Inhibition of SCUBE2, a novel VEGFR2 co-receptor, inhibits tumor angiogenesis

Abstract

This invention discloses the inhibition of SCUBE2, a novel VEGFR2 co-receptor, to suppress tumor angiogenesis. Specifically, this application relates to isolated anti-SCUBE2 antibodies or binding fragments thereof (protein 2 containing the signal peptide-complement protein C1r / C1s, Uegf, and Bmp1(CUB)-epidermal growth factor (EGF) domain). The anti-SCUBE2 antibody comprises an antigen-binding region specifically binding to the target domain located within SCUBE2 (SEQ ID NO: 66) and exhibits the property of inhibiting vascular endothelial growth factor (VEGF)-induced angiogenesis. The anti-SCUBE2 antibody or its binding fragment can be used in individuals with this need to treat diseases associated with VEGF-induced angiogenesis, or to treat tumors or inhibit tumor angiogenesis and cancer cell growth.

Description

[0001] This application is a divisional application of patent application No. 201780023573.8 (International Application No.: PCT / US2017 / 026855), filed on April 10, 2017, entitled "Inhibiting SCUBE2, a novel VEGFR2 co-receptor, to inhibit tumor angiogenesis". Technical Field

[0002] This invention relates primarily to anti-angiogenic therapy, and more specifically, to an anti-SCUBE2 antibody for use in anti-angiogenic therapy. Background Technology

[0003] Vascular endothelial growth factor (VEGF) is a major regulator of angiogenesis; it binds to its receptor VEGFR2 (receptor tyrosine kinase, RTK) on the surface of endothelial cells (ECs) and triggers dimerization and transphosphorylation to activate a signaling cascade amplification, including p44 / 42 mitogen-activated protein kinase (MAPK) and AKT, which are required for EC migration, proliferation, and tubulogenesis. These VEGF responses can be further promoted by a few VEGFR2 co-receptors (such as neurofeedin, heparin sulfate proteoglycans, CD44, and CD146). However, other potential VEGFR2 co-receptors regulating VEGF-induced angiogenesis remain to be discovered.

[0004] SCUBE2 is the second member of a small, evolutionarily conserved gene family initially composed of three distinct genes (SCUBE1, 2, and 3) identified from human endocrine disruptors (ECs). This gene encodes a ~1,000 amino acid polypeptide, structured as a modular protein with five protein regions: an NH2-terminal signal peptide, nine consecutive EGF-like motifs, a spacer region, three cysteine-rich (CR) repeat regions, and a CUB region at the COOH terminus. These SCUBE proteins bind to the cell surface via their spacer and CR repeat regions through two distinct membrane anchoring mechanisms (i.e., electrostatic and lectin-glycan interactions) as peripheral membrane proteins and co-receptors for various growth factors. In addition to expression in normal endothelium, SCUBE2 is also highly expressed in hypoxic tumor microvessels. However, whether SCUBE2 can act as a co-receptor for VEGFR2 and its role in VEGF-induced angiogenesis remains to be investigated. Summary of the Invention

[0005] In one aspect, the present invention relates to an isolated anti-SCUBE2 antibody or a binding fragment thereof, comprising an antigen-binding region specifically bound to a target domain located within SCUBE2 (SEQ ID NO: 66), and exhibiting properties that inhibit VEGF-induced angiogenesis. The target domain is selected from the group consisting of: EGF-like motifs 4 to 6 in the range of aa positions 175 to 323 of SCUBE2 (SEQ ID NO: 66), or a spacer region in the range of aa positions 441 to 659, or a first cys-rich motif in the range of aa positions 668 to 725.

[0006] In one embodiment, the isolated anti-SCUBE2 antibody or its binding fragment of the present invention exhibits properties that inhibit tumor angiogenesis and VEGF-induced endothelial cell proliferation and capillary formation.

[0007] In another embodiment, the isolated anti-SCUBE2 antibody or its binding fragment of the present invention comprises a heavy chain variable domain (V... H ) and light chain variable domain (V L ),in:

[0008] (a) V H Contains the amino acid sequence of SEQ ID NO: 17, and V L Containing the amino acid sequence of SEQ ID NO: 18; or

[0009] (b) V H Contains the amino acid sequence of SEQ ID NO: 1, and V L Contains the amino acid sequence of SEQ ID NO: 2; or

[0010] (c) V H Contains the amino acid sequence of SEQ ID NO: 33, and V L The amino acid sequence containing SEQ ID NO: 34; or

[0011] (d) V H Contains the amino acid sequence of SEQ ID NO: 49, and V L The amino acid sequence containing SEQ ID NO: 50.

[0012] In another embodiment, the V of the anti-SCUBE2 antibody of the present invention H and V L Each contains the following complementary determinant regions (CDRs): CDR1, CDR2, and CDR3:

[0013] (i) V H The CDR1 contains the amino acid sequence of SEQ ID NO: 19, the CDR2 contains the amino acid sequence of SEQ ID NO: 20, and the CDR3 contains the amino acid sequence of SEQ ID NO: 21; and V L The CDR1 containing the amino acid sequence of SEQ ID NO: 22, the CDR2 containing the amino acid sequence of SEQ ID NO: 23, and the CDR3 containing the amino acid sequence of SEQ ID NO: 24; or

[0014] (ii) V H The CDR1 contains the amino acid sequence of SEQ ID NO: 3, the CDR2 contains the amino acid sequence of SEQ ID NO: 4, and the CDR3 contains the amino acid sequence of SEQ ID NO: 5; and V L The CDR1 containing the amino acid sequence of SEQ ID NO: 6, the CDR2 containing the amino acid sequence of SEQ ID NO: 7, and the CDR3 containing the amino acid sequence of SEQ ID NO: 8; or

[0015] (iii) V H The CDR1 contains the amino acid sequence of SEQ ID NO: 35, the CDR2 contains the amino acid sequence of SEQ ID NO: 36, and the CDR3 contains the amino acid sequence of SEQ ID NO: 37; and V L The CDR1 containing the amino acid sequence of SEQ ID NO: 38, the CDR2 containing the amino acid sequence of SEQ ID NO: 39, and the CDR3 containing the amino acid sequence of SEQ ID NO: 40; or

[0016] (iv) V H The CDR1 contains the amino acid sequence of SEQ ID NO: 51, the CDR2 contains the amino acid sequence of SEQ ID NO: 52, and the CDR3 contains the amino acid sequence of SEQ ID NO: 53; and V L The CDR1 contains the amino acid sequence of SEQ ID NO: 54, the CDR2 contains the amino acid sequence of SEQ ID NO: 55, and the CDR3 contains the amino acid sequence of SEQ ID NO: 56.

[0017] In another embodiment, the isolated anti-SCUBE2 antibody or its binding fragment of the present invention is a monoclonal antibody, is humanized, is a fusion protein, or is selected from the group consisting of: Fv fragments, fragment antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fd' fragments, and Fd fragments and single-chain antibody variable fragments (scFv).

[0018] In another embodiment, the isolated anti-SCUBE2 antibody or binding fragment of the present invention is encapsulated in liposomes.

[0019] In another aspect, the present invention relates to a pharmaceutical composition comprising: (i) a therapeutically effective amount of the isolated anti-SCUBE2 antibody of the present invention or a binding fragment thereof; and (ii) a therapeutically effective amount of a VEGF antibody specifically against VEGF.

[0020] In one embodiment, the V of the anti-SCUBE2 antibody in the pharmaceutical composition H and V L It contains the following amino acid sequence: (i) V H Contains the amino acid sequence of SEQ ID NO: 17, and V L The amino acid sequence containing SEQ ID NO: 18, or (ii) V H The CDR1 contains the amino acid sequence of SEQ ID NO: 19, the CDR2 contains the amino acid sequence of SEQ ID NO: 20, and the CDR3 contains the amino acid sequence of SEQ ID NO: 21; and V L The CDR1 contains the amino acid sequence of SEQ ID NO: 22, the CDR2 contains the amino acid sequence of SEQ ID NO: 23, and the CDR3 contains the amino acid sequence of SEQ ID NO: 24.

[0021] In another embodiment, the anti-SCUBE2 antibody in the pharmaceutical composition may be a monoclonal antibody, a chimeric antibody, or a humanized antibody.

[0022] In another embodiment, the isolated anti-SCUBE2 antibody or its binding fragment of the present invention has specific binding affinity for the cross-reactive epitopes shared by SCUBE2 expressed on human vascular endothelial cells or SCUBE2 expressed on mouse vascular endothelial cells.

[0023] In another aspect, the present invention relates to the use of isolated anti-SCUBE2 antibodies or their binding fragments in the manufacture of a medicament for treating VEGF-induced angiogenesis-related diseases in individuals with this need. In one embodiment, the VEGF-induced angiogenesis-related diseases are selected from at least one of the following groups: tumors, neovascular ocular diseases, persistent proliferative vitreous syndrome, diabetic retinopathy, retinopathy of preterm birth, choroidal neovascularization, endometriosis, uterine bleeding, ovarian cysts, and ovarian hyperstimulation. In another embodiment, the use of the present invention combined with the use of VEGF antibodies specifically anti-VEGF is used to manufacture a medicament for treating VEGF-induced angiogenesis-related diseases in individuals with this need.

