Targeted Degradation of VEGFR2 by LYTACs Suppresses Angiogenesis and Tumor Growth

Targeted Degradation of VEGFR2 by LYTACs Suppresses Angiogenesis and Tumor Growth | 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 Targeted Degradation of VEGFR2 by LYTACs Suppresses Angiogenesis and Tumor Growth Rui Wang, Yundi Ren, Mingyan Liu, Yuting Lou, Guangyong Li, Xiaohong Chu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9310166/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Vascular endothelial growth factor receptor 2 (VEGFR2) is a key regulator of tumor angiogenesis and an important therapeutic target. Current VEGFR2-targeted strategies rely on monoclonal antibodies or tyrosine kinase inhibitors, which often face limitations including acquired resistance and off-target toxicity. Here, we developed two lysosome-targeting chimeras (LYTACs), ZV2-IGF2 and ZV2-EndoTag, designed to degrade cell surface VEGFR2 on endothelial cells. Both molecules were constructed by fusing a high-affinity VEGFR2 affibody with either the IGF2 domain or the engineered EndoTag module as lysosomal targeting ligands. Following prokaryotic expression and purification, both LYTACs were efficiently internalized into HUVECs and colocalized with lysosomes. Western blot and flow cytometry confirmed concentration-dependent VEGFR2 degradation, with ZV2-EndoTag exhibiting approximately 13% higher efficiency than ZV2-IGF2. Mechanistically, VEGFR2 degradation suppressed downstream p-AKT and p-MEK signaling. Functional assays revealed that both LYTACs inhibited HUVEC proliferation (∼46% at 500 nM), migration (60–70% reduction), and tube formation (42% reduction). Aortic ring sprouting was inhibited by 89–92%, and Matrigel plug assays confirmed anti-angiogenic activity in vivo . In a 4T1 murine breast cancer model, intratumoral administration of ZV2-IGF2 or ZV2-EndoTag significantly suppressed tumor growth, with tumor volumes reduced by 42% and 47%, respectively. Immunofluorescence analysis revealed that the VEGFR2/CD31 ratio within tumor tissues decreased by approximately 38% in both treatment groups, confirming effective target degradation and reduced microvascular density. Together, these results establish LYTAC-mediated VEGFR2 degradation as a promising anti-angiogenic strategy and provide proof-of-concept for targeting membrane proteins via lysosomal degradation pathways. Angiogenesis Tumour Angiogenesis LYTACs Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1 Introduction Tumor angiogenesis is a critical process in the growth and metastasis of solid tumors. When tumor volume reaches 1–2 mm³ and diffusion can no longer provide sufficient nutrients, pro-angiogenic factors released by the tumor microenvironment (such as VEGF and FGF2) activate endothelial cells, ultimately forming a new microvascular network[ 1 – 4 ]. The core regulator VEGF binds to VEGFR2 (also known as KDR/Flk-1) on endothelial cell surfaces, activating downstream signaling pathways including MAPK/ERK and PI3K/AKT/mTOR[ 5 – 7 ]. This potently promotes endothelial cell proliferation, migration, and survival, ultimately forming an abnormal tumor vascular network[ 8 , 9 ]. Consequently, VEGFR2 has become a primary target for anti-angiogenic therapies[ 10 – 13 ]. Monoclonal antibodies, fusion proteins, and small-molecule tyrosine kinase inhibitors (e.g., sorafenib, sunitinib) developed against this protein are currently used clinically. However, monotherapy targeting the VEGF/VEGFR2 pathway often leads to acquired drug resistance and adverse reactions[ 6 ]. These limitations underscore the urgent need for alternative strategies that achieve sustained target inhibition without the drawbacks of conventional occupancy-driven inhibitors[ 14 – 17 ]. Lysosome-Targeting Chimeras (LYTACs) represent an emerging class of protein degradation tools[ 18 ]. Their core mechanism involves simultaneously binding to endocytic receptors on the cell surface such as the mannose-6-phosphate receptor, M6PR and the target protein, thereby directing membrane-associated or extracellular proteins (POIs) to lysosomes for efficient degradation[ 19 – 25 ]. By enabling the physical elimination rather than temporary blockade of target proteins, this technology offers a paradigm shift in addressing traditionally hard-to-target extracellular and membrane proteins, potentially overcoming the resistance mechanisms that limit current anti-angiogenic agents[ 25 – 28 ]. LYTACs rely on lysosome-targeting receptors on the cell membrane surface to exert their effects. POI forms a chimeric structure with LYTACs and LTR, enabling entry into endosomes and subsequent transport to lysosomes for degradation. In this study, we developed two distinct LYTACs targeting VEGFR2 based on different lysosomal targeting ligands. A high-affinity VEGFR2 affibody (ZVEGFR2_Bp21), hereinafter referred to as ZV2, was previously reported to bind human VEGFR2 with picomolar affinity[ 17 ], was selected as the targeting module. For lysosomal targeting, we pursued two strategies. The first involved fusing the VEGFR2 affibody to the insulin-like growth factor 2 (IGF2) domain, which binds to the widely expressed cation-independent mannose-6-phosphate receptor (CI-M6PR/IGF2R) and enables efficient endocytosis and lysosomal delivery[ 26 , 27 ]. The second strategy leveraged the Rosetta de novo protein design tool, jointly developed by Professor David Baker's team and Professor Carolyn Bertozzi's team. The second strategy utilized an engineered EndoTag module, computationally designed using the Rosetta platform to bind IGF2R without competing with endogenous ligands, thereby potentially minimizing off-target effects[ 29 ]. Notably, the parallel comparison of a natural ligand (IGF2) and an engineered module (EndoTag) within the same LYTAC framework represents a unique opportunity to evaluate the relative advantages of these distinct targeting mechanisms, providing critical insights for future LYTAC optimization. By comparing the degradation efficiency, specificity, and functional outcomes of these two LYTACs, we aimed to establish a dual-validation strategy for VEGFR2-targeted protein degradation and to evaluate its therapeutic potential in angiogenesis-dependent diseases. Here, we demonstrate that both ZV2-IGF2 and ZV2-EndoTag efficiently internalize into endothelial cells, colocalize with lysosomes, and induce concentration-dependent degradation of VEGFR2. This leads to suppression of downstream AKT and MEK signaling, inhibition of endothelial cell proliferation, migration, and tube formation, and reduced angiogenesis in ex vivo aortic ring assays and in vivo Matrigel plug models. Finally, we show that intratumoral administration of these LYTACs significantly suppresses tumor growth in a murine 4T1 breast cancer model, accompanied by reduced intratumoral VEGFR2 expression and microvascular density. Together, these findings establish LYTAC-mediated VEGFR2 degradation as a promising anti-angiogenic strategy that addresses the limitations of current therapies by enabling sustained target elimination. Moreover, the successful application of both IGF2 and EndoTag modules provides a versatile platform for developing next-generation LYTACs targeting a broad range of membrane proteins, highlighting the translational potential of this degradation-based approach. 2 Materials and methods 2.1 Materials All plasmids were synthesized by GenScript (Nanjing, China), and sequence verified. The host strain E. coli . M15 cells were purchased from Shanghai Weidi Biotechnology company (China). RPMI 1640 (Cellmax), DMEM (Hyclone) and Fetal Bovine Serum (FBS) were purchased from qualified vendors. LysoTracker™ Red DND-26 and Hoechst 33342 were purchased from Invitrogen. 6-carboxyfluorescein (6-FAM) NHS ester were purchased from Ruixi Biotechnology Company (Xi’ an, China). 2.2 Cell culture Human Umbilical Vein Endothelial Cells (HUVECs) and The breast cancer cell line 4T1 were obtained from the American Type Culture Collection (ATCC). These cells were meticulously cultured in specialized endothelial cell medium (ScienCell catalog number 1001) and Dulbecco's Modified Eagle Medium (DMEM, Thermo Fisher), respectively, at an optimal temperature of 37°C in a humidified chamber with 5% CO 2 . Subconfluent cells, specifically those between passages 2 to 6, were carefully selected for the experiments to ensure optimal growth and response. 2.3 Evaluation of lysosomal colocalization Lysosomal colocalization was evaluated using confocal laser scanning microscopy (CLSM). HUVECs (2×10⁴ cells/well) were seeded onto 12-well plates with integrated coverslips and cultured overnight in ECM medium. After discarding the medium and washing twice with PBS, cells were treated with different FAM-labeled samples (1 µM, including ZV2, ZV2-IGF2, ZV2-EndoTag) at 37°C for 2 h. Cells were then washed with PBS and incubated with LysoTracker Red at 37°C according to the manufacturer's instructions. Following staining, cells were washed again and fixed with 4% formaldehyde at room temperature for 20 min. Finally, nuclei were stained with DAPI for 10 min, followed by removal of the staining solution and three washes with PBS. Cover slips were sealed in the dark and imaged using CLSM. 2.4 In Vitro Cell Proliferation Experiment of LYTAC The CCK-8 assay kit (C0038, Beyotime) was used to detect the inhibitory effect of LYTAC on VEGF165 (P5561, Beyotime)-stimulated proliferation in HUVECs. Cells were seeded at a density of 5×10³ cells per well in a 96-well plate and cultured overnight in ECM medium. Subsequently, cells were starved with 0.5% ECM for 6 h, followed by addition of a mixture containing VEGF165 (50 ng mL⁻¹) and varying concentrations of LYTAC. Cells were further cultured for 24 h in medium supplemented with 2% FBS. Then, 10 µL of CCK-8 solution was added to each well. After incubation for 4 h, directly measure the absorbance of each well at 450 nm wavelength using a BioTek microplate reader. $$\:\begin{array}{c}Cell\:Viability\:\left(\text{%}\right)=\left({A}_{treatment}-{A}_{blank}\right)/\left({\text{A}}_{\text{C}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}}-{\text{A}}_{\text{b}\text{l}\text{a}\text{n}\text{k}}\right)\times\:100\%\#\left(1\right)\end{array}$$ 2.5 Wound Healing Assay of LYTAC This study evaluated the effects of different samples on VEGF165-induced migration of HUVECs using a cell scratch assay. HUVECs were seeded at a density of 1×10⁵ cells per well in a 6-well plate and cultured overnight in ECM medium. A scratch was created on the monolayer using a 200 µL pipette tip to form a wound area. After washing cells twice with PBS, different samples containing VEGF165 (50 ng mL⁻¹) were added to each well. Cells were cultured for 24 h in medium supplemented with 2% FBS. The scratch area was photographed using an inverted microscope, and cell migration rates were calculated using ImageJ software. $$\:\begin{array}{c}Wound\:Closure\:Rate\:\left(\text{%}\right)=\left({\text{A}\text{r}\text{e}\text{a}}_{0\text{h}}-{\text{A}\text{r}\text{e}\text{a}}_{\text{t}\text{h}}\right)/{\text{A}\text{r}\text{e}\text{a}}_{0\text{h}}\times\:100\%\#\left(2\right)\end{array}$$ 2.6 Transwell migration assay Transwell migration assays were employed to evaluate endothelial cell (EC) migration capacity. First, HUVECs were co-treated with different LYTAC protein samples for 24 h. Subsequently, 1.5 × 10⁵ treated cells were resuspended in 500 µL endothelial basal medium (ECM) and seeded into the upper chamber of a 24-well Transwell plate (polycarbonate membrane, 8 µm pore size; Corning; Merck KGaA, Darmstadt, Germany). The lower chamber of each well was filled with 750 µL of ECM containing 1% fetal calf serum (FCS). After incubation for 5 h, non-migrated cells were removed, and cells migrating to the membrane surface were stained with crystal violet (C0121, Beyotime). Using a confocal laser scanning microscope (CLSM; Leica TCS SP8 STED 3X), 20 random fields of view were selected to count migrating cells. Cell migration rates were expressed as percentages relative to the control group. $$\:\begin{array}{c}Migration\:Rate\:\left(\text{%}\right)={\text{N}}_{\text{t}\text{r}\text{e}\text{a}\text{t}\text{m}\text{e}\text{n}\text{t}}/{\text{N}}_{\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}}\times\:100\%\#\left(3\right)\end{array}$$ 2.7 Protein Degradation Analysis using Western Blot HUVECs (1×10⁵) were seeded into 6-well plates and starved for 6 h in ECM medium supplemented with 0.5% FBS. Subsequently, cells were treated with a mixture of VEGF165 (50 ng mL⁻¹) and LYTAC protein (1 µM) for 24 h. Following treatment, cells were lysed using RIPA buffer containing protease and phosphatase inhibitors (P1045, Beyotime) to extract total protein, which was quantified using the BCA assay kit (P0010, Beyotime). Protein samples were mixed with loading buffer, denatured by heating at 100°C for 5 min, and separated by electrophoresis on a 4–12% SDS-PAGE gel (ET12412, ACE Biotechnology). Proteins were then transferred to a polyvinylidene difluoride (PVDF) membrane and blocked at room temperature with TBST containing 5% nonfat milk for 1 h. After three washes with TBST, the membrane was incubated at 4°C for 24 h with either the VEGFR2 primary antibody (1:1000, 2479, CST) or the β-Actin primary antibody (1:2000, 60004-1-Ig, Proteintech). After three additional washes, membranes were incubated at room temperature for 2 h with the corresponding secondary antibody (1:5000, Proteintech). Finally, after thorough washing with TBST, membranes were developed using ECL chemiluminescence. 