragment\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eSEC\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eSize exclusion chromatography\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eTie-2\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eTyrosine-protein kinase receptor\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eVEGF\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eVascular endothelial growth factor\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eVHH\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eVariable heavy domain of heavy-chain antibody\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003cbr/\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-translational-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jtrm","sideBox":"Learn more about [Journal of Translational Medicine](http://translational-medicine.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jtrm/default.aspx","title":"Journal of Translational Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Neovascular eye disease, VEGF, Ang-2, Antibody fusion protein, VEGF-Trap, VHH","lastPublishedDoi":"10.21203/rs.3.rs-5135499/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5135499/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eIn clinical practice, anti-vascular endothelial growth factor (VEGF) therapies have been successfully applied to patients with neovascular eye diseases. However, unmet clinical needs have not yet been fully addressed, as about 20% of patients do not response to anti-VEGF monotherapies, meanwhile, the high frequency of intravitreal (IVT) injections imposes a significant burden on patients. To overcome these challenges, we developed a novel antibody fusion protein GB10 consisting of a VEGF-Trap and an anti-angiopoietin 2 (Ang-2) variable heavy domain of heavy-chain antibody (VHH) to inhibit the pro-angiogenic pathways of VEGF and Ang-2 simultaneously for enhanced and more enduring efficacy. The activity and developability of GB10 were characterized.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe first explored two categorical formats for molecular construction and selected the format that demonstrated the best activity and CMC-related properties for the generation of GB10. Subsequently, we evaluated the multi-targeting capability of GB10 using bridging enzyme-linked immunosorbent assay (ELISA) and bio-layer interferometry (BLI), followed by a side-by-side comparison of the \u003cem\u003ein vitro\u003c/em\u003e activities of GB10 and faricimab, the only marketed bispecific antibody for neovascular eye diseases, through assays such as VEGF reporter assay, human umbilical vein endothelial cells (HUVEC) proliferation, Ang-2 blocking ELISA, and Tie-2 phosphorylation. The \u003cem\u003ein vivo\u003c/em\u003e efficacy of GB10 and faricimab was next evaluated using a non-human primate model of laser-induced choroidal neovascularization (CNV). Finally the developability of GB10 was evaluated by intraocular pharmacokinetics and stress test.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eGB10 bound VEGF and Ang-2 simultaneously with high affinity, and exhibited superior activity \u003cem\u003ein vitro\u003c/em\u003e in inhibiting the VEGF and Ang-2 signaling pathways compared to faricimab. \u003cem\u003eIn vivo\u003c/em\u003e, GB10 demonstrated greater efficacy and durability compared to faricimab in a CNV model. GB10 also possessed a longer half-life in vitreous measured in a rabbit model. Moreover, GB10 showed excellent injectability and stability at a high-concentration of 140 mg/mL.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe superb efficacy and favorable developability profile make GB10 a potential best-in-class therapy for patients with neovascular eye diseases, warranting further evaluation in clinical settings.\u003c/p\u003e","manuscriptTitle":"GB10, a best-in-class antibody fusion protein targeting VEGF/Ang-2, exhibits promising therapeutic efficacy for neovascular eye diseases","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-18 21:38:42","doi":"10.21203/rs.3.rs-5135499/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2024-12-25T23:22:26+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-10-20T05:33:32+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-10-16T20:21:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-14T14:22:47+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Translational Medicine","date":"2024-09-23T02:13:35+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-translational-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jtrm","sideBox":"Learn more about [Journal of Translational Medicine](http://translational-medicine.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jtrm/default.aspx","title":"Journal of Translational Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"33d4ecb8-ae6b-45f4-a8df-69ae3e80bc37","owner":[],"postedDate":"November 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-03-06T20:33:15+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-18 21:38:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5135499","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5135499","identity":"rs-5135499","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}