[0024] In another aspect, the present invention relates to the use of isolated anti-SCUBE2 antibodies or their binding fragments in the manufacture of medicaments for treating tumors or inhibiting tumor angiogenesis and cancer cell growth in individuals in need. Alternatively, the present invention relates to isolated anti-SCUBE2 antibodies or their binding fragments for treating diseases associated with VEGF-induced angiogenesis in individuals in need, or for treating tumors or inhibiting tumor angiogenesis and cancer cell growth. The anti-SCUBE2 antibodies or their binding fragments of the present invention can be used in combination with anti-VEGF antibodies (such as bevacizumab) for treating diseases associated with VEGF-induced angiogenesis in individuals in need, or for treating tumors or inhibiting tumor angiogenesis and cancer cell growth.

[0025] This invention also relates to methods for treating diseases associated with VEGF-induced angiogenesis in individuals with this need, or for treating tumors or inhibiting tumor angiogenesis and cancer cell growth. The method comprises administering to the individual with this need a therapeutically effective amount of the anti-SCUBE2 antibody of the present invention or a binding fragment thereof. The method may further comprise administering to the individual with this need a therapeutically effective amount of the anti-VEGF antibody.

[0026] These and other aspects will become apparent from the following description of preferred embodiments in conjunction with the accompanying drawings, but variations and modifications may be made without departing from the spirit and scope of the novel concept of this disclosure.

[0027] The accompanying drawings illustrate one or more embodiments of the invention and, together with the written description, explain the principles of the invention. Wherever possible, the same reference numerals will be used throughout the drawings to refer to the same or similar components of the embodiments. Attached Figure Description

[0028] Figure 1This study demonstrates SCUBE2 expression on the cell surface of human umbilical vein endothelial cells (HUVECs) and its regulation of VEGF-induced HUVEC proliferation and lumen formation. A and B: SCUBE2 immunofluorescence staining (green) and flow cytometry measurements. Arrows indicate SCUBE2 expression on the EC membrane. Cell nuclei are stained blue with DAPI. C: Western blot analysis of SCUBE2 protein expression in subconfluent (proliferating) and confluent (non-proliferating) HUVECs. D to F: SCUBE2 overexpression enhances VEGF-induced HUVEC proliferation and lumen formation. Exogenous SCUBE2 expression in HUVECs transduced with empty lentivirus or recombinant lentivirus encoding FLAG-labeled SCUBE2 (D). Effect of SCUBE2 overexpression on VEGF-induced HUVEC cell growth (E). Data are mean ± SD from three independent experiments and represent percentage increases relative to unstimulated cells. **, P <0.01. In vitro MATRIGEL™ angiogenesis analysis (F) of the effect of SCUBE2 overexpression on VEGF-induced lumen formation. Representative images are shown above and quantified by counting the total number of lumens in each region. Data are mean ± SD, ** calculated from 3 independent experiments. P <0.01. GI, SCUBE2 gene knockdown reduced VEGF-induced HUVEC proliferation and lumen formation. SCUBE2 expression was downregulated in HUVECs by two independent SCUBE2-targeting shRNA lentiviruses (SCUBE1-shRNA #1 and #2). Luciferase shRNA lentivirus served as a negative control (control shRNA). Effect of SCUBE2 gene knockdown on VEGF-induced (H) HUVEC cell growth and (I) lumen formation. Data are mean ± SD from three independent experiments and represent percentage increases relative to unstimulated cells. **, P <0.01. In vitro MATRIGEL™ angiogenesis analysis of the effect of SCUBE2 gene knockdown on VEGF-induced lumen formation. Representative images are shown above and quantified by counting the total number of lumens in each region (I). Data are mean ± SD,** calculated from 3 independent experiments. P <0.01.

[0029] Figure 2Demonstrates hypoxia-mediated HIF-1α-induced upregulation of SCUBE2 in HUVECs. A and B show that SCUBE2 expression is upregulated in HUVECs in response to hypoxia. HUVECs were cultured for 12 hours under normoxic or hypoxic (1%) conditions. SCUBE2 mRNA and protein expression levels were validated by quantitative RT-PCR (A) and Western blot analysis (B). Data are mean ± SD from three independent experiments. **, compared to normoxic conditions. P <0.01. C, schematic diagram of the SCUBE2 promoter reporter gene construct containing natural (WT) or mutant (M1, M2 and M3) HIF-1α binding sites. SCUBE2 The location of the gene promoter (-1500 to +1) is indicated by a horizontal line. HIF-1α binding sites are marked with diamonds, and mutation sites are marked with "×". Primers and amplicon regions used for ChIP analysis are also marked in the figure (top). D, HIF-1α transactivates the luciferase activity driven by the SCUBE2 promoter. Transfected into HUVECs SCUBE2 Quantitative plots of the activity of WT or mutant promoter constructs. Luciferase activity was normalized to Renilla luciferase activity (pRL-TK) to control transfection efficiency. Under hypoxic conditions, HIF-1α directly binds to the SCUBE2 promoter. In vivo binding of HIF-1α protein to HUVECs was analyzed by PCR using specific primers for amplifying the 238-bp fragment. SCUBE2 ChIP analysis of the promoter. Cell lysate deficiency (-) served as a negative control. Data are mean ± SD of three independent experiments. P <0.01.

[0030] Figure 3 exhibit Scube2 Attenuation of adult angiogenesis in EC-KO mice. A to D, MATRIGEL™ embedding analysis. Representative images (A) of MATRIGEL™ embeddings containing saline (-) or VEGF (+) removed from control and EC-KO mice 7 days after injection. Heme content (B) of MATRIGEL™ embeddings supplemented with saline (-) and VEGF (+) from control and EC-KO mice. Data are mean ± SD (n = 5). P <0.01. Anti-CD31 staining (C) of MATRIGEL™ inlay sections removed from control and EC-KO mice containing saline (-) or VEGF (+). Scale bar = 40 μm. Angiogenesis of MATRIGEL™ inlays was determined by IMAGEJ™ quantitative anti-CD31 positivity (D). Data are mean ± SD (n = 5). ** P<0.01. E to H, hindlimb ischemia-induced angiogenesis. Representative laser Doppler images (A) and quantifications (B) of hindlimb blood flow before and after right femoral artery ligation in mice. Representative images (C) and quantifications (D) of anti-CD31 immunostaining of lower leg vessels 21 days after femoral artery ligation in control and EC-KO mice. Scale bar = 100 µm. Data are mean ± SD (n = 6). P <0.01, compared to the control.

[0031] Figure 4 This study demonstrates how SCUBE2 modulates VEGFR2 phosphorylation and downstream signaling in HUVECs. A and B show that SCUBE2 knockdown reduces VEGF signaling in HUVECs. Western blot analysis (A) and quantification (B) of VEGF signaling in HUVECs with VEGF-induced phosphorylation at Tyr1059 (VEGFR2), Thr202 / Tyr204 (p44 / 42 mitogen-activated protein kinase (MAPK)), and Ser473 (AKT) in both control and SCUBE2 shRNA knockdown HUVECs. Data are presented as mean ± SD from three independent experiments. * P <0.05;** P <0.01, compared to control. C and D, Western blot analysis (C) and quantification (D) of VEGF signaling in ECs with VEGF-induced phosphorylation of VEGFR2 Tyr1059, p44 / 42 MAPK Thr202 / Tyr204, and AKT Ser473 in control and SCUBE2-overexpressing ECs. Data are expressed as mean ± SD from three independent experiments. * P <0.05;** P <0.01, compared to the carrier.

[0032] Figure 5 Demonstrates the VEGF response and signal transduction attenuation in the lung ECs (MLECs) of EC-KO mice. A and B, VEGF-induced lumen formation (A) and proliferation (B) in EC-KO MLECs. Data are mean ± SD from three independent experiments. P <0.01, compared to the control group. C and D, Western blot analysis (C) and quantification (D) of VEGF signaling in MLECs with VEGF-induced phosphorylation of VEGFR2 Tyr1059, p44 / 42MAPK Thr202 / Tyr204, and AKT Ser473, in the control group and EC-KO MLECs. Data are mean ± SD of three independent experiments. * P <0.05;**P <0.01, compared to the control group. E, Micrograph showing the vascular response of collagen-embedded thoracic aortic rings from control and EC-KO mice after 5 days of culture. Scale bar = 1 mm. F, Quantification of the number and length of microvessel sprouts. The vascular response of individual aortic rings was determined by quantifying the number of growing microvessels (left) and by measuring the total length occupied by newly generated microvessels (right).

[0033] Figure 6 This image shows the high expression of SCUBE2 in tumor endothelial cells (ECs). Immunohistochemical staining results of SCUBE2-enriched EC expression (brown as indicated by red arrows) in mouse (mammary tumor, lung, melanoma) (A) and human (prostate, sarcoma, bladder) tumors (B). Spontaneous mouse mammary tumors derived from lung metastases were obtained using a transgenic mouse mammary tumor virus polyoma middle T (MMTV-PyMT) animal model. Two xenograft tumors were generated by subcutaneous injection of syngeneic melanoma (B16F10) or Lewis lung carcinoma (LLC) cells. Endothelial cell immunoreactivity appears red (not brown) in melanoma tumors due to the melanin background of the melanoma cells. Human normal and tumor tissues were purchased from commercially available tissue microarray sections.

[0034] Figure 7 Demonstrating the absence of endothelial cells Scube2 Inhibition of tumor angiogenesis and tumor growth in EC-KO mice. A to D: Subcutaneous injection of B16F10 melanoma cells or Lewis lung cancer (LLC) tumor cells into control and EC-KO mice. Representative photographs of B16F10 (A) and LLC (B) tumors at 16 days post-implantation and tumor growth rates measured at specified time points (C and D). Scale bar = 10 mm. E to H: Reduced tumor microvascular distribution in EC-KO mice. Anti-CD31 staining of tumor sections shows a reduced number of ECs and vascular structures in EC-KO mice (E and F). Quantitative mapping of tumor angiogenesis at 16 days post-implantation (G and H). Scale bar = 40 µm. Data are mean ± SD (n = 6). P <0.01.