2.8 Protein Degradation Analysis by Flow Cytometry HUVECs (1×10⁵) were seeded into 6-well plates and starved for 6 h in ECM medium supplemented with 0.5% fetal bovine serum. Cells were then treated with a mixture of VEGF165 (50 ng mL⁻¹) and LYTAC protein (1 µM) for 24 and 48 h, respectively. Following treatment, adherent cells were harvested via trypsin digestion, washed three times with staining buffer, and fixed with 4% paraformaldehyde at room temperature for 20 min. Subsequently, cells were blocked with staining buffer containing 5% bovine serum albumin at room temperature for 30 min, then incubated with primary antibody (67407-1-Ig, Proteintech) at 4°C for 30 min. After washing with staining buffer, cells were incubated with FITC-labeled secondary antibody (abs20012, Absin). Finally, cells were washed and resuspended in 200 µL staining buffer for analysis using a flow cytometer (LSRFortessa, Becton Dickinson Immunocytometry Systems). 2.9 Tube formation assay The tube formation assay was employed to analyse the tubule-forming capacity of endothelial cells (ECs). 1.5 × 10⁴ HUVECs were resuspended in ECM medium containing different protein samples and seeded into 96-well plates pre-coated with Matrigel. Following 24 h of culture, newly formed vascular-like structures were imaged using phase-contrast microscopy. The number of luminal networks per well was quantified using ImageJ software and its Angiogenesis Analysis plugin (National Institutes of Health, Bethesda, MD, USA), with endothelial tube-forming capacity expressed as a percentage relative to the control group. $$\:\begin{array}{c}Relative\:Tube\:Length\:\left(\text{%}\right)={\text{L}}_{\text{t}\text{r}\text{e}\text{a}\text{t}\text{m}\text{e}\text{n}\text{t}}/{\text{L}}_{\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}}\times\:100\%\#\left(4\right)\end{array}$$ 2.10 Aortic ring assay Thoracic aortas were harvested from BALB/c mice, sectioned into 0.5 mm annular segments, and embedded in Matrigel matrix gel (Corning; Merck KGaA). A single aortic ring per well was placed in a 96-well plate. Following a 15 min incubation, wells were treated with LYTAC proteins under varying conditions. After a further 6 days of culture, aortic rings were imaged using a phase-contrast microscope. Vascular sprouting was quantified by measuring the area of newly formed sprouting regions, expressed as a percentage relative to the control group. $$\:\begin{array}{c}Relative\:Sprouting\:Area\:\left(\text{%}\right)={\text{A}}_{\text{t}\text{r}\text{e}\text{a}\text{t}\text{m}\text{e}\text{n}\text{t}}/{\text{A}}_{\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}}\times\:100\%\#\left(5\right)\end{array}$$ 2.11 Matrigel plug assay A Matrigel mixture containing mouse VEGF (1 µg/mL; R&D Systems), mouse FGF2 (1 µg/mL; R&D Systems), heparin (60 IU/mL), and 1 µM LYTAC protein (or control) was subcutaneously injected into the lateral abdomen of 8-week-old BALB/c mice (Janvier-Labs), with seven mice per group. After 7 days, the Matrigel plugs were retrieved for subsequent immunohistochemical analysis. 2.12 Animal experiments Female BALB/c J mice (6–8 weeks old) were provided by Jinan Pengyue Experimental Animal Breeding Co, Ltd. (Jinan, China). Mice were maintained under specific pathogen-free(SPF) conditions. All animal procedures for pharmacokinetic studies were performed following protocols approved by The Special Committee of Scientific Research Ethic of Liaocheng University (Approval Number: AP2026031535; Date: March 15, 2026). 2.13 Tumor Model Establishment To evaluate the in vivo antitumor efficacy of LYTAC-mediated VEGFR2 degradation, a subcutaneous 4T1 murine breast cancer model was established in female BALB/c mice (6–8 weeks old). 4T1 cells were harvested, resuspended in sterile PBS at 1×10 7 cells/mL, and 1×10 6 cells(100 µL) were injected subcutaneously into the right dorsal flank of each mouse. When tumor volume reached 60–80 mm³ (5–7 days post-inoculation), mice were randomly assigned to four groups (n = 6 per group): PBS control, ZV2, ZV2-IGF2, and ZV2-EndoTag. They received five intratumoral injections of 100 µg protein in 100 µL PBS every other day, and were then maintained until day 20 post‑tumor inoculation. Tumor volume was measured every two days using a digital caliper, and body weight was monitored to assess systemic toxicity. At the experimental endpoint (day 20), when tumors in the control group reached 500–600 mm³, mice were euthanized; tumor tissues were excised, weighed, and either frozen at − 80°C or fixed in 4% paraformaldehyde for subsequent analysis. $$\:\begin{array}{c}Volume=\text{L}\text{e}\text{n}\text{g}\text{t}\text{h}\times\:\text{W}\text{i}\text{d}\text{t}{\text{h}}^{2}/2\#\left(6\right)\end{array}$$ 2.14 Histology and immunohistochemistry Formalin-fixed paraffin-embedded Matrigel plugs and tumor tissues were serially sliced into 3-µm sections. For the analysis of tumor size, sections with the largest area for each tumor were selected, stained with hematoxylin and eosin (H&E), imaged using a microscope, and subjected to planimetric tumor area measurements by means of an image analysis software. 2.15 Statistics Analysis Data were presented as mean ± SEM. Student's t test was used to compare 2 groups of samples. The difference among multiple groups was evaluated using a one-way analysis of variance (ANOVA) with Bonferroni's post hoc test. * p < 0.05 was considered as statistically significant. 3 Results and Discussion 3.1 Design and Construction of Recombinant Protein LYTACs To establish a novel strategy for targeted degradation of VEGFR2, we designed two recombinant LYTACs based on distinct lysosomal targeting mechanisms. As illustrated in Fig. 1 a, the proposed working mechanism of these LYTACs involves simultaneously binding to cell surface VEGFR2 and the CI-M6PR lysosomal targeting receptor via an IGF2 ligand, thereby inducing VEGFR2 internalization and subsequent lysosomal degradation. For molecular construction, we employed a modular design strategy (Fig. 1 b) A high-affinity VEGFR2 affibody (ZVEGFR2_Bp21), previously reported to bind human VEGFR2 with picomolar affinity (KD 241 ± 4 pM)[ 17 ], served as the targeting module. This affibody was fused via a flexible (GGGGS)₄ linker to either the insulin-like growth factor 2 (IGF2) domain or the engineered EndoTag module, which function as lysosomal targeting ligands. An N-terminal 6×His tag was incorporated into both constructs to facilitate protein purification and detection. The resulting recombinant proteins were designated as ZV2-IGF2 and ZV2-EndoTag. Following subcloning of the synthetic genes into the pQE30 prokaryotic expression vector, both recombinant proteins were expressed in E. coli M15 cells upon IPTG induction. The expression and purification processes were monitored by SDS-PAGE analysis, which confirmed successful expression and efficient purification of both proteins via Ni²⁺ affinity chromatography. The purity of the final products was assessed by SDS-PAGE (Fig. 1 c), revealing single bands at the expected molecular weights (approximately 23.2 kDa for ZV2-IGF2 and 30.1 kDa for ZV2-EndoTag) for the purified ZV2, ZV2-IGF2, and ZV2-EndoTag proteins. Western blot analysis using an anti-6×His antibody further confirmed the identity of the purified proteins (Fig. 1 d). Together, these results demonstrate the successful design, prokaryotic expression, and purification of two novel LYTAC molecules targeting VEGFR2, providing the necessary material basis for subsequent functional validation studies. 3.2 Lysosome Colocalization and Cellular Uptake of LYTAC Having confirmed the VEGFR2 degradation efficacy of our LYTACs, we next investigated the mechanism by which IGF2 and EndoTag mediate lysosomal targeting. To verify this core function, ZV2-IGF2, ZV2-EndoTag, and the control ZV2 molecule were chemically conjugated with the FAM green fluorescent probe and co-incubated with HUVEC cells. As shown in Fig. 2 a, the control ZV2-FAM exhibited only faint non-specific intracellular signals with minimal overlap with the red fluorescence of lysosomes; whereas both ZV2-IGF2-FAM and ZV2-EndoTag-FAM experimental groups exhibited strong green fluorescence signals. These signals highly overlapped with LysoTracker Red-labelled lysosomal regions, producing distinct yellow colocalisation signals. This colocalisation phenomenon was particularly pronounced in the cytoplasmic region, indicating that both recombinant LYTACs proteins were efficiently internalised and successfully transported to lysosomes. To quantify this phenomenon, we employed ImageJ software to measure and plot the intensity distribution curves of FAM green fluorescence and LysoTracker red fluorescence (Fig. 2 b). The results revealed a substantial region of overlap between the fluorescence curves of ZV2-IGF2 and ZV2-EndoTag. IGF2, as a natural ligand, exhibits a well-established binding mechanism with CI-M6PR/IGF2R[ 22 , 25 , 27 , 29 ]. This receptor is widely expressed across diverse cell types, efficiently mediating the endocytosis of the ligand-receptor complex and directing its transport towards lysosomes. The present findings further validate the feasibility of incorporating IGF2 into LYTAC constructs, demonstrating that the IGF2 domain retains its receptor-binding activity when fused to the VEGFR2 affinity fragment, thereby effectively directing the entire fusion protein into the lysosomal degradation pathway. This provides robust support for LYTAC design strategies based on natural ligands. As a module engineered through de novo protein design, EndoTag's core advantage lies in achieving specific receptor recognition and endocytosis induction independently of natural ligands[ 29 ]. Our findings validate this design principle: when fused to the VEGFR2 phage display construct, EndoTag demonstrated lysosomal targeting efficiency comparable to IGF2, with fluorescence curves exhibiting high overlap. This not only confirms EndoTag's utility as a lysosomal targeting module but, more significantly, represents an engineered strategy that opens broader application prospects for developing novel LYTAC molecules. This is particularly relevant for targets or tissue types lacking known natural endocytic ligands. 3.3 Protein Degradation Capability of LYTAC After validating the lysosomal targeting capabilities of IGF2 and EndoTag, we further assessed the degradation efficiency of recombinant LYTACs on VEGFR2 expressed on the cell membrane surface. Results shown in Figs. 3 a and 3 c indicated that both recombinant LYTACs reduced VEGFR2 levels in a concentration-dependent manner. Quantitative analysis revealed that at equivalent concentrations, ZV2-EndoTag achieved approximately 13% higher degradation efficiency than ZV2-IGF2 (Fig. 3 c), demonstrating its superior VEGFR2 clearance capacity. To validate these findings, we further quantified changes in cell surface VEGFR2 expression via flow cytometry. As shown in Fig. 3 b, HUVEC cells treated with LYTACs for 24 h exhibited significantly reduced fluorescence intensity upon binding with VEGFR2-specific antibodies, with the ZV2-EndoTag-treated group demonstrating a markedly greater decrease than the ZV2-IGF2 group. This result further confirms that ZV2-EndoTag exhibits superior efficacy in degrading cell surface VEGFR2. The disparity in degradation efficacy between the two LYTACs may stem from multiple factors. First, as a de novo engineered protein module, EndoTag may possess superior binding affinity for IGF2R or more efficient endocytosis triggering compared to the native IGF2 ligand. Second, EndoTag's structural design may facilitate sustained effective interaction with the receptor post-binding to the target protein, or exhibit enhanced stability within endosomal vesicles. Additionally, differences may exist in their lysosomal sorting signals or the kinetics of receptor dissociation. Flow cytometry results further corroborate this disparity, confirming ZV2-EndoTag's superiority through in situ detection of membrane proteins. This disparity in degradation efficiency correlates with subsequent observations of cellular functional suppression including proliferation, migration, and tubule-forming capacity establishing a preliminary positive correlation between degradation efficiency and functional inhibition. These findings not only validate the molecular design strategy employed herein but also point towards future optimisation of LYTAC molecules: engineered targeting modules may outperform natural ligands in functionality, demonstrating greater developmental potential. 3.4 The effect of VEGFR2 degradation on downstream AKT and MEK phosphorylation levels Building upon confirmation that LYTACs effectively degrade cell surface VEGFR2, this study further investigates their impact on downstream signalling pathway activity. As a core regulatory receptor for angiogenesis, VEGFR2 activation primarily mediates endothelial cell proliferation, survival, and migration via the PI3K-AKT and MAPK-MEK signalling pathways[ 6 , 7 , 15 ]. To evaluate the impact of VEGFR2 degradation on these pathways, we collected lysates from HUVECs treated with different LYTACs and assessed changes in the protein levels of phosphorylated AKT (p-AKT) and phosphorylated MEK (p-MEK) by Western blot analysis. As depicted in Fig. 4 a, compared to the control group, both p-AKT and p-MEK band intensities were markedly diminished in the ZV2-IGF2 and ZV2-EndoTag treatment groups. Quantitative analysis of grey values further confirmed that expression levels of both phosphorylated proteins were significantly downregulated following LYTACs treatment. Notably, the degree of downregulation of p-AKT and p-MEK in the ZV2-EndoTag group was slightly higher than in the ZV2-IGF2 group, consistent with the previously observed higher VEGFR2 degradation efficiency of ZV2-EndoTag. These findings provide molecular evidence that LYTACs-mediated VEGFR2 degradation effectively inhibits downstream PI3K-AKT and MAPK-MEK signaling pathways. The suppression of these pathways is particularly significant given their central role in transducing VEGFR2 signals to regulate endothelial cell functions[ 5 , 14 ]. The observed reduction in p-AKT levels suggests impaired cell survival signaling, while decreased p-MEK indicates disruption of the proliferative MAPK cascade[ 7 , 11 , 31 ]. Together, these results mechanistically explain the functional inhibition of endothelial cell proliferation, migration, and tube formation observed in our previous experiments. Furthermore, the correlation between degradation efficiency and signaling suppression across the two LYTACs reinforces the mechanistic link between target protein clearance and pathway inhibition. These findings establish that the anti-angiogenic effects of our LYTACs are mediated through specific disruption of VEGFR2-dependent signaling networks, validating the therapeutic potential of this degradation-based approach. 3.5 Effects of LYTACs Recombinant Proteins on the Proliferative Capacity of HUVEC Cells Building upon the established role of VEGFR2 in promoting cell proliferation through downstream signaling pathways such as AKT activation, we next evaluated the impact of LYTACs-mediated VEGFR2 degradation on the biological behavior of HUVEC cells. Results shown in Fig. 5 demonstrate that both LYTAC molecules inhibited HUVEC proliferation in a concentration-dependent manner. At a concentration of 500 nM, treatment with ZV2, ZV2-IGF2, or ZV2-EndoTag suppressed cell viability by approximately 46% compared to untreated controls. Notably, at equivalent concentrations, ZV2-IGF2 and ZV2-EndoTag exhibited significantly stronger anti-proliferative effects than the ZV2 affinity control, which lacks the fused lysosomal targeting module, indicating that VEGFR2 degradation effectively blocks its pro-proliferative signaling pathway. The observed anti-proliferative effects correlate well with the differential VEGFR2 degradation efficiencies of the two LYTACs. The approximately 46% reduction in cell viability achieved by all three proteins at 500 nM likely reflects the contribution of VEGFR2 binding alone, as ZV2 can sterically hinder receptor dimerization and activation. However, the enhanced inhibition mediated by ZV2-IGF2 and ZV2-EndoTag demonstrates that active degradation of the receptor provides additional therapeutic benefit beyond simple antagonism. This finding underscores the mechanistic advantage of the LYTAC approach: by physically eliminating the target protein rather than temporarily blocking its activity, degradation-based strategies can achieve more profound and sustained functional suppression. Furthermore, the slightly stronger inhibition observed with ZV2-EndoTag compared to ZV2-IGF2 aligns with its superior degradation efficiency documented earlier, reinforcing the relationship between target clearance and functional outcome. These results establish that VEGFR2 degradation translates directly into impaired endothelial cell proliferative capacity, providing a functional rationale for pursuing this strategy in anti-angiogenic therapy. 3.6 Effects of LYTACs Recombinant Proteins on the Migration Capacity of HUVEC Cells After verifying the effects of recombinant LYTACs proteins on cell proliferation, we further investigated their regulatory role in HUVEC cell migration. As shown in Fig. 6 , this study employed wound healing assays and Transwell migration assays to evaluate two-dimensional planar migration and three-dimensional transmembrane migration potential, respectively. Figure 6 a showed significantly reduced scratch closure rates (p < 0.01) in ZV2-IGF2 and ZV2-EndoTag-treated groups compared to controls and ZV2-treated groups, indicating LYTACs effectively inhibit HUVEC two-dimensional lateral migration. Concurrently, Transwell assay results revealed that HUVEC cells treated with ZV2-IGF2 and ZV2-EndoTag exhibited approximately 60%-70% fewer transmembrane cells compared to the control group (p < 0.001), with ZV2-EndoTag demonstrating a stronger migration inhibitory trend (Fig. 6 c). Collectively, these data demonstrate that recombinant LYTACs proteins significantly inhibit HUVEC migration by degrading VEGFR2, providing robust evidence for further investigation of their anti-angiogenic effects. Live-cell tracking analysis revealed that the motility range of LYTACs-treated cells was markedly restricted, as shown in Fig. 6 e. The inhibitory effects of LYTACs on HUVEC migration were confirmed across multiple assays, demonstrating that VEGFR2 degradation impairs endothelial cell motility. Migration represents a critical step in angiogenesis, as endothelial cells must coordinately migrate toward angiogenic stimuli to form new vascular sprouts[ 8 , 9 ]. The reduction in both two-dimensional wound closure and three-dimensional transmembrane migration following LYTACs treatment confirms that VEGFR2 degradation disrupts this essential process. Notably, the approximately 60%-70% reduction in Transwell migration exceeds the degree of proliferation inhibition at equivalent concentrations, suggesting endothelial cell migration is particularly sensitive to VEGFR2 signaling perturbations. This heightened sensitivity likely reflects the acute dependence of directed cell movement on precise spatiotemporal regulation of receptor activity, which is more readily disrupted by receptor degradation than by simple antagonism[ 6 , 13 ]. Live-cell tracking further revealed restricted motility range in LYTACs-treated cells, indicating sustained VEGFR2 signaling is required for maintaining migration capacity. The stronger inhibitory effects of ZV2-EndoTag align with its superior degradation efficiency, reinforcing the link between target clearance and functional outcome. Together with proliferation and tube formation data, these results establish that VEGFR2 degradation produces multifaceted disruption of endothelial cell functions essential for angiogenesis. 3.7 Effects of LYTACs Recombinant Proteins on the Angiogenesis Capacity of HUVEC Cells Building upon the established ability of recombinant LYTACs to inhibit HUVEC migration, we next evaluated their impact on angiogenesis using complementary in vitro and in vivo models. In vitro experiments employed both the Matrigel tube formation assay and the mouse aortic ring sprouting model to simulate early-stage tubulogenesis and sprouting from mature vessels, respectively[ 32 – 34 ]. Quantitative analysis revealed that HUVEC cells treated with ZV2-IGF2 and ZV2-EndoTag exhibited significantly reduced tubular network formation on Matrigel. As shown in Fig. 7 b, compared to the control group, total tube length was reduced by approximately 42% in both LYTAC-treated groups, representing a 14% decrease relative to the ZV2 control group. Additionally, both the length and number of microvascular sprouts in mouse aortic rings were markedly suppressed (Fig. 7 a-d). Quantification of sprouting area showed that ZV2-EndoTag treatment reduced sprouting by approximately 92% compared to controls, while ZV2-IGF2 achieved an 89% reduction, confirming that both LYTACs effectively disrupt critical steps in angiogenesis. To validate the anti-angiogenic efficacy of LYTACs in vivo , we employed the mouse Matrigel plug angiogenesis model. Matrigel plugs pre-mixed with different treatment proteins were subcutaneously implanted into C57BL/6 mice and retrieved after 7 days for histological analysis. Consistent with the in vitro findings, assessment via H&E staining and immunohistochemical analysis (Fig. 7 e-f) revealed that microvascular density in the ZV2-IGF2 and ZV2-EndoTag-treated groups was significantly lower than that in the control group, with CD31-positive area quantification (Fig. 7 g) further corroborating the reduction in neovascularization. These results demonstrate that recombinant LYTACs can effectively inhibit angiogenesis in vivo , consistent with our in vitro observations. The consistent superiority of ZV2-EndoTag across these models further aligns with its higher VEGFR2 degradation efficiency, reinforcing the relationship between target clearance and functional anti-angiogenic outcomes. Collectively, the anti-angiogenic effects of LYTACs were robustly validated across complementary ex vivo and in vivo models. The Matrigel tube formation and aortic ring assays collectively demonstrated that VEGFR2 degradation disrupts both the initial assembly of endothelial cells into capillary-like structures and the sprouting capacity of mature vessels. Notably, the approximately 42% reduction in tube length and the near-complete inhibition of aortic ring sprouting (89–92%) indicate that angiogenesis is highly dependent on sustained VEGFR2 signaling, and its depletion produces profound functional consequences. The slightly superior efficacy of ZV2-EndoTag in suppressing sprouting aligns with its higher VEGFR2 degradation efficiency, further supporting the mechanistic link between target clearance and functional outcome. Importantly, the in vivo Matrigel plug assay confirmed that these effects translate to a physiologically relevant setting, where LYTACs treatment significantly reduced neovascularization. Taken together with the earlier observations of impaired endothelial cell proliferation and migration, these findings establish that targeted degradation of VEGFR2 produces a multifaceted blockade of angiogenesis, positioning this LYTAC-based strategy as a promising therapeutic approach for diseases driven by pathological angiogenesis. The consistent superiority of ZV2-EndoTag across these models further reinforces the relationship between target clearance and functional anti-angiogenic outcomes. 3.8 LYTAC-Mediated VEGFR2 Degradation Suppresses Tumor Growth and Angiogenesis To evaluate the in vivo antitumor efficacy of LYTAC-mediated VEGFR2 degradation, we established a subcutaneous 4T1 murine breast cancer model in BALB/c mice. Following tumor establishment, mice were randomly assigned to treatment groups and received five intratumoral injections of PBS, ZV2, ZV2-IGF2, or ZV2-EndoTag every other day, and were then maintained until day 20. As shown in Fig. 8 c and 8 d, both ZV2-IGF2 and ZV2-EndoTag treatment groups exhibited significantly reduced tumor volume and weight compared to the PBS control group, with inhibition effects markedly superior to the ZV2 monotherapy group. Quantitative analysis revealed that tumor volumes in the ZV2-IGF2 and ZV2-EndoTag groups were reduced by approximately 42% and 47%, respectively, relative to the control group, demonstrating that both LYTACs effectively suppress tumor growth in vivo . Body weight monitoring throughout the treatment period revealed no significant differences among groups (Fig. 8 b), indicating minimal systemic toxicity. Histological analysis of tumor sections by H&E staining (Fig. 8 e) and immunofluorescence staining for CD31 and VEGFR2 (Fig. 8 f) revealed substantial reductions in both VEGFR2 expression and microvascular density in tumors treated with ZV2-IGF2 and ZV2-EndoTag. Specifically, quantitative analysis of VEGFR2/CD31 dual staining showed that the ratio of VEGFR2-positive area normalized to CD31-positive area decreased by approximately 38% in both treatment groups compared to controls, indicating effective reduction of VEGFR2 signaling within the tumor vasculature. These results demonstrate that recombinant LYTACs achieve effective target protein degradation and angiogenesis inhibition in vivo , leading to suppressed tumor growth. The in vivo antitumor efficacy of our LYTACs validates the therapeutic potential of VEGFR2 degradation in a clinically relevant model. The significant tumor growth suppression achieved by both ZV2-IGF2 and ZV2-EndoTag, coupled with the absence of overt systemic toxicity, supports the safety and efficacy of this targeted degradation approach. Notably, the superior antitumor activity of ZV2-EndoTag compared to ZV2-IGF2 aligns with its higher in vitro degradation efficiency and stronger suppression of endothelial cell functions, reinforcing the relationship between target clearance and therapeutic outcome. Immunofluorescence analysis provided critical mechanistic insight by confirming that reduced tumor growth correlates directly with decreased intratumoral VEGFR2 expression and microvascular density[ 35 , 36 ]. This establishes that the observed antitumor effects arise from on-target degradation of VEGFR2 and subsequent inhibition of tumor angiogenesis, rather than off-target effects. Compared to conventional VEGFR2 inhibitors that merely block receptor signaling, our LYTAC strategy achieves physical elimination of the target protein, potentially offering more sustained effects and reduced opportunity for compensatory signaling. These findings provide proof-of-concept for LYTAC-based membrane protein degradation as a viable antitumor strategy and support further development of this approach for cancer therapy. 