[0035] Figure 8This image illustrates the oxygen and nutrient deficiency in EC-KO tumors, leading to apoptosis. H&E staining (A), TUNEL assay (C), and Ki-67 staining (E) are shown in B16F10 or LLC tumor sections from control or EC-KO mice. Quantitative graphs of tumor necrosis (B), tumor cell apoptosis (D), and proliferation (F) are presented. Scale bar = 100 µm. Data are mean ± SD (n = 6). P <0.01.

[0036] Figure 9 Demonstrating the absence of endothelial cells Scube2 Inhibition of tumor angiogenesis and tumor growth in EC-KO mice. A and B: Subcutaneous injection of MLTC testicular interstitial tumor cells in control and EC-KO mice. Representative photographs of MLTC testicular interstitial tumors at 60 days post-implantation (A) and tumor growth rates measured at specified time points (B). Scale bar = 10 mm. C and D: Anti-CD31 staining of tumor sections showing a reduction in the number of ECs and vascular structures in EC-KO mice (C). Quantitative map of tumor angiogenesis 16 days after tumor cell implantation (D). Scale bar = 40 µm. Data are mean ± SD (n = 6). P <0.01.

[0037] Figure 10 Demonstration of endothelial cell knockout Scube2 Effects on retinal vascular development outcomes. A, Whole specimens from control and EC-KO juvenile mice, embedded P5 retina stained with pan-endothelial marker CD31. White dashed circles represent angiogenesis in the control group (left), and orange dashed circles represent angiogenesis in the EC-KO group (right). B, High-magnification (100×) view of angiogenesis. Yellow dots mark filopodia. C and D, Quantification of retinal angiogenesis by measuring the radial distance from the optic nerve (C) and the number of filopodia (D) (percentage relative to control). Data are mean ± SD (n = 5 for each group). P <0.01.

[0038] Figure 11This diagram illustrates how the absence of SCUBE2 in endothelial cells reduces oxygen-induced retinopathy (OIR). A, Schematic diagram of the OIR animal model. Newborn mice were exposed to 75% oxygen from day 7 (P) to day 12 after birth, and returned to room air from day 12 to day 18, with pathological angiogenesis observed at day 18. B and C, Total RNA (n = 3) was isolated from the retinas of OIR-exposed or age-matched normoxic control mice at day 18, and the mRNA expression levels of angiogenesis marker genes (B) or the SCUBE gene family (C) were subsequently analyzed by Q-PCR or RT-PCR. D, Control group after OIR exposure. or EC-KO Whole specimen embedding analysis of retinal vessels in mice. The size of the central avascular or neovascularized region at p18 (6 days after 75% oxygen removal) is indicated in red or blue, respectively. E and F, compared to controls undergoing the OIR pattern (n = 5), EC-KO In mice (n = 5), the size (as a percentage) of the neovascularized region (E) or the central avascular region (F) relative to the entire retina was significantly reduced. P <0.01.

[0039] Figure 12 Demonstrating the anti-SCUBE2 mAb and its anti-angiogenic effects. A, Anti-SCUBE2 mAb clones and their target domains. B, Evaluation of the anti-SCUBE2 mAb's ability to inhibit angiogenesis by in vitro lumenogenesis analysis. A summary of anti-SCUBE2 mAbs with specific and anti-angiogenic effects. h, Human; m, Mouse; z, Zebrafish.

[0040] Figure 13 This study demonstrates the inhibition of VEGF-induced responses and signaling in HUVECs by anti-SCUBE2 mAb SP.B1. A and B show the properties of anti-SCUBE2 mAb SP.B1. SP.B1 mAb can identify human and mouse SCUBE2 (non-1 and 3) proteins expressed in HEK-293T cells (A) and SCUBE2 expression on the surface of HUVEC cells (B) using Western blotting and flow cytometry. CI shows that SP.B1 mAb blocks VEGF-stimulated cellular responses and VEGF-induced VEGFR2 phosphorylation and MAPK / Akt activation. The addition of SP.B1, rather than control IgG, inhibits VEGF-induced HUVEC proliferation (C) and angiogenesis (D and E) in a dose-dependent manner. F shows VEGF-induced phosphorylation and total VEGFR2, MAPK, and Akt in HUVECs in the presence of SP.B1 or control IgG, and a quantification plot (GI). Data are mean ± SD (GI) of three independent experiments. P <0.01.

[0041] Figure 14 Demonstrates the additive antitumor effect of combining anti-SCUBE2 mAb SP.B1 and anti-VEGF bevacizumab (AVASTIN®) on lung cancer tumor growth and angiogenesis. A, Mean tumor volume measured at time points in each treatment group after injection of LLC lung cancer cells (n = 6). Arrows indicate antibody treatment time points. B, Representative LLC lung cancer tumors in xenograft mice for each treatment. Scale bar = 1 cm. C, Resected and weighed tumors (n = 6 / group). (D and E) Tumor microvessel density. D and E, Representative anti-CD31 immunostaining (D) and quantified tumor angiogenesis (E) of lung cancer tumor sections quantified by IMAGEJ™. Scale bar = 40 µm. * P <0.05;** P <0.01.

[0042] Figure 15 This study demonstrates the additive antitumor effect of combining anti-SCUBE2 mAb SP.B1 and anti-VEGF bevacizumab (AVASTIN®) on the growth and angiogenesis of pancreatic ductal carcinoma. A, Mean tumor volume (n = 6) measured at time points after injection of PANC-1 pancreatic ductal tumor cells in each treatment group. Arrows indicate antibody treatment time points. B, Representative PANC-1 pancreatic carcinomas from xenograft mice in each treatment. Scale bar = 1 cm. C, Resected and weighed tumors (n = 6 / group). (D and E) Microvessel density of the tumor. D and E, Representative anti-CD31 immunostaining (D) and quantified tumor angiogenesis (E) of pancreatic ductal carcinoma sections quantified by IMAGEJ™. Scale bar = 40 µm. * P <0.05;** P <0.01.

[0043] Figure 16 illustrates the additive antitumor effect of combined anti-SCUBE2 mAb SP.B1 and anti-VEGF bevacizumab treatment on colorectal adenocarcinoma growth and angiogenesis. A, Mean tumor volume (n = 6) measured at time points after injection of LS 174T colon cancer cells in each treatment group. Arrows indicate antibody treatment time points. B, Representative LS 174T colorectal adenocarcinomas in xenograft mice for each treatment. Scale bar = 1 cm. C, Resected and weighed tumors (n = 6 / group). (D and E) Microvessel density of the tumor. D and E, Representative anti-CD31 immunostaining (D) and quantified tumor angiogenesis (E) of colorectal adenocarcinoma sections quantified by IMAGEJ™. Scale bar = 40 µm. * P <0.05;** P <0.01. Detailed Implementation

[0044] The invention is described more specifically in the following examples, which are intended for illustrative purposes only, as many modifications and variations will be apparent to those skilled in the art. Various embodiments of the invention are now described in detail. Referring to the accompanying drawings, similar numbers indicate similar components throughout the drawings. As used in this specification and throughout the claims, unless the context clearly indicates otherwise, the terms “a / an” and “the” include a plurality of references. Furthermore, as used in this specification and throughout the claims, unless the context clearly indicates otherwise, the term “in” includes both “in” and “on”. Additionally, headings or subheadings may be used in this specification for the reader's convenience, which should not affect the scope of the invention. Furthermore, some terms used in this specification are defined more specifically below.

[0045] definition

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In case of conflict, this document (including the definitions) shall prevail.

[0047] For the purposes of this specification and claims, the term "antibody fragment" or "fraction thereof" is used to mean a portion or fragment of a complete antibody molecule, wherein the fragment retains antigen-binding function; that is, F(ab')2, Fab', Fab, Fv, single-chain Fv ("scFv"), Fd', and Fd fragments. Methods for generating various fragments from mAbs are well known to those skilled in the art.

[0048] The term “specific binding affinity or binding specificity for a cross-reactive epitope shared by SCUBE2 expressed on human vascular endothelial cells and SCUBE2 expressed on mouse vascular endothelial cells” (or “binding specificity for a cross-reactive epitope between human and mouse SCUBE2”) is used herein to mean the property of an anti-SCUBE2 antibody to bind to a cross-reactive epitope present on both human and mouse SCUBE2, wherein (a) the epitope is accessible on the surface of vascular endothelial cells expressing SCUBE2; and (b) the binding to the SCUBE2 epitope present on mouse endothelial cells is greater than the binding exhibited by isotype control immunoglobulin, and “greater than” can be quantitatively measured because the anti-SCUBE2 antibody… The binding of mAb minus one standard deviation must be greater than the binding of isotype control immunoglobulin plus one standard deviation, as will be more evident in the following examples; and (c) the binding of anti-SCUBE2 antibody to SCUBE2 expressed on mouse endothelial cells is at least twice as low as the binding of anti-SCUBE2 antibody to SCUBE2 expressed on human endothelial cells, as detected in standard assays of immunoreactivity.

[0049] Co-receptors are cell surface receptors that bind to signal transduction molecules in addition to the primary receptor, in order to facilitate ligand recognition and initiate biological processes.

[0050] Humans SCUBE2 Nucleotide sequence of cDNA (SEQ ID NO: 65); amino acid sequence of human SCUBE2 (SEQ ID NO: 66).

[0051] Abbreviations: EC, endothelial cells; EC-KO mice, EC-specific Scube2 Gene knockout mice; LLC, Lewis lung cancer; AP, alkaline phosphatase; HIF, hypoxia-inducible factor; HUVEC, human umbilical vein endothelial cells; KO, gene knockout; MAPK, mitogen-activated protein kinase; MLEC, mouse lung endothelial cells; RTK, receptor tyrosine kinase; SCUBE2, protein 2 containing the signal peptide-complement protein C1r / C1s, Uegf, and Bmp1(CUB)-epidermal growth factor (EGF) domains; shRNA, short hairpin RNA; VEGF, vascular endothelial growth factor; VEGFR2, vascular endothelial growth factor receptor 2.

[0052] Efficacy / Indications

[0053] (1) Pathological tumor angiogenesis; and

[0054] (2) Neovascular ocular diseases

[0055] SCUBE2 is a peripheral membrane protein expressed in both normal and tumor vascular endothelial cells (ECs); however, its role in angiogenesis remains poorly understood. We discovered that SCUBE2 acts as a co-receptor for VEGFR2 and plays a role in VEGF-induced angiogenesis. SCUBE2 expression increases in response to hypoxia via hypoxia-inducible factor (HIF-1α) and interacts with VEGFR2 in human ECs. In human ECs, SCUBE2 acts as a co-receptor for VEGFR2, facilitating VEGF binding and enhancing downstream VEGF signaling, thus promoting VEGF-induced angiogenesis and tumor angiogenesis. This SCUBE2-VEGFR2 interaction and enhanced signaling can be mediated by endothelial cells. Scube2 Gene deactivation, SCUBE2 shRNA-mediated gene knockdown, and SCUBE2 mAb neutralization in vitro and in vivo. Endothelial cells. Scube2 Gene knockout (EC-KO mice) did not affect angiogenesis, but MATRIGEL™ implants showed reduced VEGF-induced angiogenesis and restored blood flow after induced hindlimb ischemia. SCUBE2, a novel co-receptor of VEGFR2, influences VEGF-induced lumen formation and EC proliferation by fine-tuning VEGFR2-mediated signaling, particularly during postpartum angiogenesis induced by ischemia or hypoxia. Targeting this SCUBE2 function in tumor ECs presents a potential anti-tumor modality by inhibiting tumor angiogenesis.

[0056] We found that SCUBE2 is a co-receptor for VEGFR2, enhancing VEGF-VEGFR2 binding and amplifying its signaling, including VEGFR2 phosphorylation and p44 / 42 mitogen-activated protein kinase (MAPK) / Akt activation, thus promoting cell proliferation and lumen formation in endometrium. In mice, endothelial cell removal... Scube2 In this context, physiological angiogenesis is normal, while pathological angiogenesis in experimental tumors is altered, showing inhibition of tumor growth and reduction of microvessel density. To simulate the angiogenesis environment of tumors, isolate... Scube2 Defective ECs proliferate in vitro via VEGF. Mutant ECs exhibit significantly reduced VEGF binding, proliferation, budding response to VEGF, and downstream signaling activation. Furthermore, anti-SCUBE2 and anti-VEGF monoclonal antibodies showed additive inhibitory effects against xenograft lung tumors. In conclusion, our findings identify SCUBE2 as a key regulator of VEGF response in tumor ECs and demonstrate the significant potential of anti-SCUBE2 strategies in combination with anti-angiogenic cancer therapies.

[0057] Example

[0058] The scope of this invention is not intended to be limited. Exemplary instruments, apparatuses, methods, and related results according to embodiments of the invention are provided below. It should be noted that headings or subheadings may be used in the examples for ease of reading, but these should in no way limit the scope of the invention. Furthermore, certain theories are presented and disclosed herein; however, whether correct or incorrect, they should in no way limit the scope of the invention, provided that the invention is practiced according to the invention, without regard to any particular theory or operational procedure.

[0059] Materials and Methods

[0060] Antibodies and reagents. Anti-SCUBE2, anti-HIF-1α, and anti-phospho-tyrosine polyclonal antibodies were from GeneTex (Irvine, CA). Anti-phospho-VEGFR2 (Thr1059), anti-phospho-MAPK p44 / p42 (Thr202 / Tyr204), anti-MAPK p44 / p42, anti-phospho-AKT (Ser473), anti-AKT, and anti-EGFR antibodies were from Cell Signaling Technology (Danvers, MA). Anti-VEGFR2 and anti-VEGF antibodies were from Thermo Scientific (Rockford, IL) and Santa Cruz Biotechnology (Santa Cruz, CA), respectively. Anti-CD31, anti-phospho-serine / threonine, anti-VEGFR1, and anti-neuroflavin-1 antibodies were from Abcam (Cambridge, MA). Anti-Ki67 and anti-β-actin antibodies were derived from DAKO Cytomatation (Glostrup, Denmark) and NOVUS Biologicals (Littleton, CO), respectively. Recombinant VEGF 165 The protein is derived from R&D Systems (Minneapolis, MN).

[0061] Generation of anti-SCUBE2 monoclonal antibodies. Anti-SCUBE2 specific antibodies were generated as follows: Spinal cells from BALB / c mice immunized with purified recombinant SCUBE2 spacer region (amino acids 445 to 667) produced from HEK-293T cells were fused with myeloma cells (SP2 / O) to generate hybridomas. The hybridoma cell line was prepared and subcloned as previously described (Hadas et al., “Production of monoclonal antibodies. The effect of hybridoma concentration on the yield of antibody-producing clones”). J Immunol Methods 1987; 96:3-6). Hybridoma cell lines that are positive for SCUBE2 (not SCUBE1 and 3) should be identified as described (Tu et al., “Localization and characterization of a novel protein SCUBE1 in human platelets”, Cardiovasc Res. 2006;71:486-95; Cheng et al., “SCUBE2 suppresses breast tumor cell proliferation and confers a favorable prognosis in invasive breast cancer”, Cancer Res. 2009; 69:3634-41).

[0062] conditional Scube2 Flox / Flox and endothelial specificity Scube2 Generation of gene knockout (EC-KO) mice. As described, this generates two 65 kb genomic sequences separated by covered exons 2 through 21. lox Conditionality of P site Scube2 Flox Alleles. To generate endothelium-specific conditional KO mice, [the following will be used]. Tie2 promoter 2 Controlled expression of Cre recombinase and targeting Scube2 Heteromorphic transgenic mice ( Tie2-Cre; Scube2 + / - )and Scube2 Flox / Flox Animal hybridization to obtain experimental results Tie2-Cre ; Scube2 Flox / + (Comparison) and Tie2-Cre ; Scube2 Flox / - (EC-KO) mice. Male mice are primarily used in each genotype group (e.g., specifying n = 5 or greater). All animal experiments are approved by animal management societies and the institutions using the animals.

[0063] Tumor xenografting. This was achieved by subepithelial injection of 1 × 10⁻⁶ tumor cells into the flank of control and EC-KO mice. 6 Xenograft tumors were generated using melanoma cells (B16F10) or Lewis lung cancer cells (LLC). Tumor size was measured twice weekly using a digital diameter gauge and calculated as length × width × height × 0.5236 (in mm). 3(Units). After 16 days of tumor growth, the tumors were fixed and embedded in paraffin for tissue sectioning. Pimonidazole and APO-BRDU™ (TUNEL) apoptosis reagent were used according to the manufacturer's instructions. Angiogenesis and proliferation of tissues from LLC and B16F10 tumors were observed using CD31 and Ki-67 antibodies, respectively.

[0064] MATRIGEL™ angiogenesis assay. The angiogenesis model is based on the use of MARTIGEL™ implants in control or EC-KO mice. 0.5 ml of growth factor-depleted MARTIGEL™ (with or without 100 ng / ml VEGF) is injected into the flank of the mouse. The injection is performed rapidly using a 26G needle to ensure that the syringe contents are delivered as a single plug. One week later, the plugs are collected and homogenized in RIPA lysate buffer. After removing the residue by centrifugation, heme concentration is measured using Drabkin's reagent (Sigma-Aldrich). Alternatively, the plugs are fixed overnight in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with anti-CD31 antibody.

[0065] A hindlimb ischemia model. As previously described. 3 A hindlimb ischemia model was established. In short, the femoral artery of control and EC-KO mice (8 weeks old) was exposed, separated from the femoral nerve and vein, and ligated at two points 5 mm apart. One point was close to the inguinal ligament, and the second point was far from the first and close to the popliteal artery. The skin was closed with interrupted 4-0 sutures. Mice were anesthetized at different times before and after surgery, and laser Doppler flow imaging was performed using a Moor infrared laser Doppler imaging system. Three weeks after proximal femoral artery ligation, calf muscle was harvested from anesthetized mice and stained with CD31.

[0066] Aortic ring sprouting assay. The mouse aortic ring sprouting assay was performed largely according to standard procedures. Aortas were collected from mice. All excess fat, tissue, and branching vessels were removed using forceps and a scalpel. The aorta was transferred to a culture dish containing OPTI-MEM medium, and a ring approximately 0.5 mm thick was cut from the aorta. The aortic ring was embedded in 1 mg / ml type I collagen and then incubated at 37°C / 5% CO2 for 1 h. OPTI-MEM medium supplemented with 2.5% FBS and 30 ng / ml VEGF was added and surrounding the aortic ring. The number and length of microvessel sprouts were counted on day 5.