4 Conclusions To overcome the limitations of conventional VEGFR2 inhibitors, this study successfully designed and constructed two novel LYTACs, ZV2-IGF2 and ZV2-EndoTag, based on distinct lysosomal targeting mechanisms. These recombinant proteins achieve specific targeting and efficient degradation of VEGFR2 by binding to different IGF2R ligands via a high-affinity VEGFR2 affibody. Mechanistically, both LYTACs induce lysosomal degradation of VEGFR2, thereby effectively blocking downstream signaling pathways and inhibiting the expression and function of angiogenesis-related genes. Through systematic evaluation, we confirmed that both LYTAC molecules exhibit significant biological effects at the cellular and animal levels. In vitro experiments demonstrated that ZV2-IGF2 and ZV2-EndoTag effectively reduced VEGFR2 expression on human umbilical vein endothelial cells (HUVECs) and significantly inhibited cell proliferation, migration, and angiogenesis. In animal models, LYTACs treatment markedly suppressed the growth and progression of mouse xenograft tumors, with histological analysis confirming reduced vascular density within the tumors. Notably, the engineered EndoTag module exhibited superior degradation efficiency and functional outcomes compared to the natural IGF2 ligand, highlighting the potential of rationally designed targeting modules in LYTAC development. These findings not only validate the efficacy of LYTAC technology in targeting membrane protein degradation but also provide crucial experimental evidence for developing novel anti-angiogenic therapeutic approaches that circumvent the drug resistance commonly associated with traditional inhibitors. Together, these results establish LYTAC-mediated VEGFR2 degradation as a viable therapeutic strategy that addresses the limitations of current anti-angiogenic agents by enabling sustained target elimination. The successful application of both natural and engineered lysosomal targeting modules further provides a versatile platform for developing next-generation LYTACs against a broad range of membrane proteins, offering new avenues for treating cancer and other angiogenesis-related diseases[ 37 , 38 ]. Declarations Acknowledgments This work was supported by a grant from the Shandong Provincial Natural Science Foundation (ZR2021MC017, ZR2021LSW001, ZR2024QB069). Funding This work was supported by a grant from the Shandong Provincial Natural Science Foundation (ZR2021MC017, ZR2021LSW001, ZR2024QB069). Ethical approval All animal procedures were approved by The Special Committee of Scientific Research Ethic of Liaocheng University (Approval No. AP2026031535, date: March 15, 2026) and were performed in accordance with the National Institute Guide for the Care and Use of Laboratory Animals. Conflicts of Interest The authors declare no competing interests. Clinical trial number This study is not a clinical trial. Therefore, clinical trial registration is not applicable. Consent to Participate declaration Consent to participate is not applicable because this study did not involve human subjects. Consent to Publish declaration Consent to publish is not applicable as no individual person’s data or images are presented in this manuscrip. Data availability The datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request. The uncropped original images of Western blots and gels have been provided as Supplementary Information. 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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-9310166","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":633587448,"identity":"ea20be09-20b0-4d96-b79f-b52c50893f96","order_by":0,"name":"Rui Wang","email":"","orcid":"","institution":"Liaocheng University","correspondingAuthor":false,"prefix":"","firstName":"Rui","middleName":"","lastName":"Wang","suffix":""},{"id":633587449,"identity":"e81f634c-8644-4166-a3ec-1c02271a8b99","order_by":1,"name":"Yundi Ren","email":"","orcid":"","institution":"Liaocheng University","correspondingAuthor":false,"prefix":"","firstName":"Yundi","middleName":"","lastName":"Ren","suffix":""},{"id":633587450,"identity":"fdf10854-e4c5-43b9-89b6-3c6536e731aa","order_by":2,"name":"Mingyan Liu","email":"","orcid":"","institution":"Liaocheng University","correspondingAuthor":false,"prefix":"","firstName":"Mingyan","middleName":"","lastName":"Liu","suffix":""},{"id":633587451,"identity":"7c84a02c-0e42-482b-8551-4f6b28de4133","order_by":3,"name":"Yuting Lou","email":"","orcid":"","institution":"Liaocheng University","correspondingAuthor":false,"prefix":"","firstName":"Yuting","middleName":"","lastName":"Lou","suffix":""},{"id":633587452,"identity":"66e24408-e593-48cb-aa90-80bcde134d64","order_by":4,"name":"Guangyong Li","email":"","orcid":"","institution":"Liaocheng University","correspondingAuthor":false,"prefix":"","firstName":"Guangyong","middleName":"","lastName":"Li","suffix":""},{"id":633587453,"identity":"f01ec894-fe9d-4791-8427-6e04dd3cb630","order_by":5,"name":"Xiaohong Chu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6UlEQVRIiWNgGAWjYDACCSBOACMGxgcPwEIJxGthNkggWgtUGZsEUVr4Z/eYSTyoscnjl26/VpFQc5iBnz3HgOHnDjyW3DljbJBwLK1Ycs6ZshsJxw4zSPa8MWDsPYNbi4FEjuGDBLbDiRtu5KTdSGw4zGBwI8eAmbENrxaDAwn/IFoKQFrsidBi+CCxDaQl/RgD2BYJAlokbqQVGyT2pSXOnJHDLJFwLJ1H4syzgoO9eLTwz0jeJvnjm01iv0T6ww8faqzl+NuTNz74iUcLEuAxAJMg4gBRGhgY2B8QqXAUjIJRMApGGgAAwyRUZBXEvFQAAAAASUVORK5CYII=","orcid":"","institution":"Liaocheng University","correspondingAuthor":true,"prefix":"","firstName":"Xiaohong","middleName":"","lastName":"Chu","suffix":""},{"id":633587454,"identity":"178cd5da-20d5-445f-99b2-47c0ad24eba5","order_by":6,"name":"Dianlong Jia","email":"","orcid":"","institution":"Liaocheng University","correspondingAuthor":false,"prefix":"","firstName":"Dianlong","middleName":"","lastName":"Jia","suffix":""},{"id":633587455,"identity":"8ea4e350-2204-4293-a495-82d64e4b91bc","order_by":7,"name":"Jun Li","email":"","orcid":"","institution":"Liaocheng University","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2026-04-03 07:40:52","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9310166/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9310166/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108408970,"identity":"3b613845-7e50-4980-9922-7deb58269200","added_by":"auto","created_at":"2026-05-04 09:56:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":7339867,"visible":true,"origin":"","legend":"\u003cp\u003eDesign, construction, and purification of recombinant LYTACs targeting VEGFR2. (a). Schematic of LYTAC mechanism: VEGFR2 affibody-IGF2 chimera bridges VEGFR2 and CI-M6PR, inducing internalization and lysosomal degradation. (b). Modular design of ZV2-IGF2 and ZV2-EndoTag constructs. (c, d). SDS-PAGE analysis of expression and purification for ZV2-IGF2 (c). SDS-PAGE of purified ZV2, ZV2-IGF2, and ZV2-EndoTag. (d). Western blot detection using anti-6×His antibody. (n=3)\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-9310166/v1/bec4e41ee50c3041d9c2e462.png"},{"id":108408966,"identity":"1521cd93-2bf4-4a24-9178-f674030dac8b","added_by":"auto","created_at":"2026-05-04 09:56:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1293995,"visible":true,"origin":"","legend":"\u003cp\u003eLysosomal colocalization analysis. (a). Representative CLSM images demonstrate lysosomal colocalization of various FAM-labeled substances within HUVECs. Scalebar: 5 μm (b). FI distribution curves across CLSM image cross-sections quantitatively reveal the intracellular distribution of these compounds.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-9310166/v1/6254446b8fdbf946f7105d93.png"},{"id":108409029,"identity":"7dbb993e-b026-4a10-9405-1e99f732c502","added_by":"auto","created_at":"2026-05-04 09:56:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":9233780,"visible":true,"origin":"","legend":"\u003cp\u003eDegradation capacity of LYTACs. (a). Western blot analysis of ZV2-IGF2 and ZV2-EndoTag-mediated degradation of cell surface VEGFR2. (b). Flow cytometry detection of cell surface VEGFR2 levels in HUVEC cells following treatment with 1 μM LYTAC protein. (c). Quantitative analysis of Western blot results from (a). Data are presented as mean ± SD (n = 3). *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001 vs. control group.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-9310166/v1/c500575a6011230f32e2c4a0.png"},{"id":108408968,"identity":"5838e8e1-748b-4ec6-8652-bb0bce71fead","added_by":"auto","created_at":"2026-05-04 09:56:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1035879,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of LYTACs degradation of VEGFR2 on downstream signalling pathways. (a) Western blot analysis demonstrating the effects of 1 μM LYTACs treatment on downstream p-AKT, p-MEK, and GAPDH proteins in HUVEC cells. (b-d) Quantitative analysis of VEGFR2 (b), p-AKT (c), and p-MEK (d) protein band intensities relative to GAPDH from Western blots shown in (a). Data are presented as mean ± SD (n = 3). *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 vs. control group.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-9310166/v1/045cadf3adf49dccb261fff7.png"},{"id":108409023,"identity":"7e55c709-c88e-4385-b06c-2c431b8b27bb","added_by":"auto","created_at":"2026-05-04 09:56:39","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1332295,"visible":true,"origin":"","legend":"\u003cp\u003eAnti-proliferative effects of recombinant LYTACs in HUVECs mediated by VEGFR2 degradation. (a). Dose-response curves of cell viability in HUVECs treated with ZV2 for 24 h. (b). Concentration-dependent inhibition of proliferation by ZV2-IGF2. (c). ZV2-EndoTag inhibits HUVEC growth in a concentration-dependent manner. Data represent mean ± standard deviation (n=6). *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 compared to untreated controls.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-9310166/v1/95734c5624ca9a9c808c2e34.png"},{"id":108493251,"identity":"614ee3d0-eec9-4665-ad44-33e1c8202983","added_by":"auto","created_at":"2026-05-05 09:59:47","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2904772,"visible":true,"origin":"","legend":"\u003cp\u003eLYTACs inhibit HUVEC cell migration capacity. (a). Representative images of wound healing assays showing HUVEC migration at 0 and 24 h after treatment. (b). Quantitative analysis of wound closure rate in HUVECs following 24 h treatment. (c). Representative micrographs of Transwell migration assays after 24 h incubation. (d). Statistical analysis of migrated cell numbers per field across treatment groups. (e). Analysis of migration trajectories of HUVEC cells treated with 1 μM LYTACs protein over 48 h, processed using ImageJ software. Data are presented as mean ± SD (n = 3). *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001 vs. control group.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-9310166/v1/275bb7b6dba991d2c8971af6.png"},{"id":108408973,"identity":"787702d9-cd1a-4aab-9f9b-99c22a727f70","added_by":"auto","created_at":"2026-05-04 09:56:31","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2418893,"visible":true,"origin":"","legend":"\u003cp\u003eLYTACs inhibit vascular network formation \u003cem\u003ein vitro\u003c/em\u003e. (a). Representative images of HUVEC tube formation on Matrigel after 24 h treatment. (b). Quantitative analysis of total tube length from panel a. (c). Representative bright-field images of mouse aortic ring sprouts following treatment. (d). Quantification of vascular sprout length and number from panel c. (e). The Matrigel plug assay further confirms the inhibitory effect of the LYTAC protein on \u003cem\u003ein vivo\u003c/em\u003e angiogenesis. (f). Figures show the H\u0026amp;E staining and immunohistochemical analysis results of the Matrigel plugs in Figure E, respectively. (g). Quantitative analysis of CD31-positive area from immunohistochemical staining in panel f. Data are presented as mean ± SD (n = 3). *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001 vs. control group.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-9310166/v1/4a7f111f99b63f97366da437.png"},{"id":108408975,"identity":"0b4381c2-4cf1-4fa9-8042-09a16300bca6","added_by":"auto","created_at":"2026-05-04 09:56:31","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":2588543,"visible":true,"origin":"","legend":"\u003cp\u003eLYTACs inhibit tumor growth in mice \u003cem\u003ein vivo\u003c/em\u003e. (a). Schematic diagram of the treatment experiment design in the 4T1 tumor-bearing mouse model. (b). Body weight change curves of mice in each group during treatment to assess systemic toxicity. (c). Dynamic curves of tumor volume growth over time in different treatment groups. (d). Representative macroscopic appearance photographs of tumors collected after treatment. and statistical analysis of tumor weights at the endpoint. (e). Representative images of microvascular density in tumor sections demonstrated by CD31/VEGFR2 immunofluorescence staining. (f). Area of VEGFR2 signal normalized to CD31 area (% of control) in tumors. Data are presented as mean ± SD (n = 3). *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 vs. control group.