[0067] Immunohistochemistry. Tissue sections (5 μm thick) were dewaxed with xylene, rehydrated in a series of ethanol solutions, treated with 3% H₂O₂ for 20 min, washed with PBS, incubated with blocking solution (PBST supplemented with 2% BSA and 2% normal goat serum) for 1 h, and then incubated overnight at room temperature with primary antibody. Antibody binding was detected using peroxidase-bound antibodies and stable 3,3'-diaminobenzidine (DAB) peroxidase substrates. Hematoxylin was used for contrast staining.

[0068] Isolation, characterization, and culture of primary mouse lung ECs (MLECs). As described. 6 Primary MLECs were isolated from lung tissue of control and EC-KO mice. Each mouse (6 weeks old) was first injected intramuscularly with 100 µl of heparin (140 U / ml). Ten minutes later, the mice were anesthetized and their thoracic cavity exposed. Blood was flushed from the lungs by injecting low-temperature M199 medium into the right ventricle. Then, 1 ml of collagenase A (1 mg / ml) was rapidly instilled into the lungs via the trachea. The lungs were removed and incubated with 5 ml of collagenase A at 37°C for 30 min. The cell suspension was filtered through a 70 µm filter and then centrifuged at 1000 rpm for 5 min. The cell pellet was resuspended in growth medium (M199 medium supplemented with 20% fetal bovine serum, 50 μg / ml EC growth factor, 100 μg / ml heparin, 2 mM glutamine, 100 IU / ml penicillin, and 100 μg / ml streptomycin) and cultured in gelatin-coated tissue culture dishes for 2 days. Cells were removed using trypsin, and the cell suspension was used for anti-CD31-PE antibody staining and FACS sorting.

[0069] Hypoxia treatment and lentiviral SCUBE2 overexpression / knockdown in HUVECs. HUVECs were purchased from the Bioresource Collection and Research Center and cultured according to the supplier's recommendations. For hypoxia treatment, HUVECs were exposed to hypoxia (1% O2) by incubation in a CO2 incubator aerated with a 95% N2 / 5% CO2 mixture. HUVECs were modified using a self-inactivating lentiviral transduction system with either a full-length SCUBE2 expression vector or an empty vector. We used vector-based hairpin RNA (shRNA) generated by the RNAi Consortium to knock down the endogenous SCUBE2 gene in HUVECs.

[0070] Chromatin immunoprecipitation (ChIP). ChIP analysis is as described in the EZ-MAGNACHIP™ G kit instructions. In short, HUVEC (1 × 10⁻⁶)7 Cells were cross-linked with 1% formaldehyde, dissolved in 500 μl of lysing buffer, and ultrasonically agitated to form fragments of approximately 500 bp. ChIP was performed using antibodies against HIF-1α or IgG. Control DNA or immunoprecipitated DNA was amplified in a 50 μl reaction volume consisting of 2 μl of DNA template using primers (Table 1). PCR involved Taq polymerase: 35 cycles of 94°C for 30 sec, 63.5°C for 30 sec, and 72°C for 40 sec, followed by a 5 min at 72°C. 10 µl samples from each PCR reaction were separated on a 1.5% agarose gel.

[0071] Luciferase reporter gene assay. HUVECs were transiently transfected using NUCLEOFECTOR™ with 4.5 µg of SCUBE2 promoter luciferase reporter gene plasmids (WT, M1, M2, and M3) and an internal control (0.45 µg of pRL-TKRenilla luciferase plasmid) according to the manufacturer's instructions. Cells were cultured for 2 days, and reporter gene assays were collected and prepared for use in a dual-luciferase reporter gene assay system.

[0072] EC proliferation assay. The effect of SCUBE2 on EC proliferation was determined as described by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, ECs were treated with trypsin and cultured at 2000 cells / well in 100 µl of complete medium in 96-well cell culture dishes. The following day, cells were stimulated with VEGF (100 ng / ml) or control medium for 4 days, and cell counts were determined.

[0073] EC lumen formation measurement. EC (3 × 10⁻⁶) was used. 3 Cells per well were seeded onto MATRIGEL™ plates in 15-well µ-Slide angiogenesis discs containing VEGF (100 ng / ml). Lumenization was determined by counting vessels in three random regions in each well after 16 hours.

[0074] VEGF alkaline phosphatase (AP) binding assay. The generation and binding of AP-labeled VEGF proteins were performed essentially as described in the literature. AP-VEGF chimeric ligands were constructed by PCR amplification of a portion of the VEGF cDNA using the following primers: CCG CTC GAG GCA CCC ATG GCA GAA GGA (SEQ ID NO: 67) and GCT CTA GAT TAT CAC CGCCTC GGC TTG TCA CA (SEQ ID NO: 68). The 498 bp amplified fragment was then cloned into the XhoI and XbaI restriction sites of APtag-5. The AP-VEGF fusion protein and control AP protein were generated by transient transfection into HEK-293T cells. The supernatant from transfected HEK-293T cells was collected, concentrated, and stored. HEK-293T cells overexpressing VEGFR2 alone or together with SCUBE2 were incubated with ECs in the supernatant for 4 h and washed three times with PBS at 4°C. Cells were then lysed on ice in lysing buffer (25 mM Hepes, pH 7.6, 150 mM NaCl, 5 mM EDTA, 10 µg / ml aprotinin, 5 µg / ml leucopeptide, 10% glycerol, and 1% Triton X-100) for 5 min. The cell lysate was collected by centrifugation at 10,000 xg for 20 min at 4°C. AP activity was measured using p-nitrophenyl phosphate substrate.

[0075] Pull-down assay. Myc-labeled VE-cadherin-FL, D1, and D2 proteins (Myc.VE-cadherin-FL, -D1, -D2) were generated using in vitro transcription / translation. Recombinant GST-labeled SCUBE2-CUB protein (GST.SCUBE2-CUB) was bound to glutathione-SEPHAROSE™ beads from the soluble fraction of bacterial lysate. FLAG-labeled SCUBE2-FL, EGF, spacer region, CR, and CUB proteins (FLAG.SCUBE2-FL, -EGF, -spacer, -CR, -CUB) were generated via overexpression in HEK-293T cells. Recombinant VEGF 165 Proteins were purchased from R&D Systems. Regarding SCUBE2 and VEGF... 165 Interaction analysis, recombinant VEGF 165Proteins bound to anti-FLAG M2 antibody-agarose beads or control beads, including FLAG.SCUBE2-FL, EGF, spacers, CR, and CUB proteins, were mixed in 0.5 ml binding buffer [40 mM HEPES (pH 7.5), 100 mM KCl, 0.1% NONIDET™ P-40, and 20 mM 2-mercaptoethanol]. After incubation at 4°C for 4 h, the beads were thoroughly washed, and the interacting proteins were observed using an anti-VEGF antibody via Western blotting. For the SCUBE2 and VEGFR2 interaction assay, GST-labeled SCUBE2-CUB protein was mixed with Myc.VEGFR2-FL, -D1, and -D2 proteins in 0.5 ml binding buffer. After incubation at 4°C for 4 h, the protein solution was incubated with glutathione-SEPHAROSE™ for 2 h with gentle shaking. After washing three times with binding buffer, the precipitated binding protein was observed using an anti-Myc antibody via immunoblotting.

[0076] Confocal immunofluorescence microscopy analysis. Endothelial cells were fixed in 4% formaldehyde, treated with 2% fetal bovine serum for 1 h, and incubated with chicken anti-SCUBE2 and mouse anti-VEGFR2 antibodies for 1 h. After washing three times with PBS, the cells were stained with ALEXA FLUOR® 488-labeled anti-mouse IgG antibody and Alexa Fluor 594-labeled anti-chicken IgY antibody for 1 h, followed by washing three times with PBS and mounting with VECTASHIELD® mounting medium containing DAPI. Fluorescence images were captured under a confocal microscope at room temperature.

[0077] RNA extraction, cDNA synthesis and RT-PCR

[0078] Total RNA was prepared from cultured cells using the TRIZOL® method. 5 μg of RNA was used for first-strand cDNA synthesis using SUPERSCRIPT™ II reverse transcriptase. One-tenth of the first-strand cDNA was used as a template for each PCR. PCR products were analyzed on a 1% agarose gel. Primers are listed in Table 1.

[0079] Immunoprecipitation and Western blot analysis

[0080] Myc-labeled VEGFR2 expression constructs, alone and in combination with a series of expression plasmids encoding the specified FLAG-labeled SCUBE2 protein, were transfected into HEK-293T cells. Two days after transfection, cells were washed once with PBS and incubated on ice in cell lysis buffer (25 mM Hepes, pH 7.6, 150 mM NaCl, 5 mM EDTA, 10 µg / ml aprotinin, 5 µg / ml leucopeptide, 10% glycerol, and 1% TRITON® X-100) for 5 min. Cell lysates were collected by centrifugation at 10,000 x g for 20 min at 4 °C. The sample was incubated with 1 µg of the specified antibody and 20 µl of 50% (v / v) Protein A agarose for 2 h with gentle shaking. After washing three times with buffer, the precipitated complex was dissolved in sample buffer by boiling, separated by SDS-PAGE, and transferred to a PVDF membrane. The membrane was reacted with phosphate-buffered saline (pH 7.5) containing 0.1% gelatin and 0.05% TWEEN® 20 and analyzed using a specified antibody. After two washes, the blot was incubated with peroxidase-bound goat anti-mouse IgG for 1 h. Following membrane washing, the blot was observed using the VISGLOW™ chemiluminescent immunoassay and HRP (Visual Protein) system.