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-9310166/v1/3f0d1d75a0e6fec8c0a14f6d.png"},{"id":108809214,"identity":"2dea39c4-e38c-4c4e-8fd3-6e5744262cb2","added_by":"auto","created_at":"2026-05-08 15:50:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":28502054,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9310166/v1/56e7867a-18c3-47d2-977c-67faae45c032.pdf"},{"id":108803899,"identity":"ea6e2c05-0f90-43da-8497-31f0788064ec","added_by":"auto","created_at":"2026-05-08 15:10:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":937632,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformationOriginalBlots.pdf.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9310166/v1/c6f87cd3da984096dfbc1fe2.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Targeted Degradation of VEGFR2 by LYTACs Suppresses Angiogenesis and Tumor Growth","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eTumor angiogenesis is a critical process in the growth and metastasis of solid tumors. When tumor volume reaches 1\u0026ndash;2 mm\u0026sup3; and diffusion can no longer provide sufficient nutrients, pro-angiogenic factors released by the tumor microenvironment (such as VEGF and FGF2) activate endothelial cells, ultimately forming a new microvascular network[\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The core regulator VEGF binds to VEGFR2 (also known as KDR/Flk-1) on endothelial cell surfaces, activating downstream signaling pathways including MAPK/ERK and PI3K/AKT/mTOR[\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. This potently promotes endothelial cell proliferation, migration, and survival, ultimately forming an abnormal tumor vascular network[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Consequently, VEGFR2 has become a primary target for anti-angiogenic therapies[\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Monoclonal antibodies, fusion proteins, and small-molecule tyrosine kinase inhibitors (e.g., sorafenib, sunitinib) developed against this protein are currently used clinically. However, monotherapy targeting the VEGF/VEGFR2 pathway often leads to acquired drug resistance and adverse reactions[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. These limitations underscore the urgent need for alternative strategies that achieve sustained target inhibition without the drawbacks of conventional occupancy-driven inhibitors[\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLysosome-Targeting Chimeras (LYTACs) represent an emerging class of protein degradation tools[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Their core mechanism involves simultaneously binding to endocytic receptors on the cell surface such as the mannose-6-phosphate receptor, M6PR and the target protein, thereby directing membrane-associated or extracellular proteins (POIs) to lysosomes for efficient degradation[\u003cspan additionalcitationids=\"CR20 CR21 CR22 CR23 CR24\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. By enabling the physical elimination rather than temporary blockade of target proteins, this technology offers a paradigm shift in addressing traditionally hard-to-target extracellular and membrane proteins, potentially overcoming the resistance mechanisms that limit current anti-angiogenic agents[\u003cspan additionalcitationids=\"CR26 CR27\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLYTACs rely on lysosome-targeting receptors on the cell membrane surface to exert their effects. POI forms a chimeric structure with LYTACs and LTR, enabling entry into endosomes and subsequent transport to lysosomes for degradation. In this study, we developed two distinct LYTACs targeting VEGFR2 based on different lysosomal targeting ligands. A high-affinity VEGFR2 affibody (ZVEGFR2_Bp21), hereinafter referred to as ZV2, was previously reported to bind human VEGFR2 with picomolar affinity[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], was selected as the targeting module. For lysosomal targeting, we pursued two strategies. The first involved fusing the VEGFR2 affibody to the insulin-like growth factor 2 (IGF2) domain, which binds to the widely expressed cation-independent mannose-6-phosphate receptor (CI-M6PR/IGF2R) and enables efficient endocytosis and lysosomal delivery[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The second strategy leveraged the Rosetta de novo protein design tool, jointly developed by Professor David Baker's team and Professor Carolyn Bertozzi's team. The second strategy utilized an engineered EndoTag module, computationally designed using the Rosetta platform to bind IGF2R without competing with endogenous ligands, thereby potentially minimizing off-target effects[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Notably, the parallel comparison of a natural ligand (IGF2) and an engineered module (EndoTag) within the same LYTAC framework represents a unique opportunity to evaluate the relative advantages of these distinct targeting mechanisms, providing critical insights for future LYTAC optimization. By comparing the degradation efficiency, specificity, and functional outcomes of these two LYTACs, we aimed to establish a dual-validation strategy for VEGFR2-targeted protein degradation and to evaluate its therapeutic potential in angiogenesis-dependent diseases.\u003c/p\u003e \u003cp\u003eHere, we demonstrate that both ZV2-IGF2 and ZV2-EndoTag efficiently internalize into endothelial cells, colocalize with lysosomes, and induce concentration-dependent degradation of VEGFR2. This leads to suppression of downstream AKT and MEK signaling, inhibition of endothelial cell proliferation, migration, and tube formation, and reduced angiogenesis \u003cem\u003ein ex vivo\u003c/em\u003e aortic ring assays and \u003cem\u003ein vivo\u003c/em\u003e Matrigel plug models. Finally, we show that intratumoral administration of these LYTACs significantly suppresses tumor growth in a murine 4T1 breast cancer model, accompanied by reduced intratumoral VEGFR2 expression and microvascular density. Together, these findings establish LYTAC-mediated VEGFR2 degradation as a promising anti-angiogenic strategy that addresses the limitations of current therapies by enabling sustained target elimination. Moreover, the successful application of both IGF2 and EndoTag modules provides a versatile platform for developing next-generation LYTACs targeting a broad range of membrane proteins, highlighting the translational potential of this degradation-based approach.\u003c/p\u003e"},{"header":"2 Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials\u003c/h2\u003e \u003cp\u003eAll plasmids were synthesized by GenScript (Nanjing, China), and sequence verified. The host strain \u003cem\u003eE. coli\u003c/em\u003e. M15 cells were purchased from Shanghai Weidi Biotechnology company (China). RPMI 1640 (Cellmax), DMEM (Hyclone) and Fetal Bovine Serum (FBS) were purchased from qualified vendors. LysoTracker\u0026trade; Red DND-26 and Hoechst 33342 were purchased from Invitrogen. 6-carboxyfluorescein (6-FAM) NHS ester were purchased from Ruixi Biotechnology Company (Xi\u0026rsquo; an, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Cell culture\u003c/h2\u003e \u003cp\u003eHuman Umbilical Vein Endothelial Cells (HUVECs) and The breast cancer cell line 4T1 were obtained from the American Type Culture Collection (ATCC). These cells were meticulously cultured in specialized endothelial cell medium (ScienCell catalog number 1001) and Dulbecco's Modified Eagle Medium (DMEM, Thermo Fisher), respectively, at an optimal temperature of 37\u0026deg;C in a humidified chamber with 5% CO\u003csub\u003e2\u003c/sub\u003e. Subconfluent cells, specifically those between passages 2 to 6, were carefully selected for the experiments to ensure optimal growth and response.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Evaluation of lysosomal colocalization\u003c/h2\u003e \u003cp\u003eLysosomal colocalization was evaluated using confocal laser scanning microscopy (CLSM). HUVECs (2\u0026times;10⁴ cells/well) were seeded onto 12-well plates with integrated coverslips and cultured overnight in ECM medium. After discarding the medium and washing twice with PBS, cells were treated with different FAM-labeled samples (1 \u0026micro;M, including ZV2, ZV2-IGF2, ZV2-EndoTag) at 37\u0026deg;C for 2 h. Cells were then washed with PBS and incubated with LysoTracker Red at 37\u0026deg;C according to the manufacturer's instructions. Following staining, cells were washed again and fixed with 4% formaldehyde at room temperature for 20 min. Finally, nuclei were stained with DAPI for 10 min, followed by removal of the staining solution and three washes with PBS. Cover slips were sealed in the dark and imaged using CLSM.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 \u003cem\u003eIn Vitro\u003c/em\u003e Cell Proliferation Experiment of LYTAC\u003c/h2\u003e \u003cp\u003eThe CCK-8 assay kit (C0038, Beyotime) was used to detect the inhibitory effect of LYTAC on VEGF165 (P5561, Beyotime)-stimulated proliferation in HUVECs. Cells were seeded at a density of 5\u0026times;10\u0026sup3; cells per well in a 96-well plate and cultured overnight in ECM medium. Subsequently, cells were starved with 0.5% ECM for 6 h, followed by addition of a mixture containing VEGF165 (50 ng mL⁻\u0026sup1;) and varying concentrations of LYTAC. Cells were further cultured for 24 h in medium supplemented with 2% FBS. Then, 10 \u0026micro;L of CCK-8 solution was added to each well. After incubation for 4 h, directly measure the absorbance of each well at 450 nm wavelength using a BioTek microplate reader.\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\begin{array}{c}Cell\\:Viability\\:\\left(\\text{%}\\right)=\\left({A}_{treatment}-{A}_{blank}\\right)/\\left({\\text{A}}_{\\text{C}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}}-{\\text{A}}_{\\text{b}\\text{l}\\text{a}\\text{n}\\text{k}}\\right)\\times\\:100\\%\\#\\left(1\\right)\\end{array}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Wound Healing Assay of LYTAC\u003c/h2\u003e \u003cp\u003eThis study evaluated the effects of different samples on VEGF165-induced migration of HUVECs using a cell scratch assay. HUVECs were seeded at a density of 1\u0026times;10⁵ cells per well in a 6-well plate and cultured overnight in ECM medium. A scratch was created on the monolayer using a 200 \u0026micro;L pipette tip to form a wound area. After washing cells twice with PBS, different samples containing VEGF165 (50 ng mL⁻\u0026sup1;) were added to each well. Cells were cultured for 24 h in medium supplemented with 2% FBS. The scratch area was photographed using an inverted microscope, and cell migration rates were calculated using ImageJ software.\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:\\begin{array}{c}Wound\\:Closure\\:Rate\\:\\left(\\text{%}\\right)=\\left({\\text{A}\\text{r}\\text{e}\\text{a}}_{0\\text{h}}-{\\text{A}\\text{r}\\text{e}\\text{a}}_{\\text{t}\\text{h}}\\right)/{\\text{A}\\text{r}\\text{e}\\text{a}}_{0\\text{h}}\\times\\:100\\%\\#\\left(2\\right)\\end{array}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Transwell migration assay\u003c/h2\u003e \u003cp\u003eTranswell migration assays were employed to evaluate endothelial cell (EC) migration capacity. First, HUVECs were co-treated with different LYTAC protein samples for 24 h. Subsequently, 1.5 \u0026times; 10⁵ treated cells were resuspended in 500 \u0026micro;L endothelial basal medium (ECM) and seeded into the upper chamber of a 24-well Transwell plate (polycarbonate membrane, 8 \u0026micro;m pore size; Corning; Merck KGaA, Darmstadt, Germany). The lower chamber of each well was filled with 750 \u0026micro;L of ECM containing 1% fetal calf serum (FCS). After incubation for 5 h, non-migrated cells were removed, and cells migrating to the membrane surface were stained with crystal violet (C0121, Beyotime). Using a confocal laser scanning microscope (CLSM; Leica TCS SP8 STED 3X), 20 random fields of view were selected to count migrating cells. Cell migration rates were expressed as percentages relative to the control group.\u003cdiv id=\"Equc\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equc\" name=\"EquationSource\"\u003e\n$$\\:\\begin{array}{c}Migration\\:Rate\\:\\left(\\text{%}\\right)={\\text{N}}_{\\text{t}\\text{r}\\text{e}\\text{a}\\text{t}\\text{m}\\text{e}\\text{n}\\text{t}}/{\\text{N}}_{\\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}}\\times\\:100\\%\\#\\left(3\\right)\\end{array}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Protein Degradation Analysis using Western Blot\u003c/h2\u003e \u003cp\u003eHUVECs (1\u0026times;10⁵) were seeded into 6-well plates and starved for 6 h in ECM medium supplemented with 0.5% FBS. Subsequently, cells were treated with a mixture of VEGF165 (50 ng mL⁻\u0026sup1;) and LYTAC protein (1 \u0026micro;M) for 24 h. Following treatment, cells were lysed using RIPA buffer containing protease and phosphatase inhibitors (P1045, Beyotime) to extract total protein, which was quantified using the BCA assay kit (P0010, Beyotime). Protein samples were mixed with loading buffer, denatured by heating at 100\u0026deg;C for 5 min, and separated by electrophoresis on a 4\u0026ndash;12% SDS-PAGE gel (ET12412, ACE Biotechnology). Proteins were then transferred to a polyvinylidene difluoride (PVDF) membrane and blocked at room temperature with TBST containing 5% nonfat milk for 1 h. After three washes with TBST, the membrane was incubated at 4\u0026deg;C for 24 h with either the VEGFR2 primary antibody (1:1000, 2479, CST) or the β-Actin primary antibody (1:2000, 60004-1-Ig, Proteintech). After three additional washes, membranes were incubated at room temperature for 2 h with the corresponding secondary antibody (1:5000, Proteintech). Finally, after thorough washing with TBST, membranes were developed using ECL chemiluminescence.