[0081] Statistical analysis. Data are presented as mean ± SD and analyzed using a two-tailed paired approach. t The test results were analyzed. P A difference of <0.05 is considered statistically significant.

[0082] Table 1 lists the primer sequences and their corresponding SEQ ID NOs for RT-PCR (SEQ ID NO: 69-82; 85-86); ChIP (SEQ ID NO: 83-84); Q-PCR (SEQ ID NO: 87-94); and genotyping (SEQ ID NO: 95-102).

[0083] Table 1

[0084]

[0085] * SCUBE1 to 3 and GAPDH It is human genes; Scube1 to 3 and Gapdh It is a mouse gene.

[0086] result

[0087] SCUBE2 is expressed in human ECs and regulates VEGF-induced EC proliferation and tube formation.

[0088] We use immunostaining ( Figure 1 A) Flow cytometry ( Figure 1 B) and Western blot analysis ( Figure 1 C) First, it was confirmed that SCUBE2 protein is expressed in HUVECs. Confocal immunofluorescence staining and flow cytometry analysis revealed that SCUBE2 is expressed on the EC cell membrane. Figure 1 (A and 1B). Furthermore, SCUBE2 expression levels were higher in proliferative sub-confluent ECs than in growth-arrested confluent ECs (A and B). Figure 1 C).

[0089] We then investigated the potential role of SCUBE2 in regulating the VEGF response by overexpressing and knocking it out in human ECs. Full-length FLAG-labeled SCUBE2 or two independent short hairpin RNAs (shRNAs 1 and 2) targeting SCUBE2 were expressed in HUVECs using recombinant lentivirus. Overexpression and knockout of SCUBE2 were verified by RT-PCR or Western blot analysis. Figure 1 D and 1G). SCUBE2 overexpression was significantly increased (D and 1G). Figure 1 E and 1F) while SCUBE2 gene knockout ( Figure 1 H and 1I significantly reduced VEGF-induced EC growth and capillary-like network formation on MATRIGEL™. In summary, these data support the ability of SCUBE2 to regulate VEGF-induced proliferation and lumen formation in HUVECs.

[0090] SCUBE2 in HUVECs is mediated by the upregulation of HIF-1α.

[0091] Because hypoxia-induced VEGF expression via HIF-1α is crucial for postpartum angiogenesis, we subsequently investigated whether endothelial cell SCUBE2 expression also increases through a similar mechanism. After 12 hours of hypoxia exposure to HUVECs, SCUBE2 (but not SCUBE1 or SCUBE3) expression significantly increased at both the mRNA and protein levels, and its expression was positively correlated with HIF-1α. Figure 2 A and 2B). Furthermore, ChIP analysis and promoter mutation analysis confirmed that under hypoxic conditions, endogenous HIF-1α can interact with the HIF-binding region (A / GCTGA) of the SCUBE2 promoter, leading to increased SCUBE2 expression in HUVECs. Figure 2 C-2E).

[0092] EC specificity Scube2 Production of gene knockout (EC-KO) mice

[0093] To further study endothelial cells Scube2 Regarding the effect of VEGF on in vivo response, we used methods to... Scube2 The conditional "Floxed" allele [encodes 9 EGF-like repeat units, a spacer region, 3 cysteine-rich motifs, and has loxP Mice with exons in the CUB domain were crossed with transgenic mice expressing phage recombinase Cre (pan-endothelial expression) under the control of the angiopoietin receptor (Tie2) promoter, specifically knocking out exons in the EC. Scube2 .male Tie2-Cre ; Scube2 + / - With female Scube2 Flox / Flox Mice mating to obtain Tie2-Cre ; Scube2 Flox / + (Named "Control Group") and Tie2-Cre ; Scube2 Flox / - (Named EC-KO) mouse.

[0094] To determine Scube2 Flox Endothelial-specific recombination efficiency of alleles was assessed by isolating primary MLECs from control and EC-KO mice. Flow cytometry analysis revealed that this cell population was highly enriched in ECs, as approximately 95% of these cells expressed CD31. Furthermore, mRNA and protein analysis showed… Scube2 Specifically removed from mouse endothelial cells without affecting EC-KO MLECs Scube1 and Scube3 Expression levels. Similarly, immunostaining of adult mouse lungs validated the complete removal of endothelial cells (but not bronchial epithelial cells) in EC-KO mice. Scube2 Expression. Therefore, effective knockout in the endothelial cells of adult EC-KO mice. Scube2 .

[0095] Both control and EC-KO mice recovered at the expected Mendelian rates and exhibited normal weight gain patterns, indicating that angiogenesis and vasculature were sufficient for normal development. We observed no significant differences in overall health or behavior between the control and EC-KO mice. EC-KO females reproduced normally and their offspring were normal, suggesting that regenerative angiogenesis was sufficient for population proliferation. In conclusion, these data indicate that endothelial cells… Scube2 Gene knockout does not affect physiological angiogenesis.

[0096] Scube2 EC-KO mice showed a reduced response to exogenous VEGF and adult angiogenesis.

[0097] To investigate endothelial cells Scube2 To investigate the potential function of VEGF-stimulated angiogenesis in vivo, we used MATRIGEL™ injections, in which MATRIGEL™ mixtures with VEGF(+) or saline(-) were subcutaneously administered to age- and sex-matched EC-KO and control mice, and recovered after 7 days. Figure 3 On the naked eye, MATRIGEL™ plugs containing VEGF from control mice are pale reddish-brown. Figure 3 A). However, thiamin containing VEGF from EC-KO mice was less effective than thiamin in the control group ( Figure 3 A). Similarly, in EC-KO mice, the total heme content (a measure of the absence of ruptured blood vessels, correlated with the amount of newly formed capillary network) in VEGF-containing MATRIGEL™ plugs was reduced by approximately 50%. Figure 3 B). To ensure that this heme difference is due to reduced microvascular density, vascular proliferation in the thrombus was quantified by immunostaining with anti-CD31 antibody (a marker of endothelium). Figure 3 C and 3D). Compared with control mice implanted with MATRIGEL™, EC-KO mice showed reduced angiogenesis, even in septic tanks containing saline solution. Figure 3 C). Furthermore, the microvessel density containing VEGF plugs in EC-KO mice was 50% lower than in control mice. Figure 3 D), and no large blood vessels were observed ( Figure 3 C) This indicates that VEGF-induced adult angiogenesis is influenced by endothelial cells in vivo. Scube2 adjust.

[0098] To further evaluate endothelial cells Scube2 To investigate the pro-angiogenic effect, we used a second adult angiogenesis animal model (i.e., hind limb ischemia): the common femoral artery was ligated in both control and EC-KO mice, and blood flow and angiogenesis were monitored over time using laser Doppler imaging and CD31 immunostaining, respectively. Laser Doppler analysis revealed a similar reduction in blood flow in the ligated limbs of both the control and EC-KO animals after surgery compared to the unligated contralateral limb. Figure 3 E and 3F indicate that postoperative local ischemia was similar in both strains. In control mice, blood flow recovered to near baseline levels 21 days postoperatively. However, in EC-KO animals, blood flow recovery was significantly reduced ( Figure 3 E and 3F). Similarly, histological examination of the gastrocnemius (calf) muscle showed that at 21 days, the density of anti-CD31 positive capillaries induced by ligation in EC-KO mice was lower than that in control mice. Figure 3 (G and 3H). Therefore, after local ischemia induced by femoral artery ligation, Scube2 It plays an indispensable role in the functional angiogenesis of endothelial cells.

[0099] SCUBE2 co-localizes with VEGFR2 in HUVECs and enhances the binding of VEGF to VEGFR2.

[0100] Because SCUBE2 can regulate VEGF response both in vitro and in vivo ( Figure 1 and 3 Furthermore, SCUBE protein can serve as a co-receptor for signal transduction receptors such as serine / threonine kinases or RTKs. Therefore, we analyzed whether SCUBE2 co-localizes with and interacts with VEGFR2 in HUVECs. Confocal microscopy and co-immunoprecipitation experiments showed that VEGF enhances the binding of SCUBE2 to VEGFR2 in HUVECs, reaching a peak 10 min after VEGF stimulation. This result was further described in detail in HEK-293T cells transfected with SCUBE2 and VEGFR2 expression plasmids, showing that the CUB structural region of SCUBE2 can interact with VEGFR2. In addition, this SCUBE2-VEGFR2 interaction is specific because anti-SCUBE2 immunoprecipitation did not reduce other RTKs (such as VEGFR1 and EGFR) or another VEGFR2 co-receptor, neurofeline-1. Most importantly, in addition to binding to VEGFR2, SCUBE2 can directly bind to VEGF-A via its EGF-like repeat structural region. 165 Binding. We performed additional co-immunoprecipitation assays to verify whether SCUBE1 or SCUBE3 could also bind to VEGF or VEGFR2. Unlike SCUBE2, which specifically binds to VEGF, SCUBE1 and SCUBE3 did not interact with VEGF, indicating that SCUBE1 or SCUBE3 cannot act as co-receptors for VEGF (see Lin et al., “Endothelial SCUBE2 Interacts With VEGFR2 and Regulates VEGF-Induced Angiogenesis”). Arterioscler Thromb Vasc Biol. 2017;37:144-155).