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Protein Degradation Analysis by Flow Cytometry\u003c/h2\u003e \u003cp\u003eHUVECs (1\u0026times;10⁵) were seeded into 6-well plates and starved for 6 h in ECM medium supplemented with 0.5% fetal bovine serum. Cells were then treated with a mixture of VEGF165 (50 ng mL⁻\u0026sup1;) and LYTAC protein (1 \u0026micro;M) for 24 and 48 h, respectively. Following treatment, adherent cells were harvested via trypsin digestion, washed three times with staining buffer, and fixed with 4% paraformaldehyde at room temperature for 20 min. Subsequently, cells were blocked with staining buffer containing 5% bovine serum albumin at room temperature for 30 min, then incubated with primary antibody (67407-1-Ig, Proteintech) at 4\u0026deg;C for 30 min. After washing with staining buffer, cells were incubated with FITC-labeled secondary antibody (abs20012, Absin). Finally, cells were washed and resuspended in 200 \u0026micro;L staining buffer for analysis using a flow cytometer (LSRFortessa, Becton Dickinson Immunocytometry Systems).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Tube formation assay\u003c/h2\u003e \u003cp\u003eThe tube formation assay was employed to analyse the tubule-forming capacity of endothelial cells (ECs). 1.5 \u0026times; 10⁴ HUVECs were resuspended in ECM medium containing different protein samples and seeded into 96-well plates pre-coated with Matrigel. Following 24 h of culture, newly formed vascular-like structures were imaged using phase-contrast microscopy. The number of luminal networks per well was quantified using ImageJ software and its Angiogenesis Analysis plugin (National Institutes of Health, Bethesda, MD, USA), with endothelial tube-forming capacity expressed as a percentage relative to the control group.\u003cdiv id=\"Equd\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equd\" name=\"EquationSource\"\u003e\n$$\\:\\begin{array}{c}Relative\\:Tube\\:Length\\:\\left(\\text{%}\\right)={\\text{L}}_{\\text{t}\\text{r}\\text{e}\\text{a}\\text{t}\\text{m}\\text{e}\\text{n}\\text{t}}/{\\text{L}}_{\\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}}\\times\\:100\\%\\#\\left(4\\right)\\end{array}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Aortic ring assay\u003c/h2\u003e \u003cp\u003eThoracic aortas were harvested from BALB/c mice, sectioned into 0.5 mm annular segments, and embedded in Matrigel matrix gel (Corning; Merck KGaA). A single aortic ring per well was placed in a 96-well plate. Following a 15 min incubation, wells were treated with LYTAC proteins under varying conditions. After a further 6 days of culture, aortic rings were imaged using a phase-contrast microscope. Vascular sprouting was quantified by measuring the area of newly formed sprouting regions, expressed as a percentage relative to the control group.\u003cdiv id=\"Eque\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Eque\" name=\"EquationSource\"\u003e\n$$\\:\\begin{array}{c}Relative\\:Sprouting\\:Area\\:\\left(\\text{%}\\right)={\\text{A}}_{\\text{t}\\text{r}\\text{e}\\text{a}\\text{t}\\text{m}\\text{e}\\text{n}\\text{t}}/{\\text{A}}_{\\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}}\\times\\:100\\%\\#\\left(5\\right)\\end{array}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Matrigel plug assay\u003c/h2\u003e \u003cp\u003eA Matrigel mixture containing mouse VEGF (1 \u0026micro;g/mL; R\u0026amp;D Systems), mouse FGF2 (1 \u0026micro;g/mL; R\u0026amp;D Systems), heparin (60 IU/mL), and 1 \u0026micro;M LYTAC protein (or control) was subcutaneously injected into the lateral abdomen of 8-week-old BALB/c mice (Janvier-Labs), with seven mice per group. After 7 days, the Matrigel plugs were retrieved for subsequent immunohistochemical analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 Animal experiments\u003c/h2\u003e \u003cp\u003eFemale BALB/c J mice (6\u0026ndash;8 weeks old) were provided by Jinan Pengyue Experimental Animal Breeding Co, Ltd. (Jinan, China). Mice were maintained under specific pathogen-free(SPF) conditions. All animal procedures for pharmacokinetic studies were performed following protocols approved by The Special Committee of Scientific Research Ethic of Liaocheng University (Approval Number: AP2026031535; Date: March 15, 2026).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13 Tumor Model Establishment\u003c/h2\u003e \u003cp\u003eTo evaluate the in vivo antitumor efficacy of LYTAC-mediated VEGFR2 degradation, a subcutaneous 4T1 murine breast cancer model was established in female BALB/c mice (6\u0026ndash;8 weeks old). 4T1 cells were harvested, resuspended in sterile PBS at 1\u0026times;10\u003csup\u003e7\u003c/sup\u003e cells/mL, and 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells(100 \u0026micro;L) were injected subcutaneously into the right dorsal flank of each mouse. When tumor volume reached 60\u0026ndash;80 mm\u0026sup3; (5\u0026ndash;7 days post-inoculation), mice were randomly assigned to four groups (n\u0026thinsp;=\u0026thinsp;6 per group): PBS control, ZV2, ZV2-IGF2, and ZV2-EndoTag. They received five intratumoral injections of 100 \u0026micro;g protein in 100 \u0026micro;L PBS every other day, and were then maintained until day 20 post‑tumor inoculation. Tumor volume was measured every two days using a digital caliper, and body weight was monitored to assess systemic toxicity. At the experimental endpoint (day 20), when tumors in the control group reached 500\u0026ndash;600 mm\u0026sup3;, mice were euthanized; tumor tissues were excised, weighed, and either frozen at \u0026minus;\u0026thinsp;80\u0026deg;C or fixed in 4% paraformaldehyde for subsequent analysis.\u003cdiv id=\"Equf\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equf\" name=\"EquationSource\"\u003e\n$$\\:\\begin{array}{c}Volume=\\text{L}\\text{e}\\text{n}\\text{g}\\text{t}\\text{h}\\times\\:\\text{W}\\text{i}\\text{d}\\text{t}{\\text{h}}^{2}/2\\#\\left(6\\right)\\end{array}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.14 Histology and immunohistochemistry\u003c/h2\u003e \u003cp\u003eFormalin-fixed paraffin-embedded Matrigel plugs and tumor tissues were serially sliced into 3-\u0026micro;m sections. For the analysis of tumor size, sections with the largest area for each tumor were selected, stained with hematoxylin and eosin (H\u0026amp;E), imaged using a microscope, and subjected to planimetric tumor area measurements by means of an image analysis software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.15 Statistics Analysis\u003c/h2\u003e \u003cp\u003eData were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Student's t test was used to compare 2 groups of samples. The difference among multiple groups was evaluated using a one-way analysis of variance (ANOVA) with Bonferroni's post hoc test. *\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered as statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results and Discussion","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Design and Construction of Recombinant Protein LYTACs\u003c/h2\u003e \u003cp\u003eTo establish a novel strategy for targeted degradation of VEGFR2, we designed two recombinant LYTACs based on distinct lysosomal targeting mechanisms. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, the proposed working mechanism of these LYTACs involves simultaneously binding to cell surface VEGFR2 and the CI-M6PR lysosomal targeting receptor via an IGF2 ligand, thereby inducing VEGFR2 internalization and subsequent lysosomal degradation. For molecular construction, we employed a modular design strategy (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) A high-affinity VEGFR2 affibody (ZVEGFR2_Bp21), previously reported to bind human VEGFR2 with picomolar affinity (KD 241\u0026thinsp;\u0026plusmn;\u0026thinsp;4 pM)[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], served as the targeting module. This affibody was fused via a flexible (GGGGS)₄ linker to either the insulin-like growth factor 2 (IGF2) domain or the engineered EndoTag module, which function as lysosomal targeting ligands. An N-terminal 6\u0026times;His tag was incorporated into both constructs to facilitate protein purification and detection. The resulting recombinant proteins were designated as ZV2-IGF2 and ZV2-EndoTag.\u003c/p\u003e \u003cp\u003eFollowing subcloning of the synthetic genes into the pQE30 prokaryotic expression vector, both recombinant proteins were expressed in E. coli M15 cells upon IPTG induction. The expression and purification processes were monitored by SDS-PAGE analysis, which confirmed successful expression and efficient purification of both proteins via Ni\u0026sup2;⁺ affinity chromatography. The purity of the final products was assessed by SDS-PAGE (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec), revealing single bands at the expected molecular weights (approximately 23.2 kDa for ZV2-IGF2 and 30.1 kDa for ZV2-EndoTag) for the purified ZV2, ZV2-IGF2, and ZV2-EndoTag proteins. Western blot analysis using an anti-6\u0026times;His antibody further confirmed the identity of the purified proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). Together, these results demonstrate the successful design, prokaryotic expression, and purification of two novel LYTAC molecules targeting VEGFR2, providing the necessary material basis for subsequent functional validation studies.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Lysosome Colocalization and Cellular Uptake of LYTAC\u003c/h2\u003e \u003cp\u003eHaving confirmed the VEGFR2 degradation efficacy of our LYTACs, we next investigated the mechanism by which IGF2 and EndoTag mediate lysosomal targeting. To verify this core function, ZV2-IGF2, ZV2-EndoTag, and the control ZV2 molecule were chemically conjugated with the FAM green fluorescent probe and co-incubated with HUVEC cells. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, the control ZV2-FAM exhibited only faint non-specific intracellular signals with minimal overlap with the red fluorescence of lysosomes; whereas both ZV2-IGF2-FAM and ZV2-EndoTag-FAM experimental groups exhibited strong green fluorescence signals. These signals highly overlapped with LysoTracker Red-labelled lysosomal regions, producing distinct yellow colocalisation signals. This colocalisation phenomenon was particularly pronounced in the cytoplasmic region, indicating that both recombinant LYTACs proteins were efficiently internalised and successfully transported to lysosomes. To quantify this phenomenon, we employed ImageJ software to measure and plot the intensity distribution curves of FAM green fluorescence and LysoTracker red fluorescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). The results revealed a substantial region of overlap between the fluorescence curves of ZV2-IGF2 and ZV2-EndoTag.\u003c/p\u003e \u003cp\u003eIGF2, as a natural ligand, exhibits a well-established binding mechanism with CI-M6PR/IGF2R[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. This receptor is widely expressed across diverse cell types, efficiently mediating the endocytosis of the ligand-receptor complex and directing its transport towards lysosomes. The present findings further validate the feasibility of incorporating IGF2 into LYTAC constructs, demonstrating that the IGF2 domain retains its receptor-binding activity when fused to the VEGFR2 affinity fragment, thereby effectively directing the entire fusion protein into the lysosomal degradation pathway. This provides robust support for LYTAC design strategies based on natural ligands.\u003c/p\u003e \u003cp\u003eAs a module engineered through de novo protein design, EndoTag's core advantage lies in achieving specific receptor recognition and endocytosis induction independently of natural ligands[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Our findings validate this design principle: when fused to the VEGFR2 phage display construct, EndoTag demonstrated lysosomal targeting efficiency comparable to IGF2, with fluorescence curves exhibiting high overlap. This not only confirms EndoTag's utility as a lysosomal targeting module but, more significantly, represents an engineered strategy that opens broader application prospects for developing novel LYTAC molecules. This is particularly relevant for targets or tissue types lacking known natural endocytic ligands.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Protein Degradation Capability of LYTAC\u003c/h2\u003e \u003cp\u003eAfter validating the lysosomal targeting capabilities of IGF2 and EndoTag, we further assessed the degradation efficiency of recombinant LYTACs on VEGFR2 expressed on the cell membrane surface. Results shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec indicated that both recombinant LYTACs reduced VEGFR2 levels in a concentration-dependent manner. Quantitative analysis revealed that at equivalent concentrations, ZV2-EndoTag achieved approximately 13% higher degradation efficiency than ZV2-IGF2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec), demonstrating its superior VEGFR2 clearance capacity. To validate these findings, we further quantified changes in cell surface VEGFR2 expression via flow cytometry. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, HUVEC cells treated with LYTACs for 24 h exhibited significantly reduced fluorescence intensity upon binding with VEGFR2-specific antibodies, with the ZV2-EndoTag-treated group demonstrating a markedly greater decrease than the ZV2-IGF2 group. This result further confirms that ZV2-EndoTag exhibits superior efficacy in degrading cell surface VEGFR2.\u003c/p\u003e \u003cp\u003eThe disparity in degradation efficacy between the two LYTACs may stem from multiple factors. First, as a de novo engineered protein module, EndoTag may possess superior binding affinity for IGF2R or more efficient endocytosis triggering compared to the native IGF2 ligand. Second, EndoTag's structural design may facilitate sustained effective interaction with the receptor post-binding to the target protein, or exhibit enhanced stability within endosomal vesicles. Additionally, differences may exist in their lysosomal sorting signals or the kinetics of receptor dissociation. Flow cytometry results further corroborate this disparity, confirming ZV2-EndoTag's superiority through in situ detection of membrane proteins. This disparity in degradation efficiency correlates with subsequent observations of cellular functional suppression including proliferation, migration, and tubule-forming capacity establishing a preliminary positive correlation between degradation efficiency and functional inhibition. These findings not only validate the molecular design strategy employed herein but also point towards future optimisation of LYTAC molecules: engineered targeting modules may outperform natural ligands in functionality, demonstrating greater developmental potential.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.4 The effect of VEGFR2 degradation on downstream AKT and MEK phosphorylation levels\u003c/h2\u003e \u003cp\u003eBuilding upon confirmation that LYTACs effectively degrade cell surface VEGFR2, this study further investigates their impact on downstream signalling pathway activity. As a core regulatory receptor for angiogenesis, VEGFR2 activation primarily mediates endothelial cell proliferation, survival, and migration via the PI3K-AKT and MAPK-MEK signalling pathways[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. To evaluate the impact of VEGFR2 degradation on these pathways, we collected lysates from HUVECs treated with different LYTACs and assessed changes in the protein levels of phosphorylated AKT (p-AKT) and phosphorylated MEK (p-MEK) by Western blot analysis. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, compared to the control group, both p-AKT and p-MEK band intensities were markedly diminished in the ZV2-IGF2 and ZV2-EndoTag treatment groups. Quantitative analysis of grey values further confirmed that expression levels of both phosphorylated proteins were significantly downregulated following LYTACs treatment. Notably, the degree of downregulation of p-AKT and p-MEK in the ZV2-EndoTag group was slightly higher than in the ZV2-IGF2 group, consistent with the previously observed higher VEGFR2 degradation efficiency of ZV2-EndoTag.\u003c/p\u003e \u003cp\u003eThese findings provide molecular evidence that LYTACs-mediated VEGFR2 degradation effectively inhibits downstream PI3K-AKT and MAPK-MEK signaling pathways. The suppression of these pathways is particularly significant given their central role in transducing VEGFR2 signals to regulate endothelial cell functions[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The observed reduction in p-AKT levels suggests impaired cell survival signaling, while decreased p-MEK indicates disruption of the proliferative MAPK cascade[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Together, these results mechanistically explain the functional inhibition of endothelial cell proliferation, migration, and tube formation observed in our previous experiments. Furthermore, the correlation between degradation efficiency and signaling suppression across the two LYTACs reinforces the mechanistic link between target protein clearance and pathway inhibition. These findings establish that the anti-angiogenic effects of our LYTACs are mediated through specific disruption of VEGFR2-dependent signaling networks, validating the therapeutic potential of this degradation-based approach.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Effects of LYTACs Recombinant Proteins on the Proliferative Capacity of HUVEC Cells\u003c/h2\u003e \u003cp\u003eBuilding upon the established role of VEGFR2 in promoting cell proliferation through downstream signaling pathways such as AKT activation, we next evaluated the impact of LYTACs-mediated VEGFR2 degradation on the biological behavior of HUVEC cells. Results shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e demonstrate that both LYTAC molecules inhibited HUVEC proliferation in a concentration-dependent manner. At a concentration of 500 nM, treatment with ZV2, ZV2-IGF2, or ZV2-EndoTag suppressed cell viability by approximately 46% compared to untreated controls. Notably, at equivalent concentrations, ZV2-IGF2 and ZV2-EndoTag exhibited significantly stronger anti-proliferative effects than the ZV2 affinity control, which lacks the fused lysosomal targeting module, indicating that VEGFR2 degradation effectively blocks its pro-proliferative signaling pathway.\u003c/p\u003e \u003cp\u003eThe observed anti-proliferative effects correlate well with the differential VEGFR2 degradation efficiencies of the two LYTACs. The approximately 46% reduction in cell viability achieved by all three proteins at 500 nM likely reflects the contribution of VEGFR2 binding alone, as ZV2 can sterically hinder receptor dimerization and activation. However, the enhanced inhibition mediated by ZV2-IGF2 and ZV2-EndoTag demonstrates that active degradation of the receptor provides additional therapeutic benefit beyond simple antagonism. This finding underscores the mechanistic advantage of the LYTAC approach: by physically eliminating the target protein rather than temporarily blocking its activity, degradation-based strategies can achieve more profound and sustained functional suppression. Furthermore, the slightly stronger inhibition observed with ZV2-EndoTag compared to ZV2-IGF2 aligns with its superior degradation efficiency documented earlier, reinforcing the relationship between target clearance and functional outcome. These results establish that VEGFR2 degradation translates directly into impaired endothelial cell proliferative capacity, providing a functional rationale for pursuing this strategy in anti-angiogenic therapy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Effects of LYTACs Recombinant Proteins on the Migration Capacity of HUVEC Cells\u003c/h2\u003e \u003cp\u003eAfter verifying the effects of recombinant LYTACs proteins on cell proliferation, we further investigated their regulatory role in HUVEC cell migration. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, this study employed wound healing assays and Transwell migration assays to evaluate two-dimensional planar migration and three-dimensional transmembrane migration potential, respectively.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea showed significantly reduced scratch closure rates (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) in ZV2-IGF2 and ZV2-EndoTag-treated groups compared to controls and ZV2-treated groups, indicating LYTACs effectively inhibit HUVEC two-dimensional lateral migration. Concurrently, Transwell assay results revealed that HUVEC cells treated with ZV2-IGF2 and ZV2-EndoTag exhibited approximately 60%-70% fewer transmembrane cells compared to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), with ZV2-EndoTag demonstrating a stronger migration inhibitory trend (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). Collectively, these data demonstrate that recombinant LYTACs proteins significantly inhibit HUVEC migration by degrading VEGFR2, providing robust evidence for further investigation of their anti-angiogenic effects. Live-cell tracking analysis revealed that the motility range of LYTACs-treated cells was markedly restricted, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee.\u003c/p\u003e \u003cp\u003eThe inhibitory effects of LYTACs on HUVEC migration were confirmed across multiple assays, demonstrating that VEGFR2 degradation impairs endothelial cell motility. Migration represents a critical step in angiogenesis, as endothelial cells must coordinately migrate toward angiogenic stimuli to form new vascular sprouts[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The reduction in both two-dimensional wound closure and three-dimensional transmembrane migration following LYTACs treatment confirms that VEGFR2 degradation disrupts this essential process. Notably, the approximately 60%-70% reduction in Transwell migration exceeds the degree of proliferation inhibition at equivalent concentrations, suggesting endothelial cell migration is particularly sensitive to VEGFR2 signaling perturbations. This heightened sensitivity likely reflects the acute dependence of directed cell movement on precise spatiotemporal regulation of receptor activity, which is more readily disrupted by receptor degradation than by simple antagonism[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Live-cell tracking further revealed restricted motility range in LYTACs-treated cells, indicating sustained VEGFR2 signaling is required for maintaining migration capacity. The stronger inhibitory effects of ZV2-EndoTag align with its superior degradation efficiency, reinforcing the link between target clearance and functional outcome. Together with proliferation and tube formation data, these results establish that VEGFR2 degradation produces multifaceted disruption of endothelial cell functions essential for angiogenesis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Effects of LYTACs Recombinant Proteins on the Angiogenesis Capacity of HUVEC Cells\u003c/h2\u003e \u003cp\u003eBuilding upon the established ability of recombinant LYTACs to inhibit HUVEC migration, we next evaluated their impact on angiogenesis using complementary \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e models. \u003cem\u003eIn vitro\u003c/em\u003e experiments employed both the Matrigel tube formation assay and the mouse aortic ring sprouting model to simulate early-stage tubulogenesis and sprouting from mature vessels, respectively[\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Quantitative analysis revealed that HUVEC cells treated with ZV2-IGF2 and ZV2-EndoTag exhibited significantly reduced tubular network formation on Matrigel. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb, compared to the control group, total tube length was reduced by approximately 42% in both LYTAC-treated groups, representing a 14% decrease relative to the ZV2 control group. Additionally, both the length and number of microvascular sprouts in mouse aortic rings were markedly suppressed (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea-d). Quantification of sprouting area showed that ZV2-EndoTag treatment reduced sprouting by approximately 92% compared to controls, while ZV2-IGF2 achieved an 89% reduction, confirming that both LYTACs effectively disrupt critical steps in angiogenesis.\u003c/p\u003e \u003cp\u003eTo validate the anti-angiogenic efficacy of LYTACs \u003cem\u003ein vivo\u003c/em\u003e, we employed the mouse Matrigel plug angiogenesis model. Matrigel plugs pre-mixed with different treatment proteins were subcutaneously implanted into C57BL/6 mice and retrieved after 7 days for histological analysis. Consistent with the \u003cem\u003ein vitro\u003c/em\u003e findings, assessment via H\u0026amp;E staining and immunohistochemical analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee-f) revealed that microvascular density in the ZV2-IGF2 and ZV2-EndoTag-treated groups was significantly lower than that in the control group, with CD31-positive area quantification (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eg) further corroborating the reduction in neovascularization. These results demonstrate that recombinant LYTACs can effectively inhibit angiogenesis \u003cem\u003ein vivo\u003c/em\u003e, consistent with our \u003cem\u003ein vitro\u003c/em\u003e observations. The consistent superiority of ZV2-EndoTag across these models further aligns with its higher VEGFR2 degradation efficiency, reinforcing the relationship between target clearance and functional anti-angiogenic outcomes.\u003c/p\u003e \u003cp\u003eCollectively, the anti-angiogenic effects of LYTACs were robustly validated across complementary \u003cem\u003eex vivo\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e models. The Matrigel tube formation and aortic ring assays collectively demonstrated that VEGFR2 degradation disrupts both the initial assembly of endothelial cells into capillary-like structures and the sprouting capacity of mature vessels. Notably, the approximately 42% reduction in tube length and the near-complete inhibition of aortic ring sprouting (89\u0026ndash;92%) indicate that angiogenesis is highly dependent on sustained VEGFR2 signaling, and its depletion produces profound functional consequences. The slightly superior efficacy of ZV2-EndoTag in suppressing sprouting aligns with its higher VEGFR2 degradation efficiency, further supporting the mechanistic link between target clearance and functional outcome. Importantly, the \u003cem\u003ein vivo\u003c/em\u003e Matrigel plug assay confirmed that these effects translate to a physiologically relevant setting, where LYTACs treatment significantly reduced neovascularization. Taken together with the earlier observations of impaired endothelial cell proliferation and migration, these findings establish that targeted degradation of VEGFR2 produces a multifaceted blockade of angiogenesis, positioning this LYTAC-based strategy as a promising therapeutic approach for diseases driven by pathological angiogenesis. The consistent superiority of ZV2-EndoTag across these models further reinforces the relationship between target clearance and functional anti-angiogenic outcomes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.8 LYTAC-Mediated VEGFR2 Degradation Suppresses Tumor Growth and Angiogenesis\u003c/h2\u003e \u003cp\u003eTo evaluate the \u003cem\u003ein vivo\u003c/em\u003e antitumor efficacy of LYTAC-mediated VEGFR2 degradation, we established a subcutaneous 4T1 murine breast cancer model in BALB/c mice. Following tumor establishment, mice were randomly assigned to treatment groups and received five intratumoral injections of PBS, ZV2, ZV2-IGF2, or ZV2-EndoTag every other day, and were then maintained until day 20. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ec and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ed, both ZV2-IGF2 and ZV2-EndoTag treatment groups exhibited significantly reduced tumor volume and weight compared to the PBS control group, with inhibition effects markedly superior to the ZV2 monotherapy group. Quantitative analysis revealed that tumor volumes in the ZV2-IGF2 and ZV2-EndoTag groups were reduced by approximately 42% and 47%, respectively, relative to the control group, demonstrating that both LYTACs effectively suppress tumor growth \u003cem\u003ein vivo\u003c/em\u003e. Body weight monitoring throughout the treatment period revealed no significant differences among groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb), indicating minimal systemic toxicity. Histological analysis of tumor sections by H\u0026amp;E staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ee) and immunofluorescence staining for CD31 and VEGFR2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ef) revealed substantial reductions in both VEGFR2 expression and microvascular density in tumors treated with ZV2-IGF2 and ZV2-EndoTag. Specifically, quantitative analysis of VEGFR2/CD31 dual staining showed that the ratio of VEGFR2-positive area normalized to CD31-positive area decreased by approximately 38% in both treatment groups compared to controls, indicating effective reduction of VEGFR2 signaling within the tumor vasculature. These results demonstrate that recombinant LYTACs achieve effective target protein degradation and angiogenesis inhibition \u003cem\u003ein vivo\u003c/em\u003e, leading to suppressed tumor growth.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003ein vivo\u003c/em\u003e antitumor efficacy of our LYTACs validates the therapeutic potential of VEGFR2 degradation in a clinically relevant model. The significant tumor growth suppression achieved by both ZV2-IGF2 and ZV2-EndoTag, coupled with the absence of overt systemic toxicity, supports the safety and efficacy of this targeted degradation approach. Notably, the superior antitumor activity of ZV2-EndoTag compared to ZV2-IGF2 aligns with its higher \u003cem\u003ein vitro\u003c/em\u003e degradation efficiency and stronger suppression of endothelial cell functions, reinforcing the relationship between target clearance and therapeutic outcome. Immunofluorescence analysis provided critical mechanistic insight by confirming that reduced tumor growth correlates directly with decreased intratumoral VEGFR2 expression and microvascular density[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. This establishes that the observed antitumor effects arise from on-target degradation of VEGFR2 and subsequent inhibition of tumor angiogenesis, rather than off-target effects. Compared to conventional VEGFR2 inhibitors that merely block receptor signaling, our LYTAC strategy achieves physical elimination of the target protein, potentially offering more sustained effects and reduced opportunity for compensatory signaling. These findings provide proof-of-concept for LYTAC-based membrane protein degradation as a viable antitumor strategy and support further development of this approach for cancer therapy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4 Conclusions","content":"\u003cp\u003eTo overcome the limitations of conventional VEGFR2 inhibitors, this study successfully designed and constructed two novel LYTACs, ZV2-IGF2 and ZV2-EndoTag, based on distinct lysosomal targeting mechanisms. These recombinant proteins achieve specific targeting and efficient degradation of VEGFR2 by binding to different IGF2R ligands via a high-affinity VEGFR2 affibody. Mechanistically, both LYTACs induce lysosomal degradation of VEGFR2, thereby effectively blocking downstream signaling pathways and inhibiting the expression and function of angiogenesis-related genes. Through systematic evaluation, we confirmed that both LYTAC molecules exhibit significant biological effects at the cellular and animal levels. \u003cem\u003eIn vitro\u003c/em\u003e experiments demonstrated that ZV2-IGF2 and ZV2-EndoTag effectively reduced VEGFR2 expression on human umbilical vein endothelial cells (HUVECs) and significantly inhibited cell proliferation, migration, and angiogenesis. In animal models, LYTACs treatment markedly suppressed the growth and progression of mouse xenograft tumors, with histological analysis confirming reduced vascular density within the tumors. Notably, the engineered EndoTag module exhibited superior degradation efficiency and functional outcomes compared to the natural IGF2 ligand, highlighting the potential of rationally designed targeting modules in LYTAC development.\u003c/p\u003e \u003cp\u003eThese findings not only validate the efficacy of LYTAC technology in targeting membrane protein degradation but also provide crucial experimental evidence for developing novel anti-angiogenic therapeutic approaches that circumvent the drug resistance commonly associated with traditional inhibitors. Together, these results establish LYTAC-mediated VEGFR2 degradation as a viable therapeutic strategy that addresses the limitations of current anti-angiogenic agents by enabling sustained target elimination. The successful application of both natural and engineered lysosomal targeting modules further provides a versatile platform for developing next-generation LYTACs against a broad range of membrane proteins, offering new avenues for treating cancer and other angiogenesis-related diseases[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by a grant from the Shandong Provincial Natural Science Foundation (ZR2021MC017, ZR2021LSW001, ZR2024QB069).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by a grant from the Shandong Provincial Natural Science Foundation (ZR2021MC017, ZR2021LSW001, ZR2024QB069).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal procedures were approved by The Special Committee of Scientific Research Ethic of Liaocheng University (Approval No. AP2026031535, date: March 15, 2026) and were performed in accordance with the National Institute Guide for the Care and Use of Laboratory Animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study is not a clinical trial. Therefore, clinical trial registration is not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConsent to participate is not applicable because this study did not involve human subjects.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConsent to publish is not applicable as no individual person\u0026rsquo;s data or images are presented in this manuscrip.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request. The uncropped original images of Western blots and gels have been provided as Supplementary Information.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eR. Wang, Y. Ren, D. Jia, J. Li, and X. Chu designed the research; R. Wang, Y. Ren, M. Liu, Y. Lou, and G. Li performed the experiments; R. Wang, Y. Ren, M. Liu, and Y. Lou analyzed the data; D. Jia, J. Li, and X. Chu drafted the manuscript. All authors have reviewed and approved the final version of the manuscript for publication.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eYe B, Lei H, Jing X, Ding Q, Yao S, Wang H, Meng C, Guo X, Wu B, Wu Y, Sun T. Vascular Environment-Responsive DNA Nanoswitch Controls the Positive Feedback System for Spatiotemporal Coupling of Angiogenesis and Osteogenesis. 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Mol Divers. 2026;30(1):571\u0026ndash;93. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11030-025-11185-8\u003c/span\u003e\u003cspan address=\"10.1007/s11030-025-11185-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\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":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"discover-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"dion","sideBox":"Learn more about [Discover Oncology](https://www.springer.com/12672)","snPcode":"","submissionUrl":"","title":"Discover Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Angiogenesis, Tumour Angiogenesis, LYTACs","lastPublishedDoi":"10.21203/rs.3.rs-9310166/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9310166/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eVascular endothelial growth factor receptor 2 (VEGFR2) is a key regulator of tumor angiogenesis and an important therapeutic target. Current VEGFR2-targeted strategies rely on monoclonal antibodies or tyrosine kinase inhibitors, which often face limitations including acquired resistance and off-target toxicity. Here, we developed two lysosome-targeting chimeras (LYTACs), ZV2-IGF2 and ZV2-EndoTag, designed to degrade cell surface VEGFR2 on endothelial cells. Both molecules were constructed by fusing a high-affinity VEGFR2 affibody with either the IGF2 domain or the engineered EndoTag module as lysosomal targeting ligands. Following prokaryotic expression and purification, both LYTACs were efficiently internalized into HUVECs and colocalized with lysosomes. Western blot and flow cytometry confirmed concentration-dependent VEGFR2 degradation, with ZV2-EndoTag exhibiting approximately 13% higher efficiency than ZV2-IGF2. Mechanistically, VEGFR2 degradation suppressed downstream p-AKT and p-MEK signaling. Functional assays revealed that both LYTACs inhibited HUVEC proliferation (\u0026sim;46% at 500 nM), migration (60\u0026ndash;70% reduction), and tube formation (42% reduction). Aortic ring sprouting was inhibited by 89\u0026ndash;92%, and Matrigel plug assays confirmed anti-angiogenic activity \u003cem\u003ein vivo\u003c/em\u003e. In a 4T1 murine breast cancer model, intratumoral administration of ZV2-IGF2 or ZV2-EndoTag significantly suppressed tumor growth, with tumor volumes reduced by 42% and 47%, respectively. Immunofluorescence analysis revealed that the VEGFR2/CD31 ratio within tumor tissues decreased by approximately 38% in both treatment groups, confirming effective target degradation and reduced microvascular density. Together, these results establish LYTAC-mediated VEGFR2 degradation as a promising anti-angiogenic strategy and provide proof-of-concept for targeting membrane proteins via lysosomal degradation pathways.\u003c/p\u003e","manuscriptTitle":"Targeted Degradation of VEGFR2 by LYTACs Suppresses Angiogenesis and Tumor Growth","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-04 09:55:58","doi":"10.21203/rs.3.rs-9310166/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"9279545119960304987515495859506237947","date":"2026-05-13T00:11:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"36714361574735102993168419354234681029","date":"2026-05-12T04:41:55+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-06T07:46:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"87145716924542376109768650201404883127","date":"2026-04-27T01:24:18+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-23T10:50:23+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-04-21T17:39:52+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-18T08:35:19+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-15T07:28:04+00:00","index":"","fulltext":""},{"type":"submitted","content":"Discover Oncology","date":"2026-04-15T06:55:02+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"discover-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"dion","sideBox":"Learn more about [Discover Oncology](https://www.springer.com/12672)","snPcode":"","submissionUrl":"","title":"Discover Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6948e2d2-5216-40c2-aa8a-6566f7654bd8","owner":[],"postedDate":"May 4th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"9279545119960304987515495859506237947","date":"2026-05-13T00:11:56+00:00","index":53,"fulltext":""},{"type":"reviewerAgreed","content":"36714361574735102993168419354234681029","date":"2026-05-12T04:41:55+00:00","index":51,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-06T07:46:40+00:00","index":39,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-04T09:55:58+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-04 09:55:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9310166","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9310166","identity":"rs-9310166","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}