[0101] To assess whether SCUBE2 actually enhances VEGF-VEGFR2 binding, as described, we first generated a functional alkaline phosphatase (AP)-VEGF fusion protein. In HUVECs, the AP-VEGF protein was able to bind to endogenous VEGFR2 compared to the control AP protein. Interestingly, SCUBE2 overexpression increased AP-VEGF binding compared to the corresponding control HUVECs, while SCUBE2 gene knockdown or deletion decreased AP-VEGF binding. Similarly, Scatchard analysis showed that in HEK-293T cells, the binding affinity of VEGF to VEGFR2 was approximately 3-fold increased (Kk) in the case of co-expression of VEGFR2 and SCUBE2 compared to the case of VEGFR2 alone. d = 0.21 nM vs. 0.58 nM). In summary, these data indicate that SCUBE2 acts as a co-receptor for VEGFR2 and enhances the binding of VEGF to VEGFR2.

[0102] SCUBE2 modulates VEGFR2 phosphorylation and downstream signaling in HUVECs.

[0103] We then investigated whether SCUBE2 contributes to VEGF activation signaling in HUVECs. To this end, we used VEGF as a stimulator and SCUBE2 shRNA gene knockdown or SCUBE2 overexpression to assess the function of SCUBE2 in VEGF-induced signaling. VEGF can induce VEGFR2 Tyr1059 phosphorylation, p44 / 42 MAPK signaling cascade, and AKT activation. However, SCUBE2 shRNA gene knockdown reduced (…). Figure 4 (A to B) while SCUBE2 is overexpressed ( Figure 4 C through D) enhance VEGF-induced VEGFR2 phosphorylation, p44 / 42 MAPK signaling cascade, and AKT activation. Our data strongly suggest that SCUBE2, as a VEGFR2 co-receptor, is capable of regulating VEGF-induced downstream VEGFR2 signaling in HUVECs. We determined the phosphorylation status of SCUBE2 in HUVECs after VEGF treatment. Following VEGF stimulation, anti-SCUBE2 immunoprecipitates were blotted using either anti-phosphotyrosine (p-Tyr) or anti-phosphoserine / threonine (p-Ser / Thr) panspecific antibodies. After VEGF treatment for up to 30 min, SCUBE2 appeared to be unphosphorylated. However, further phosphoproteomic analysis is needed to confirm whether SCUBE2 is phosphorylated after VEGF stimulation.

[0104] Scube2 Modulation of VEGF signaling in primary mouse lung ECs (MLECs)

[0105] Compared with our gene knockdown experiments in HUVECs ( Figure 1 Similar to G-1I, VEGF-induced cell proliferation and lumen formation were significantly reduced in EC-KOMLECs compared to control MLECs. Figure 5 (A to B). We further evaluated the endothelial cells. Scube2 The effect of gene deletion on VEGF-induced signal transduction. We stimulated control and EC-KO MLEC with 50 ng / ml VEGF for 0, 10, 20, and 30 min. Consistent with SCUBE2 shRNA knockdown in HUVECs, VEGF signal transduction in EC-KO, measured by phosphorylation of VEGFR2, p44 / 42 MAPK, and AKT, was significantly lower than that in control MLECs. Figure 5 (C to D)

[0106] Scube2 EC-KO mice exhibit reduced aortic ring microvascularization capacity.

[0107] We further investigated the function of SCUBE2 in angiogenesis using isolated aortic ring analysis, comparing the angiogenic capacity of aortic rings derived from control and EC-KO mice. Aortic rings isolated from control and EC-KO mice were treated with PBS or VEGF, and the angiogenic response of individual aortic rings was determined by quantifying the number of blood vessels grown and measuring the total length of newly formed vessels. On day 5, quantitative results of the number of tubular structures (buds) responding to VEGF (30 ng / ml) and the length of buds showed that both parameters were significantly lower in EC-KO aortic rings compared to the control group. Figure 5 (E to 5F). Therefore, these results further confirm the in vitro analysis results, demonstrating the importance of SCUBE2 in VEGFR2-related angiogenesis.

[0108] Reduced tumor growth in EC-KO mice

[0109] SCUBE2 is highly expressed in tumor endothelial cells. Figure 6 The results of immunohistochemical staining show that SCUBE2 is highly expressed in ECs (arrows) in breast, lung, melanoma (A), prostate, sarcoma, and bladder cancer (B). Endothelial cells Scube2 It plays an important role in adult angiogenesis. Figure 3 We then studied Scube2The effect of endothelial cell inactivation (EC-KO) on angiogenesis and tumor growth in pathological tumors. Control and EC-KO mice were subcutaneously injected with syngeneic melanoma (B16F10) or Lewis lung cancer (LLC) cells. Tumor growth and size in EC-KO mice were significantly lower than in control mice. Figure 7 (A to D). With the removal of endothelial cells Scube2 The adult angiogenesis was consistent, and the microvessel density in EC-KO (as observed by anti-CD31 staining) was significantly lower than that in the control group tumor. Figure 7 E to H), showing endothelial cells Scube2 It plays an important role in promoting tumor angiogenesis and growth. Importantly, the vascular density in the skin of non-tumor adult animals was not different from that in the control group and EC-KO animals (data not shown).

[0110] Defective angiogenesis observed in EC-KO mice suggests that tumor cells may lack nutrients and oxygen, thus undergoing apoptosis and necrosis. Consistent with this concept, hematoxylin and eosin staining revealed necrotic tissue in EC-KO tumors, but the necrotic area was much smaller in control tumors. Figure 8 (A to B). Furthermore, compared to control tumors, terminal deoxynucleotidyl transferase (dUTP) nick end marker (TUNEL) analysis and Ki-67 immunostaining showed increased apoptosis and significantly reduced tumor cell proliferation in EC-KO tumors. Figure 8 CF). Furthermore, these results show endothelial cells Scube2 Angiogenesis is essential for maintaining the survival of tumor cells.

[0111] Figure 9 Displaying missing endothelial cells Scube2 Inhibition of tumor angiogenesis and tumor growth in EC-KO mice. Representative photographs are shown 60 days after implantation of MLTC testicular interstitial tumors, along with tumor growth rates measured at specified time points (A and B). EC-KO mice show decreased tumor vascular distribution. Anti-CD31 staining of tumor sections reveals a reduction in the number of ECs and vascular structures in EC-KO mice (C). Quantitative map of tumor angiogenesis 60 days after tumor cell implantation (D).

[0112] Reduced retinal angiogenesis in EC-KO mice

[0113] Figure 10 Demonstration of endothelial cell knockout Scube2 Its effect on the development and growth of retinal blood vessel distribution.

[0114] EC-KO mice showed reduced oxygen-induced retinopathy

[0115] Figure 11 This study demonstrates that the absence of SCUBE2 in endothelial cells reduces oxygen-induced retinopathy (OIR).

[0116] Anti-SCUBE2 antibody

[0117] SCUBE2 has at least 5 distinct structural regions ( Figure 12 A): The sequence consists of an NH2-terminal signal peptide, nine repeating EGF-like repeat regions (E), a spacer region, three cysteine-rich motifs (CR), and a COOH-terminal CUB region. The amino acid composition of each region is as follows: (1) SP: aa 1-37; (2) EGF-like repeat region: aa 49-442; (3) spacer region: aa 443-669; (4) CR: aa 70-803; and (5) CUB: aa 838-947. The SCUBE2 protein (aa 38 to aa 1028) can be secreted into the extracellular medium.

[0118] Anti-SCUBE2 antibody was generated. Figure 12 A shows the specific targeting domains of each anti-SCUBE2 mAb clone. EGF-C3 recognizes EGF-like repeat regions 4-6 (aa 175-323), while SP-A1 (aa 441-659), B1 (aa 441-659), and B2 (aa 441-659) bind to the SCUBE2 spacer region. The CR-#5 clone detects the first cysteine-rich motif of SCUBE2 (aa 668-725). The EGF-C3 clone was obtained by immunization with a recombinant protein containing the EGF-like repeat region of SCUBE2, and SP-A1, B1, and B2 were obtained by immunization with a recombinant protein containing the SCUBE2 spacer region. Similarly, the CR-#5 clone was obtained by immunization with a recombinant protein containing the first cysteine-rich motif.

[0119] These antibodies, when incubated with endothelial cells in an in vitro lumen formation assay, showed inhibitory properties against angiogenesis. Figure 12 B). Tables 2 to 6 show the CDR sequences of SCUBE2mAb. (V is also shown.) H With V L The complementary determining regions 1 to 3 (CDR1 to 3) and the framework regions 1 to 4 (FW1 to 4) of the domain.

[0120] Treatment of neovascular eye diseases

[0121] In a mouse model of oxygen-induced retinopathy, intravitreal injection of SCUBE2 antibodies was performed to observe their effect on retinal angiogenesis. Our preliminary data, obtained using in vitro endothelial cell tube formation assays, showed that these SCUBE2 antibodies inhibited angiogenesis. For example, the SP.B1 clone was able to inhibit VEGF-stimulated endothelial cell proliferation and capillary formation. Figure 13 (C to E).

[0122] Anti-SCUBE2 (SP.B1) and anti-VEGF (bevacizumab) antibodies synergistically inhibit the growth of lung, pancreatic, and colorectal cancers.

[0123] Since our results indicate that membrane-associated SCUBE2 plays a crucial role in tumor angiogenesis, we evaluated the potential of neutralizing mAbs to inhibit SCUBE2 as a treatment for solid tumors. An mAb specific to SCUBE2 (clone SP.B1) was generated; this mAb did not cross-react with SCUBE1 or 3. Figure 13 (A to B), which showed no significant off-target effects. SCUBE2 has pro-angiogenic activity, and we first evaluated the in vitro effect of this mAb on the response to VEGF stimulation in HUVECs. Incubation with SP.B1 mAb instead of control IgG blocked VEGF-induced signaling, including VEGFR2 phosphorylation and p44 / 42 MAPK / Akt activation (A to B). Figure 13 F to I). The functional activity of this mAb was verified by its ability to inhibit EC proliferation and capillary formation stimulated by VEGF. Figure 13 CE).

[0124] Furthermore, we established a tumor model with LLC cells of lung cancer that did not express SCUBE2 or VEGFR2. Co-incubation with SP.B1 mAb did not affect cell growth (data not shown), therefore SP.B1 mAb does not target actual tumor cells; thus, any reduction in tumor growth is attributed to its anti-angiogenic effect. Since SCUBE2 is a co-receptor for VEGFR2 and bevacizumab inhibits tumor angiogenesis, we also investigated whether the combination therapy with SP.B1 and bevacizumab (AVASTIN®) had a cumulative effect on inhibiting lung tumor growth. When the lung tumor reached 50 mm... 3 Therapeutic injections were initiated at that time. Compared with mouse or human IgG treatment, LLC growth was significantly inhibited after SP.B1, bevacizumab, and combination therapy (SP.B1 + bevacizumab). Figure 14 (A to C). Furthermore, compared to SP.B1 (29%) or bevacizumab (40%) alone, SP.B1 + bevacizumab showed greater inhibitory effect on tumor growth (56%). Figure 14C), therefore, these two agents may act on different pathways in tumor angiogenesis. Furthermore, immunohistochemical analysis showed that SP.B1 combined with bevacizumab significantly reduced microvessel density. Figure 14 (D to E). Importantly, compared with SP.B1 and bevacizumab alone, the number of blood vessels was reduced by 49% and 31% in the case of SP.B1 + bevacizumab, respectively. The combination therapy of SP.B1 and bevacizumab has the potential to target tumor angiogenesis in clinical practice.

[0125] Figure 15 This demonstrates the additive antitumor effect of combined anti-SCUBE2 SP.B1 and anti-VEGF bevacizumab (AVASTIN®) treatment on pancreatic ductal carcinoma growth and angiogenesis. Figure 16 illustrates the additive antitumor effect of combined anti-SCUBE2 SP.B1 and anti-VEGF bevacizumab treatment on colorectal adenocarcinoma growth and angiogenesis.

[0126] Table 2

[0127]

[0128] Table 3

[0129]

[0130] Table 4

[0131]

[0132] Table 5

[0133]

[0134] Table 6

[0135]

[0136] In summary, we demonstrate that SCUBE2 (but not SCUBE1 or 3) expression in HUVECs is increased under hypoxia regulated by HIF-1α. This unique hypoxia-inducible effect of SCUBE2 was observed in a blood flow restoration experiment following hindlimb ischemia. SCUBE2 regulation of angiogenesis is not limited to VEGF action. Further research is needed to clarify whether SCUBE2 is involved in postpartum angiogenesis mediated by sonic hedgehog or VE cadherin signaling. Here, our data reveal that SCUBE2, as a novel VEGFR2 co-receptor, regulates VEGF-induced angiogenesis and cell proliferation in ECs by finely modulating VEGFR2-mediated signaling. SCUBE2 may have clinical importance in the pathogenesis of various angiogenesis-related diseases, such as atherosclerosis, diabetic retinopathy, and age-related macular degeneration. In addition to expression in normal organ ECs, SCUBE2 is also highly expressed in ECs of a wide range of human cancers and xenograft tumors (our unpublished data). Although further research is needed to confirm whether endothelial cell SCUBE2 is involved in tumor angiogenesis, SCUBE2 is a potential target molecule for cancer therapy due to its anti-angiogenic effects. Pharmacological blockade of endothelial cell SCUBE2 represents a novel therapeutic strategy applicable to angiogenesis-related diseases. Examples of diseases characterized by abnormal or excessive angiogenesis or those resulting from it are listed in Table 7.

[0137] Table 7

[0138]

[0139] The foregoing description of exemplary embodiments of the present invention is presented for illustrative and descriptive purposes only and is not intended to be exhaustive or to limit the invention to the precise forms disclosed. Many modifications and variations are possible based on the foregoing teachings. References are cited and discussed in the description of the invention, including patents, patent applications, and various publications. Such citations and / or discussions are provided merely to illustrate the description of the invention and do not imply that any such references are "prior art" to the invention described herein. All references cited and discussed in this specification are incorporated herein by reference in their entirety as if each reference had been individually incorporated by reference.

Claims

1. An isolated anti-SCUBE2 monoclonal antibody or antigen-binding fragment comprising an antigen-binding region specifically binding to a target domain located within SCUBE2 of sequence SEQ ID NO: 66 and exhibiting the property of inhibiting VEGF-induced angiogenesis, wherein the target domain is located at EGF-like motifs 4 to 6 within the range of amino acid residue positions 175 to 323 of SEQ ID NO: 66, or a spacer region within the range of amino acid residue positions 441 to 659, or a first cysteine-rich motif within the range of amino acid residue positions 668 to 725, wherein the isolated anti-SCUBE2 monoclonal antibody or antigen-binding fragment comprises a heavy chain variable domain (V H ) and light chain variable domain (V L ), the V H Includes complementary determinant region V H CDR1, V H CDR2 and V H CDR3, and the V L Includes complementary determinant region V L CDR1, V L CDR2 and V L CDR3, where: (i) The V H CDR1, V H CDR2 and V H CDR3 are the amino acid sequences of SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 53, respectively; and the V L CDR1, V L CDR2 and V L CDR3 represents the amino acid sequences of SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 56, respectively, wherein the target domain is located at the first cysteine-rich motif within the range of amino acid residue positions 668 to 725 of SEQ ID NO: 66; or (ii) The V H CDR1, V H CDR2 and V H CDR3 represents the amino acid sequences of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively; and the V L CDR1, V L CDR2 and V L CDR3 represents the amino acid sequences of SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, wherein the target domain is located at EGF-like motifs 4 to 6 within the amino acid residue range of SEQ ID NO: 66, from amino acid residue 175 to 323; or (iii) The V H CDR1, V H CDR2 and V H CDR3 represents the amino acid sequences of SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37, respectively; and the V L CDR1, V L CDR2 and V L CDR3 are the amino acid sequences of SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40, respectively, wherein the target domain is located in the interval region between amino acid residue positions 441 to 659 of SEQ ID NO:

66.

2. The isolated anti-SCUBE2 monoclonal antibody or antigen-binding fragment as described in claim 1, wherein: (a) The V H Contains the amino acid sequence of SEQ ID NO: 49, and the V L Containing the amino acid sequence of SEQ ID NO: 50, wherein the target domain is located at the first cysteine-rich motif in the range of amino acid residue positions 668 to 725 of SEQ ID NO: 66; or (b) The V H Contains the amino acid sequence of SEQ ID NO: 1, and the V L Containing the amino acid sequence of SEQ ID NO: 2, wherein the target domain is located at EGF-like motifs 4 to 6 within the range of amino acid residues 175 to 323 of SEQ ID NO: 66; or (c) The V H Contains the amino acid sequence of SEQ ID NO: 33, and the V L The amino acid sequence comprising SEQ ID NO: 34, wherein the target domain is located in the spacer region between amino acid residue positions 441 to 659 of SEQ ID NO:

66.

3. The isolated anti-SCUBE2 monoclonal antibody or antigen-binding fragment as described in claim 1 is humanized.

4. The isolated anti-SCUBE2 monoclonal antibody or antigen-binding fragment as described in claim 1, selected from the group consisting of: Fv fragment, Fab fragment, F(ab')2 fragment, Fab' fragment, and single-chain antibody variable fragment (scFv).

5. A liposome comprising the isolated anti-SCUBE2 monoclonal antibody or antigen-binding fragment as described in claim 1, wherein the isolated anti-SCUBE2 monoclonal antibody or antigen-binding fragment is encapsulated within the liposome.

6. A single-chain variable fragment (scFv) comprising the heavy chain variable domain (V) of an isolated anti-SCUBE2 monoclonal antibody or antigen-binding fragment as described in claim 1 or 2. H ) and light chain variable domain (V L ) consists of, wherein the heavy chain variable domain (V H ) and light chain variable domain (V L They form a fusion protein.

7. A pharmaceutical composition comprising: (i) the isolated anti-SCUBE2 monoclonal antibody or antigen-binding fragment as described in any one of claims 1 to 4, the liposome as described in claim 5, or the single-chain variable fragment as described in claim 6; and (ii) Bevacizumab.

8. The pharmaceutical composition of claim 7, wherein the anti-SCUBE2 monoclonal antibody is a chimeric antibody.

9. The pharmaceutical composition of claim 7, wherein the anti-SCUBE2 monoclonal antibody is a humanized antibody.

10. Use of the isolated anti-SCUBE2 monoclonal antibody or antigen-binding fragment as described in any one of claims 1 to 4, or the liposome as described in claim 5, in the manufacture of a medicament for the treatment in individuals of need of melanoma, lung, pancreatic, colorectal cancer, and neovascular eye diseases associated with VEGF-induced angiogenesis.

11. The use as described in claim 10, wherein the neovascular ocular disease is a retinal disease.

12. Use of the pharmaceutical composition of any one of claims 7 to 9 in the manufacture of a medicament for treating melanoma, lung, pancreatic, colorectal cancer and neovascular eye diseases associated with VEGF-induced angiogenesis in individuals of need.

13. The use as described in claim 12, wherein the neovascular ocular disease is a retinal disease.

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